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DESK ENCYCLOPEDIA OF
MICROBIOLOGY
SECOND EDITION

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DESK ENCYCLOPEDIA OF
MICROBIOLOGY
SECOND EDITION
EDITOR-IN-CHIEF

MOSELIO SCHAECHTER
San Diego State University
San Diego, CA, USA

AMSTERDAM  BOSTON  HEIDELBERG  LONDON  NEW YORK  OXFORD
PARIS  SAN DIEGO  SAN FRANCISCO  SINGAPORE  SYDNEY  TOKYO
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Forensic Microbiology

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PREFACE

The field of Microbiology encompasses highly diverse life forms – bacteria, archaea, fungi, protists, and viruses. Their
influence on this planet is profound: they play an essential role in the cycles of matter in nature, affect all biological
environments, interact in countless ways with other living beings, and play a crucial role in agriculture and industry.
The field of Microbiology is so broadly encompassing that, of necessity, literature associated with it tends to be
specialized and focused. For that reason, it is difficult to find sources that provide a broad perspective on a wide
range of microbiological topics. This is the aim of The Desk Encyclopedia of Microbiology.
The purpose of this venture is to provide a single reference volume with appeal to microbiologists on all levels and
fields, including those working in industry, health-related institutions, academia, and government. We believe that this
book will be especially helpful for accessing material in areas in which the reader is not a specialist. It is intended to
facilitate preparing lectures, grant applications and reports, and to satisfy curiosity regarding microbiological topics.
The Desk Encyclopedia of Microbiology is principally a synthesis from the comprehensive multi-volume Encyclopedia of
Microbiology. Our intention is to provide affordable and ready access to a large variety of topics within one set of covers.
To this end, we have chosen subjects that, in our opinion, will be of greatest interest to the largest number of readers.
Included are the most general chapters from the Encyclopedia of Microbiology.
The result is a volume where coverage is extensive but not overly long in specific details. We believe this will be a
most appropriate reference for anyone with an interest in the intriguing field of Microbiology.
Moselio Schaechter, 2009

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EDITOR-IN-CHIEF

Professor Schaechter did his graduate work at the University of Kansas and the University of Pennsylvania. He worked
on the biology of rickettsiae at Walter Reed Army Institute of Research, and was a postdoctoral fellow for two years in
Copenhagen, in the laboratory of Ole Maaløe. Professor Schaechter’s research interest concerned various aspects of the
regulation of bacterial growth. He discovered the existence of polyribosomes in bacteria and was among the first to
elucidate aspects of polyribosome metabolism and the role of the cell membrane in DNA synthesis and chromosome
segregation.
Professor Schaechter spent most of his career at Tufts University in Boston, MA, where he chaired the department of
Molecular Biology and Microbiology for 23 years. Since 1995 he has resided in San Diego, California, where he teaches
and continues to write books and a blog, ‘‘Small Things Considered.’’ He has written nine books, including several
textbooks and reference works, and he has served as president of the American Society of Microbiology and in many
advisory capacities to agencies and organizations.

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LIST OF CONTRIBUTORS

S T Abedon J M Bower
The Ohio State University, Mansfield, OH, USA University of Utah, Salt Lake City, UT, USA

G N Agrios P J Brennan
University of Florida, Gainesville, FL, USA Colorado State University, Fort Collins, CO, USA

S-I Aizawa B Budowle
Prefectural University of Hiroshima, Hiroshima, Japan Federal Bureau of Investigation, Laboratory Division,
Quantico, VA, USA
B E Alber
Ohio State University, Columbus, OH, USA L Cegelski
Washington University, School of Medicine, St. Louis,
O Amster-Choder MO, USA
The Hebrew University Medical School, Jerusalem, Israel
Yung-Chi Cheng
A M Arvin Yale University School of Medicine, New Haven, CT, USA
Stanford University School of Medicine, Stanford, CA,
USA
S F Chen
Stanford University School of Medicine, Stanford, CA,
F K Bahrani-Mougeot
USA
Carolinas Medical Center, Charlotte, NC, USA

J T Barbieri P J Christie
Medical College of Wisconsin, Milwaukee, WI, USA University of Texas Medical School at Houston, Houston,
TX, USA
N G Barnaby
Federal Bureau of Investigation, Laboratory Division, S Chugani
Quantico, VA, USA University of Washington, Seattle, WA, USA

A J Bendich J A Coker
University of Washington, Seattle, WA University of Maryland Biotechnology Institute, Baltimore,
MD, USA
G W Blakely
University of Edinburgh, Institute of Cell Biology, J W Costerton
Edinburgh, UK University of Southern California, Los Angeles, CA, USA

M J Blaser P Courvalin
New York University School of Medicine and VA Medical Institut Pasteur, Paris, France
Center, New York, NY, USA
P DasSarma
A Böck University of Maryland Biotechnology Institute, Baltimore,
University of Munich, Munich, Germany MD, USA

W Bodemer S DasSarma
Deutsches Primatenzentrum GmbH (DPZ), Leibniz- University of Maryland Biotechnology Institute, Baltimore,
Institut für Primatenforschung, Goettingen, Germany MD, USA

ix

x List of Contributors

M A de Pedro A Garcı́a-Sastre
CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain Mount Sinai School of Medicine, New York, NY, USA

J W Deming F Garcia-Pichel
University of Washington, Seattle, WA, USA Arizona State University, Tempe, AZ, USA

B K Dhakal A F Gillaspy
University of Utah, Salt Lake City, UT, USA University of Oklahoma Health Sciences Center,
Oklahoma City, OK, USA
B Ding
Ohio State University, Columbus, OH, USA R T Gill
University of Colorado, Boulder, CO, USA
A E Douglas
University of York, York, UK J H Golbeck
The Pennsylvania State University, University Park, PA,
K Drlica USA
University of Medicine and Dentistry of New Jersey,
Newark, NJ, USA E C Gotschlich
The Rockefeller University, New York, NY, USA
P V Dunlap
University of Michigan, Ann Arbor, MI, USA D A Haake
University of California at Los Angeles, Los Angeles, CA,
L Eggeling USA
Institute of Biotechnology, Research Center Jülich, Jülich,
Germany J Handelsman
University of Wisconsin-Madison, Madison, WI, USA
T Egli
P Handke
Swiss Federal Institute for Aquatic Science and
University of Colorado, Boulder, CO, USA
Technology, Dübendorf, Switzerland
T M Henkin
H Eilers
The Ohio State University, Columbus, OH, USA
Georg-August-University Göttingen, Göttingen, Germany
D L Heymann
N C Engleberg
World Health Organization, Geneva, Switzerland
University of Michigan Medical School, Ann Arbor, MI,
USA
J F Holden
University of Massachusetts, Amherst, MA, USA
L E Erickson
Kansas State University, Manhattan, KS, USA
S J Hultgren
Washington University, School of Medicine, St. Louis,
A Espinel-Ingroff MO, USA
Virginia Commonwealth University Medical Center, VA,
USA S Y Hunt
Federal Bureau of Investigation, Laboratory Division,
M Filutowicz Quantico, VA, USA
University of Wisconsin-Madison, Madison, WI, USA
P Hyman
S M Finegold Medcentral College of Nursing, Mansfield, OH, USA
VA Medical Center West Los Angeles and UCLA School
of Medicine, Los Angeles, CA, USA J J Iandolo
University of Oklahoma Health Sciences Center,
P C Fineran Oklahoma City, OK, USA
University of Cambridge, Cambridge, UK
L O Ingram
L S Frost University of Florida, Gainesville, FL, USA
University of Alberta, Edmonton, AB, Canada
B Jagannathan
M Y Galperin The Pennsylvania State University, University Park, PA,
National Institutes of Health, Bethesda, MD, USA USA

List of Contributors xi

L R Jarboe M A Mulvey
University of Florida, Gainesville, FL, USA and Iowa State University of Utah, Salt Lake City, UT, USA
University, Ames, IA, USA
N E Murray
C A Jerez University of Edinburgh, Institute of Cell Biology,
University of Chile and ICDB Millennium Institute, Edinburgh, UK
Santiago, Chile
N Nanninga
P J Johnsen Universiteit van Amsterdam, Amsterdam, The
University of Tromsø, Tromsø, Norway Netherlands

D B Johnson K M Nielsen
Bangor University, Bangor, UK University of Tromsø, Tromsø, Norway

O J Johnson H Nikaido
University of Southern California, Los Angeles, CA, USA University of California, Berkeley, CA, USA

D M Karl E Paintsil
University of Hawaii, Honolulu, HI, USA Yale University School of Medicine, New Haven, CT, USA

S Kaushik
S Parekh
University of California, San Francisco, CA, USA
Dow AgroSciences, Indianopolis, IN, USA

D M Knipe
I T Paulsen
Harvard Medical School, Boston, MA, USA
Macquarie University, Sydney, NSW, Australia
A K Korgaonkar
B Périchon
The University of Texas at Austin, Austin, TX, USA
Institut Pasteur, Paris, France
J G Kuenen
N K Petty
Delft University of Technology, Department of
University of Cambridge, Cambridge, UK
Biotechnology, The Netherlands

J H Leamon P J Piggot
RainDance Technologies, Guilford, CT, USA Temple University School of Medicine, Philadelphia, PA,
USA
R E Lenski
Michigan State University, East Lansing, MI, USA J S Poindexter
Barnard College, Columbia University, NY, USA
R Letelier
Oregon State University, Corvallis, OR, USA J A Poupard
Pharma Institute of Philadelphia, Inc., Philadelphia, PA,
J A Levy USA
University of California, San Francisco, CA, USA
M M Ramsey
P Martin The University of Texas at Austin, Austin, TX, USA
University of Miami Miller School of Medicine, Miami, FL,
USA J L Ray
University of Tromsø, Tromsø, Norway
A Matin
Stanford University School of Medicine, Stanford, CA,
Q Ren
USA
J. Craig Venter Institute, Rockville, MD, USA
L A Miller
GlaxoSmithKline Collegeville, PA, USA W S Reznikoff
Marine Biological Laboratory, Woods Hole, MA, USA
S Morse
Centers for Disease Control and Prevention, Atlanta, GA, M R Rondon
USA University of Wisconsin-Madison, Madison, WI, USA

xii List of Contributors

J M Rothberg J M Struble
The Rothberg Institute for Childhood Diseases, Guilford, University of Colorado, Boulder, CO, USA
CT, USA
J Stülke
R Sá-Leão Georg-August-University Göttingen, Göttingen, Germany
Universidade de Lisboa, Lisboa, Portugal and
Universidade Nova de Lisboa, Oeiras, Portugal S Suerbaum
Hannover Medical School, Hannover, Germany
Z L Sabree
University of Wisconsin-Madison, Madison, WI, USA A Teske
University of North Carolina at Chapel Hill, Chapel Hill,
H Sahm NC, USA
Institute of Biotechnology, Research Center Jülich, Jülich,
Germany A Tomasz
The Rockefeller University, New York, NY, USA
M H Saier Jr.
A X Tran
University of California, San Diego, CA, USA
University of Guelph, Guelph, ON, Canada
G P C Salmond
A Ul-Hassan
University of Cambridge, Cambridge, UK
University of Warwick, Coventry, England, UK
A A Salyers A J Uriel
University of Illinois, Urbana, IL, USA Mount Sinai School of Medicine, New York, NY, USA

P J Sansonetti A L van Lint
Institut Pasteur, Paris, France Harvard Medical School, Boston, MA, USA

M Schaechter G M Walker
San Diego State University, San Diego, CA, USA University of Abertay Dundee, Dundee, Scotland

S R Schmidl E M Wellington
Georg-August-University Göttingen, Göttingen, Germany University of Warwick, Coventry, England, UK

M W Scobey M Whiteley
Carolinas Medical Center, Charlotte, NC, USA The University of Texas at Austin, Austin, TX, USA

K T Shanmugam C Whitfield
University of Florida, Gainesville, FL, USA University of Guelph, Guelph, ON, Canada

N B Shoemaker M J Wiser
University of Illinois, Urbana, IL, USA Michigan State University, East Lansing, MI, USA

C L Smith A Zago
Washington University, School of Medicine, St. Louis, Northwestern University, Chicago, IL, USA
MO, USA
X Zhong
Ohio State University, Columbus, OH, USA
M P Spector
University of South Alabama Mobile, AL, USA

CONTENTS

ACTINOBACTERIA A Ul-Hassan and E M Wellington 1
ADHESION, MICROBIAL L Cegelski, C L Smith and S J Hultgren 20
AGROBACTERIUM AND PLANT CELL TRANSFORMATION P J Christie 29
AMINO ACID PRODUCTION L Eggeling and H Sahm 44
ANTIBIOTIC RESISTANCE B Périchon and P Courvalin 53
ANTIFUNGAL AGENTS A Espinel-Ingroff 65
ANTIVIRAL AGENTS E Paintsil and Yung-Chi Cheng 83
ARCHAEA (OVERVIEW) S DasSarma, J A Coker and P DasSarma 118
AUTOTROPHIC CO2 METABOLISM B E Alber 140

BACILLUS SUBTILIS P J Piggot 154
BACTERIOPHAGE (OVERVIEW) P Hyman and S T Abedon 166
BIOFILMS, MICROBIAL J W Costerton 183
BIOLOGICAL WARFARE J A Poupard and L A Miller 189
BIOLUMINESCENCE, MICROBIAL P V Dunlap 202
BIOREACTORS L E Erickson 219

CAULOBACTER J S Poindexter 225
CELL CYCLES AND DIVISION, BACTERIAL N Nanninga 242
CELL MEMBRANE, PROKARYOTIC M H Saier Jr. 251
CELL STRUCTURE, ORGANIZATION, BACTERIA AND ARCHAEA N Nanninga 266
CHROMOSOME, BACTERIAL K Drlica and A J Bendich 284
CONJUGATION, BACTERIAL L S Frost 294
CONTINUOUS CULTURES (CHEMOSTATS) J G Kuenen and O J Johnson 309
CYANOBACTERIA F Garcia-Pichel 327

DEEP-SEA HYDROTHERMAL VENTS A Teske 346
DNA RESTRICTION AND MODIFICATION G W Blakely and N E Murray 357
DNA SEQUENCING AND GENOMICS J H Leamon and J M Rothberg 369

EMERGING INFECTIONS D L Heymann 383
ENDOSYMBIONTS AND INTRACELLULAR PARASITES A E Douglas 391
ENTEROPATHOGENIC INFECTIONS F K Bahrani-Mougeot, M W Scobey and P J Sansonetti 405
ESCHERICHIA COLI M Schaechter 420
ETHANOL L R Jarboe, K T Shanmugam and L O Ingram 428
EVOLUTION, THEORY AND EXPERIMENTS WITH MICROORGANISMS R E Lenski and M J Wiser 438

xiii

xiv Contents

EXOTOXINS J T Barbieri 453
EXTREMOPHILES: ACIDIC ENVIRONMENTS D B Johnson 463
EXTREMOPHILES: COLD ENVIRONMENTS J W Deming 483
EXTREMOPHILES: HOT ENVIRONMENTS J F Holden 495

FERMENTATION A Böck 515
FLAGELLA, PROKARYOTIC S-I Aizawa 528
FORENSIC MICROBIOLOGY S Y Hunt, N G Barnaby, B Budowle and S Morse 539

GASTROINTESTINAL MICROBIOLOGY IN THE NORMAL HOST S M Finegold 552
GENOME SEQUENCE DATABASES: GENOMIC, CONSTRUCTION OF LIBRARIES J M Struble, P Handke and R T Gill 574
GRAM-NEGATIVE COCCI, PATHOGENIC E C Gotschlich 585

HELICOBACTER PYLORI S Suerbaum and M J Blaser 597
HEPATITIS VIRUSES A J Uriel and P Martin 603
HERPESVIRUSES A L van Lint and D M Knipe 625
HIV/AIDS S Kaushik and J A Levy 640
HORIZONTAL GENE TRANSFER: UPTAKE OF EXTRACELLULAR DNA BY BACTERIA K M Nielsen, J L Ray and
P J Johnsen 663

INFLUENZA A Garcı́a-Sastre 673

LEGIONELLA, BARTONELLA, HAEMOPHILUS N C Engleberg 680
LIPOPOLYSACCHARIDES (ENDOTOXINS) A X Tran and C Whitfield 692

MARINE HABITATS D M Karl and R Letelier 708
METABOLISM, CENTRAL (INTERMEDIARY) M P Spector 728
METAGENOMICS Z L Sabree, M R Rondon and J Handelsman 751
METAL EXTRACTION AND BIOMINING C A Jerez 762
MYCOPLASMA AND SPIROPLASMA J Stülke, H Eilers and S R Schmidl 776

NUTRITION, MICROBIAL T Egli 788

GRAM-NEGATIVE OPPORTUNISTIC ANAEROBES: FRIENDS AND FOES A A Salyers and N B Shoemaker 805
OUTER MEMBRANE, GRAM-NEGATIVE BACTERIA H Nikaido 813

PEPTIDOGLYCAN (MUREIN) M A de Pedro 827
PHOTOSYNTHESIS: MICROBIAL B Jagannathan and J H Golbeck 844
PILI, FIMBRIAE B K Dhakal, J M Bower and M A Mulvey 861
PLANT PATHOGENS AND DISEASE: GENERAL INTRODUCTION G N Agrios 881
PLASMIDS, BACTERIAL M Filutowicz 915
POSTTRANSCRIPTIONAL REGULATION T M Henkin 937
PRIONS W Bodemer 952
PSEUDOMONAS A Zago and S Chugani 971

QUORUM-SENSING IN BACTERIA M M Ramsey, A K Korgaonkar and M Whiteley 987

SENSORY TRANSDUCTION IN BACTERIA M Y Galperin 1005
SPIROCHETES D A Haake 1022
STAPHYLOCOCCUS A F Gillaspy and J J Iandolo 1037

Contents xv

STRAIN IMPROVEMENT S Parekh 1048
STREPTOCOCCUS PNEUMONIAE R Sá-Leão and A Tomasz 1061
STRESS, BACTERIAL: GENERAL AND SPECIFIC A Matin 1075

TRANSCRIPTIONAL REGULATION O Amster-Choder 1091
TRANSDUCTION: HOST DNA TRANSFER BY BACTERIOPHAGES P C Fineran, N K Petty and G P C Salmond 1107
TRANSPORT, SOLUTE Q Ren and I T Paulsen 1121
TRANSPOSABLE ELEMENTS W S Reznikoff 1137
TUBERCULOSIS: MOLECULAR BASIS OF PATHOGENESIS P J Brennan 1147

VACCINES, VIRAL A M Arvin and S F Chen 1154
VIROIDS/VIRUSOIDS B Ding and X Zhong 1163

YEASTS G M Walker 1174

INDEX 1189

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Actinobacteria
A Ul-Hassan and E M Wellington, University of Warwick, Coventry, England, UK
ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Genome Structure and Evolution
Introduction Industrially Important Phenotypes of Actinobacteria
Systematics of Actinobacteria Concluding Remarks
Phylogeny of Actinobacteria Further Reading

Glossary phylogeny Evolutionary history of a group of
co-metabolism The metabolic transformation of a organisms.
substance by one organism to a second substance, pseudogene A gene that has lost its protein-coding
which serves as a primary source of carbon for another ability.
organism. taxonomy Science of classification.
mycelium The mass of hyphae that forms the
vegetative and aerial parts of the streptomycete colony
before sporulation.

Abbreviations MLST multilocus sequence typing
ARDRA Amplified ribosomal DNA Restriction PAHs polycyclic aromatic hydrocarbons
analysis PCDOs Polychlorinated dibenzo-p-dioxins
CGH comparative genomic hybridization PCR-RAPD PCR-randomly Amplified Polymorphic
DF dibenzofuran DNA
DR direct repeat PFGE pulse-field gel electrophoresis
FAME fatty acid methyl ester analysis PyMS pyrolysis mass spectrometry
GITs gastrointestinal tracts RFLP restriction fragment length
HGT horizontal gene transfer polymorphism
hsp heat shock protein rRNA ribosomal RNA
IS insertion sequence TEs transposable elements
ISP International Streptomyces Project VNTRs variable number tandem repeats
LFRFA Low-frequency restriction fragment
analysis

Defining Statement and Actinomyces bovis in 1877 by Carl Otto Harz. Harz
described A. bovis, causing a disease of cattle called
The Actinobacteria form a distinct clade of Gram-positive ‘lumpy jaw’, as having thin filaments that ended in club-
bacteria which contains a large number of genera. This shaped bodies that he considered to be ‘gonidia’ resem-
diverse and important group encompasses key antibiotic- bling those of fungi, hence the name Actinomyces (Latin for
producers, many soil bacteria critical for decomposition ray fungus). In hindsight, though, the ‘gonidia’ were
and resilient species capable of growing in hostile, pol- almost certainly host cells and so the resemblance to
luted environments. A few are medically important typical fungi was false. A number of other microorganisms
pathogens including the causal agent of TB. were isolated, which had some of the same properties and
were thought to belong to the same group as those men-
tioned above, including the causative agents of leprosy
Introduction and tuberculosis. This group of organisms was officially
recognized as Actinomycetales in 1916 and it became appar-
Some of the earliest descriptions of Actinobacteria were ent that they comprised a large heterogeneous group,
those of Streptothrix foersteri in 1875 by Ferdinand Cohn which vary greatly in their physiological and biochemical

1

2 Actinobacteria

properties, though their phylogenetic position as true Systematics of Actinobacteria
bacteria was not established until the 1960s. The
Traditional Phenotypic Analysis
Actinobacteria are now considered to be one of the largest
phyla of the bacterial kingdom. The GC content of these The best-studied members of the Actinobacteria class
organisms ranges from 54% in some corynebacteria to belong to the genus Streptomyces, which was proposed by
more than 70% in streptomycetes. Tropheryma whipplei, Waksman and Henrici in 1943. Members of this genus
the causative agent of Whipple’s disease, has a GC con- have high GC content DNA, being highly oxidative and
tent of 46.3%, which is the lowest so far reported for forming extensive branching substrate and aerial hyphae.
Actinobacteria. They also produce a variety of pigments responsible for
The Actinobacteria are morphologically diverse and can the color of the substrate and aerial hyphae (Bergey’s
range from coccoids (Micrococcus) and rods (Mycobacterium) Manual of Systematic Bacteriology). Streptomyces species are
to branching mycelium (Streptomyces), many of which can prolific producers of antibiotics, and since the discovery
also form spores. Actinobacterial species are ubiquitous in of actinomycin and streptomycin produced by S. antibio-
the environment and can be isolated from both aquatic and ticus and S. griseus respectively during the early 1940s, the
terrestrial habitats. New species of Actinobacteria have been interest in streptomycetes grew very rapidly. The discov-
recovered from a diverse range of environments, including ery of Streptomyces species as rich sources of commercially
medieval wall paintings, desert soil, butter, marine important antibiotics led to new techniques for the culti-
sponges, and radon-containing hot springs. The ability of vation of these organisms. However, due to the lack of
Actinobacteria to inhabit varied environments is due to their standards for the classification and identification of new
ability to produce a variety of extracellular hydrolytic species, the new strains were described based on only
enzymes, particularly in the soil, where they are responsi- small differences in morphological and pigmentation
ble for the breakdown of dead plant, animal and fungal properties. This, along with the belief that one strain
material, thus making them central organisms in carbon
produced only one antibiotic, led to overspeciation of
recycling. Some species can break down more complex,
the genus, resulting in over 3000 species being described
recalcitrant compounds, of which Rhodococcus species are a
by the late 1970s. A number of methods were developed
good example; they can degrade nitro-, di-nitrophenol,
to overcome this problem, with the earliest being based
pyridine, and nitroaromatic compounds.
on only a few subjectively chosen characters focusing
Actinobacteria are well-known for their ability to produce
largely on morphological and pigmentation properties
secondary metabolites, many of which have antibacterial
that were rarely tested under standardized conditions.
and antifungal properties. Of all the antibiotics produced
Subsequently, biochemical, nutritional, and physiological
by Actinobacteria, Streptomyces species are responsible for
characters were included, but as these were only 
80%, with smaller contributions by Micromonospora,
Saccharopolyspora, Amycolatopsis, Actinoplanes, and Nocardia. A applied to selected species they did not necessarily reflect
number of species have developed complex symbiotic rela- the phylogeny of streptomycetes. The International
tionships with plants and insects. A unique relationship has Streptomyces Project (ISP) was established in 1964 with an
been reported between the European bee-wolf wasp, in aim to describe and classify extant type strains of
which the female wasps carry Streptomyces species in Streptomyces using traditional tests under standardized
their antennae and apply them to the brood cells. The conditions. This study resulted in more than 450 species
bacteria are taken up by the larva and colonize the walls of being redescribed, but an attempt to delineate the genera
the cocoon, where they protect it from fungal infestation. was futile.
Streptomyces species also share beneficial relationships with The data collected by the ISP project were used by
plants, with S. lydicus being found to enhance pea root several researchers to develop computer-assisted identifi-
nodulation by Rhizobium species. The best-studied example cation systems. In 1962, Silvestri was the first to apply
of a Streptomyces-eukaryotic relationship is between patho- numerical taxonomy to the genus Streptomyces, where
genic strains such as Streptomyces scabies that cause scab in nearly 200 strains were tested for 100 unit characters.
potatoes, carrots, beets, and other plants. Strains of Frankia This study highlighted the fact that many of the characters
can fix nitrogen and are responsible for the nodulation of used to describe Streptomyces species are highly variable
many dicotyledonous plants. Some members of the and can be erroneously interpreted. Williams and collea-
Actinobacteria are important human and animal plant gues carried out a more comprehensive study of the
pathogens. These include Mycobacterium leprae (leprosy), streptomycetes. The majority of the strains studied were
Mycobacterium tuberculosis (tuberculosis in humans), Myco- from the ISP project, along with soil isolates and repre-
bacterium bovis (tuberculosis in cattle), Corynebacterium sentative strains from 14 other Actinobacterial genera.
diphtheriae (diphtheria), Propionibacterium acnes (acne), and Each strain was tested for 139 unit characters, including
Streptomyces somaliensis, and Actinomadura and Nocardia species spore chain morphology, spore chain ornamentation, color
(actinomycetomas). of aerial mycelium, color of substrate and extracellular

Actinobacteria 3

pigments, production of extracellular enzymes, carbon and taxonomic studies have enabled the description of each
nitrogen source utilization, and resistance and sensitivity species to be made and facilitated the development of new
to certain antibiotics. Strains were clustered according to methods that would allow closely related species to be
the observed similarities and this resulted in 19 major, 40 distinguished.
minor, and 18 single-strain clusters being identified, where
the single-strain clusters were considered as species and
Characterization Based on Chemical
the major clusters were referred to as species groups. The
Constituents
largest species group is cluster 1 S. albidoflavus, containing 
70 strains. This cluster is divided into three subclusters. One of the drawbacks of numerical taxonomy is that it
Subcluster 1A contains strains like S. albidoflavus, S. limosus, measures phenotypic similarities and differences and these
and S. fellus, which form hooked or straight chains of do not always correlate with the genotype and thus only
smooth, yellow, or white spores and are melanin-negative. provide an estimate of relatedness between strains.
Subcluster 1B strains are similar in morphology and pig- Numerical taxonomy has largely been superseded by
mentation to those in subcluster 1A and comprise strains chemo- and molecular taxonomy. Chemotaxonomic meth-
such as S. griseus, S. anulatus, and S. ornatus. Strains in ods have long been used to determine the relatedness
subcluster 1C produce gray, smooth spores and are between organisms. Goodfellow and colleagues used com-
melanin-negative; S. olivaceus, S. griseolus, and S. halstedii parisons of fatty acid methyl ester analysis (FAME) between
represent this group. bacterial genera. Members of the Streptomycetaceae family
As in streptomycete taxonomy, early attempts to differ- have been described as having major amounts of either
entiate mycobacterial species were based on phenotypic LL-diaminopimelic acid (LL-A2pm) (Streptacidiphilus and
properties such as growth rate and pigmentation. With the Streptomyces) or meso-diaminopimelic acid (meso-A2pm)
discovery of new species and the fact that pigment production (Kitasatospora) in their substrate mycelium and LL-A2pm as
can be temperature-dependent and that not all strains of a the major diamino acid in aerial or submerged spores.
species share pigment-producing abilities, classification of Analysis of whole-cell sugar patterns revealed galactose or
mycobacterial species became less reliable. An alternative galactose and rhamnose (Kitasatospora and Streptacidiphilus).
scheme was based on the pathogenic potential of a species, The presence of LL-A2pm and glycine, with the absence of
although this too was constantly changing as pathogenicity characteristic sugars, is typical of the Streptomyces cell wall
was being discovered in more species of Mycobacterium. type, which is characterized as Type I. These members also
The International Working Group on Mycobacterial taxon- contain saturated iso- and anteiso- fatty acids with either
omy was set up in 1967 with an aim to standardize techniques seven or eight hydrogenated menaquinones with nine iso-
used for the classification of these strains. This led to a prene units as the predominate isoprenologues. They
numerical taxonomic approach to study mycobacterial strains. lack mycolic acids but contain the lipids phosphatidylgly-
Closely related strains of Corynebacterium, Rhodococcus, and cerol, phosphatidylethanolamine, phophatidylinositol, and
Nocardia were also included in these studies, which revealed phosphatidylinositol mannosides. Chemotaxonomic charac-
that strains belonging to these four genera form distinct clades teristics of other families of the Actinobacteria class are
significantly separate from each other, with many strains summarized in Table 1.
being reclassified. Strains identified as Corynebacterium equi Curie-point pyrolysis mass spectrometry (PyMS) has
and C. hoagii were found to belong to Rhodococcus, and further been applied for typing Actinobacteria. This method pro-
chemical and genetic analysis confirmed the reclassification as vides a fingerprint of the organisms, which can be used
they conformed to the original description of the genus. quantitatively to analyze differences between strains.
Bacterionema matruchotti was included in this study as its generic Both FAME and PyMS require stringent standardization
position was unresolved, being originally classified as a mem- as changes in culture media and incubation can affect
ber of the Actinomycetaceae family. This strain was found to the results. Many examples exist in the literature where
have all the characteristics of true corynebacteria and was chemotaxonomy has been used successfully for the rapid
renamed Corynebacterium matruchotti. characterization of new species as well as confirming the
Attempts were made to resolve the confusion sur- integrity of existing taxonomic clusters.
rounding the classification of Nocardia asteroides and its
relationship to N. farcinica. One hundred and forty-nine
Genotypic Approaches to Determining
randomly selected Nocardia strains from various sources
Relatedness
were analyzed. This study recovered seven major, nine
minor, and twelve single-strain clusters. Two apparently DNA–DNA hybridization
identical strains of N. farcinica (NTCC 4524 and ATCC Streptomyces coelicolor A3(2) and S. lividans 66 are members
3318) were found to group in two separate clusters. of the cluster 21 Streptomyces violaceoruber species group as
Strain NTCC 4524 clustered with Mycobacterium species, defined by Williams and colleagues, which represents one
whereas 3318 grouped with N. asteroides. Numerical of the well-defined species groups of the genus. Cluster 21

Table 1 Chemotaxonomic characteristics of selected families belonging to the class Actinobacteria

Phospholipid
Family pattern Fatty acids Menaquinone Diamino acid Interpeptide bridge Cell wall sugars

Dermatophilaceae PG, DPG, PI, C16:0, C15:0, C14:0, MK-8(H4) meso-A2pm None
PE, PC C17:0C17:1 C18:1
Dermacoccaceae PG, DPG, PI, C17:0, C18:0, C18:1, MK-8(H4), MK-8(H2), MK-8MK-9, L-lysine L-Ser1-2-D-Glu/ L-Ser1-2-L-Ala-D-
PE, PC i-C17:0i-C17:1, ai-C17:0 MK-10 Glu,D-Glu2, L-Ser-D-Asp
Cellulomonadaceae PG, DPG, PI ai-C15:0, C16:0 MK-9(H4) L-lysine/ornithine L-Thr D-Asp/L-Thr D-Glu,D- Rhamnose
Asp, D-Glu
Micrococcaceae CL, PG, DPG, i-C15:0, ai-C15:0,ai- MK-7(H2), MK-8(H2), MK-9(H2)MK- meso-A2pm, L-lysine, L-alanine Galactosamine,
PI, PL C17:0i-C16:0 8, MK-9, MK-10 Lysine LL-A2pm, glucosamine
ornithine
Corynebacteriaceae DPG, PG, PI, C16:0, C18:1, C18:0 MK-8(H2), MK-9(H2) meso-A2pm Arabinose/
PIM, PG galactose
Micromonosporaceae PC, PE i-C15:0, i-C15:1, C17:1 MK-9(H4), MK-10(H4), MK-10(H6) meso- or LL-A2pm L-glycine Xylose/arabinose
MK-12(H4), MK-12(H6), MK-
12(H8)
Streptosporangiaceae PG, PI, PE i-C16:0, C17:0, i-C18:0 MK9(H4), MK-10(H4) meso-A2pm Madurose,
glucose, ribose,
mannose
Nocardioidaceae PG, DPG, PE, i-C16:0, C16:0, C18:0, MK-8(H4), MK-9(H4), MK-9(H6) LL-A2pm L-glycine Glucose, ribose,
PC PI, PIM C18:1 ai-C15:0, i-C14:0, MK-9(H8), MK-10(H4) mannose,
C18:1 galactose
Intersporangiaceae PI, PIM, PG, i-C15:0, ai-C15:0, i-C14:0, MK-8, MK-8(H4) LL-A2pm or meso- L-glycine Glucose
DPG, PE i-C16:0, C17:0 A2pm

DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PE, phosphatidylethanolamine; PC, phosphatidylcholine; CL, cardiolipin; PL, unknown
phospholipids; MK, menaquinone; meso-A2pm, meso-diaminopimelic acid; LL-A2pm, LL-diaminopimelic acid. Taken from Dworkin et al., 2006.

Actinobacteria 5

strains produce smooth gray spores and diffusible pig- they shared high DNA relatedness values (>80%). S. colom-
ments, which are blue or red depending on the pH biensis was reduced to a synonym of S. lavendulae as it
of the medium. S. coelicolor A3(2) and S. lividans 66 are showed 83% DNA homology to S. lavendulae type strain.
model representatives of this cluster as they have been A number of strains showed <45% relatedness and were
genetically, biochemically, and physiologically character- therefore considered to belong to a different species group.
ized. However, both strains have had a long and confused The DNA relatedness studies discussed above outline the
taxonomic history. importance of evaluating numerical taxonomic clusters
In 1908, Muller isolated an actinomycete as a contami- using taxonomic criteria. This is particularly important in
nant that produced a soluble blue pigment and named it the case of the Actinobacteria that comprise many species. In
Streptothrix coelicolor. In 1916, Waksman and Curtis inde- situations such as this there is a risk of grouping unrelated
pendently isolated an actinomycete culture from the or partially related strains together in clusters using insuffi-
soil, which also produced a red and blue pigment and cient properties.
was named Actinomyces violaceoruber. For a long time Members of the M. tuberculosis complex include the
the two strains were considered to be synonyms and strains M. tuberculosis, M. bovis, M. africanum, M. canettii,
when the Streptomyces genus was established both were M. microti, M. caprae, and M. pinnipedii. DNA–DNA hybri-
named S. coelicolor (Muller) Waksman and Henrici in the dization analysis reveals that this complex comprises a
fourth edition of Bergey’s Manual of Systematic Bacteriology. single species. Subsequent analysis of the genomes has
Subsequently isolated strains that produced blue pigments revealed little sequence variation among the members.
were either considered to be S. coelicolor (Muller) Waksman DNA–DNA hybridization has been used to study Nocardia
and Henrici or as different species altogether. Kutzner and species and this allowed the differentiation between species
Waksman reexamined all the strains that produced a blue, of N. asteroides and other members of the genus.
red, and purple pigment and clarified that the strains iso- Stackebrandt and Fiedler studied 16 strains of Arthrobacter
lated by Muller in 1906 (S. coelicolor (Muller)) and and two strains of Brevibacterium. The DNA of these strains
Waksman and Curtis in 1916 (S. violaceoruber) are distinctly was hybridized to the Arthrobacter type strain A. globiformis.
different species. Analysis of the blue pigments produced This analysis indicates that Arthrobacter species share little
by these strains showed that they are chemically very homology between themselves, with values ranging
different. S. coelicolor (Muller) is a member of cluster 1 strep- from 11 to 55%. However, Brevibacterium sulfureum and
tomycetes, showing similarity to S. griseus and is not a Brevibacterium protophormiae showed relatively high homol-
member of the S. violaceoruber clade. Monson and colleagues ogy to the Arthrobacter strains. Based on these results it was
(1969) confirmed the results of Kutzner and Waksman by recommended that the two Brevibacterium strains be reclas-
DNA–DNA hybridization between S. violaceoruber and sified as Arthrobacter strains. DNA–DNA hybridization is
S. coelicolor (Muller). routinely used to aid the characterization of novel species
DNA–DNA hybridization experiments are an acknowl- of Actinobacteria with many examples in the literature.
edged approach in determining the integrity of taxonomic
clusters defined by numerical taxonomy, and the study of Comparative genomic analysis
Monson and colleagues (1969) was one of the first to use The availability of whole genome sequences has allowed
this technique. Research based on numerical phenetic and the development of microarrays, which have revolutio-
DNA–DNA hybridization data has revealed high levels of nized functional and genomic analysis of organisms.
congruence as the same taxonomic groups are recovered. Microarrays have been extensively used to analyze gene
The cluster 18 S. cyaneus species group is highly hetero- expression and regulation and have application in a num-
geneous, with 9 out of 18 type strains being assigned to two ber of disciplines, including immunology, oncology,
DNA relatedness groups defined at or above the 70% forensic science, pharmacogenomics, and drug discovery.
relatedness level. The use of DNA–DNA hybridization More recently, microarrays have been used to investigate
experiments also demonstrated that the cluster 32 S. viola- the genome-wide analysis between closely related strains,
ceusniger species group encompasses several genomic in particular those of pathogens, with an aim to identify
species when type strains are examined. The DNA related- pathogenicity determinants. This comparative genomic
ness groups were defined at similarity levels >70%, seven hybridization (CGH) microarray analysis has been
of which consisted of single members. The multimembered used to investigate a number of bacterial pathogenic spe-
clusters were equated with S. hygroscopicus and S. violaceus- cies in relation to pathogenesis and host specificity. To
niger. The latter two species were redescribed and a number date, there are 115 actinobacterial genomes that are
of strains carrying different specific names reduced to syno- either completed or at various stages of completion
nyms of the newly redescribed taxa. A high degree of (www.genomesonline.org). This has led to the develop-
heterogeneity in the Streptomyces lavendulae (cluster 61) clus- ment of microarrays for some sequenced genomes,
ter was reported by Labeda and Labeda and Lyons. including those for S. coelicolor, and Mycobacterium and
A number of strains were related at the species level as Corynebacterium species. To date, many of the microarray

6 Actinobacteria

studies have focused on expression analysis of RNA, par- of Arthrobacter and Microbacterium. With the use of
ticularly with the pathogenic strains of Mycobacterium and ARDRA in conjunction with PFGE, strain-specific
Corynebacterium. Microarrays can also be used for DNA– restriction patterns for Arthrobacter and Microbacterium
DNA comparative genomic analysis between closely have been obtained. RFLP of the rRNA gene has been
related strains, as was done for members of the cluster 21 developed as a tool for identification of corynebacterial
S. violaceoruber strains. Using PCR-based microarrays strains. The strains could be grouped based on the band-
Weaver and colleagues were able to identify 1-Mb ampli- ing patterns and generally strains belonging to the same
fication of the terminal regions of a number of laboratory species clustered together. PCR-restriction enzyme
strains of S. coelicolor A3(2) compared to the sequenced pattern analysis (PRA) of Hsp65 (a heat shock protein)
strain M145. The comparison of the cluster 21 streptomy- has been developed as a tool for differentiating
cetes also revealed 14 regions that were present in between Nocardia species. However, when PRA analysis
S. coelicolor M145 but absent in the other members. These was applied to Nocardia species, the same banding patterns
regions encoded genes for biosynthesis of secondary meta- were recovered, demonstrating this technique not to be as
bolites and heavy metal resistance. All 14 regions were useful for identification or differentiation of these species.
associated with transposon and insertion sequence (IS) Sequence analysis of the Hsp65 gene displays sufficient
elements and the fact they showed a much lower GC polymorphic sites to allow identification. For methods
content than the rest of the Streptomyces chromosome which generate banding patterns that are subsequently
strongly suggests these regions to have been acquired by used for identification, it is important to stress that a
horizontal gene transfer (HGT) by S. coelicolor M145. different banding pattern does not necessarily represent
Ward and Goodfellow have reported a core set of genes a new species. The differences may be attributed to gen-
from the comparative analysis of the Cluster 21 strains. ome rearrangements such as amplifications or deletions,
These genes included those already identified as house- which occur frequently.
keeping genes and also those for some secondary PCR-randomly amplified polymorphic DNA (PCR-
metabolites; for example, genes for biosynthesis of acti- RAPD) involves PCR of the genome with an arbitrary set
norhodin were found to be conserved among the cluster 21 of primers to generate a characteristic fingerprint profile.
strains. Ul-Hassan (2006) used oligo-based microarrays for The advantage of this method is that prior knowledge of
the comparative genomic analysis of soil strains identified the chromosomal sequence is not necessary, although
as S. violaceoruber. The results revealed the strains to have stringent standardization is required. The drawback of
undergone extensive deletions that could be correlated this technique is that it is highly sensitive and variability
with their observed phenotypes. CGH microarray has in banding patterns may be observed depending on the
been used to investigate the molecular taxonomy of a type of reaction mixture, primers, and concentration of
number of organisms, including Saccharomyces and target DNA. This was used to analyze the relationship
Yersinia species and Clostridia. As more actinobacterial between S. lavendulae and Streptomyces virginiae, which
genomes are becoming available there is the potential to were reported by Williams and colleagues to be syno-
develop CGH microarray as a taxonomic tool to study nyms. Although consistent results were obtained when
environmentally and clinically important Actinobacteria. comparing to DNA–DNA hybridization, LFRFA, and
biochemical properties, the interspecific relationship of
Restriction digestion analysis of total S. lavendulae and S. virginiae remained unresolved.
chromosomal DNA Analysis of the genomes of members of the M. tubercu-
Restriction digestion analysis of total chromosomal DNA losis complex demonstrates a number of repeat sequences,
provides a fingerprint of the organisms being analyzed. of which the best studied is the direct repeat (DR) region.
Low-frequency restriction fragment analysis (LFRFA), This region consists of DR sequences (36 bp) interspersed
restriction fragment length polymorphism (RFLP), and with spacer sequences (34–41 bp), collectively termed
pulse-field gel electrophoresis (PFGE) have all been used direct variable repeat sequences. Spoligotyping was devel-
to provide an indication of relatedness between strains. oped as a tool for analyzing the structure of the DR region.
However, the results of these methods can be misleading Spoligotyping patterns are produced by hybridization of
if the strains have undergone large chromosomal sample DNA to oligonucleotides based on the specific
deletions or amplifications. Amplified ribosomal DNA sequences in the DR region. An international spoligotype
restriction analysis (ARDRA) requires the amplification database was set up and now consists of 39 000 entries
of parts of the rRNA operon, including part of the 16S from research groups worldwide. The spoligotype pat-
rRNA, the 16S–23S rRNA spacer region, and part of the terns can be aligned, enabling researchers to group
23S rDNA. The amplified PCR products are subjected to isolates based on these alignments into clades or strain
restriction enzyme digestion and electrophoresis, provid- families. Filliol and colleagues and Molhuizen and collea-
ing specific banding patterns for the strains being gues (1998) were able to identify distinct spoligotype
analyzed. ARDRA has been used to differentiate strains patterns for nearly all of the defined M. tuberculosis

Actinobacteria 7

complex strains. However it could only provide limited similarities of the 16S rRNA gene, Wellington and col-
discrimination of M. bovis strains. Analysis of multiple leagues included Kitosatospora into the Streptomyces genus.
genomic regions that contain variable number tandem This was contested by Zhang and colleagues, who
repeats (VNTRs) of different families of genetic elements demonstrated that strains of Kitosatospora always form a
has been proposed as an alternative tool to examine stable monophyletic clade away from the streptomycetes
M. bovis strains. The studies of Roring and colleagues when the entire 16S rDNA sequences were used.
and Allix and colleagues have shown VNTR to be more Streptomycete-specific primers have also been designed
discriminatory than both spoligotyping and RFLP for for the 23S and 5S rRNA gene. Sequence analysis of the
M. bovis. 5S rDNA gene sequences were used to confirm that
Chainia, Elytrosporangium, Kitasatoa, Microellobosporia, and
Streptoverticillium as members of the Streptomyces genus.
Phylogeny of Actinobacteria Stackebrandt and colleagues proposed a new hierarchic
system of classification for Actinobacteria, which was based
Molecular Analysis of 16S rRNA Sequences
exclusively on 16S rRNA-rDNA sequences.
Molecular methods are now used together with numer- A comprehensive study on the relationships of members
ical and chemotaxonomic techniques to improve the of the Actinobacteria class has not been done prior to this
understanding of species relatedness. Woese and Fox work. 16S rDNA sequences from representative strains from
used molecular systematic analysis of ribosomal RNA each genus of Actinobacteria were used to construct a phylo-
(rRNA) molecules to provide an evolutionary classifica- genetic tree (Figure 1). It is important to note that, as an up-
tion of organisms. All 16S rRNAs have conserved to-date authoritative list of validly described Actinobacteria is
primary structures, which allowed Edwards and collea- not available (e.g., Bergey’s Manual of Systematic Bacteriology),
gues, to design universal primers to amplify the entire orders, families, and genera that have been identified were
16S rRNA gene. Analysis of the 16S rRNA gene taken from those available in the public database. Four
revealed regions that are genus-specific and more vari- subclasses were recognized: Actinobacteridae, Acidimicrobidae,
able regions that can be used to infer relationships at a Coriobacteridae, and Rubrobacteridae, with Actinobacteridae
lower taxonomic order. Stackebrandt and colleagues being the largest, consisting of 11 suborders and 43 families
identified three regions within the 16S rRNA gene that (Table 2). When generating the tree, care was taken to use
show variation: -region (nt 982–998) (S. ambofaciens sequences of good quality and the cutoff for the length of the
nomenclature) and -region (nt 1102–1122), which can sequence was 1400 bp. Any sequences below this were not
be used to resolve species to the genus level. The most included in the phylogenetic analysis. Sequences containing
variable region is the -region (nt 150–200), which is ambiguous bases were also excluded from the study. The
species-specific. Inferred relationships based on gene phylogenetic tree was constructed with PHYLIP, the neigh-
sequences are subject to a number of assumptions with bor-joining method using the kimura-2-parameter model of
the major condition being that the gene analyzed must sequence evolution. Bootstrap was used to calculate the
not be subject to gene transfer. It is well-known that confidence in the groupings of strains and the significant
bacteria contain multiple copies of the 16S rRNA gene bootstrap values (>80) are indicated on the nodes
but transfer of 16S rRNA has not been determined (Figure 1).
definitively. It has been suggested that high levels of The phylogenetic tree shows clear, distinct groupings
similarity can facilitate recombination between closely of strains of particular genera in their respective families
related species, resulting in strains containing chimeric that were expected; however, some irregularities can be
molecules of 16S rRNA. Phylogenetic analysis using 16S observed. The genus Amycolatopsis was proposed by
rRNA genes can pose problem due to intraspecific var- Lechevalier and colleagues and, based on phylogenetic
iation and intragenomic heterogeneity. Another problem and chemotaxonomic properties, this genus is placed
with using 16S rRNA genes is that it is a slowly evolving within the family Pseudonocardiaceae. The genus cur-
gene, making it more difficult to resolve the relationships rently contains ten validated species that include
between strains to the species level. Katakoa and collea- A. fastidiosa. However, Figure 1 clearly demonstrated
gues was able to examine the -region of a number of A. fastidiosa to group with Actinokineospora diospyrosa,
streptomycete strains and, although being too variable which belongs to the family Actinosynnemataceae, with
for determining generic relationships, the inter- and a significant bootstrap value. In previous phylogenetic
intraspecies relationships were resolved. When Hain studies of the Amycolatopsis genus, A. fastidiosa consistently
and colleagues investigated the S. albidoflavus group it grouped outside other members of Amycolatopsis. In these
was apparent that use of the 16S rDNA sequences were studies only Amycolatopsis strains have been used occasion-
useful for species delimitation but not strain differentia- ally alongside Pseudonocardia, but members of the closely
tion. The 16S–23S intergenic region was better in related family Actinosynnemataceae were excluded. It is
determining intraspecific relationships. Based on the recommended that further work be done to establish to

8 Actinobacteria

Family

Bacillus subtilis
Arthrobacter viscosus Conexibacteraceae, Patulibacteraceae
Rubrobacter radiotolerans Robrobacteraceae, Solirubrobacteraceae,
Thermoleophilum minutum Thermoleophilaceae
Patulibacter minatonensis
82 Conexibacter woesei
100
85 Solirubrobacter pauli
Olsenella profusa
98 Atopobium fossor
86 Atopobium rimae
Collinsella intestinalis
Coriobacterium glomerans Coriobacteriaceae
100 Slackia faecicanis
Cryptobacterium curtum
Eggerthella hongkongensis
Denitrobacterium detoxificans
Acidimicrobium ferrooxidans Acidimicrobiaceae
Bifidobacterium bifidum
Bifidobacterium breve
100 Bifidobacterium longum
Bifidobacterium gallinarum
Bifidobacterium coryneforme
100 100 Bifidobacteriaceae
Bifidobacterium aerophilum
Metascardovia tsurumii
Gardnerella vaginalis
Alloscardovia omnicolens
Parascardovia denticolens
99 Scardovia inopinata
Catenulispora acidiphila Actinospicaceae,
Actinospica acidiphila Catenulisporaceae
98
100 Actinospica robiniae
Corynebacterium glutamicum
94 Corynebacterium mastitidis Corynebacteriaceae
100 Corynebacterium diphtheriae
100 Corynebacterium ulcerans
Dietzia cinnamea
100 Dietzia daqingensis (smooth)
100 Dietzia daqingensis (rough) Dietziaceae
96 Dietzia kunjamensis
100 Dietzia maris
Tsukamurella tyrosinosolvens
Tsukamurella pseudospumae
100 Tsukamurella poriferae Tsukamurellaceae
Tsukamurella spp
Tsukamurella pulmonis
Williamsia muralis
83 100 Gordonia westfalica
Gordonia hydrophobica
99 Gordonia sihwensis
97
Gordonia otitidis
100 Gordonia rhizosphera Williamsiaceae, Gordoniaceae
Williamsia serinedens
96 Williamsia deligens
Williamsia maris
Millisia brevis
Skermania piniformis
Mycobacterium paratuberculosis
Mycobacterium leprae
100 Mycobacterium tuberculosis
Mycobacterium bovis Mycobacteriaceae
100 Mycobacterium africanum
100 Mycobacterium microti
Mycobacterium fortuitum
83 93 Mycobacterium smegmatis
Nocardia africana
Nocardia beijingensis
Nocardia asteroides
100 Nocardia flavorosea
Rhodococcus kunmingensis Nocardiaceae
93 Rhodococcus equi
Rhodococcus jostii
Rhodococcus corynebacterioides
Rhodococcus phenolicus
Segniliparus rotundus
Segnilisporaceae
100 Segniliparus rugosus
80 Actinokineospora diospyrosa
Amycolatopsis fastidiosa
97 Saccharothrix longispora Actinosynnemataceae
Actinosynnema mirum
Lechevalieria flava
83 Lentzea albida
Kibdelosporangium albatum
Kutzneria kofuensis
Streptoalloteichus hindustanus
Crossiella cryophila
Goodfellowia coeruleoviolacea
Actinoalloteichus alkalophilus
100 Actinoalloteichus cyanogriseus
Saccharopolyspora flava
98 Actinostreptospora chiangmaiensis
Pseudonocardia alni Pseudonocardiaceae
96 Pseudonocardia sulfidoxydans
Thermocrispum municipale
99 Amycolatopsis orientalis
Amycolatopsis sacchari
Prauserella alba
90 Saccharomonospora azurea
Actinopolyspora halophila
100 Actinopolyspora salina

Figure 1 (Continued)

Actinobacteria 9 Jiangella gansuensis Actinocorallia glomerata Spirillospora albida 100 Parvopolyspora pallida 82 Actinomadura citrea Thermomonosporaceae 81 Actinomadura livida Thermomonospora chromogena Thermobispora bispora Thermopolyspora flexuosa Microtetraspora fusca 92 88 Microbispora corallina 83 Planotetraspora mira 80 Herbidospora cretacea Acrocarpospora corrugata Astrosporangium hypotensionis 87 Sphaerosporangium melleus Streptosporangiaceae Clavisporangium rectum Nonomuraea rubra Planobispora longispora Planomonospora alba Streptosporangium violaceochromogenes Streptosporangium brasiliensis Streptosporangium longisporum Thermobifida fusca Haloactinomyces albus 100 Prauseria hordei 100 Nocardiopsis Nocardiopsis tropica trehalosi Nocardiopsaceae Nocardiopsis chromatogenes Streptomonospora alba Actinocatenispora sera Glycomyces harbinensis Glycomyces illinoisensis 100 Glycomyces algeriensis Glycomyces lechevalierae 100 Glycomyces rutgersenis Glycomycetaceae 100 Glycomyces arizonensis 100 Glycomyces tenuis 100 Stakebrandtia flavoalba 89 Stakebrandtia nassauensis Longispora albida Pilimelia anulata Actinoplanes cyaneus Actinoplanes lobatus Catenuloplanes caeruleus 96 Catenuloplanes crispus Actinoplanes violaceus Couchioplanes caeruleus Krasilnikovia cinnamoneum Asanoa ferruginea Asanoa iriomotensis Spirilliplanes yamanashiensis Polymorphospora ruber Verrucosispora gifhornensis 100 Verrucosispora shenzhensis Micromonosporaceae Micromonospora echinospora 97 Micromonospora globosa 92 Micromonospora fulvopurpurea 93 Micromonospora aquatica Salinispora pacifica 100 Salinispora arenicola Dactylosporangium fulvum 100 Dactylosporangium roseum Virgisporangium aurantiacum 100 Virgisporangium ochraceum Catellatospora citrea 100 Catellatospora coxensis 90 Luedemannella flava 100 Luedemannella helvata Actinopolymorpha singaporensis 97 Kribbella lupini Aeromicrobioides flava Aeromicrobium fastidiosum Marmoricola aurantiacus 94 Nocardioides oleivorans 94 Nocardioides simplex Nocardioidaceae 94 Nocardioides albus 100 Nocardioides luteus 81 Friedmanniella capsulata 90 Microlunatus phosphovorus Micropruina glycogenica 83 83 Propionicicella superfundia 95 Propionicimonas paludicola Propioniferax innocua Propionibacterium acnes Tessaracoccus bendigoensis Brooklawnia cerclae Propionibacteriaceae 83 Propionimicrobium lymphophilum Aestuariimicrobium kwangyangensis Luteococcus japonicus Parastreptomyces abscessus Streptacidiphilus carbonis Kitasatosporia azatica 91 Streptomyces aureofaciens 100 Streptomyces roseus Streptomyces avermitilis 94 Streptoallomorpha polyantibiotica Streptomyces fradiae Streptomyces platensis Streptomycetaceae 100 Streptomyces rimosus Streptomyces lavendulae Streptomyces coelicolor Streptomyces albidoflavus 82 Streptomyces griseus Streptomyces glaucescens Streptomyces albus Streptomyces cyaneus Streptomyces violaceoruber Acidothermus cellulolyticus Frankia spp. 2215 Frankia alni Sporichthya brevicatena Sporichthyaceae 100 Sporichthya polymorpha Cryptosporangium arvum Cryptosporangium minutisporangium Kineosporiaceae 100 Cryptosporangium aurantiacum Cryptosporangium japonicum 91 Actinotelluria brasiliensis Geodermatophilus obscurus 83 Blastococcus saxobsidens 99 Blastococcus aggregatus Blastococcus jejuensis Geodermatophilaceae 86 93 Modestobacter multiseptatus Modestobacter versicolor 99 Modestobacter spp. CNJ793 99 Modestobacter spp. Frankiaceae 93 96 Frankia spp. 49136 Acidothermaceae. Ellin164 Nakamurella multipartita 100 Solicoccus flavidus Nakamurellaceae Figure 1 (Continued) .

fastidiosa belongs. Serinicoccus marinus Ornithinimicrobium caseinolyticus Intersporangiaceae 90 Kytococcus schroeteri Dermacoccaceae 100 Kytococcus sedentarius 95 Isoptericola variabilis Isoptericola dokdonensis Xylanibacterium ulmi 85 89 Xylanimicrobium pachnodae 97 Xylanimonas cellulosilytica Myceligenerans xiligouense Promicromonosporacea Promicromonospora citrea 100 Promicromonospora sukumoe Cellulosimicrobium terreum 83 Cellulosimicrobium cellulans 100 Cellulosimicrobium funkei Cellulomonas fermentans Cellulomonas uda 85 Cellulomonas biazotea 100 Cellulomonas chitinilytica Cellulomonas parahominis Cellulomonadaceae Oerskovia enterophila Oerskovia turbata 100 Oerskovia paurometabola 97 Oerskovia jenensis Rarobacter faecitabidus Rarobacteriaceae 100 Rarobacter incanus Sanguibacter suarezii Sanguibacteraceae 100 Sanguibacter keddieii Arthrobacter casei Citricoccus alkalitolerans 100 Citricoccus muralis Micrococcus lylae Micrococcus antarcticus 100 Micrococcus luteus 93 Micrococcus indicus Liuella qinghaiensis Arthrobacter globiformis Micrococcaceae 100 Arhtrobacter ramosus Arthrobacter agilis Acariicomes phytoseiuli Arthrobacter cumminsii Nesterenkonia halobia Nesterenkonia lutea 100 Nesterenkonia halotolerans 100 Nesterenkonia sandarakina 99 100 Yania flava Yaniaceae Yania halotolerans Kocuria marina Kocuria polaris 100 Kocuria rosea 88 Kocuria halotolerans Micrococcaceae Rothia amarae Rothia nasimurium 100 Rothia dentocariosa 100 Rothia mucilaginosa Dermabacter hominis Brachybacterium rhamnosum 100 Brachybacterium muris 93 Brachybacterium arcticum Dermabacteraceae Brachybacterium alimentarium 98 Brachybacterium sacelli Brachybacterium conglomeratum Brachybacterium faecium Figure 1 (Continued) which family A. The genus Parvopolyspora was methods. since Parvopolyspora gues showed P. however. pallida to be closely related to Actinomadura pallida has not been described efficiently according to the with the latter study proposing P. and DNA–DNA hybridization should be included. Using 16S rRNA gene . morpho- Amycolatopsis strains members of Actinosynnemataceae logical. pallida to be transferred rules of Bacterial Nomenclature. Itoh and colleagues and Miyadoh and collea- described by Liu and Lian. 100 Arsenicicoccus bolidensis Ornithinicoccus hortensis Oryzihumus leptocrescens Terrabacter aerolatum Terrabacter terrae 99 Terrabacter tumescens 82 Teracoccus luteus Intersporangium calvum 81 Janibacter anophelis Janibacter limosus 85 84 Janibacter terrae Janibacter marinus Intersporangiaceae Janibacter melonis 100 Knoellia sinensis 99 Knoellia subterranea Nostocoida limicola 80 Tetrasphaera jenkinsii 80 Tetrasphaera australiensis Tetrasphaera nostocoidensis 99 Tetrasphaera japonica Tetrasphaera elongata Lochheadia duodecas Nostocoida aromativora 100 Phycicoccus jejuensis Kribbia dieselivorans Dermatophilus spp. Dermatophilus congolensis Dermatophilaceae Dermacoccus nishinomiyaensis Demetria terragena Dermacoccaceae 86 Dermacoccus spp. physiological. Using chemotaxonomic. and in future studies of a valid genus/species.10 Actinobacteria Kineococcus aurantiacus 93 Kineosporia rhamnosa Kineosporia rhizophila Kineosporiaceae 100 Kineosporia succinea 100 Kineosporia aurantiaca 83 Quadrisphaera granulorum Kineococcus marinus Brevibacterium albus Brevibacterium samyangensis Bogoriellaceae 100 Bravibacterium lutescens 86 Brevibacterium aureum 99 Brevibacterium epidermidis 100 Dermatophilus chelonae Dermabacter spp. it is not considered to be to the genus Actinomadura.

Salana multivorans 0. pallida. The scale bar indicates 10% nucleotide dissimilarity (10% nucleotide substitutions per 100 nucleotides). further made to accommodate P. . In the current emended description of the genus Actinomadura to be study. Actinobacteria 11 Actinobaculum spp. Numbers on the branches indicate the percentage bootstrap value of 100 replicates. P. and calls for an that favored the transfer to Actinomadura.1 Figure 1 Phylogenetic analysis of species belonging to the class Actinobacteria using the near entire 16S rDNA gene sequences. pallida groups with Actinomadura strains. The phylogenetic tree was construced using the neighbour-joining method and Kimura-2-parameter of sequence evolution. Tamura and Hatano confirmed previous work confirming earlier work on P. pallida as Actinomadura pallida. sequences. 87 Actinobaculum massiliae Actinobaculum suis 95 Actinobaculum schaalii Actinobaculum urinale Arcanobacterium bonasi Arcanobacterium hippocoleae 92 Arcanobacterium haemolyticum Actinomycetaceae Actinomyces nasicola Actinomyces bowdenii 99 Actinomyces denticolens Actinomyces ruminocola 82 98 Varibaculum cambriense 96 Mobiluncus mulieris Mobiluncus curtisii 100 Jonesia denitrificans 100 Mobiluncus holmesii Jonesia quinghaiensis Jonesiaceae 100 Zimmermannella alba Pseudoclavibacter helvolus 95 Zimmermannella faecalis Gulosibacter molinativorax 100 Brevibacterium equis 96 Zimmermannella bifida Tropheryma whipplei Twist 100 Tropheryma whipplei TW08/27 82 Leucobacter alluvii Leucobacter komagatae Leucobacter albus 99 Leucobacter aridicollis 100 Leucobacter solipictus Leucobacter luti Agromyces rhizospherae Agromyces albus Agromyces fucosus 83 Agromyces subbeticus 100 Agromyces luteolus Agromyces bracchium Agromyces ulmi 83 Agromyces neolithicus Agromyces terreus Agromyces allium 100 Agromyces lapidis Frigoribacterium faeni Subtercola frigoramans Subtercola boreus Agreia bicolorata 80 100 Agreia pratensis Leifsonia aurea 100 97 Leifsonia rubra Rhodoglobus vestalii Leifsonia poae Leifsonia shinshuensis 99 97 Leifsonia xyli Leifsonia aquatica 100 Leifsonia naganoensis Agrococcus casei Agrococcus lahaulensis Agrococcus baldri 84 Agrococcus citreus 100 Agrococcus jenensis Cryobacterium psychrophilum Microbacteriaceae 96 Cryocola antiquus 100 Labedella kawkjii Parkia alkaliphila Salinibacterium aquaticus 86 100 Microcella alkaliphila Microcella putealis Clavibacter insidiosus Clavibacter sepedonicus Clavibacter nebraskensis 100 Clavibacter michiganensis Clavibacter tessellarius Crustibacterium reblochoni Okibacterium fritillariae Curtobacterium fangii Plantibacter elymi Plantibacter flavus 100 Plantibacter cousiniae Plantibacter auratus Plantibacter agrosticola 100 Mycetocola tolaasinivorans Mycetocola lacteus 100 Mycetocola saprophilus Microbacterium indica Microbacterium halotolerans 80 Microbacterium aurum Microbacterium aerolatum Microbacterium liquefaciens 83 100 Microbacterium oxydans Microbacterium chocolatum Microbacterium thalassium 90 Microbacterium flavescens Rathayibacter toxicus Rathayibacter tritici 91 Rathayibacter rathayi 100 Rathayibacter festucae Rathayibacter caricis 95 Curtobacterium albidum Curtobacterium citreum 100 Curtobacterium ammoniigenes Curtobacterium herbarum Curtobacterium luteum Curtobacterium pusillum Bogoriellaceae Bogoriella caseolytica 91 Georgenia muralis 100 Georgenia ruanii Beutenbergia cavernosa Beutenbergiaceae 100 Beutenbergia spp.

so re-examination of these strains is T. M. which belongs to advised to determine their exact positions within the three .12 Actinobacteria Table 2 Members comprising the class Actinobacteria Class Subclass Order Suborder Family Actinobacteria Acidimicrobidae Acidimicrobiales Acidimicrobineae Acidimicrobiaceae (1) Actinobacteridae Actinomycetales Actinomycineae Actinomyceaceae (5) Catenulisporineae Actinospicaceae (1) Catenulisporaceae (1) Corynebacterineae Corynebacteriaceae (1) Dieziaceae (1) Gordoniaceae (3) Mycobacteriaceae (1) Nocardiaceae (3) Segniliparaceae (1) Tsukamurellaceae (1) Williamsiaceae (1) Frankineae Acidothermaceae (1) Frankiaceae (1) Geodermatophilaceae (4) Kineosporiaceae (3) Nakamurellaceae (1) Sporichthyaceae (1) Glycomycineae Glycomycetaceae (2) Micrococcineae Beutenbergiaceae (3) Bogoriellaceae (1) Brevibacteriaceae (1) Cellulomonadaceae (3) Dermabacteraceae (2) Dermacoccaceae (3) Dermatophilaceae (2) Intrasporangiaceae (14) Jonesiaceae (1) Microbacteriaceae (24) Micrococcaceae (10) Promicromonosporaceae (7) Rarobacteraceae (1) Sanguibacteraceae (1) Yaniaceae (1) Micromonosporineae Micromonosporaceae (18) Propionibacterineae Nocardioidaceae (11) Propionibacteriaceae (8) Pseudonocardineae Actinosynnemaaceae (6) Pseudonocardiaceae (17) Streptomycineae Streptomycetaceae (8) Streptosporangineae Nocardiopsaceae (4) Streptosporangiaceae (13) Thermomonosporaceae (4) Bifidobacteriales Bifidobacteriaceae (3) Coriobacteridae Coriobacteriales Coriobacterineae Coriobacteriaceae (8) Rubrobacteridae Rubrobacterales Rubrobacterineae Conexibacteraceae (1) Paulibacteraceae (1) Rubrobacteraceae (1) Solirubrobacteraceae (1) Thermoleophilaceae (1) Reproduced from sequences available from NCBI. family Streptosporangiaceae. Numbers in parenthesis indicate the number of genera in each family. flexuosa is Thermomonospora chromogena. bispora and chemotaxonomic traits. yet in Figure 1 form a separate group from the Pseudonocardiaceae. bispora is next to Thermopolyspora flexuosa within the Thermomonosporaceae. The three strains also share many Pseudonocardiaceae. These three strains member of the Pseudonocardiaceae family. distinctly separate from being closer to the last. Grouping outside both M. Microbispora bispora is currently recognized as a the family Thermomonosporaceae. and Streptosporangiaceae families.

5%. Chater and further resolution of the intrageneric relationships Chandra constructed a phylogenetic tree using the 16S between the closely related species. with a high boot. whipplei has been under strains formed a tight monophyletic cluster in the partial some considerable debate. recA. The last accommodates two genera. clpC. but it was more difficult to needs to be studied again to clarify their taxonomic do so between subspecies. T. and trpB. thus providing better gues set out to analyze whether large linear chromosomes resolution of closely related strains. Phylogenetic histories for the housekeeping Only recently. than 16S rRNA. and which has been used in molecular epidemiology for phy- Demetria make up the family Dermacoccaceae and 14 gen. sodA. The The phylogenetic position of T. whipplei. In many of these studies.7 to and Dermabacter. In this study. lividans and has subsequently been seen possibility of HGT. trpB. for the genotypic characterization between closely related ceae. whipplei grouped closer to S. gyrB. most species within the genus. Arsenicicoccus bolidensis. Brachybacterium similarity of the 16S rDNA gene ranging from 87. although not placed within it. with the 16S sely related species. La Scola and colleagues gave a detailed genes were generated and the relative separation in phy- description of the Whipple’s disease bacillus with regard to logenetic tree space was examined using the Robinson– its cultivation and morphology. Linearity of . who included two strains of T. These results were rRNA genes of sequenced Actinobacterial genomes. therefore reducing the determined in S. strains from these families have been misclassified. violaceoruber cluster are member of the Microbacteriaceae family. For MLST analysis the genes positions. The genes chosen for phylogenetic analysis must fulfill certain criteria in that they must be essential The linearity of the streptomycete chromosome was first and distributed among the genera. dnaG. Redenbach and collea- rate higher than the 16S rRNA gene. This has largely been due to the 16S rRNA gene tree. secA1. T. the use of placed it among the Nocardiopsaceae to a strain of alternative genes resolved the relationships between clo- Nocardiopsis with a bootstrap value of 100. as indicated by highly homogeneous. whipplei used the entire 16S rRNA sequence to study type mem- grouped with Leifsonia and also showed a close relationship to bers of the S. species using the allelic mismatch of a number of house- philaceae. recA. whipplei was placed as a topology of the trees and grouping of the strains were deep-branching strain of the Cellulomonadaceae family. and recA show a faster evolutionary rate sequence data as it was not possible to study any chemotaxo. which in agreement with those of Duangmal and colleagues. and therefore being good choices for nomic traits. Examples of the were a distinct feature of Streptomyces species. and Dermato. Actinobacteria 13 families. and trpB lack of chemotaxonomic studies as this organism has been analysis correlated with the 16S rRNA analysis as the difficult to culture. Initially. The new genus and species Foulds distance metric. Figure 1 demonstrates that T. purF. violaceoruber strains with recA. Kytococcus. MLST is a method Attention is also drawn to the families Dermabactera. as well as soil isolates. This is a powerful tool and Kineosphaera. Analysis using strap value. but is not as close to the Streptomyces The study of Ul-Hassan was similar to a multilocus genus as shown in their tree. rpoC. whipplei is a and showed that members of the S. Analysis of the Practical Streptomyces Handbook. with some strains possessing identical sequences. They must also have an evolutionary in other members of the genera. the 16S rRNA sequences allowed the discrimination of in particular Dermatophilaceae and Dermacoccaceae. Ul-Hassan examined the rDNA sequences of the two strains showing 99% sequence S. trpB. The genera Dermacoccus. Analysis of its 16S rRNA gene has hsp65. gyrB. violaceoruber cluster. dnaB. dnaJi. Genes A number of studies have made use of other housekeeping genes to support the phylogeny derived from the 16S Genome Structure and Evolution rRNA gene. Dermatophilus keeping genes (usually seven). and xlp were used. whipplei was based solely on the 16S rRNA gene genes gyrB. Prauseria hordei is also recognized as a member of housekeeping genes that have been used in conjunction Pseudonocardiaceae. identical. Streptomyces strains. gyrB. though this strain has not been validly with the 16S rRNA gene include recA. sequence typing (MLST) approach. It is evident from Figure 1 that some 99. The classification of members of these families. logenetic analysis of bacterial pathogens. Ventura and colleagues developed MLST to Intrasporangiaceae. This confirmed that the name of T. described in the literature. rpoB. study strains of the Bifidobacteria genus. The results of the gyrB. For thus making it difficult to identify and characterize example. Bifidobacterium era have been described for Intrasporangiaceae with strains have been shown to have high levels of sequence Dermabacteraceae containing two genera. T. The phylogenetic tree generated from the concatenated sequences showed a significant increase in the discrimi- Speciation of Genera Using Protein-Coding natory power between the strains. Chater and Chandra. and trpB showed no Microbacteriaceae. violaceoruber cluster and phylogenetic analysis was similarity. Dermacoccaceae. In a phylogenetic tree presented in The use alongside the 16S rRNA gene. Intrasporangiaceae. done using 16S rRNA. and trpB genes. a Dermabacter species groups with a member of strains.

and annotated. and translation are located in The ability to easily acquire and lose DNA without causing the core region whereas those with a nonessential func. A process of reductive evolution is seen to occur of the S. become redundant as the host will supply these needs. that were absent in its close relatives and of these regions. A gene for a particular function will be The extreme variability of Actinobacteria is a well-known made redundant when the functional constraint is relaxed. and it was only after the deletion of the terminal regions that the circular chromosomes became stable structures. These include nucleases. geosmins.14 Actinobacteria the chromosomes was determined by PFGE. tuberculosis and C. Genomes of 19 medically and industrially genome. chromosome and the distribution of the essential and which are antiparasitic agents used in human and veter. 14 organic. Significant synteny between the core of the contrast to this. Approximately 40% Of the streptomycetes. and mation. Prior Streptomycetes have a saprophytic lifestyle and their to sequencing of the S.7 and 9 Mb respectively. coelicolor A3(2) chromosome consisted of in these pathogens where a number of gene functions acquired DNA. with a similar pattern being genetically the best-known representative of the genus as seen in S. coelicolor A3(2) genome. Analysis of the types and location of the genes in desferrioxamines. Sequencing of the chromosome revealed 20 more xylanases. circular be up to 2 Mb.g. Initially. lyzed. 104 to 102 per spore. thus making it prone to inactivating mutations. avermitilis organism in that it allows a certain amount of plasticity to have linear chromosomes of 8. avermitilis is an important organism in the pharmaceu. coelicolor A3(2) and S. thus making strep. The structure of the Streptomyces tical industry as it is a major producer of avermectins. coelicolor A3(2) genome. and chitinases. which exceeds the size of a small bacterial chromosomes. Actinoplanes. When these and is clearly evident when examining culture plates.g. whereas the arms organisms. and the whiE cluster had been ana- enzymes. genetic instability has a pleiotropic effect of this study concluded that members of the genera which and can result in the loss of antibiotic biosynthesis and undergo a complex cycle of morphological differentiation resistance. diphtheriae was observed. the genome. In mutations become fixed in a population the gene becomes a . Both S. Lin and Chen (1997) proposed the high numbers of The Streptomyces Genome transposable elements (TEs) in the terminal regions to be responsible for genetic instability. and gene transfer events. particularly in the upper layers of the soil profile. deletions. Analyses of whole genome sequences have However artificially circularized. gene ability to successfully colonize the soil is due to clusters for actinorhodin. 11 were located in the arms of the chromosome. coelicolor A3(2) is S. to 25% of the genome. These tomycetes central organisms in decomposition. include clusters for coelichelin. chromosomes were provided an insight into how different Actinobacteria have found to be more unstable than the parent chromosome become adapted to their particular ecological niches. coelicolor A3(2) genome must withstand extremes of temperature and moisture. avermitilis. these deletions can remove up nobacterial strains with simpler life cycle (Mycobacterium.. coelicolor A3(2) chromosome suggests that it com- prises a central core region and a pair of chromosomal arms. ments in the form of transpositions. and aerial mycelium for- (e. coelicolor of TEs are located in the terminal regions of the A3(2) and S. phenomenon first demonstrated by the work of Lieske. The results nearly all cases. Genes with an essential function such as in DNA Reductive Genomes replication. lipases. S. insertions. nonessential genes and TEs provides some benefit to the inary medicine. Genes can be lost in various frequencies from Nocardia) possess large linear chromosomes. transcription. secondary metabolism) are located in the arm the evolution of these free-living saprophytic bacteria. The presence of pseudogenes in a genome gives an estima- Genetic instability of the Streptomyces genome tion of gene decay. and Rhodococcus) have smaller. S. whereas acti. For a chromosome of 8 Mb this can Corynebacterium.. amylases. Organisms regions were identified in the S. proteinases. Streptomyces. which is a highly heterogeneous matrix composed of ments and acquisitions. TEs can be found in multiple copies nearly all major achievements in streptomycete genetics in the genome and this can often lead to DNA rearrange- and physiology have been done in this model organism. avermitilis are available. pigment production. inorganic. clusters encoding putative secondary metabolites. undecylprodigiosin. genetic instability was considered to be important Actinobacteria have been completely sequenced a consequence of the linear structure of the chromosome. coelibactin. obligate intracellular pathogens occupy a S. coelicolor A3(2) genome and the whole genomes of stable environmental niche. the S. Micromonospora. detrimental effects to the organisms plays a major role in tion (e. complete genomes of S. suggest. and hopanoids (Table 3). such a crucial role in the evolution and adaptation of these ing these to have a common ancestor. As the terminal regions contain only a few Streptomyces species are the predominant Actinobacteria in essential genes they are more tolerant to DNA rearrange- soil. and gaseous material. so gene transfer does not play M. Using DNA microarrays. In regions. calcium- the production of a variety of extracellular hydrolytic dependent antibiotic.

In contrast. acnes are compared they show a large difference in GC content P. in M. coelicolor A3(2) It is now proposed that M. Pigments T. which is Other molecules considerably lower than that of streptomycetes and myco- Butyrolactones SCO6266 Geosmin SCO6073 bacterial strains and is in contrast to the reduced M. tuberculosis is 65. in information processing (DNA/RNA polymerases and SCO7222 gyrases) are present. The most striking feature of the nistic pathogen and can cause acne. leprae genome is that it contains 49. This Siderophore synthetase SCO5799-SCOSCO5801 implies that amino acids are obtained from the host. leprae is 1116. The aver- Hopanoids SCO6759-SCO6771 age GC content of the genome is 46%. genes for biosynthesis of Deoxysugar synthases/ amino acids were present. Unknown structure Analysis of the genome has shown that enzymes involved Chalcone synthases SCO7669-SCO7671. The complete genomes of M. suggesting that these are limit- glycosyl transferases SCO0381-SCO0401 Nonribosomal peptide ing in their environment. The genome and be subjected to further mutations to such an extent that shows little evidence of gene acquisition. has a limited metabolic repertoire and host range.5 Mb encoding 2333 genes. leprae having an average GC of 57. but in T. The number of pseudogenes 20% sharing no significant similarity to any database entries. high numbers of pseudogenes are present. indicating that no further genome M. but it can be an opportu- M. In other examples of reduced genomes like those of M. When the two mycobacterial strains P. Reduction in the genome size is also connected with Known structures the observation that intracellular pathogens make exten- Antibiotics sive use of host cellular processes. leprae. than the streptomycete genomes: 3. unlike Location on Secondary metabolites chromosome M. tuberculosis. T. mon property of reduced genomes.4 Mb (M. whipplei is the most extreme example of Methylenomycin SCP1 plasmid an Actinobacterium that has undergone genome reduc- Siderophores tion. coelicolor are involved in sporulation. which is a com- they are no longer recognizable or are completely removed. Actinobacteria 15 Table 3 Secondary metabolites produced by S. whipplei was also difficult to culture and it was only in Isorenieratene SCO0185-SCO0191 2000 that it was grown in human fibroblasts and exhibits a Tetrahydroxynaphthalene SCO1206-SCO1208 slow doubling time of 17 days comparable to 14 days for TW95a (whiE) spore pigment SCO5314-SCO5320 M. leprae. whipplei is the causative agent of Whipples disease. acnes contains a single M. with only six being found in The presence of 35 pseudogenes containing frameshift M. whipplei genome contains a seen in M. genes (1604 genes) compared to 90. tuberculosis.8% while that of preference to sebaceous follicles. (2002) and Challis and Hopwood (2003). Actinorhodin SCO5071-SCO5092 Calcium dependant antibiotic SCO3210-SCO3249 T. In M.5% protein-coding circular chromosome of 2. whipplei Twist Lipids strain has recently become available and shows it to have Eicosapentaenoic acid SCO0124-SCO0129 a small circular chromosome of only 0. whipplei Prodiginines SCO5877-SCO5898 The genome of T. P. leprae and few pseudogenes. leprae. identification of whiA and whiB. M. Like M.8% (3959) protein. Putative functions were assigned to 68% of the genes with coding genes in M. mutations or premature stop codons leads the author to . leprae. leprae has evolved to have the natural minimal gene set for Mycobacteria and. Spores have not been reported in T. leprae and Rickettsia prowazekii. whipplei. indicating massive gene decay in M. leprae) and 4. leprae suggesting that these genomes are still in the process of The best documented example of reductive evolution is downsizing. These genes will either remain in the genome may perhaps arise in the environment. least two amino acids and peptide ABC transport systems synthases SCO6826-SCO6827 Type II fatty acid synthase SCO1265-SCO1273 were identified in the genome of T. The genome sequence of the T. tuberculosis. tuberculosis have been sequenced and are much smaller decay is occurring. leprae. although they pseudogene.2 Mb (M.6%. whipplei under laboratory conditions. tuberculosis). acnes is a commensal bacterium found on human skin with with M. whipplei it became synthetases SCO6429-SCO6438 apparent that complete and partial losses of some amino Sesquiterpene cyclase SCO5222-SCO5223 acid biosynthesis gene clusters have occurred. which in S. the T. leprae.92 Mb. At Type I polyketide SCO6273-SCO6288. An interest- ing observation from the genome included the Taken from Bentley et al. Coelibactin SCO7681-SCO7691 Coelichelin SCO0489-SCO0499 which is characterized by malabsorption and is a systemic Desferrioxamines SCO2782-SCO2785 infection affecting any part of the body.

the uptake of structurally diverse oligosaccharides.16 Actinobacteria suggest this gene decay to be a recent event. longum to be a strict fermentative recognized as an extracellular triacylglycerol lipase. Three enzymes with putative hemolytic have been predicted in the genome and these cover a wide activities show some resemblance to hemolysin III of range of substrates including di-. More than 20 putative peptidases have acid transport proteins. 59end. Industrially Important Phenotypes of Bifidobacteria longum Actinobacteria Members of the Bifidobacterium genus comprise 3–6% of the adult fecal flora and the presence of these organisms is The best-studied example of an important industrial thought to provide health benefits. longum to ferment a large variety of sugars. further confirming the esterases have been identified. Thus a complex balance the 1960s. by streptomycete strains. and the microflora in the upper GIT. Three putative of 2-hydroxyacid and other predicted deaminases and sialidase enzymes have been identified. Sequencing the genome of B. being of foreign origin. pounds. programs in a hope of finding novel antibiotic com- when Bacteroides take over. As mentioned previously.3 Mb and P. the promoting foods. Homologs of the enzymes needed for the fer- which degrades skin tissue components. The genome of B. Analysis of the system. This resulted in a rapid increase in the rate at nization is thought to play a major role in the build-up of the discovery of new compounds between the 1940s and immune system tolerance. which is consis- ing. and enzymes required for this have been identified. longum to obtain sialoglycoconjugants to obtain sialic acid as a source of amino acids from proteinaceous material in the GIT carbon and energy. with nearly cytosine residues. suggesting that P. along with sialic dehydratases. are pre- duction of fatty acids that assist in bacterial adhesion and sent.5% of the predicted proteins are dedicated to carbohy- rabbits. Homologs of CAMP factors have also where carbohydrates are less abundant. acnes. Unlike An interesting feature of some of the genes is the most bacteria. acnes can grow anaerobically on a number of substrates. acnes cleaves been predicted and these enable B.and disaccharides as they are taken up by the host are secreted proteins that are known as pathogenic deter. which tent with the need of B. which exist B. The ability of mentation of glucose. These years are now considered to be the of microflora is needed for a normal and healthy digestive Golden Age of antibiotic discovery. Other genes identified were of the organisms and the mechanism of host-microbe inter- involved in substrate uptake and pathogenicity. As a result. GITs of humans. anaerobe. as after 1960s the . which may aid B. longum to compete for response that is seen during acne. one of which contained genes for the However. whole genome led to ten regions being identified as possibly where the incidence of GIT disorders greatly increases. shunt and a partial Embden–Meyerhof pathway. Bifidobacterium spe- been recognized in P. The length of this poly(C)/(G) tract is variable and Negative repressors are thought to allow for a more precise is generated during replication by slipped-strand mispair. longum is 2. and higher order Bacillus cereus. B. confirming B. This has led to the application of Actinobacteria is the production of antibiotics increase in the use of Bifidobacterium species in health. longum makes great use of negative tran- presence of continuous stretches of 12–16 guanine or scriptional control to regulate gene expression. Many surface proteins that can act as anti. Bifidobacterium species are obligate anae. including endoglycocera. They are among ferent parts of the world to initiate large screening the first to colonize GITs of newborn babies until weaning. Enzymes that are needed to feed many sugars into the colonization of the follicles. Many glycosyl hydrolases forming toxins. drate transport and metabolism. discovery of actinomycin and streptomycin in the early robes and the majority of strains have been isolated from 1940s led many large pharmaceutical companies in dif- mammalian gastrointestinal tracts (GITs). it is estimated that 86% of the genome is protein coding. which tends to be poor in been detected in streptococcal species. little is known about the physiology and genetics biosynthesis of lanthionine. acnes and these had only previously cies colonize the lower GIT. This system of successive colo. No aerobic or anaerobic respiratory components were Prior to the genome being sequenced. longum is able to ferment amino acids through the use in the cell membranes of all vertebrates. longum to adapt to the constantly serves as an adaptation mechanism whereby the organism changing conditions of the GIT. This becomes evident after antimicrobial therapy. GehA was identified. response to changes in the environment. CAMP factors mono. including the fructose-6-phosphate lipases to degrade human skin lipids results in the pro. tri-. can change its phenotype to evade immune responses or rapidly adapt to environmental changes. Many other lipases and fructose-6-phosphate are present. With regard action. either in the promoter region or at the 70% of its transcriptional regulators being repressors. Oligosaccharide transporters have also gens have been identified and can trigger an inflammatory been identified. midase that breaks down glycosphingolipids. CAMP factors have been suggested to act as pore. ability of B. longum has provided to the physiology of P. These tracts are involved in phase variation. all genes of the Embden– important clues into the adaptation of Bifidobacterium to Meyerhof and pentose-phosphate pathway are present. oligosaccharides. more that minants and have lethal effects when given to mice and 8.

containing vinyl chloride than without. It is solvents such as perchloroethylene and trichloroethene. This rationale. was isolated from polluted water taken from the inside of Another strain of Mycobacterium vanbaalenii PYR-1 was a deteriorated rubber tyre. increasing the likelihood of isolating new and Shewanella. family as Corynebacteria (Corynebacteriaceae). Inoue compounds under marine-specific nutritional conditions. Carbazole is an a novel bioactive compound. and Takahashi obtained when Nocardioides sp. It is widely used as a raw material for the normal culture media are not sufficient for these organ. lactofermen- genera. Alcaligenes faecalis. Agrobacterium tumefaciens. including 2. physiology of marine Actinobacteria to develop effective The sequences showed similarities to genes found in techniques for their isolation. The extract was then plated soil. these include Cenibacterium arsenoxi- common marine organisms that tend to outgrow any rare dans. and tum) are resistant to arsenic and genes involved in this Mycobacterium. to accumulate as an end-product of dechlorination of lated and studied further for antibiotic production. Streptosporangium. glutamicum for biore- but the genomes of two nonpathogenic mycobacterial mediation of arsenic from heavily contaminated water strains have been sequenced. medicines. Members bioactive compounds. By using chemotaxonomy. Many organisms have been documented to be improved method for detecting marine Actinobacteria. Pseudomonas and Sphingamonas strains. Actinophage can be used for host of the genus Corynebacterium are of great biotechnological identification at the genus and the species level. PAH com- sharply. One method of discovering new ide and 1. KMS was sites. although amino acids such as L-glutamate and L-lysine. Initial methods used in isolating new compounds pounds are toxic and have carcinogenic properties. Higher growth yields were compounds is by isolating new organisms. Actinobacteria 17 rate at which new compounds were discovered decreased the ring component of these compounds. genetically engineered strains of C. production of dyes. ars1 and ars2. the strain is lation methods that have been developed based on this unusually sensitive to vinyl chloride starvation. Kurtboke (2005) developed an its increase. . strain possess a 300-kb plasmid carrying genes encoding Considerable interest has been applied in screening monooxygenases and epoxyalkane: coenzyme M transfer- marine organisms for the discovery of novel compounds. Bacillus. found to possess the remarkable ability to degrade DNA–DNA hybridization and 16S sequence analysis this PAHs. and plastics. isolated from soil sites that had been polluted by creosols. thought to be involved in degradation of vinyl The study of Okazaki and colleagues and subsequent chloride. Actinobacteria. Members of some cross-reactivity has also been detected with other the coryneform bacteria (C. Representative colonies were iso. This study highlighted the fact that and mutagenic.6-trinitrophenol. Actinobacteria-specific bac. which contain up to four aromatic rings. importance. and dibenzofuran (DF). process are contained in two operons. Sequencing of mycobacterial strains is done mainly Research of Mateos and colleagues aims to make use of because they are important human and animal pathogens. Both these mycobacterial species use Gordonia and was given the name Gordonia westfalica. ase. including Nocardia. An arsenic-defence mechanism is present actinobacterial strains that potentially produce novel in all organisms studied for arsenic degradation.and nitro-substituted PAHs such strain was found to represent a new species within the genus as naphthalene. JS614 was grown on media  and Omura provide an excellent review on different iso. although anthropogenic action has led to of actinobacterial strains. including alkyl. through either reduction or method uses the actinophage to reduce the number of oxidation reactions.2-dichloroethane. Nocardioides species are known to degrade other research by the group has reported the isolation of an aromatic compounds. Nocardioides isms as they have adapted to producing bioactive aromaticivorans IC177 is able to degrade carbazole. JS614 was isolated from an secondary metabolism and occurs only under certain industrial soil site that was contaminated with vinyl chlor- nutritional conditions. A strain bons (PAHs). and were based on simple plating procedures where the soil microbial degradation of these compounds is the most samples were mixed with water and subsequent filtering effective method of remediation of the contaminated to remove large soil particles. In gen. Mycobacterium sp. and polycyclic aromatic hydrocar. Members of the genus Gordonia belong to the same pentachlorophenol. Vinyl chloride is a potential carcinogen and tends on nutrient medium. This able to transform arsenic. It could dioxygenases and monooxygenases for the oxidation of utilize natural and synthetic components of rubber. glutamicum and C. This isolate would only N-heterocyclic aromatic compound derived from creo- produce this compound in selective sea water containing sote and crude and shade oil and is known to be both toxic Japanese seaweed. actinobacterial strain from the Sagami bay area producing phenanthrene. now well known that antibiotic production is part of A strain of Nocardioides sp. teriophage have been successfully used for isolation and Arsenic is a highly toxic metal and its presence in the identification of novel or rare actinobacterial strains from environment is largely from a geochemical source (rocks terrestrial environments and to determine the relatedness and minerals). especially for the large-scale production of eral. and colleagues were able to clone and partially sequence It is therefore important to study and understand the the car genes responsible for carbazole degradation. streptomycetes phage are genus-specific.4.

and indene. acidogensis. which converts glucose into fructose. terrae. and tryptophan  Fermentative production of nucleotides which are used as flavor enhancers in foods Microbispora rosea  Produces D-xylose isomerase. It is for this capabilities to either utilize these compounds as sources reason they have been referred to as ‘masters of catabolic of energy or break them down to simpler forms. in particular the genus Streptomyces. environment and are released as contaminants in pesticides s-trazines. Members of Actinobacteria play a major role in to accumulate in the body fat of animals. Actinobacteria. flavor and keeping quality  Synthesis of long-chain aliphatic hydrocarbons that have the potential to be processed into lubricating oils or other petroleum substitutes Rhodococcus species  Rhodococcal nitrile converting enzymes used to convert nitriles into their corresponding higher value acids and amides. isopropylbenzene. YK3. and sulfonylur- and herbicides. which utilizes DF It is clear from the examples discussed above that in a similar manner. terrae contains the pound though co metabolism. Table 4 Uses of actinobacterial strains in biotechnology Organism Biotechnological uses Mycobacterium(nonmedical  Biotransformation of steroids strains)  Removal of vinyl chloride from industrial waste  Production of optically active epoxides which are subsequently used for chemical synthesis of optically active pharmaceutical compounds Corynebacterium  C. tylosine. As mentioned before members of the biphenyls. also encoded on plasmids. including halogenated and long chain as well as aromatic compounds  Biopurification of coal and crude oil by removing contaminating organosulfur compounds Frankia  Along with other actinorhizal organisms are used where it is necessary to rapidly establish a plant cover Cellulomonas species  Used for single-cell protein production from a variety of waste products  Mixed cultures can be used to convert xylan into methane via hydrolysis. are ciated with virulence in pathogenic strains (R. These strains can (nonmedical strains) also be modified to produce threonine.18 Actinobacteria including cis-1.  Capacity to degrade a diverse range of hydrocarbons. which can be used as polymers in dispersants. and superabsorbents  Commercial production of biosurfactants. flocculants. Many of the genes turn can be used by other organisms (cometabolism). Sekine and colleagues. De Schrijver and De Mot dbdA (DF dioxygenase) gene cluster and sequence analysis provide a comprehensive review on the degradation of demonstrated it to be nearly identical to the cluster found pesticides by Actinobacteria. required for degradation of xenobiotic compounds are Actinobacteria also have an enormous biotechnological encoded on plasmids. A DF-degrading biotransformation and biodegradation of these chemicals.4-polyisoprene. glutamicum used for the large-scale production of L-glutamic acid and L-lysine. emediation and biodegradation of complex xenobiotic known for their ability to biodegrade and transform a compounds in the environment. the overall metabolic diversity and genetics of these Polychlorinated dibenzo-p-dioxins (PCDOs) and polycho. These are highly toxic compounds and tend eas. which can use DF as a sole source of where a single strain can degrade more than one com- carbon and energy. aroma. and methanogensis  Photoevolution of molecular hydrogen using cellulose as sole carbon source Micromonospora  Commercial scale production of amylases and cellulases  Vitamin B12 production Brevibacterium  Used for cheese ripening Nocardioides  Used for their ability to perform chemical and enzymatic modifications of complex compounds and production of industrially important enzymes . organophosphates. Readers are recommended the Examples of other uses of Actinobacteria are listed in articles by Larkin and colleagues. was also isolated. which in versatility’ by Larkin and colleagues. Pesticides are composed of compounds with lorinated DF (PCDFs) are common pollutants in the varying chemical structures. Rhodococcus species are well. Genes asso. organisms. Janibacter terrae strain XJ-1 and Gurtler and colleagues for the detailed analysis of was found to have the ability to degrade DF. isoleucine. equis) are major producers of medically important antibiotics. members of the Actinobacteria play a major role in bior- Among the Actinobacteria. strain of J. which is subsequently used to produce high-fructose syrup Micrococcus species  Used for processing of fermented meats to improve color. They have the genetic wide range of complex organic compounds. triazinones. J. Table 4. including those for polychlorinated potential. phenylalanine. including organochlorides. on a large plasmid of Terrabacter sp.

Rosenberg E. 3rd edn. Rainey FA. Williams ST. 115: 225–233.0 . Journal of General Bacterial genomes are under constant selection pressure and Applied Microbiology 49: 141–154. New Further Reading advances in DNA technology have contributed consider. Molecular Microbiology study also highlights the importance of using a combination 26: 709–719. et al. Goodfellow M. numerical taxonomy. Relevant Website citrant compounds. for the screening of new bioactive compounds. comprising the Actinobacteria class have been studied in Goodfellow M. chemicals. pp. Actinomycetes. closely related families is essential to allow a more accurate Takahashi Y and Omura S (2003) Isolation of new actinomycete strains discrimination between strains. vol. Kers J. and Ward-Rainey NL (1997) Proposal for a the phylogeny of strains. 23–41. and clusters that have already been Lin YS and Chen CW (1997) Instability of artificially circularized defined by numerical taxonomy were retained. The aim of this chapter Antibiotic Makers New York. Kulakov LA. (1983) Numerical pathogenic bacteria (Mycobacterium and Tropheryma) have classification of Streptomyces and related genera. vol. Bacteria: Firmicutes. 129: 1743–1813. living a saprophytic lifestyle in the soil. 3. Sequence analysis of the 16S rRNA Microbiology Reviews. Falkow S. The results of this study correlated well with criterion in the classification of aerobic Actinomycetes. or as Wellington EM and Toth IK (1996) Studying the ecology of actinomycetes in the soil rhizosphere. Alderson G. and Stackebrandt E newly isolated strains. and Alderson G (1989) Genus Streptomyces Waksman and Henrici 1943. and DNA–DNA hybridization methods have provided a basis for studying the taxonomy of Actinobacteria. Actinobacteria 19 Concluding Remarks ongoing analysis has revealed the enormous genetic capabil- ities of this important group of bacteria. Loria R. previous analyses. Also when generating Stackebrandt E. 9: 279–286. This chromosomes of Streptomyces lividans. Degradation of pesticides by is routinely used in conjunction with analysis of chemo. Sharpe ME. and Allen CC (2005) Biodegradation and Rhodococcus – masters of catabolic versatility.org – GOLD Genomes OnLine genome sequences of actinobacterial strains and their Database v 2. classification and identification of Streptomyces species – a review. however. in many of these studies members of other closely Gene.) (2006) The Prokaryotes. In: Hall GS (ed. adapt to their particular ecological niches. such as Streptomyces have made use of HGT. 339AL. taxonomic traits to identify and describe existing and Dworkin M. The application of chemotaxonomy. and Joshi M (2006) Evolution of plant pathogenicity in of traditional methods along with newer molecular based Streptomyces. Ferguson EV. Critical Reviews Microbiology. FEMS of small subunit rRNA. Schleifer K. Current Opinion genera that have been described as Actinobacteria using the Biotechnology 16: 282–290. Annual Reviews Phytopathology 44: 469–487. Archaea. CAB International. 4. Inc. Free-living species Williams ST. Actinobacteria classis nov. and Holt JG (eds. tolerating acqui. in particular sequence analysis Streptomyces analysed by genome comparisons. New York: Springer. the analysis of more strains from new hierarchic classification system. In: Williams ST. Ward AC and Bora N (2006) Diversity and biogeography of marine whether they are in the environment in the presence of toxic Actinobacteria. techniques for taxonomic purposes. was to examine the phylogenetic relationships of all the Larkin MJ. 30: 651–672.) Methods for obligate intracellular pathogens. Current Opinion Microbiology. Wilkins. UNESCO and IUB. The increasing availability of whole http://www. thus allowing them to acclimatize to Bacteriology.. In contrast. Actinobacteria represent an important group of organisms for the bioremediation of water and soil sites that have been polluted with toxic recal. 2452–2492.) Bergey’s Manual of Systematic sition and loss of genes. Journal of General chosen the path of reductive evolution where nearly all Microbiology. Actinobacteria represent a Examination of Organismal Diversity in Soils and Sediments heterogeneous group of organisms that have the ability to pp. genes not essential for growth are lost. Hopwood DA (2007) Streptomyces in nature and medicine: The related families are not included. Baltimore: Williams and their fluctuating environment more rapidly. International Journal of Systematic Bacteriology 20: 435–443. and Sanglier JJ (1992) Numerical great detail with regard to the genera they accommodate. 25: 85–119. Journal of Systematic Bacteriology 47: 479–491. Phylogenetic analyses of families (eds. De Schrijver A and De Mot R (1999). Chater KF and Chandra G (2006) The evolution of development in ably to bacterial taxonomy. entire 16S rDNA gene sequences available in the public Lechevalier MP and Lechevalier H (1970) Chemical composition as a database.genomesonline. Goodfellow M. Oxford University Press. Actinomycetes.

20 . Thus. Abbreviations IBCs intracellular bacterial communities CF cystic fibrosis UPEC uropathogenic E. leading to cellular injury and disease. Microbes also Microbial adhesion is crucial to the survival and lifestyle adhere to the hulls of ships and to machinery in food- of many microorganisms. Bacteria. microbial roots and the human intestine. teichoic acids A class of negatively charged polymers microbial ecology The study of interactions and expressed on the cell surface of Gram-positive bacteria relationships between microorganisms and their that are either linked covalently to the peptidoglycan environment. functional implications of microbial adhesion is crucial for generating complete descriptions of our ecosystems. Louis. Defining Statement Consequences of Microbial Adhesion in Human Introduction Disease Biological Significance of Microbial Adhesion Targeting Adhesion to Inhibit Bacterial Virulence Mechanisms of Microbial Adhesion Further Reading Selected Survey of Specific Adhesion Strategies Glossary planktonic cells Cells grown predominantly in biofilm A community of microorganisms associated suspension as individual cells in a liquid medium. and attempting to con- trol and prevent the unfortunate and often devastating From the center of the earth and deep-sea vents to plant consequences of infectious diseases. This article will focus on the adhesive include bacteria. and S J Hultgren. coli DAF decay-accelerating factor UTI urinary tract infection ECM extracellular matrix Tafi thin aggregative fimbriae GbO4 globotetraosylceramide Defining Statement niche and adhere to and infect other sites and host tissues. resulting in contamination and relationships forged between microbe and host depend on adverse circumstances. This article highlights evolved in order to facilitate microbial attachment to the highly evolved microbial adhesion mechanisms and diverse substrata in both symbiotic and pathogenic asso- discusses the prevalence and implications of adhesion in ciations. for example. archaea. C L Smith. Understanding the molecular mechanisms and diverse ecosystems. and are strategies employed specifically by bacteria. resulting light several exciting and up-to-date scientific discoveries in mutual benefit to both microbe and host. microorganisms occupy adhesion is a fundamental component of the field of remarkably diverse niches on our planet. Pathogenic as a platform to illustrate the biological significance and and unwelcome bacteria can egress from their native implications of microbial adhesion. Both beneficial and pathogenic processing factories. Specific adhesion strategies have adhesive events and colonization. USA ª 2009 Elsevier Inc. though many found attached to rocks and soil particles. (wall teichoic acids) or linked to lipids in the cytoplasmic membrane (lipoteichoic acids). Adhesion. All rights reserved. fungi. MO. harbored by the other classes of microbes. with a surface. These microbes microbial ecology. School of Medicine. We will high- nize plants and humans as well as fish and squid. and parallels can be found in the arsenal of adhesive strategies ocean sponges. Washington University. St. understanding and predicting ecosystem stability due to Introduction globalization and climate change. Microbial L Cegelski. corals. and protista. symbiotically colo.

now referred to as Bt transgenic which bacterial adhesion (often followed by biofilm forma. human cells. rhizobia structurally complex and dynamic bacterial communities. important for their own energy conditions. Attachment to surfaces allows bacteria to persist in advantageous locations where there may be high nutrient Rhizobia are Gram-negative soil bacteria that adhere to concentrations or to provide protection from hostile envir. mollusks and urchins. and development. including flies. bacteria disperse from the biofilm. environmental conditions is a major benefit of life in a Rhizobia are particularly important to plants in nitro- biofilm and the first line of defense is provided by members gen-deficient soils. The attractive chemical signals and ultimate adhesive and the significantly smaller microorganisms with which interactions of Agrobacterium tumefaciens with wounded they cohabit. marine life. mosquitoes. B. Under certain rich organic compounds. Upon entry into a root hair. the hallmark of Crown sites in coral tissue including the surface mucous layer Gall diseases. microcolony. In return. and costly consequences. Microbial 21 Biological Significance of Microbial deterioration and corrosion of the steel surfaces. some plants and exists naturally in some caterpillars. gen (N2) from the air into ammonia. where the prolif. upon ingestion. growth. a protei- naceous crystal that is lethal to several pests. while key questions being addressed in the emerging field of enhancing adhesive interactions between bacteria in the coral microbiology. including onments. increases drag. and beetles. This bacterium is an important Gram-positive pathogen tion. Adhesive plant proteins called vitronectins tions that promote health versus those that cause disease are also implicated in the adhesion process. and provide organic the rhizosphere. decompose chitin. rhizobia receive carbon- residing at the edges of the community. new niches. In a two-step adhesive process. including beautiful fish. initial compounds. living freely in a plank- Adhesion to Plants tonic state. Microbial fouling is an economic and environmental bur. Adhesion. Attachment is the first step in the patho- and porous components in the coral skeleton where they genic cascade and takes place in the soil around the roots – fix nitrogen. together with proliferating plant cells form a nodule. traverse a distance to the center of the root hair cell and The metabolic labor of acquiring nutrients is divided. They colonize distinct on the lower stems and main roots. Understanding the microbial interac. bacteria form biofilms – soybeans and alfalfa. Bacteria are. an integral constituent plants leads to the unfortunate development of tumors of the microbiota of healthy corals. some. Protection from harsh nitrogenous compounds to support plant metabolism. thuringiensis colonizes the surface of tion) takes place. ing or abiotic) in nearly all ecosystems far exceeds the tendency to persist in suspension. Adhesion events are crucial to biofilm forma. and thus Adhesion in the Human Host decreases the efficiency of movement through the water. In numerous instances. DNA (the transfer DNA) from the bacterium to a plant Adhesion of bacteria in many water environments cell results in the expression of several oncogenic genes inevitably has detrimental consequences. from the plant. and the formation of tumors. in fact. sion and biofilm formation on the hulls of ships creates resistance to water flow. and colonize the root cells of leguminous plants. times according to spatial coordinates in the community. In the wild. . The normal and healthy human body is composed of den in this way and in several industrial settings including approximately ten times more bacterial cells than drinking water pipes and oil pipelines. Subsequent is important in efforts to preserve and prevent further DNA transfer and integration of a specific fragment of destruction of coral reefs worldwide. Adhesion Understanding the mechanisms of adhesion is key to developing strategies to control and prevent these adverse The propensity for bacteria to associate with surfaces (liv. We set the stage for whose insecticidal properties have gained attention in the discussing the mechanisms of microbial adhesion by first development of crops genetically modified to express the illustrating a few examples across a broad landscape in bacterium’s potent toxin. The molecular mechanisms of adhesion and weak binding interactions are followed by the bacterial the sustained interactions between bacteria and their expression of multiple gene products to synthesize cellu- coral hosts are currently not well understood but are lose and anchor the microbe to the host tissue. crops. These bacteria comprise our microbiota eration of sulfide-producing bacteria leads to the and are colonized in distinct sites throughout the body. and other tecture of community members. rhizobia fix nitrogen. This sym- Adhesion in the Water biosis with plants is dependent on initial host–microbe The coral reefs are home to an enormous diversity of adhesion events. Here. converting molecular nitro- and distribution is promoted through an organized archi. The bacterium produces a unique kind of endotoxin. Bacterial adhe. nitrates. new environment and potentially readhere and colonize Other beneficial symbionts include Bacillus thuringiensis. to seek a production.

Nonspecific interactions are the primary form of attach. initiate attachment when conditions are favorable and to ing medium affects the electrostatic interactions.5–2 nm). where Mechanisms of Microbial Adhesion one adhesive strategy may be more effective than another. The ionic strength of the surround. creating extracellular fibers called pili or fimbriae. Bacteria can produce a diverse array of adhesins with General Physicochemical Factors Affecting varying specificities for a wide range of host receptor Adhesion molecules. Microbial including the skin and mouth. interface. for example. Other ment to abiotic surfaces in aquatic and soil environments. for example. In the mouth and small intestine.22 Adhesion. into the host cell also serve as attractive binding platforms face. and the permit detachment when necessary. Escherichia coli adhesion in the urinary tract and intestine. Indeed. Our microbiota is. and other acidic groups. is negative due to cell wall and cell membrane compo. adhesins recognize specific amino acid-recognition motifs Van der Waals interactions are attractive. When ides into carbon and energy sources. electrostatic for many bacteria. Indeed. On many biotic surfaces. and the small intestine and most aquatic environments due to high ionic strength colon. in such fibers have been described in Gram-negative . the microbiota is dynamic and shifts in balance that alter the sizes of dif. At smaller distances (10–20 nm). Streptococcus pneumoniae adhesion in the respiratory tract. in general. the term ascribed to the surface-exposed bacterial mole- particularly in the hospital setting. respectively. in addition to sur. bacteria like to adhere to Gram-positive bacteria. ties of both the bacterium and the substratum. Bacteria in the gut. stronger interactions includ- results indicate that the balance of bacterial populations in ing hydrogen bonding and the formation of salt bridges the gut influence caloric intake through complex inter. In the range of is critical to maintenance of microbial populations. Adhesin is environment is a common source of infectious disease. usually weak. Recent separated by less than 1 nm. pH. Thus. and Helicobacter pylori adhesion in the stomach. displacement of water molecules at that surface. Most adhesins are incorporated into heteropolymeric however. Typical binding surfaces. hundreds of aforementioned repulsion is eliminated. A successful adhesive event depends on proper. or nutrient status. bacterial adhesion resulting from high salt concentration. the asso- the nonadherent bacteria. the adhesive forces and interac- ferent bacterial populations can also lead to proliferation tions described above promote the formation of an initial of disease-causing opportunistic pathogens. The entire binding process is to present adhesins at the right time and the right place to akin to a tug-of-war. and adhesion. where near contact (0. Hallmark examples of carbohydrate recognition include multiple mechanisms can act cooperatively to promote Pseudomonas aeruginosa. have a net negative surface charge. naceous molecules such as lipopolysaccharides and carboxyls. hydrophobic interactions are either salivary flow or movement of contents eliminates important for bacterial adhesion. attach to hydrophobic surfaces compensates for the unfavorable undigested by-products and degrade some polysacchar. It is not unusual for bacteria to harbor numerous and will be addressed in more detail after the several types of specific adhesive machinery to provide description of specific adherence mechanisms. adhesive proteins on the bacterial cell surface. often to enable host cell internalization. Of course. positively charged surfaces. Bacteria invest electrostatic repulsion that must be overcome by other enormous cellular resources to assemble fimbriae in order physicochemical factors. Energetically. ciation of nonpolar groups on a bacterial surface with beneficial. contribute to surface adhesion. Many diverse and can be generally classified as either general bacterial adhesins function as lectins and the interactions nonspecific interactions or specific molecular-recognition between bacterial lectins and host cell carbohydrates are binding events that involve the presentation of specific among the best-characterized attachment processes. teichoic acids. Adhesion mechanisms can be classified Mechanisms of microbial attachment are incredibly according to the type of adhesin–receptor pair. and numerous adhesins bind to these interactions participate and compete as attractive and components in order to indirectly hijack the host signal- repulsive forces. Extracellular noncovalent forces that can operate at large separation matrix (ECM) proteins that are not directly integrated distances (>50 nm) between the bacterium and the sur. Haemophilus influenzae. Another general category of adhesins includes nonprotei- nents including negatively charged phosphate groups. The consequences of cule that mediates specific binding to a receptor or ligand bacterial adhesion in human infectious diseases are on a target cell. synthesized by Gram-negative and face-exposed proteins. in proteins expressed on host cell surfaces. but require concomitant or subsequent specific the inoculation of the human host with bacteria from the adhesive interactions to enable firm adhesion. adhesive capacity to multiple receptor molecules or to permit adhesion under changing environmental condi- tions such as temperature. bacterial metabolic networks and further study may help to understand and potentially control (decrease or Specific Adhesin–Receptor Mechanisms increase) caloric uptake. for example. The net surface charge of most bacteria ing pathways. In addition.

Adhesive autotransporters FimH-presenting type 1 pili are required for E. including species presenting P pili are associated with pyelonephritis. fibronectin. or infection of the bladder. Bordetella. and joined to a thicker rigid helical rod and both are pneumonia. bronchitis. FimH and PapG adhesins are and Dr-mediated adhesion is important for binding in the presented at the tips of type 1 and P pili. Helicobacter. Adhesion. and 100 chaperone–usher systems have been identified collagen. with a distal tip fibrillum a causative agent of sinusitis. infec- of Rickettsia. newborn meningitis CooD CooB/CooC CS1 pili Diarrhea CsgA CsgB (nucleator). protein Mycobacterium bovis Haemophilus influenzae HifB/HifC Hif pilus Otitis media. and many tion of the kidney. the remarkable diversity. Microbial 23 organisms. and survival of patho. Gram-negative counterparts. expresses an adhesive autotransporter termed assembled by the chaperone–usher system. Neisseria. Adhesins of the Dr Uropathogenic E. influenzae. heteropolymeric structures. genital. either as symbionts or as secretion pathway. Type 1 and P pili are composite members of the family Enterobacteriaceae. DAF is found adhesins and adhesive organelles to mediate adhesion in in the respiratory. adhesion have emerged from studies of pathogenic bac- genic microorganisms in the human host. the extracellular nucleation–precipita. coli strains and mediate recog- the genitourinary tract and produce numerous important nition of decay-accelerating factor (DAF). host–microbe interactions as well as some involved in the died and include the chaperone–usher pathway. A representa- Selected Survey of Specific Adhesion tive set of fimbrial adhesins is provided in Table 1. Unlike their among microbial adhesive strategies. and digestive tracts. invasion. respectively. Four distinct teria involved in infectious diseases. pathogens. These are expressed as monomeric proteins or Pilus-Mediated Adhesion to Carbohydrates protein complexes that assemble at the cell surface and in the Urinary Tract recognize host cell surface elements. the general attachment of bacteria to plants. many The most comprehensive descriptions of bacterial appear critical to binding. Strategies Some bacteria present afimbrial adhesins on their sur- face. Pilin Cholera Vibrio cholerae. CsgE/CsgF (assembly). For example. and the alternate chaperone pathway. More than Hap. urinary. Gram. Examples of these assembly mechanisms have emerged as the most well stu. are described in more detail below to highlight tion pathway. all components of the ECM. coli (UPEC) colonize the gut as well as family are expressed by E. Curli Sepsis CsgG (secretion) Salmonella typhimurium PefD/PefC Pef pili Gastroenteritis LpfB/LpfC Long polar fimbriae Gastroenteritis Salmonella enteritidis AgfA AgfB (nucleator) Sef17 (thin aggregative fimbriae) Klebsiella pneumoniae MrkD MrkB/MrkC MR/K (type 3) pili Pneumonia Bordetella pertussis FimD FimB/FimC Type 2 and 3 pili Whooping cough Yersinia enterocolitica MyfB/MyfC Myf fimbriae Enterocolitis Neisseria gonorrhoea PilC General secretion apparatus Type 4 pili Gonorrhea Pseudomonas aeruginosa. and although they have diverse functions. and PapG- variety of unrelated microorganisms. intestine and urinary tract. and complexity positive pathogens also produce adhesive pili. these niches. H. Gram-positive pili are formed by covalent polymerization of pilin subunits. otitis media. meningitis . Hap mediates binding to laminin. specificity. coli to represent a class of afimbrial adhesins expressed by a cause cystitis. through comparative genome analyses and many are Table 1 Representative fimbrial adhesins and disease association Associated Organism(s) Adhesin Assembly proteins Associated fiber disease(s) Escherichia coli FimH FimC/FimD Type 1 pili Cystitis PapG PapD/PapC P pili Cystitis/ pyelonephritis PrsG PrsD/PrsC Prs pili Cystitis SfaS SfaE/SfaF S pili UTI.

and -D-galactopyranosyl-(1–4)--D-galactopyranoside moiety pilus assembly. globotetraosylceramide incorporates its neighbor’s N-terminal extension as part (GbO4). This process begins in the periplasm. and G-III) exhibit altered one is replaced by an N-terminal extension of the next specificities for three Gal(1–4)Gal-containing iso- pilus subunit. initiation. Residues defining the hydrophobic ridge around the mannose-binding pocket are labeled. FimH mediates adhesion to phobic core. Reprinted with permission from AAAS. Mannose residues are shown with carbon atoms in yellow. in which the G1  strand of the chaper. (a) (b) (d) D-mannose lle52 E. but isolates encode for a FimH variant with higher affinity for they lack the seventh  strand. Thus in the mature pilus. each subunit receptors: globotriaosylceramide. pilus assembly requires a unique protein 1 pilus. and this FimH binding specificities supports the notion that. (1998) Induction and evasion of host defenses by type 1-piliated uropathogenic E. Microbial well studied and required for the assembly of extracellu. In a process termed donor strand the monomannose-containing glycoprotein uroplakin Ia complementation. the translocation of subunits across the outer membrane. such as the adhesin. oxygen atoms in red. Journal of Bacteriology 181: 1059–1071. PapG variants (G-I. Periplasmic pilus varying specificities for monomannose and trimannose chaperones consist of two immunoglobulin (Ig)-like binding. Lopez-Boado YS. coli isolates present FimH variants (specific allelic var- subunit expression and translocation by the general secre. catalyzing the folding of the the epithelial cells that line the lumen bladder (Figure 1). mediates binding to a different carbohydrate receptor. Klebsiella. (b) Molecular surface representation in which the electrostatic potential surface with positively charged residues is shown in blue. subunit. and Yersinia. and nitrogen atoms in blue. (Top) High-resolution. (d) Type 1 pili-mediated attachment of uropathogenic E. ney. and ordered assembly of pilus subunits at the specificity of FimH is mannose.coli. Wilson CL. confers distinct roles in pilus adhesion. whereas urinary tract aggregation. the intestine exhibit a higher specificity for trimannose- active surface capped and prevent nonproductive subunit presenting glycoprotein receptors. freeze- fracture. coli Tyr137 Bladder Tyr48 epithelial Type1 Asp140 cell Lectin-binding pilus Phe1 domain lle13 Phe142 (c) Phe142 Phe1 Asn47 FimH Asp146 Pilin Glu133 domain Asn46 Asp54 Asn135 Figure 1 The FimH adhesin and type 1 pili-mediated adhesion of E. binding domain (Figure 1). Interestingly. different cell surface. Pilin subunits also have an Ig-like fold. the PapG adhesin outer membrane usher to facilitate chaperone uncapping. coli. elonga- lar adhesive organelles in pathogens including Salmonella. iations in protein sequence and structure) that exhibit tory pathway into the periplasm. after E. and globopentaosylceramide (the Forssman anti- of its own Ig fold. In the latter. termination and regulation. and neutral and hydrophobic residues in white. monomannose. consists of a pilin subunit and the receptor- pair (a chaperone and a usher) to facilitate the folding. deep-etch electron micrograph is from Mulvey MA.24 Adhesion. incorporated at the tip of the type usher system. tion. G-II. Science 282: 1494–1497. thus exposing the hydro. Chaperone–subunit complexes are targeted to an Presented at the tips of P pili. The demonstrated allelic variation in PapG and other interactive subunits. (a) The ribbon representation of FimH (from the crystal structure of the FimCH complex). negatively charged residues in red. (Bottom) Scanning electron micrograph of a bacterium entering the membrane of bladder epithelial cells is reprinted from Soto GE and Hultgren SJ (1999) Bacterial adhesins: Common themes and variations in architecture and assembly. This occurs via a process termed donor of glycolipids presented by cells predominantly in the kid- strand exchange. Haemophilus. et al. FimH expressed by most commensal isolates of domains and bind to folded subunits to keep their inter. Subunits have distinct specificity for gen). . The primary carbohydrate transport. the chaperone’s G1  strand serves as that is expressed on the surface of superficial facet cells – the pilin’s seventh strand. coli (UPEC) to the luminal surface of the bladder epithelium. D-mannose is located at the top of the molecule. (c) The mannose-binding site with FimH residues. In each chaperone– The FimH adhesin.

CsgB. The highly homologous fibers produced by organisms exploit to gain a foothold in host tissue. Streptococcus pyo. coli grown on Congo red-containing agar medium take up the dye and stain red. aureus expresses the fibro. attachment to host proteins including fibronectin. selected for increasing the fitness of pathogenic organisms YadA. leading to integrin clustering and eventual internalization. bind to fibronectin and bridge the bacteria to integ- in distinct niches in the host. The Salmonella species are called Tafi (thin aggregative fim- ability to adhere to ECM components is a primary adhesion briae). coli. Prevalent components include collagen. CsgA into -sheet-rich amyloid fibers depends on the nucleating activity of the minor subunit. and proteins and functions to provide struc. Proteins CsgE. Fibronectin is present in most tissues and fluids of the body and helps to create a Curli are a unique class of adhesive extracellular amyloid cross-linked network between cells by presenting binding fibers produced by Gram-negative bacteria. particular bacterial pathogen. Curliated E. Invasin is a Yersinia adhesin that bypasses the ECM and binds directly to integrin transmembrane Adhesion to ECM Components receptors. Pinkner JS. a process that pathogenic E. rins. CsgF. as well as molecules such as heparan Curli-Mediated Multipurpose Adhesion sulfate and chondroitin sulfate. pyogenes adhesin. and plasminogen. and CsgG are assembly factors required for the stabilization and transport of CsgA and CsgB to the cell surface. This crucial binding event leads to host cell Curli are assembled by the nucleation–precipitation path- cytoskeletal rearrangements and invasion. . Curli are amyloid fibers and bind the hallmark amyloid dyes Congo red and thioflavin T. the The major subunit protein (CsgA) and the nucleator (a) (b) A A Curli (–) Curli (+) A A A A A A A B A Outer membrane B F B G G (c) E A B A Inner membrane Sec G F E D B A Figure 2 Curli biogenesis and biology. lami- genic microorganisms. sites of expression often relate to the tissue tropism of a tural support and adhesive interactions among cells. coli. Like the FnBP-A adhesin in S. (a) Current model of curli biogenesis. serve as binding receptors for bacterial adhesins and their proteoglycans. Tafi contribute to a remarkable aggregative phenotype nectin-binding proteins FnBP-A and FnBP-B that permit characterized by a patterned assembly of cells radiating adherence to fibronectin that are bridged to cellular integ. Staphylococcus aureus is a significant nin. Polymerization of the major curli subunit protein. From Chapman MR. rins. Science 295: 851–855. The fibers mediate biofilm formation and mechanism that contributes to the virulence of many patho. and have been implicated in human cause of nosocomial and often persistent infections. Reprinted with permission from AAAS. including sites for other ECM components. and assembly requires specific molecular machinery genes is armed with more than 12 fibronectin. Among sepsis. way. Noncurliated cells do not. lami- nin. Curli assemble through the nucleation–precipitation pathway. Adhesion. (c) High-resolution deep-etch electron micrographs of curliated E. curli and other ECM-binding proteins. et al. (2002) Role of Escherichia coli curli operons in directing amyloid fiber formation. pathoadaptive mutations are major S.and collagen. S. from the center when grown on a surface such as agar. Robinson LS. and the Yersinia adhesin. (b) Congo red-binding phenotype. and vitronectin. encoded by the csgBA and csgDEFG operons (Figure 2). aureus. binding proteins. When expressed together with cellulose. SfbI. Microbial 25 through bacterial evolution. fibronectin. Other less-ubiquitous ECM components also The ECM contains a diverse array of oligosaccharides.

coli strains bacteria form large. particularly colonize the urinary tract. In this intracellular niche. Spread to new cells Consequences of Microbial Adhesion in Human Disease The critical first step in most infectious diseases requires physical contact between a bacterium and host cell. Curli are also implicated in the binding of E. CsgA. UPEC can persist for months in a quiescent bladder E. coli O157:H7. external conditions in the environment initiate another round of IBC formation in other urothe- and in the host may differ as a function of time. pH. UPEC bind to and invade the important in the pathogenesis of chronic infections includ. with many CsgB. activate a complex developmental cascade. and bacteria lial cells (Figure 3). ficial facet cell.26 Adhesion. curli production is sufficient to permit times life-long cycle of interactions between pathogen laboratory strains of E. sion of host bladder epithelial cells. and other world. certain circumstances. UPEC coordinate highly organized temporal and spatial events to the cascading sequelae of infectious diseases. UPEC even- and under the conditions tested. Quiescent bacteria can reemerge as UPEC engage in an incredibly coordinated and regulated pathogens from their protected intracellular niche and can be a genetic and molecular cascade to assemble type 1 pili. where they ing urinary tract infection (UTI). The sticky nature of curli amyloid fibers is like that of amyloid aggregates and plaques asso- ciated with eukaryotic amyloid disorders such as Binding Alzheimer’s and Parkinson’s diseases. coli. Microbial protein (CsgB) are secreted to the cell surface in a CsgG. Thus. Binding events can lead to Biofilm dispersion and cell exit extracellular colonization and invasion into underlying host cells. Some fluxing bacteria form filaments. biofilm-like intracel- to plant surfaces and are expressed by many strains asso. ongoing curli research that aims to elucidate structural features of curli assembly and the functional implications of curli-mediated Invasion adhesion may also provide valuable information to the and replication exciting field of amyloid fiber biogenesis and aggregation. from the IBC to adhesion. densely packed. initiating complex signaling cascades in both the formation pathogen and the host. Upon entry into superficial facet cells. which has caused several food. Even after considerable attention since the discovery of curli as acute infection is resolved and the urothelium is amyloid fibers in 2002. as source of recurrent urinary tract infections (UTIs). Transcriptional regulation of the curli operons is experiencing recurrent UTIs. and chronic lung infections. superficial umbrella cells that line the bladder lumen. among pathogenic strains such as E. . are resistant to antibiotics. may depend more on one adhesive system than another in which are resistant to neutrophil phagocytosis. including the prototype coccoid bacteria. the pathogens borne outbreaks in the United States and around the are protected from antibodies. UTIs are among the most common dependent fashion. chronic otitis media rapidly replicate to form a biofilm-like intracellular bacterial (middle ear infection). coli (UPEC). enabling them to bind the including temperature. coli for E. coli and UTI reservoir following acute infection. In the IBC. IBC formation is not an end point or dead end alfalfa. Inside urothelial cells. and challenge current antimicrobial therapies. and osmolarity. Indeed. CsgE and CsgF are assembly factors bacterial infections and nearly 50% of women will be required for the stabilization and transport of CsgA and afflicted by at least one UTI in their lifetime. The adhesive mannose-containing host receptors. this is only the beginning of a some- investigation. there appear to be redundant adhesion systems. and subvert clearance by innate host responses. Although the exact nature of binding is still under host defenses. the flow of urine. coli to bind plant tissues. bacteria find a safe haven. Virtually all clinical UPEC complex and responds to many environmental cues isolates express type 1 pili. Bacterial adhesins mediate this binding event through the sophisticated adhesion mechanisms described above and allow the pathogen to gain a foothold in the Biofilm host. Yet. community (IBC). However. described above. or flux. Filamentation facilitates survival of the bacteria and The curli bacterial adhesive fiber machinery has gained allows them to invade other epithelial cells. curli are not required for tually detach and disperse. lular bacterial communities (IBCs) of morphologically ciated with food-borne illness. UPEC O157:H7. such as and host. Adhesion is the first step that promotes Figure 3 Pathogenic cascade of uropathogenic E. comprising up to 105 bacteria per super- strain E. which results in inva- functionality is attributed to the main fiber subunit.

Pilus-mediated adherence is important in bacteria from the biofilm and to inhibit the chemical the adhesion and early stages of epithelial colonization. Thus. This approach of using ideal strategy to combat bacterial pathogenesis because soluble carbohydrates or mimics recognized by the bac- of its importance early in the infectious process. causing nosocomial infections such development. Haemophilus. Alginate. drug develop- eventual pulmonary failure of most CF patients. The potent molecules inhibit an peutics to disarm pathogens in the host that may offer essential protein–protein interaction between chaperone reduced selection pressure for drug-resistant mutations. Electron micrographs reproduced from Pinkner JS. coli (Figure 4). inhibit the assembly of both type 1 and alternative approach to the development of new thera. aeruginosa adheres many in pathogenic cycles. adhesion is essential to the long-term persistence of organisms by tailoring the antiadhesive compounds to bacteria in the pathogenic cascade of several infectious their receptor specificities. Proceedings of the National Academy of Sciences of the United States of America 103: 17897–17902. several specific P. Targeting bacterial virulence in this way is an 2-pyridone scaffold. aeruginosa that forms a a new cell to remain undetected until drug pressure sub- matrix of ‘slime’ to surround a forming biofilm and anchors sides and conditions encourage replication and new the cells to each other and to their host. Adhesion. required for pilus biogenesis. specific times over the course of a complex infectious terial persistence within the urinary tract.S. and signaling necessary to encourage new biofilm formation. aeruginosa and CF pathogenic adhesive strategies have emerged as hallmark requirements for virulence in certain infectious diseases. . based on a bicyclic per se. Chaperone–usher In addition. standard antibiotic treatment regimens may and it is possible. Thus. U. strategies to prevent alginate. yet targeting adhesion holds to the respiratory epithelium. bacteria can remain within the bladder As emphasized earlier. the bacteria are protected from the host defenses microbial adhesion are being considered in combination and are often resistant to treatment with antibiotics. and Yersinia biota. terial lectin can be readily extended to other adherent tion. Surrounded by intracellular biofilm formation. responsible for the biofilm-associated infections. A new class of pilicides.A. Nevertheless. the fluxing bacteria are capable of readhering to persistence in the lung. and bacteremia. perone function. for example. et al. against several Gram-negative pathogens. Klebsiella. No compound Plus pilicide S N N O CO2Li Pilicide O Figure 4 Targeting microbial adhesion. Thus. gaining a foothold and potentially invading exopolysaccharide produced by P. Adhesion is sometimes just the first step of as pneumonia. (2006) Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. Remaut H. Indeed. Rationally designed ‘pilicides’ inhibit pilus biogenesis by disrupting chaperone–usher protein interactions and reduce piliation levels dramatically. is a mucoid new host cells. diseases. that pili- lead to the loss of symbiotic benefits and the proliferation cides may exert broad-spectrum activity and be effective of disease-causing opportunistic pathogens. the ability of UPEC to adhere to and differentially to permit binding in specific sites and at invade bladder cells appears to facilitate long-term bac. In addi. Microbial 27 seemingly intact. aeruginosa has emerged as an opportunistic pathogen in and represent amenable targets for drug discovery and several clinical settings. P. additional virulence factors contribute to the subsequent In UTI. leading to chronic lung infec. UTIs. P pili in E. typically ment strategies include attempts to induce the dispersal of by 37 years of age. cycle. it may be difficult to develop a universal class of antiadherence drugs. and usher. Copyright (2006) National Academy of Sciences. Buelens F. Moreover. virulence-specific therapeutics could avoid systems are highly conserved among various bacteria the undesirable dramatic alterations of the host micro. including Salmonella. therapies to both prevent and treat infectious diseases. the adhesion process can be targeted ‘Pilicides’ are a class of pilus inhibitors that target cha- without placing life or death pressure on the bacterium. In tions in cystic fibrosis (CF) patients. for example. Targeting Adhesion to Inhibit Bacterial Carbohydrate derivatives of host ligands have demon- Virulence strated efficacy in blocking the adhesive properties of E. pathogens are capable of pre- for many days to weeks regardless of standard antibiotic senting multiple adhesins that can be expressed treatments. coli expressing type 1 and also P pili in biophysical The ability to impair bacterial adhesion represents an and hemagglutination assays. P. value even after an infection has been established. although not yet demonstrated.

and Hasty DL (2003) Bacterial Adhesion to Animal Cells and Tissues. and Abraham SN (2006) Bacterial adhesion. Pizarro-Cerda J and Cossart P (2006) Bacterial adhesion and entry into host cells. AlgR2/AlgR1. Marshall GR. biofilm formation. aeruginosa adherence. Nature inhibit microbial adhesion and thus prevent subsequent Reviews Microbiology 6: 17–27. 1: 75–87. disease and evolution. Koren O. Wright KJ and Hultgren SJ (2006) Sticky fibers and uropathogenesis: L Cegelski is the recipient of a Burroughs Wellcome Bacterial adhesins in the urinary tract. controls the synthesis of alginate by P. critical to P. The ability to biology and future prospects of anti-virulence therapies. and Zilber-Rosenberg I Acknowledgments (2007) The role of microorganisms in coral health. Nature Reviews Microbiology 5: 355–362.28 Adhesion. . Alginate is a key component of the protective exopoly- saccharide coat. R01AI048689. that Institutes of Health (Scor P50 DK64540/ORWH. Cell 124: 715–727. The inhibitors Further Reading of alginate synthesis could be therapeutically employed to render the pathogen more susceptible to host defenses Barnhart MM and Chapman MR (2006) Curli biogenesis and function. Microbial Compounds have been identified that target the S J Hultgren acknowledges funding from the National two-component signaling system. or to standard antibiotics currently in use. Sharon NS. Eldridge GR. numerous infectious diseases. Washington. Doyle RJ. aeruginosa. Cegelski L. Rosenberg E. pathogenic processes holds enormous therapeutic Ofek I. and thus could Annual Review of Microbiology 60: 131–147. R01AI029549. and Hultgren SJ (2008) The be effective also in combination therapy. and R01DK51406). Efrony R. Prokaryotes 2: 16–31. Reshef L. Future Microbiology Fund Career Award at the Scientific Interface. potential and promises to improve the treatment of Ofek I. and CF pathogenesis. DC: ASM Press.

activates transcription. mobilizable plasmid Conjugal plasmid that carries an origin of transfer (oriT) but lacks genes coding for its own transfer across the bacterial envelope. which. and T-DNA pro- DNA) to susceptible plant cells. transconjugant A cell that has received a plasmid from passively diffuses across the bacterial envelope and another cell as a result of conjugation. This is a multistage genome. Agrobacterium and Plant Cell Transformation P J Christie. induction of a virulence regulon. export and one or more proteins that facilitate DNA conjugation Transfer of DNA between bacteria by a delivery to recipient cells. Abbreviations NLS nuclear localization sequences AAI autoinducer OD overdrive ABC ATP-binding cassette OM outer membrane AHL acylhomoserine lactone SR substrate receptor CP coupling protein T-DNA transferred DNA Dtr DNA transfer and replication Ti tumor-inducing GFP green fluorescent protein TMS transmembrane segments GGI gonococcal genetic island TrIP transfer DNA immunoprecipitation IM inner-membrane T4S type IV secretion Mpf mating pair formation VBTs VirB2-interacting proteins Defining Statement infection process involving sensory recognition of specific plant signals. Defining Statement VirB/D4 System. to eukaryotic hosts. attachment to the plant host. under conditions of high cell density. process requiring cell-to-cell contact. and integration into the plant tumors called Crown galls. imperfect repeats that composed of a single-strand of the DNA destined for delineate the boundaries of T-DNA. Agrobacterium tumefaciens transfers oncogenic DNA (T. Houston. transfer. USA ª 2009 Elsevier Inc. University of Texas Medical School at Houston. type IV secretion system A conserved family of conjugative pilus An extracellular filament encoded by macromolecular translocation systems evolutionarily a conjugative plasmid involved in establishing contact related to conjugation systems for translocating DNA or between plasmid-carrying donor cells and recipient protein effector molecules between prokaryotic cells or cells. TX. All rights reserved. 29 . causing formation of cessing. a Member of the Type IV Secretion Introduction Family Overview of Infection Process Substrate Transfer Through the Plant Cell Ti Plasmid Agrobacterium Host Range and Genetic Engineering Chromosomally Encoded Virulence Genes Conclusions T-DNA Processing Further Reading Glossary T-DNA Segment of the Agrobacterium genome autoinducer An acylhomoserine lactone secreted from transferred to plant cells. transfer intermediate A nucleoprotein particle border sequences 25-bp direct. bacteria.

that encode most of the to synthesize as a result of T-DNA transfer. NGR234. Because of and (7) expression of T-DNA genes (see Figure 1). respectively. Agrobacterium-mediated transformation can be depicted However. (4) translocation across the bacterial new industry of plant genetic engineering. With the dual importance of Agrobacterium as a plant pathogen and the exception of attachment. the plant cell. The T-DNA is important for transformation as a model for understanding the require- infection because it codes for genes that. therefore. tumefaciens cells teria can transform plants when carrying an Agrobacterium residing in the vicinity of the plant tumor. . The other genetic information required for DNA transfer to suscep. One of these regions hairy-root appearance referred to as ‘hairy-root’ disease. and. The second region of the Ti plasmid Despite the ubiquity of Agrobacterium species in soil and involved in infection harbors the genes responsible for plant environments. Transcription of Agrobacterium species are commonly found in a variety of T-DNA in the plant cell produces 39 polyadenylated environments including cultivated and nonagricultural RNA typical of eukaryotic RNA message that is translated soils. but in fact other members of the alphaproteobac. a novel regulatory cascade involving the infection. and rice plants. The plant symbionts Rhizobium sp. and even plant vascular systems. T-DNA processing and transfer. typically a segment of 20–35 kb delimited by 25-bp directly repeated Overview of Infection Process border sequences. I will focus on Agrobacterium-mediated tumor-inducing (Ti) plasmid. Here. plant roots. gative transfer of the Ti plasmid among A. The basic infection process is similar for copies of the Ti plasmid and its associated virulence factors both the species. tumefaciens induces plants plasmids Ti and Ri. (6) integration of T-DNA into the plant genome. tumefaciens is for transfer. this oncogenic DNA can be excised from the as a multistage process involving (1) sensory perception of transferred DNA and replaced by virtually any gene of plant signals and induction of virulence genes. I will sum- marize recent findings pertaining to the mechanistic details Ti Plasmid of vir gene induction. tumefaciens-mediated engineering of a wide ment of physical contact between A. into the chromo- with the ability to infect plants through a process that somes of tobacco. Arabidopsis. the outcome of tion. emerged describing numerous aspects of the infection pro- cess and the myriad of ways this organism has been exploited for plant genetic engineering. albeit inefficiently. The transferred DNA encoded virulence (vir) genes and certain conserved (T-DNA) is a discrete region of the bacterial genome chromosomal loci among these alphaproteobacteria for delimited by 23 base pair (bp) direct repeats carried by the infection. The T-DNA harbors genes that are expressed exclusively in the plant cell. were found to Agrobacterium tumefaciens is a Gram-negative soil bacterium transfer T-DNA. disrupt plant cell growth and division events. only a small percentage of isolates are pathogenic. This involves delivery of a specific segment of its genome to discovery highlights the importance of the Ti-plasmid- the nuclei of susceptible plant cells. and substrate transfer across the bacterial envel- the causative agent of crown gall disease. in the cytoplasm. production of biomedically important proteins. A. a neoplastic ope. (2) establish- interest for A. tumefaciens and the array of plant species. T-DNA processing delivering DNA to susceptible plant cells. early stages of infection are as a DNA delivery system. The first is the T-DNA. (5) movement many diverse goals ranging from crop improvement to the of substrates through the plant cell cytoplasm to the use of plants as ‘pharmaceutical factories’ for high-level nucleus. Sinorhizobium meliloti and Mesorhizobium loti. Agrobacterium has been widely viewed as the chemical signals released both from the transformed plant only bacterial genus capable of transferring genes to cells and the infecting bacterium serves to activate conju- plants. Two additional regions of the Ti plasmid code for disease characterized by uncontrolled cell proliferation functions that are not essential for the T-DNA transfer and formation of unorganized tumors. Here. an extensive literature has mediated by genes encoded by the Ti plasmid. and T-DNA movement and integration in the plant host. Intriguingly. tumefaciens cells by a process termed as conjuga- transferred DNA differs. when expressed in ments for interkingdom DNA transfer. although the gene composition of the to other A. which today has envelope via a dedicated secretion channel. tumefaciens is a plant host. The discovery that A. Agrobacterium process per se. encodes Ti plasmid transfer functions for distributing tible plant cells. but are nevertheless intimately associated rhizogenes induces formation of hypertrophies with a with the overall infection process. Ti plasmids range in size from 180 to as many as 800 kilobases (kb). Two regions of the Ti plasmid contribute to infection (Figure 2).30 Agrobacterium and Plant Cell Transformation Introduction Ti plasmid. harbors genes involved in catabolism of novel amino acid The pathogenic strains of both the species possess large derivatives termed opines that A. (3) processing of T-DNA and protein effectors natural and efficient DNA delivery vector spawned an entire for translocation. Two species are known to infect plants by sensory recognition of plant signals.

LB. Auxins Plant: cytokinins opines LB RBO D T-DNA movement virE T-DNA T-DNA processing/transport virD Ti plasmid processing T-DNA processing & recruitment virC Tra vir activator virG Vir region Translocation virB Ti Opine Sensor kinase virA catabolism Opine uptake & degradation Trb Rep Ti plasmid Ti plasmid conjugation replication Figure 2 Regions of the Ti plasmid that contribute to infection (vir region and T-DNA). OD. delimited by 25-bp border sequences (blue boxes. overdrive sequence (red box) that enhances VirD2-dependent processing at the T-DNA border sequences. 5′ 3′ VirD5. and opines in the plant. All DNA between the The T-DNA is delimited by DNA repeats termed as border sequences can be excised and replaced with genes border sequences (Figure 2). and A. right border. cytokinins. Expression of T-DNA genes in the plant results in loss of cell growth control and tumor formation (see text for details). VirF. T-DNA. Agrobacterium and Plant Cell Transformation 31 Agrobacterium tumefaciens Plant cell Phenolics. VirE3 Figure 1 Overview of Agrobacterium tumefaciens infection process. and conjugal transfer of the Ti plasmid to recipient agrobacteria (tra and trb). sequence termed as overdrive that functions to stimulate T-DNA the T-DNA-processing reaction. Upon activation of the VirA/VirG two-component signal transduction system by signals released from wounded plant cells. acidic pH VirA VirG Signal recognition Wound Activation of vir genes Nucleus Vir pTi regulon Cell-cell T-DNA attachment VirD1 VirD2 VirE2. The various contributions of the vir gene products to T-DNA transfer are listed. Sugars. left border) codes for biosynthesis of auxins. tumefaciens will still efficiently transfer . VirE3 3′ 5′ T-DNA integration VirB/D4 T-DNA processing T4S System VirD2 T-strand VirE2 Translocation-competent Contact-dependent VirD2 nucleoprotein translocation VirF. RB. VirD5. a single-strand transferred DNA (T-DNA) is processed from the Ti plasmid and delivered as a nucleoprotein complex (T-complex) to plant nuclei. Flanking one border is a of interest. cell survival in the tumor environment (opine catabolism).

tumefaciens vate transcription of the occ operon. thus providing a source of carbon and nitro. A. Therefore. In contrast to OccR. TraR A. The pTiA6 lysis to drive the transport reaction. This shows that the AccR. synthesis of TraR under site. genes Several other genes transcribed from a single promoter encoded on the T-DNA play no role in the movement specify functions for opine transport and catabolism.32 Agrobacterium and Plant Cell Transformation the engineered T-DNA to plant cells. Cells that synthesize autoinducer molecules secrete these molecules into the environment. The Ti plasmid transfer system is related to rationalize the finding that A. Of further interest. energy-coupling proteins found associated with the so- Production of these plant hormones results in a stimula. tumefaciens evolved as a in sequence and function to other plasmid transfer sys- pathogen by acquiring the ability to transfer DNA to tems. the catabolism region of plasmid pTiA6. This regulatory cascade. For the octopine pTiA6 favorable for growth and propagation of the infecting plasmid. tumefaciens These systems are now classified as type IV secretion adapted a DNA conjugation system for interkingdom (T4S) systems (see below). Ti plasmids carry T-DNAs that code for nopalines. conjugative transfer of the Ti plasmid to bacterial recipi- ble for the active transport of opines and their ent cells (Figure 2). AccR functions as a negative border sequences are the only cis elements required for regulator of acc genes involved in nopaline catabolism. tumefaciens. Octopine modulates OccR regulatory activity by conditions of high cell density creates a positive-feedback altering both the affinity of OccR for its target site and loop whereby a TraR–AAI complex induces transcription the angle of the DNA bend. tumefaciens imports esis. involving . in turn. auxins and cytokinins. the distal end of the occR operon encodes advantage of the infecting bacterium over other a gene for a transcriptional activator termed TraR. A regulatory cascade activates Ti plasmid transfer The cotransfer of oncogenes ensures that transformed under conditions of high cell density (Figure 3). which. OccR positively regulates expression of conjunction with TraR to activate transcription of the Ti the occ genes involved in octopine uptake and catabolism plasmid tra genes as well as traI whose product mediates by inducing a bend in the DNA at the OccR-binding synthesis of AAI. results in enhanced synthesis the nopaline catabolism region of plasmid pTiC58 is of more AAI. Other enzymes catalyze the synthesis of novel of transport functions utilizing the energy of ATP hydro- amino acid derivatives termed as opines. called ATP-binding cassette (ABC) superfamily of trans- tion in cell division and a loss of cell growth control porters. Although the majority strain generally catabolizes only those opines that it of the occ operon codes for octopine transport and cata- incites plant cells to synthesize. Other sources for the bacterium. autoinducer is in low concentration. the Ti The Ti plasmid transfer (tra and trb) functions direct the plasmid carries opine catabolism genes that are responsi. At of T-DNA to plant cells. The in the surrounding environment and passively diffuses first is a regulatory function controlling expression of back into the bacterial cell to activate transcription of a opine transport and catabolism genes. a given A. therefore. plant cells. to regulate synthesis of an acylhomoserine lactone (AML) termed as autoinducer. resulting in enhanced opine synth. This plant cells proliferate. However. tumefaciens strains that are present in the vicinity of is related to LuxR. iating opine-specific binding and uptake. the regulatory autoinducer is an N-3-(oxo-octonoyl)-L-homoserine lac- protein is OccR. The ABC transporters are ubiquitous among leading to the formation of characteristic crown gall bacterial and eukaryotic cells. Typically. carries two T-DNA’s that code for the operon are genes whose products cleave opines to genes involved in synthesis of octopines – a reductive their parent compounds for use as carbon and nitrogen condensation product of pyruvate and arginine. tone termed Agrobacterium autoinducer (AAI). AAI acts in scription factors. DNA transport to incite opine synthesis in its plant host. regulatory cascade initiates when A. The regions of Ti plasmids involved in opine catabolism whereas at high cell densities this substance accumulates code for three functions related to opine catabolism. The ‘opine concept’ was developed cation system. The transfer genes of conjugative degradation. The tumor. OccR acts in conjunction with octopine to acti- A. as well as dedicated protein translocation systems. and provide a wide variety tumors. is a rich chemical environment opines released from plant cells. plasmids code for DNA-processing factors and a translo- gen for the bacterium. derived from -ketoglutarate and arginine. According to this concept. T-DNA transfer to plant cells. for example. Opine Catabolism At low cell densities. The regulatory protein for of TraI. one Oncogenes synthesize enzymes involved in the synthesis or more of these genes encode proteins homologous to of two plant growth regulators. At the distal end of plasmid. For the octopine defined set of genes. an activator shown nearly 20 years ago the tumor. Plants cannot metabolize opines. This ensures a selective bolism functions. In the case of A. and still Ti Plasmid Conjugation others code for different classes of opines. a member of the family of LysR tran. The T-DNA genes instead code the proximal end of the operon are transport genes med- for synthesis of enzymes within transformed plant cells. tumefaciens. Additionally.

Some operons have a single open reading environmental signal. which inactivates an acidic pH that are present at a plant wound site TraR and disrupts TraR–DNA complexes. the frame. TraR activity is an array of signals. low PO4. This complex regulatory system likely composed of VirB proteins and VirD4 translocates the evolved to maximize the number of Ti-plasmid-carrying T-DNA transfer intermediate and effector proteins across bacterial cells in the vicinity of the plant wound site. opine-mediated expression of traR and TraR-AAI regulatory system induces expression of the vir genes in mediated expression of Ti plasmid transfer genes under response to perception of plant-derived signals. TraR and AAI at a critical concentration activate the Ti plasmid conjugation functions (see text for details). (2) VirC conditions of high cell density. the vir Genes response regulator. For the A. The phosphorylated response regulator The Ti plasmid carries a 35-kb region harboring a coordinately activates transcription of several operons number of operons involved in T-DNA transfer whose products mediate a specific response to the inducing (Figure 1). has the net effect of and VirD proteins process T-DNA into a nucleoprotein enhancing Ti plasmid transfer in the environment of the particle for delivery to plant nuclei. and interacts with the C-terminus of TraR. Signals released from wounded plant cells initiate the infection process leading to tumor formation. TraM nolic compounds. mediates recognition of plant phenolics and sugars. including specific classes of plant phe- antagonized by two proteins. aldose monosaccharides. and (3) a T4S system plant tumor. tumefaciens vir system. conserved histidine residue. the kinase autophosphorylates at a changes in cell growth and density. a periplasmic sugar-binding protein. TrlR is a (Figure 1). Opines released from wounded plant cells activate opine catabolism functions for growth of infecting bacteria. In addition. Agrobacterium and Plant Cell Transformation 33 Agrobacterium Transformed Phenolics plant cell Sugars low pH VirA VirG Vir gene induction T-DNA T-DNA transfer Tra Nucleus Ti plasmid OccR + Opine transfer Ti T-DNA Trb Auxins cytokinins TraR Uncontrolled TraR Opines + cell + Opine AAI proliferation AAI Tral catabolism Tumors Autoinducer Cell growth/division (AAI) Figure 3 A schematic of chemical signaling events between Agrobacterium and the transformed plant cell. The members of this lactone ring of AAI. a was one of the first described of what now is recognized as a regulatory gene. TraM and TrlR. Lactonase production suppresses protein family are typically integral membrane proteins AAI-dependent expression of conjugation genes as a with an N-terminal extracytoplasmic domain. AAI-mediated activation of Ti plasmid transfer is also Infection is initiated when bacteria sense and respond to negatively controlled. the bacterial envelope (Figure 2). Opines also activate synthesis of TraR for autoinducer (AAI) synthesis. For example. The VirA/VirG signal transduction system truncated form of TraR that suppresses TraR activity together with ChvE. response regulator is VirG. VirA an AAI signal turnover system is composed of attJ. and phosphorylated VirG acti- The products of the vir region direct events within the vates transcription of six essential vir operons as well as a bacterium that must precede export of a copy of the number of other Ti plasmid and chromosomally encoded T-DNA to plant cells: (1) a VirA/VirG two-component operons whose products are probably important for . then transferring the phos- phate group to a conserved aspartate residue on the second component of this transduction pathway. through formation of inactive heterodimers. Upon sen- means of fine-tuning plasmid transfer in response to sory perception. while others code for up to 11 open reading frames. and attM whose product is an very large family of sensor kinases identified in bacteria N-acylhomoserine lactone-lactonase that hydrolyzes the and more recently in eukaryotic cells.

expression or mediate attachment to plant cells. Ros repression is counteracted domain of VirA to induce a conformational change that by the VirA/VirG induction system. and a tain vir operons. galactose. they are also negatively regulated by mediated inducing activity occurs via an interaction the Ros repressor. As such. they generally function to regulate vir gene envelope and translocation through the plant plasma . As described below. Regulators of vir Gene Expression ditions favorable for interkingdom DNA transfer.34 Agrobacterium and Plant Cell Transformation infection of certain plant species or under certain environ. Sugar. Ros pre- On the basis of recent crystallographic analysis of CheY. The molecular basis underlying the is necessary for a productive interaction with components effect of the ChvG and ChvI proteins on vir gene expres- of the transcription machinery.acting regulatory sequence and mounts a behavioral response by modulating gene (TNCAATTGAAAPy) called the vir box located upstream expression. responsible for replication of the Ti plasmid. Plant signals thus enhance Ti plasmid copy number and. sion is presently unknown. Chromosomally Encoded Virulence Genes T-DNA Processing Most studies of the A. As described above. Phospho-VirG activates transcription of the vir from the VirA/VirG system. and the promised for T-DNA transfer. Ros binds to a 9-bp inverted repeat. The VirA/VirG two-component latter activity will be described in the section titled system also activates expression of the repABC genes ‘Attachment to plant cells’. chvE mutants are severely com- ides. promoters prevents the T-DNA-processing reaction. the presence of these com. In turn. This mental conditions. though the physical addition to repressing expression of T-DNA processing mechanism of pH perception is unknown. cells and activation of vir gene expression in response to mal genes also contribute to A. tumefaciens virulence. distinct change. In cated in recognition of acidic pH. tumefaciens. the that carry an o-methoxy group. Interestingly. including glucose. a two-component regulatory system. but in the absence increases the sensitivity of VirA to phenolic inducer of plant signals. plant signals involves the processing of T-DNA into a Although mutations in these genes are often pleiotro. VirA functions as a homodimer. defects in chemotaxis toward sugars. In the ChvE. The type of substitution at periplasmic sugar-binding protein ChvE complexed the para position distinguishes strong inducers such as with any of a wide variety of monosaccharides induces acetosyringone from weaker inducers such as ferulic conformational changes in VirA allowing it to interact acid and acetovanillone. tumefaciens infection process have focused on the roles of Ti plasmid genes in T-DNA One of the early events following attachment to plant transfer. plant cells. pounds is a general feature of most plant wounds and A second locus codes for Ros. arabinose. ChvE thus appears strongly enhance vir gene induction. A periplasmic domain of VirA is also impli. ChvG is the sensor kinase and ChvI is the of each of the vir promoters. VirD operons contribute to the T-DNA processing cules that diffuse across the outer membrane (OM) into reaction. duction system. Although the promoters for these operons are the periplasm is supported by genetic experiments though subject to positive regulation by the VirA/VirG trans- direct evidence for signal binding is lacking. vents premature expression of the T-DNA oncogenes a homologue of VirG. phosphorylation of this family of in the bacterium. the VirC and model that VirA interacts directly with inducing mole. between sugars and the periplasmic sugar-binding protein the ros box residing upstream of these promoters. by promot- nolic compounds as well as the monosaccharides are ing bacterial chemotaxis toward nutrients and by secreted intermediates of biosynthetic pathways involved enhancing the efficiency of opine-encoding T-DNA to in cell wall repair. response regulator. A variety of aldose monosacchar. but they also show acidic sugars D-galacturonic acid and D-glucuronic acid. virulence potential upon perception of environmental con. VirA senses most of the plant-derived signals listed At least three groups of chromosomal genes activate or above. with phenolic inducers. Ros binding to the virC and virD molecules. The inducing phe.5. to play a dual role in the infection process. genes in the absence of a suitable plant host. The most important signal molecules are phenols repress vir gene expression. ChvE sugar interacts with the periplasmic presence of plant signals. a novel prokaryotic likely contributes to the extremely broad host range of zinc finger protein that transcriptionally represses cer- A. form that is competent for transfer across the bacterial cell pic. proteins block vir gene induction or growth of cells at indicating that a phosphorylation-dependent conformation an acidic pH of 5. but several essential and ancillary chromoso. senses environmental signals genes by interacting with a cis. Null mutations in genes for these sphorylated and phosphorylated VirG bind to the vir box. response regulators is thought to induce a conformational Finally. consequently. both nonpho.

also termed as the coupling protein (T4CP). (oriT) sequence to generate a nucleoprotein complex VirC1 is related to the ParA/MinD family of ATPases. tumefaciens adapted an ancestral conjugation C-terminal fragment of VirE2 is fused to the green fluor. it will be interesting to determine when the nick site is present on a supercoiled. double. Moreover. and VirD4 proteins are related in sequence and function to as well as another protein substrate. and nuclear membrane. VirB/D4 System. further underscoring the the VirD4 substrate receptor (SR). VirC1. the T4S family encompasses two other subfamilies. VirD1. the VirB suggested by evidence that the VirD2-T-strand complex. It recruits the VirD2-T-strand nucleoprotein oriT-like sequences located in the T-DNA border particle to the VirB/D4 transfer machine. VirD2 remains covalently bound to the 59 phos. whether these proteins provide VirC1-like functions to stranded plasmid. One. a Member of the Type IV VirD2 and other relaxases carry a motif at their extreme Secretion Family C termini that is devoid of secondary structure and rich in positively charged amino acids. . residue Tyr-29. border cleavage in vivo requires accessory pro- (T-strand) covalently attached to a nicking enzyme (see teins including VirD1. Such stimula- repeats. when the notion that A. it mediates binding of the reporter infection. protein to VirD4 in living cells. VirE2. interact with subunits of conjugation systems. Early studies supplied The VirB/D4 system and other conjugation machines evidence for 59-39 unidirectional transfer of the T-strand. Early below). suggesting that VirC1’s replication (Dtr) proteins assemble at an origin-of-transfer activity is regulated by ATP binding or hydrolysis. particularly arginines. The prevail. A mutation in an invariant Lys residue in the associated with T-DNA transfer are equivalent to those Walker A nucleotide triphosphate binding motif of mediating bacterial conjugation. The VirB proteins are mutations in the signal motif of one such substrate. couple processed DNA substrates with their cognate In addition to oriT nicking. studies showed that VirC1 binds the overdrive sequence located next to the right border repeat sequences of octopine-type Ti plasmids. the relaxase. One component of the relaxo. extrachromosomal – encode ParA/MinD homologues. A. the conjugative transfer intermediate is thought to parti- cipate in translocation of substrate DNA by supplying a signal motif recognizable by the transport machinery. from 11 VirB subunits and VirD4. and VirC2 proteins. an ancillary protein. generating processing reaction. Purified VirD2 catalyzes cleavage of but note that many mobile elements – both integrated and oligonucleotides bearing a T-DNA nick site. Very interestingly. VirC1 localizes at associated with the 59 end of the DNA strand destined cell poles. a set of proteins termed as the DNA transfer and VirC1 on T-strand production. tion channel. termed as the mating pair formation (Mpf) proteins. cytosol. as expected. VirC1 (VirC1K15Q) abolished the stimulatory effect of tion. Roles of Ancillary Processing Factors in T-DNA ing view strongly supported by molecular and genetic Processing and Transfer data is that T-DNA is transferred as a nucleoprotein Although VirD2 catalyzes nicking of T-DNA substrates particle composed of a single-stranded DNA molecule in vitro. In the generalized reac. which is also the site of VirB/D4 machine for transfer (T-strand). The charged motif likely confers VirD4 the SR. Agrobacterium and Plant Cell Transformation 35 membrane. and block translocation. cleaves and remains covalently during cell division. tumefaciens translocates the T-complex as well as effector This motif is also present at the C-termini of protein proteins through a dedicated secretion channel assembled substrates of the VirB/D4 T4S system and. system to deliver effector macromolecules to plants during escent protein (GFP). VirF. tems (Figure 4). another important function to stimulate substrate translo- In A. A recent quantitative analysis Roles of VirD2 Relaxase in T-DNA Processing established that both VirC1 and VirC2 are required and Transfer for synthesis of as many as 50 copies of the T-DNA transfer intermediate per cell within a 24-h induction It is now widely accepted that DNA-processing reactions period. of Gram-negative and -positive bacteria are members of a which is also compatible with the notion that the relaxase large family of translocation systems termed as T4S sys- serves to pilot the attached T-strand through the secre. tumefaciens. termed as the relaxosome. recognition of the substrate by the secretion channel. the relaxase component of transfer machines. as As discussed above for the Dtr-processing factors. In addition to the conjugation machines. is essential for nicking in vitro In future studies. Besides stimulating the conjugative template by a strand displacement reaction. However. The T-strand is unwound from its assembly (see below). polar-localized VirC1 supplies the translocation-competent relaxase-T-strand substrate. tory functions associated with conjugation have not been phoryl end of the nicked T-DNA via conserved tyrosine described previously for other ParA/MinD homologues. the VirD2 relaxase generates nicks at cation. which mediate partitioning of chromosomes and plasmids some.

and VirB4 are the three energetic compo- of mammals including H. to which substrates are delivered. The list of pathogens dependent on effector translo. Mutations in the Walker A motifs invariably abolish sub- cellular milieu. pylori Com IVA N. Legionella pneumophila. Each of these subunits Brucella and Bartonella species. Nevertheless. Although these systems are func- the Helicobacter pylori ComB competence system. or to tionally versatile in terms of the substrates and target cells release DNA to the milieu. as exemplified by a chromo. they share a number of somally encoded F-like transfer system carried on the common structural and functional features that distin- gonococcal genetic island (GGI) of Neisseria gonorrhoeae. these systems systems. reminis. teins have now been solved by X-ray crystallography. the ‘effector translocator’ sys. and nents of the VirB/D4 T4SS. VirB11. VirB/D4 Type IV System: Machine Architecture The third subfamily. take up DNA from the extracellular milieu or release DNA to the environment. promote genetic exchange and. as exemplified by machines (Figure 4). A third subfamily of T4S systems. The VirB/D4 system is composed of two surface structures – tems. time. drives machine assembly or function. designated as the DNA uptake/release systems. Related systems of several additional strate translocation. fairly comprehensive architectural cent of the needle complexes elaborated by type III models of the T4S system can be generated through secretion (T3S) machines. pertussis Ptl DNA uptake/release H. and thus the list of T4S effector Structures of soluble domains of two VirD4-like pro- translocators continues to grow. play indispensable roles in the infection processes a secretion channel and a conjugative pilus (Figure 5). true exporter to deliver its toxin substrate to the extra. guish them from other known bacterial translocation As with the conjugation machines. but this system functions as a site (Walker A) motif required for substrate translocation.36 Agrobacterium and Plant Cell Transformation Conjugation T-DNA VirB B1 B4 B5 B6 B8 B9 B10 B11 D4 B2 B3 B7 IVA R388 IncW RP4α IncP F IncF Effector translocation H. prowazekii IVA Brucella spp. pylori Cag R. pylori. those encoded by virB2 through virB11 and virD4 are essential for T-complex transport to plant cells. function The T4S systems are classified on the basis of exten- independently of contact with a target cell to take up sive sequence similarities with subunits of conjugation DNA from the extracellular milieu. Energy subcomplex – VirD4. At this of many prominent pathogens of plants and mammals. VirB4. A. and interaction studies of machine strates through direct contact with the eukaryotic target subunits. meliloti. and several pathogens VirD4. termed as the ‘DNA uptake and release’ systems. Effector translocation systems function to secrete proteins to eukaryotic cells during the course of infection by many medically important bacterial pathogens. there is no high-resolution structure for either struc- These machines can be viewed as ‘injectisomes’. Of the 11 VirB proteins. tumefaciens and Burkholderia cepacia. ture. also represent potential mechanisms for transfer of survival traits during infection. therefore. VirB11 plant symbionts such as S. VirB B. three stable subassemblies of VirB/D4 components. strongly indicating that ATP binding pathogens are also implicated in the trafficking of sub. cators for disease progression includes at least two phytopathogens. structural. because they deliver their sub. Ancestrally related conjugation systems mediate interbacterial transfer of DNA. one . strates to eukaryotic cells. gonorrhoeae Figure 4 Alignment of genes encoding related components of the T4S systems. topological. These studies have supplied evidence for at least cell. Bordetella pertussis uses an possesses a characteristic nucleoside triphosphate binding effector translocator as well.

and coli FtsK. The three ATPases energize machine assembly and substrate transfer and a stable ‘core’ complex nucleates machine assembly. Walker A and B nucleoside triphosphate-binding domains. VirB11 associates peripherally but tightly with the 110 Å in diameter and 90 Å in height.or dsDNA. one near the N-terminus and a second just N- shown by electron microscopy imaging. tem. The FtsK structure is slightly larger with an outer dia. TrwC. The Agrobacterium tumefaciens VirB/D4 T4S system localizes at the cell poles and is postulated to assemble as a transenvelope complex through which substrates pass to the cell surface. VirB7. predicted transmembrane segments (TMS) and a cyto- meric rings discernible by electron microscopy. the VirB proteins plus VirD4 are required for substrate secretion. last two also by X-ray crystallography. A combination of experimental studies and computer mod- meter of 120 Å and a central annulus of 30 Å. and cell localization and protein–protein molecules. Purified homologues TrbB. therefore. tumefaciens. A large central periplasmic loop. All of the VirB proteins are required to build the T pilus. is now known to play an important . functions as a receptor for the T-DNA and protein substrates of the VirB/D4 T4S sys. The eling has yielded a topology model depicting VirB4 as predicted structure is a dodecamer composed of two predominantly cytoplasmic with possible periplasmic hexamers stacked in a head-to-head arrangement. ity. and the plasmic C-terminus. unknown. The T pilus is sloughed from the cell surface and is not essential for DNA or protein translocation. Five VirB proteins are implicated as forming a ‘core’ VirB11 is a member of a large family of ATPases transenvelope structure on the basis of phylogenetic rela- associated with systems dedicated to secretion of macro. Core subcomplex – VirB6. and Brucella suis VirB11 assemble as homohexa. As loops. Agrobacterium and Plant Cell Transformation 37 B5 B2 VirB5 VirB2 VirB8 VirB7 VirB9 OM VirB2 VirB3 VirB5 B9 VirB10 B1 B7 B3 P B5 B10 B10 VirB6 VirB11 B8 VirB1 VirB4 Core complex VirD4 B6 B4 ATP ATP B11 CM D4 ATP Substrate receptor Figure 5 Topologies. and there is some evidence for ATP ture bears a striking resemblance to the F1-ATPase 33 regulation of membrane binding. The role of VirB11 in heterohexamer. VirB9. tionships. whereas the structure of the soluble T4S is still fundamentally unknown. the con- through the FtsK annulus. structures. and cellular localizations of the VirB/D4 T4S subunits. TrwC presents as six equivalent protomers C-terminal halves and a central cavity of 50 Å in dia- assembled as a spherical particle of overall dimensions meter. As with VirB11. VirB6 is highly hydrophobic with five HP0525. The overall struc. IM of A. domain closely resembles DNA ring helicases and other VirB4 subunits are large IM proteins with consensus proteins such as FtsK that translocate along ss. of TrwC encoded by plasmid R388 and one of Escherichi present as double-stacked rings formed by the N. These structures designated loop P2. dsDNA runs terminal to the Walker A motif. though this needs to be explored. VirD4. H. pylori interaction data. and it might also function as an inner-membrane VirB10 (IM) translocase. VirB8. providing a structural view of tribution VirB4 to machine assembly and function is a previously described ATP-dependent translocase activ.

tumefaciens VirB10 inter- (TrIP) was developed to trace the path of a DNA sub- acts with the IM subunits VirB8. the Pro-rich motif contributes Definition of the T-DNA translocation pathway to a rigid. VirB2 bears both sequence and struc. Furthermore. VirD4 can recruit a protein by X-ray crystallography. ments are absent from the surfaces of mutant strains This pathway provides the first glimpse of how the T4S defective in expression of one or more of the virB genes. and RP4. extended structure in the periplasm that might by TrIP permit simultaneous contacts with partner subunits at An assay termed transfer DNA immunoprecipitation the IMs and OMs. it was shown that the substrate forms close contacts establishing contact between plasmid-bearing donor cells with 6 of the 12 VirB/D4 components. The available data are consistent with a to understand how secretion substrates are recruited to proposal that VirB6 assembles as a central component of the T4S machine and the route of substrate translocation. Recently. to the cell pole. VirB8 As noted above. as deduced by the finding that VirD4 or undergo an unusual head-to-tail cyclization reaction. strate through the T4S channel. resulting in a cyclic polypeptide that accumulates in the However. a Pro- a mediator or coupling factor is not known. and it ties of T4S systems. which is composed of the that binding of both types of substrates occurs inde- VirB2 pilin protein. but not VirD4.2. and a region of sequence conservation at the C-terminal end. of periplasmic fragments of both the subunits were solved Independent of VirC1. channel might be configured across the cell envelope Furthermore. are required well as protein substrate receptor activity. chromatin immuoprecipitation assay. VirB6 interacts Dynamics of T4S System Machine Assembly with two OM proteins. Whether other protein several structural features with TonB. With the TrIP The T4S systems involved in conjugation elaborate pili for assay. The VirB7 lipoprotein forms a disulfide bridge Recruitment of secretion substrates to the with VirB9. adapted from the and with the OM-associated VirB7–VirB9 heterodimer. the secretion channel mediating substrate transfer across the IM. The steps in the pathway are as follows: cells grown at room temperature rarely possess pili. VirB11 Walker A mutations support substrate transfer. VirB2 polymerizes as the T pilus. communication between VirD4 and VirB11. and VirB7 lipopro. tural similarity to the TraA pilin subunit of the F plasmid and to the TrbB subunit of plasmid RP4. Electron microscopy studies have polytopic VirB6. These fila- ordered translocation pathway for the T-DNA substrate. involves formalde- Intriguingly. suggesting for the assembly of the T pilus. bitopic VirB8. a single TMS. and immunoprecipitation to recover channel subunits. tor. structures the processed T-complex to the polar-localized VirD4 SR. and VirB9. ParA/MinD-like VirC1 functions to recruit and VirB10 are bitopic IM subunits. VirE2. is processed from a 12-kDa propilin to a 7. For TonB. and the heterodimer sorts to the OM where it VirB/D4 T4S machine exerts stabilizing effects on other machine subunits. T4S channel for substrate translocation (see below). Over its length. rich region. in the absence of certain ‘core’ VirB proteins. A VirD4 Walker A mutant also retains T-DNA as All of the VirB proteins. pendently of ATP energy. VirB10 also functionally resembles TonB by hyde treatment of intact cells to cross-link channel linking energy at the IM to the assembly or gating of the subunits to the T-DNA substrate as it exits the cell. These studies have exploited a colocalizes with VirD4 and the T pilus at the cell poles of combination of cytological and biochemical approaches A. tumefaciens enabled formulation of a sequentially and spatially cells induced for expression of the virB genes. VirB7 is then detected by PCR amplification. TrIP. VirD4. establishing that VirD4 is the T-DNA recep- transfer of substrates to plants. tumefaciens. and and Function probably also with the other VirB ‘core’ subunits. Next. VirB2 pilin. VirB7 lipoprotein and VirB9. however. VirB10 shares effector. demonstrated the presence of long filaments approxi- Analyses of various T4S mutants with the TrIP assay mately 10 nm in diameter on the surfaces of A. and VirB4. VirD4 cannot transfer the substrate to VirB11 IM. the interesting observation was made that (Figure 5). whereas cells grown at 19  C possess these structures in • Substrate recruitment. A. VirB5.38 Agrobacterium and Plant Cell Transformation role in substrate translocation (see below). solubilization of membranes. This early transfer step also proceeds independently of kDa mature protein. VirB11. and recipient cells. The presence of T-DNA substrate in the immunoprecipitates T pilus subcomplex – VirB2. including a small substrates can also interact directly with VirD4 or require N-terminal cytoplasmic domain. VirD4 transfers the T-DNA substrate to the VirB11 ATPase. VirB6 Recent studies have begun to describe dynamic proper- exerts stabilizing effects on other VirB subunits. disruption of the cells. like TraA • Transfer to the VirB11 hexameric ATPase. suggesting tein and VirB5 associate at unspecified locations along the that these core components are important for productive T pilus. VirD4. The T-DNA substrate binds abundance. VirB2. . both VirB2 and TrbB ATP energy. This finding correlates nicely with previous VirD4 and it does so independently of other VirB findings that low temperature stimulates the virB-dependent proteins. Similarly.

a third VirB8 for polar localization. VirC1. the Ti plasmid itself localizes required for substrate transfer. tumefaciens can deliver T-DNA to unwounded VirB8 to that in the periplasm composed of VirB2 and plant tissues. VirB2 polymerizes as the T pilus. and time to mediate translocation of T-DNA and protein Therefore. whereas VirB3. and ATPase of this secretion system. (2) the VirD4 SR. tumefaciens vegetative cells. it is now known portion of the secretion channel composed of VirB6 and that A. this At this time. at least three protein complexes probably is not the case because certain mutations have been shown to localize at cell poles independently of each other: (1) the relaxosome bound at T-DNA border block pilus production without affecting substrate sequences that consists of VirD1. are protein complexes. VirB4. of entry through damaged cell walls. While TonB senses an IM electro. Several lying molecular basis for polar targeting of these various T4S subunits. possibly. therefore. including VirB3. through the lumen of the pilus to the plant cell. might be mechanical force required for a latter stage of machine phenotypically silent. the OM. the T-DNA substrate translocates from the IM tant for T-DNA integration. As noted above. VirB10 might transduce IM energy to mediate that A. VirB3. yet transformation of chemical gradient. VirB10 senses IM ATP energy. dispelling the notion that wounding is an VirB9. not channel subunits per se. As noted VirB4 and VirB11 Walker A mutants display WT locali- above. Although it is zation patterns. VirB10 for infection. Substrate to abiotic and biotic surfaces. Substrate Transfer Through the Plant Cell Energetics of DNA translocation: VirB10. VirB5. constituents and such molecules are potent vir gene indu- erodimer or multimer by a mechanism requiring ATP cer molecules. VirB6 and bitopic VirB8 proteins. the bitopic protein VirB10 interacts with infects plants at wound sites. which is important for VirB6 binding to the DNA substrate. These findings suggest that VirB10 supplies a essential prerequisite for transformation. as and VirB10 – were shown to depend on production of well as the energetic contributions of VirB4. IM energy is converted into a mation. VirB2 and VirB9 comprise the distal required for polar targeting of other VirB proteins. raising the intriguing possibility biogenesis. and VirB10 are probably substrates to target cells. Thus. tumefaciens cells have been shown to via their cell poles subsequent substrate translocation steps. In both unwounded tissues typically does not incite tumor for- the cases. the VirB/ transfer from VirB11 to VirB6 and VirB8 also require D4 T4S system also localizes at cell poles. six VirB proteins – VirB1. VirB9. However. but they do machine complexes coordinate their activities in space not form detectable interactions with the T-DNA. VirB2 might be at or near the cell poles of A. VirD2. Wounding potentially also creates portals energy use by VirD4 and VirB11. and stimulates cell tion of the transenvelope VirD4–VirB10–VirB7–VirB9 replication and division reactions considered to be impor- complex. The VirB4 and VirB11 ATPases are not • Transfer to the periplasmic and OM-associated proteins VirB2 and VirB9. Adding to this picture. a TonB-like ATP energy sensor subunit The delivery of T-DNA and protein substrates to plant Assembly and function of the conjugation machines cells requires productive contact between A. termed Spatial Positioning of the VirB/D4 T4S System P2. The most visible function similar to that described for the TonB energy manifestation of A. In recent additional ‘core’ subunits. It a component of the secretion channel extending will be interesting in future work to identify the under- through the periplasm and. VirB11 were found to localize at cell poles independently of VirB8. giving rise to a widely held VirD4 and was shown to undergo a structural transition in view that wounding establishes important preconditions response to ATP utilization by VirD4 and VirB11. tumefaciens transformation is the pro- transducer proteins. tumefaciens. A. possibly important for studies. and end of the T-DNA translocation pathway. and also to understand how these required for this step of substrate transfer. therefore. and translocation. suggesting that nucleation of the VirB formally possible that the T-DNA substrate moves proteins at the cell pole does not require ATP energy. VirB6 mutational studies identified a central periplasmic loop. and (3) the VirB channel com- mutant proteins. tumefaciens commonly A. VirB5–VirB7. In and a susceptible plant cell. VirB11 binding to these latter channel subunits. however. In strains producing the ‘uncoupling’ VirC2. VirB11 delivers the T-DNA substrate to the polytopic formation or opening of a VirB7–VirB9 channel complex to allow passage of the DNA substrate to the cell surface. duction of plant tumors. the cellular form of VirB2 is still plex. During wounding. Other domains were implicated in regulating A. Many transformation events. but rather contribute to the structural integrity of the channel. Accompanying forma. and VirB10. Agrobacterium and Plant Cell Transformation 39 • Transfer to the integral IM proteins VirB6 and VirB8. tumefaciens requires both proton motive force and ATP energy. VirB5. plants release cell wall also interacts with the OM-associated VirB7–VirB9 het. tumefaciens actually has a much broader host .

tumefaciens has been demonstrated to trans- Interestingly. therefore. VirD2 was shown to interact Recent genomic studies have begun to identify possi. Another set of genes designated the other translocated substrates function to enhance the as cel direct the synthesis of cellulose fibrils that emanate efficiency of transformation. pAtC58. a probable cell wall constitu- across the plant plasma membrane. a these cell types and. and Integration into the Host Genome Next. tumefaciens must bind plant cells to deliver T-DNA encoding arabinogalactan. and exoC (pscA). the nucleus. tumefaciens. The first is encoded by Attachment-proficient A. chv mutants accumulate low levels of port DNA to representatives of prokaryotes. tumefaciens to specific sites on the plant VirE2 binds cooperatively to the T-strand. though system. is a large nucleo- Efficient attachment of bacteria to plant cells also protein complex of an estimated size of 50  106 Da. 600 molecules of VirE2 along its length. Periplasmic -1. Binding is saturable. T-complex to the nuclear pore. Intriguingly.2 glucan plays a VirD2 also has been shown to interact with several members role in equalizing the osmotic pressure between the inside of a family of proteins termed cyclophilins. a member of a conserved family of importin/ ble plant proteins involved in attachment. and VirD5 proteins. this has been questioned because the att genes reside on a VirE3. chvA. link between the T-complex and microtubule track system. VirE2 is a single-stranded 542 kb plasmid. VirE2 is exported separately from the bacterial cell surface. whereas attachment-deficient cells do not Ti plasmid genes. Recent evidence indi. Yet other candidate receptors the given plant cell is competent for the receipt of identified to date include a rhicadhesin-binding protein. both VirD2 and VirE2 carry nuclear loca- the bacterial envelope by contributing to transporter lization sequences (NLS) that contribute to delivery of the assembly. suggestive of a VirD2–T-strand–VirE2 particle termed the T-complex. might loci is a two-step process in which bacteria first attach mediate binding of the T pilus to the plant cell surface. Binding via the chromosomally encoded attachment designated VirB2-interacting proteins (VBTs). is postulated to function as a molecular -1. All three loci are involved in synth. It has been proposed that loss of role for cyclophilins in this interaction is to supply a chaper- this form of glucan may indirectly disrupt virulence by one function at some stage during T-complex trafficking to reducing the activity or function of cell surface proteins. loosely and nonspecifically to the plant cell surface. Cyclophilins are ubiquitous proteins found in all of complex transport system (see ‘The VirB/D4 System. that -1. These fibrils are impli. from the VirD2–T-strand particle.2 glucan molecule. VirF. from plant nucleases and also facilitate T-complex move- esis of transport of a cyclic -1. This is a nonsaturable and aggregation-like mode of interac- Substrate Movement Through the Plant Cytosol tion reversible by washing with a buffered salt solution. siology of A. and several proteins virB-encoded T pilus. The postulated and outside of the cell. a T-DNA. limited number of attachment sites on the plant cell. A collection of karyopherin proteins that are known to bind NLS and . suggesting that interact with VirE2. The others identified to date include the VirE2. This binding event directs bacterial bind this molecule. human vitronectin and binding to many plant cells independently of whether or antivitronectin antibodies both inhibit the binding of not the bacterium is competent for exporting T-DNA or A. act sequentially or in tandem.2 glucan synthesis is important for the overall phy. VIP1. shown to bind the VirB2 pilin protein. the bacteria attach more specifically in a tight and saturable interaction that is resistant to washing. suggesting tance for A. A series The VirD2–T-strand complex is only one of several sub- of genes designated attachment (att) genes were impli. Some of these mutations disrupt attachment of Agrobacterium and thus might map to surface Attachment to Plant Cells receptors. and The T-complex. may be of general impor- Member of the Type IV Secretion Family’). plants. tumefaciens-mediated DNA transfer. forming a plant cell surface. a protein shown to Mutations in these genes are pleiotropic. These proteins. strates delivered to plant cells through the VirB/D4 T4S cated in mediating the latter mode of attachment. Evidence exists that VirD2 and VirE2 protect the T-strand chvB. but upon transfer to the cated in attachment of A. with AtKAPa. render the host plant recalcitrant to Agrobacterium trans- formation (rat mutants).2 glucan might influence T-DNA export across Additionally. composed of a 20-kb T-strand capped binding of virulent strains can also be prevented by the at its 59 end with a 60-kDa endonuclease and an estimated attachment of avirulent strains. tumefaciens cells bind radioactive chromosomal loci and occurs even in the absence of the vitronection. The second binding event is mediated by the cellulose synthase-like protein. yeasts. Another candidate receptor is a vitronectin-like cates that there are at least two binding events that may protein found in detergent extracts of plant cell walls. one of the proposed components of the T. tumefaciens to plant cells. ment along a microtubule network. A.40 Agrobacterium and Plant Cell Transformation range and can infect different plant cell types than mutations in Arabidopsis thaliana were generated that reported previously. and VirB10. ent. whereas dispensable for virulence. One such mutation is in the promoter of a gene A. which has been shown to be DNA-binding protein required for transformation. requires the products of three chromosomal loci.

In the case of monocot DNA transfer system for genetic engineering is its extre. and core now known to transform many nonplant species including histones that bind VIP2. reaction are not known. T-DNA S. tumefaciens excised leaves or root sections. and M. engineer nonplant organisms. the potential exists that the host range of generated as a consequence of active DNA replication. thus. known to transform a wide range of gymnosperms and lated to mediate nuclear import of VirE2. NGR234. fertile plants recovered during selective regeneration of trans- formed protoplasts. Agrobacterium is teins that bind VirD2. Agrobacterium and Plant Cell Transformation 41 mediate nuclear import. If the plasmid carries two border sequences. These different dicotyledonous plant species of agricultural importance. and maize. plant proteins. Plant specific sites in the plant genome. the entire plas- integration into the host genome. only binding of plant factors with bacterial effector proteins. might act synergistically or redundantly Additionally. the second strand of the T-DNA is replicated and annealed to A. According to a current model. tumefaciens can transform nonplant species such as yeast Increasingly. And now. another translocated that is. A. most translocated substrate. wheat. onto a plasmid that carries a single T-DNA border F-box and Skp1 are conserved components of E2 ubiquitin sequence or two T-DNA border sequences that flank ligases that mediate protein destabilization. For certain species. with the discovery that other alphaproteo- plant nuclear genome by a process termed ‘illegitimate’ bacterial species including Rhizobium sp. and surprisingly A. (2) deliver foreign DNA to ered to its site of integration in the host chromatin. Clearly. yeast. the DNA bounded by T-DNA borders is delivered to However. another plant species including rice. tumefaciens is readily manipulated such that plasmids the opposite strand of the plant DNA. but the molecular details of this Typically. species such as maize. the T-complex must be deliv. For example. immature embryos are the pre- mely broad host range. Intriguingly. a significant reduction in the number of transgenic. Once the ends of Nuts and Bolts of Genetic Engineering T-DNA are ligated to the target ends of plant DNA. Whether the armament of translocated effectors A. posed to anneal via short regions of homology to the unnicked strand of the plant DNA. with protoplast transformation there is often is an interesting question for further study. however. including CAK2M and TATA box-binding pro. T-DNA integrates into the cells. broadened even further. Interestingly. genes through T-DNA tagging. and many other fungi. the A. other transformation meth- ods have relied on transformation of plant tissues such as One of the most appealing features of the A. VIP1 that binds VirE2. which are plant homologues of yeast Skp1 proteins. and human targeting of the T-complex. meliloti. loti also deliver DNA substrates to plant invades at nicks or gaps in the plant genome possibly target cells. VirF was shown various restriction sites for cloning as well as an antibiotic to destabilize a VIP1–VirE2 complex and thus might play resistance gene to select for transformed plant cells. The frequency of stable transformation is often date participate in some way to the movement of T-DNA very high. note that all characterized effectors identified to plants. half of the protoplasts. tumefaciens with regenerating protoplasts of certain includes proteins whose functions are unrelated to T-DNA plant species can result in transformation of up to one movement and instead involved in disruption of plant phy. mimics the function of VIP1 in med. If the a role in uncoating the T-DNA prior to or during plasmid carries a single border sequence. but still harboring substrate. deleted of oncogenic T-DNA. However. Agrobacterium has long been ferred starting material for A. VirF. Consequently. DNA integration step. well-exceeding frequencies achieved by other through the plant cell or its integration into the plant gene delivery methods. Correspondingly. recombination. VIP1 is postu. of the effort in developing these transformation protocols iating VirE2 nuclear import. Both VirD2 and carrying foreign genes of interest are easily introduced VirE2 have been implicated in contributing to the T. Foreign and interact with several members of the ASK protein genes destined for delivery to plants are generally cloned family. cocultivation of genome. during the past two decades protocols have to mediate movement of the T-complex through the plant been developed for transformation of monocotyledonous cytoplasm and nuclear pore. may be important for chromatin other prokaryotes. tem is being used to (1) isolate and characterize novel plant Once inside the nucleus. strains used for gene delivery are ‘disarmed’. eukaryotic cell types transformable by bacteria can be The invading ends of the single-stranded T-DNA are pro. and (3) genetically proteins. VirE3. tumefaciens-mediated DNA . possibly explaining how has been directed toward improvement of crop traits. has been shown to possess an F-box domain intact Ti plasmid and chromosomal vir genes. So far. protoplasts can be Agrobacterium Host Range and Genetic transformed but are recalcitrant to regeneration into Engineering intact plants. tumefaciens is T-complexes and integration of T-DNA into the plant capable of delivering in excess of 180-kb of DNA to genome is a complex multistep process involving specific plants. siological processes to promote the overall infection process However. into appropriate bacterial strains for delivery to plants. movement of mid is delivered to plants. tumefaciens gene delivery sys- and human cells that lack VIP1.

tumefaciens- transform fungal species of interest means that all mediated gene transfer to fungi may not be restricted approaches developed for plants now can be applied to solely to the laboratory bench. and the progress toward this goal has involved the use of the types of selectable genes delivered to plant cells all influ. sired consequences. the kinds of vectors and bacterial strains. For directed In addition to the need to identify transformable and T-DNA insertion. This DNA transfer system is especially characterization of promoter activity. a large amount of information has into an essential gene. but further manip- dependent on a genetic interplay between A. raising the possibility that A. Today. Several variations to this methodology Saccharomyces cerevisiae genome. This underscores the notion that site. insertion can result in a modulation of gene methods. For rice. both the plant and the T-DNA are regenerable plant tissues. which. rearrangements of flanking sequences. Ideally. Further. but further efforts are needed to overcome present obstacles Gene Transfer to Yeast and Fungi impeding efficient transformation of other species of interest. Finally. A. success has been achieved with callus enhances the chances that the insertion will have unde- tissue induced from immature embryos. the mutated gene of interest can and hyphal tissue. The transformation of fila- throughout the plant genome. tumefaciens is increasingly used to characterize and for integration by homologous recombination. the type and age of plant to a restricted number of sites in the plant genome. useful tool for the genetic manipulation and characteriza- insertion downstream of a plant promoter will permit tion of fungi. a number of varieties of a engineered to carry lox sequences and the plant is also given species often need to be screened to identify the engineered to express the Cre protein. insertion is often accompanied by been assembled about the A. Conclusions Homologous or Site-Specific Recombination Although random T-DNA insertion is a boon to investi.42 Agrobacterium and Plant Cell Transformation transfer. When the isolate novel plant genes by an approach termed T-DNA lacks any extensive regions of homology with the T-DNA tagging. Additional fac. T-DNA is widely used mentous fungi with A. tumefaciens was shown to efficiently deli- phenotypes. The successful transfer of DNA to yeast depends on the presence of stabilizing sequences such as a yeast origin of replication sequence or a telomere. or regions of homol- T-DNA Tagging ogy between the transferred DNA and the yeast genome A. Upon entry of susceptible varieties. genome upon infection of bacterial cells. for P1. For rice and corn. It is also of interest to consider that both expression with potentially interesting phenotypic con. which further This information has been used to successfully . results in circularization of the P1 routine technique. tumefaciens ulation of this system should enhance its general and host cells. tumefaciens and many fungal species exist in the same sequences. Cre was shown to catalyze efficiencies is often observed depending on which cell the site-specific integration of T-DNA at the plant lox line is being tested. A large variation in transformation T-DNA into the plant cell. The early discovery that the oncogenes can be excised gators interested in characterizing plant or fungal genes. For example. bacteriophage P1 Cre/lox system for site-specific inte- ence the transformation efficiencies. The Cre site-specific most of these parameters have been optimized so that now recombinase catalyzes strand exchange between two lox the delivery of foreign DNA to these crop plants is a sites. In the way for the fast-growing industry of plant genetic addition to the potential result that T-DNA will insert engineering. This discovery extends well beyond easily be recovered in bacteria by suitable molecular academic interest because the simplicity and high effi- techniques. Fortunately. Recent tissue. tumefaciens is an exciting today as a mutagen for isolating plant genes with novel advancement. it integrates at random exist depending on the desired goals. if the valuable for species such as the mushroom Agaricus T-DNA is engineered to carry on outward reading pro. bisporus that are recalcitrant to transformation by other moter. positions by illegitimate recombination reminiscent of because insertions are generally randomly distributed T-DNA integration in plants. many of the agronomically applicability. If the mutagenic T-DNA carries a bacterial ver DNA to fungal protoplasts as well as fungal conidia origin of replication. Conversely. A. gration in the plant genome. the characterization of fungi. if the T-DNA is engineered to ciency make this gene delivery system an extremely carry a selectable or scorable gene near one of its ends. it from T-DNA and replaced with genes of interest paved is an undesired event for plant genetic engineering. the discovery that A. The frequency of directed insertion events is low interkingdom DNA transfer is a complex process compared to random insertion events. tumefaciens can soil environment. important species are readily transformable. tumefaciens infection process. T-DNA could be delivered tors such as plant genotype.

this information by diverse species of bacteria. The dis. and function of bacterial type IV secretion systems. just in quorum-sensing transcriptional regulators. mical signals comprise the words for a dynamic dialog Christie PJ (2004) Type IV secretion: The Agrobacterium VirB/D4 and related conjugation systems. Schrammeijer B. Farrand SK. covery of T-DNA transport itself supplied a mechanistic Citovsky V. The discovery that secreted che. studies have revealed that numerous Gelvin SB (2003) Agrobacterium-mediated plant transformation: The pathogens employ interkingdom transport to deliver a biology behind the ‘‘gene-jockeying’’ tool. Biochimica et Biophysica ACTA between A. Annual Review of Microbiology transduction systems in many bacterial systems. ancestrally Tzvira R and Citovsky V (2006) Agrobacterium-mediated genetic related to flagellar systems. evolution of genomes of higher organisms. Bacteriology 182: 3885–3895. and extracellular signals and characterize the cognate signal Cascales E (2005) Biogenesis. This discovery Fuqua WC. Both T3S and T4S systems transformation of plants: Biology and biotechnology. tumefaciens cells has stimulated a global effort to identify Christie PJ. Cellular Microbiology 9: 9–20. about the evolution and function of pathogenic mechan. Annual Review of Cell and Developmental Biology 22: 101–127. Hooykaas PJJ. to conjugation systems. Yeo HY and Waksman G (2004) Unveiling molecular scaffolds of the infection process will continue to spawn new applications type IV secretion system. (2005) Gene transfer to plants plants and other organisms. Nature Reviews Microbiology 1: 137–150. Jakubowski S. tumefaciens and plant cells as well as other 1694: 219–234. pathogen Agrobacterium tumefaciens. Journal of Bacteriology for this novel DNA transfer system and yield new insights 186: 1919–1926. et al. has often established a conceptual framework for initiat. Lacroix B. A. Molecular Biology Reviews 67: 16–37. Kozlovsky SV. Krishnamoorthy V. For the future. in Biotechnology 17: 147–154. tumefaciens 362: 1135–1148. Agrobacterium and Plant Cell Transformation 43 manipulate the T-DNA transfer system both to enhance Further Reading its efficiency and to broaden the range of transformable Broothaerts W. . pathway for a DNA substrate. White CE and Winans SC (2007) Cell-cell communication in the plant cell contact and. Science 304: 1170–1173. Nature 433: 629–633. which are ancestrally related transfer. it is clear that the Royal Society of London. or by the T3S systems. (2007) Biological systems of explanation for how horizontal gene transfer impacts the the host cell involved in Agrobacterium infection. Indeed. McCullen CA and Binns AN (2006) Agrobacterium and plant cell Interkingdom macromolecular translocation is mediated interactions and activities required for interkingdom macromolecular either by the T4S systems. in some cases. and Winans SC (2000) The bases of crown gall tumorigenesis. Journal of isms that are broadly distributed in nature. Microbiology and wide array of effector proteins to plant and animal hosts. Weir B. Philosophical Transactions of cellular filament or pilus. architecture. and Greenberg EP (1996) Census and also established a precedent for interkingdom transport of consensus in bacterial ecosystems: The LuxR-LuxI family of virulence factors by bacterial pathogens. et al. Current Opinion translocate substrates via processes dependent on cell-to. Biological Sciences studies of all the various aspects of the A. elaboration of an extra. the last decade. Furthermore. Zhu J. Winans SC. Cascales E and Christie PJ (2004) Definition of the type IV secretion symbiotic relationships. Michell HJ. Atmakuri K. 59: 451–485. Cascales E and Christie PJ (2003) The versatile bacterial type IV ing or extending studies of other pathogenic and secretion systems. Series B. Oger PM. Annual Review of Microbiology 50: 727–751.

Jülich. 44 . Abbreviations MSG monosodium glutamate AEC analogue aminoethyl-L-cysteine OUR oxygen uptake rate CSL corn steep liquor PGDH 3P-glycerate dehydrogenase DO dissolved oxygen SHMT serine hydroxymethyltransferase GA glutaraldehyde THF tetrahydrofolate derivative HA hexamethylene diamine Introduction feed amino acids L-lysine. in the form of the flavor enhan- groups. and (4) tives are also used in the chemical industry. and acids are essential for mammals. Amino Acid Production L Eggeling. except glycine. Research Center Jülich. tion. drug manufacturing. Research Center Jülich. All rights reserved. amino acids are used for The production methods to date are (1) microbial produc- infusions and as therapeutic agents. Eight of these protein-forming L-amino tured represent a value of roughly US$5000 million. L-threonine. Jülich. There are large demands thus trail behind antibiotics made with microorganisms. Institute of Biotechnology. and racemic mixture. Amino acid deriva. Germany H Sahm. for amino acids in the areas of food and feed additives and The market is growing steadily by about 5–10% per year. recombinant DNA technology. transport. The total amino acids manufac- L-enantiomers. They are the building blocks of peptides. cer monosodium glutamate (MSG). and function to transport substances into or out of the cell regulatory functions of the cell with the application of through the cytoplasmic membrane. Introduction Future Prospects Microbial Production Further Reading Enzymatic Production Glossary metabolic engineering The improvement of cellular carriers/transporters Membrane proteins that activities by manipulation of enzymatic. There are 20 protein-forming amino acids. At 34%. commercially valuable products. and L-aspartate and and components of other complex polymers like the cell L-phenylalanine. (3) chemical synthesis. fungicides. a volume of 56% of the other procedures give rise to optically pure L-amino the total amino acid market was occupied by the so-called acids. product. surface-active agents. and L-tryptophan. fermenter A large growth vessel used to culture regulation Processes that control the activities or microorganisms on a large scale for the production of synthesis of enzymes. Institute of Biotechnology. pesticides. Germany ª 2009 Elsevier Inc. the latter two used as starting materials wall. In medicine. such as in enzymatic synthesis. which may require additional resolution. It is estimated that in 2004. all of for the peptide sweetener L-aspartyl L-phenylalanyl which. the share of the food sector is Amino acids are simple organic compounds that contain also substantial and is essentially determined by three one or more amino groups and one or more carboxyl amino acids L-glutamate. proteins. Whereas chemical synthesis produces a synthetic leathers. are optically active and occur as methyl ester (aspartame). selection Placing organisms under conditions where immobilized cell A cell attached to a solid support over the growth of those with a particular genotype is which substrate is passed and is converted into favored. DL-methionine. (2) protein hydrolysis.

the bacteria do not over. enzymes. Corynebacterium glutamicum. phosphate. 3. (Because of L-glutamate excretion. Despite being effectively produced for over 50 years in The current repertoire of strain development includes amounts of 1. as a rule. or L-tryptophan-overproducing strains Lysine Threonine Tryptophan S-(2-aminoethyl)-L-cysteine (AEC) -Amino--hydroxyvaleric acid 5-Methyltryptophan 4-Oxalysine -Hydroxyleucine 4-Methyltryptophan L-lysine hydroxamate Norleucine 6-Methyltryptophan 2.5 mg g1 of dry cells. leading to amino acid Mutagenesis and selection is one of the most important production. further salts. This elimination of the allosteric control of biosynthetic key bacterium. for example. Identification of mutations in classical strains and their stood. Identification of genes by global microarray DNA are able to synthesize all the compounds necessary for the analysis whose expression correlates with favorable living cell from these simple nutrients.8 million tons per year. Amino acid agent in the mid-1950s. Combination of beneficial mutations of different Many bacteria are capable of growing on a simple mineral strains. bacterial cell. enzymatic. These bacteria 4. Application of intracellular flux quantifications using 13 must be able to synthesize amino acids rapidly and effi. is a short. glutamicum is maximal at a critical biotin concentration cing microorganisms. 20% nucleic controlled expression of such genes by genetic means. thus reducing Development of Amino Acid-Overproducing growth or speed of sugar conversion. classical mutagenesis has been applied to obtain mutants that are able to overproduce a specific amino acid in large amounts. acids. Manipulation of export activity to increase the export amino acids as required for growth are synthesized in the of amino acids. undirected mutagenesis. Some of the commonly used amino acid analogues – lysine. and tryptophan – are given in Table 1. L-Glutamic Acid ing mutants that are resistant to amino acid analogues. developing superior production strains. glutamicum. and glucose as the carbon and energy source. which is suboptimal for growth. Furthermore. Regulatory strains were obtained by select. the existing features of C. the molecular features the following: resulting in L-glutamate excretion are still poorly under- 1. Gram-positive rod capable of growing on a simple mineral sized via a branched pathway can be significantly increased salt medium with glucose. often based on strains derived by overproduction of metabolites. and 10% fat. the cell 5. that is. provided that biotin is also added. L-threonine-. standard method for obtaining highly productive amino tion through end products.6-Diamino-4-hexenoic acid Aminohydroxyvaleric acid 5-Fluorotryptophan 2. besides biotin limitation. thus rebuilding the producer. C-labeled substrate and 13C-NMR. threo. transport. the amount of amino acids synthe. However.) Strains 2. 10% carbohydrates. a bacterium that excreted analogue resistance may be because of derepression of the large quantities of the amino acid L-glutamic acid into enzymes involved in the biosynthesis of amino acids or the the culture medium was isolated in Japan. Amino Acid Production 45 Microbial Production undirected mutagenesis. The production of L-glutamic acid by techniques for the development of amino acid-overprodu- C. aerobic.6-Diamino-4-hexenoic acid N-Lauryl leucine 6-Fluorotryptophan "-C-Methoylysine (2. A recent monograph collected a great number of by selecting strains auxothrophic for the competing branch. like repression and feedback inhibi.5-dehydrolysine 2-Amino-3-hydroxyhexanoic acid Indolmycin . that economically control the acid-producing strains. Thus. Following the increasing demand for MSG as a flavoring nine. addition of selected Table 1 Analogues of amino acids used for the selection of L-lysine-. and the subsequent bacterial cell consists of about 60% protein. mutations that are disadvan- tageous might have accumulated. The improvement in cellular activities by directing the produce and excrete amino acids into the culture medium. However. ciently. only as much of the various 6.6-diamino-heptanoic acid) Norvaline DL-7-Azatryptophan -Hydroxylysine N-2-Thienoylmethionine 2-Azatryptophan -Chlorocaprolactam 2-Amino-3-methylthiobutyric acid 3-Inolacrylic acid Trans-4. and such mutants are used in of 0. salt medium containing ammonium. and regulatory functions of the cell The reason is that bacteria have a number of sophisticated by the use of recombinant DNA technology is now a regulatory mechanisms. Gene overexpression to overcome limiting steps. The dry matter of the producer strains or fermentations. a surprisingly introduction via recombinant DNA techniques into a large variety of other treatments also result in wild type. normally. Therefore.

glutamicum is illustrated in Figure 2. together with a eukaryotic-type serine/threonine protein kinase (PknG) Phosphoenol CO2 pyruvate and a phosphatase (Ppp). This type does not secrete L-lysine into the culture medium. or addition of ethambutol- Glucose-6P Ribulose inhibiting arabinogalactan synthesis. techniques. is mainly used as a feed supplement as it is present in most plant proteins only in low concentrations. CO2 drogenase activity.5-tetrahydrodipicolinate deficiency leads to poor glutamate yields. excellent high-yield production strains were conversion of D-glucose to L-glutamic acid. glutamicum. gluta- micum possesses a regulatory OdhI protein. As a consequence. and The pathway for the biosynthesis of L-lysine in control (usually with ammonium) are required. the genes controlling the phosphorylation status are Citrate adjacent to genes of cell wall synthesis and cell division. an amino acid essential for human and animal þ 3NADH þ 3Hþ þ CO2 nutrition. These reactions are the order of 60–70% (by weight) of the sugar supplied. The oxygen transfer rate is fairly critical: a L-lysine.3. C. glutamicum is its split pathway for the synthesis of 8. A tempera. The first enzyme in L-lysine causes accumulation of -oxoglutaric acid. glutamicum has two anaplerotic reactions for the conversion of the C3 L-glutamic acid obtained after 2–3 days of incubation is in intermediates into oxaloacetate. glutamicum. developed by mutation and by selection for antimetabolite The L-glutamic acid production is carried out in stirred resistance together with modern recombinant DNA fermenters up to the size of 500 m3. Oxaloacetate is formed via the phosphoenolpyruvate carboxylase and the pyruvate carboxylase (Figure 1). and important to always have sufficient oxalaoacetate available the final concentration is approximately 150 g l1. with lactic there are two pathways for the conversion of this precursor and succinic acid being formed instead. In C. a disordered cell wall or cell membrane might be Succinate CO 2 sensed by the system and might inhibit ketoglutarate dehy. It was found that C. At the level of L-2. pH measurement. glucose is mainly metabolized via the Figure 1 Biosynthesis of L-glutamic acid in Corynebacterium glutamicum using glucose as the carbon source. by reductive amination dehydrogenase catalyzed by the NADP-dependent glutamate dehydro- L-Glutamate genase present in C. At present. the latter two controlling the Pyruvate phosphorylation status of OdhI. Interestingly. Malate Also. A remarkable fea- ture between 30  C and 35  C and a pH between 7. glutamicum. Moreover. Thus. Thus. glycolytic pathway into C3 and C2 fragments. is regulated by concerted . as is a second serine/threonine protein kinase likely to additionally control the phosphorylation status of OdhI. -oxoglutarate is not Ketoglutarate Ketoglutarate further metabolized in the citric acid cycle. The wild L-glutamic acid per mole of glucose metabolized. all these conditions have more or less in common to target the cell wall or cell membrane.4. Unphosphorylated OdhI CO2 CO2 inhibits ketoglutarate dehydrogenase activity and the OdhI AcetylCoA protein was shown to be necessary for high L-glutamate Oxaloacetate production. C. dissolved oxygen measurement. Importantly. represents a 100% molar conversion or 81. it is known that under Fructose-6P Ribose L-glutamate-producing conditions ketoglutarate dehydro- genase activity is low. Ppp is apparently membrane bound. use of Glucose CO2 fatty acid auxotrophic strains. aspartokinase. approximately 700 000 tons per year of L-lysine Thus. but instead is dehydrogenase Glutamate converted into L-glutamic acid. The yield of biosynthesis.7% weight However.46 Amino Acid Production detergents like Tween-40. Only recently an exciting discovery Glycerolaldehyde-P was made giving at a blow an idea how all these different characteristics might be linked.0 and ture of C. addition of penicillin. Provisions for cooling. The overall reaction for L-glutamic acid production from D-glucose is as follows: L-Lysine C6 H12 O6 þ NH3 þ 3NADþ ! C5 H9 O4 N L-lysine.6-diaminopimelate. the theoretical maximal yield is 1 mol l1 of is produced using mutant strains of C. while an excess into DL-2. for condensation with acetyl-CoA and conversion of citrate to -oxoglutarate.0 are optimal.

3.52 L-methionine L-isoleucine L-2.L-2. and promotes lysine the ample supply of NADPH is important. feedback inhibition by L-threonine and L-lysine. a small reversed by L-lysine.6-diaminopimelate Cell wall Diaminopimelate decarboxylase lysA 4. glutamicum diminishes the intracel.1. glutamicum is inhibited by In addition to all the steps considered so far. This export of L-lysine into the culture medium is important.1. therefore.4 4-phospho-L-aspartate Aspartatesemialdehyde dehydrogenase asd 1.18 L.4.1. contain an aspartokinase that is LysG. By combined overex- double auxotroph of C.1. which is overproduction (15–30 g l1). pression of aspartokinase and dihydrodipicolinate synthase. regulatory mutants.1.3.2.1.1. thick arrows ¼ feedback inhibition. with five transmembrane false feedback inhibitor of aspartokinase.5-tetrahydrodipicolinate Tetrahydrodipicolinate N-succinyltransgerase dapD 2. reduces its marked feedback L-lysine production can be further increased by 10%. Another effective technique achieved by an increased flux via the pentose phosphate for obtaining L-lysine-producing strains is the selection of shunt.- L-2-succinamido-6-oxopimelate N-succinyldiaminopimelate dapC Diaminopimelate aminotransferase ddh dehydrogenase 2. LysG binds L-lysine and drives expression 1 L-lysine is overproduced (30–35 g l ). Also.4 kDa.1. inhibition is markedly enhanced by L-threonine.1.4. Hence.16 N-succinyl-L.1. lular pool of threonine. but Export is mediated by the specific exporter LysE. Growth of C.6.3-dihydrodipicolinate Dihydrodipicolinate reductase dapB 1.20 L-lysine Permease L-lysine Figure 2 The split pathway of L-lysine biosynthesis and its regulation in Corynebacterium glutamicum. At elevated intracellular L-lysine concentrations insensitive to the concerted feedback inhibition. inhibitory effect on aspartokinase. Amino Acid Production 47 L-aspartate Aspartate kinase lysC 2. Some mutants. L-Aspartate as the of the exporter gene to result in an about 20-fold precursor of L-lysine synthesis is formed from oxaloacetate increased induction. the active the L-lysine analogue aminoethyl-L-cysteine (AEC).26 L-2.3.7. a by the anaplerotic reaction of phosphoenolpyruvate and homoserine auxotroph or a threonine and methionine pyruvate carboxylation (Figure 1). of 35 mmol l1.7 DL-2. spanning helices (Figure 3).L-diaminopimelate Diaminopimelate dapF epimerase 5. Expression of this exporter is which are capable of growing in the presence of both controlled by the LysR-type transcriptional regulator AEC and L-threonine.11 L-4-aspartatesemialdehyde L-threonine Dihydrodipicolinate dapA synthase 4.5.6 diaminopimelate N-succinyldiaminopimelate desuccinylase dapE 3.2.2.1. This implies that AEC behaves as a membrane protein of 25.17 1. As a consequence. effective export .

It to reach. biochemical system. L-threonine and L-isoleucine. Waste chemical concentration gradient of L-lysine. coli strains have proved to be superior to C. the price for L-lysine  HCl feed-grade was two OH (Figure 3). A typical amounts of some hundred tons per year. involves symport of the positively charged L-lysine with In 2005. E. 1 L additive produced by fermentation. and resistance to threonine analogues such as -amino--hydroxy-vale- rate. Recently. and filtration mechanism of L-lysine excretion in Corynebacterium of precipitated salts. and reached product concentrations up to 20 g l1. by modern techniques. The components driving L-lysine mentation broth is spray dried and granulated to yield a export are the electrochemical proton potential and the feed-grade product that contains L-lysine sulfate corre- sponding to at least 60% of L-lysine hydrochloride. well designed for excretion purposes: L-lysine will continue to be the most attractive feed • Itconcentrations is only induced at eleveated intracellular -lysine of about 40 mmol l . thus preventing efflux under L-Threonine low internal lysine concentration. L-threonine was used mainly for medical pur- In fed-batch culture. which are not present in C. a new concept for lysine production was occurs. the regulation of the L-aspartate-derived amino acids involves several isoenzymes. additional brane potential. in amino acid infusion solutions. and C+ C+ – OH Lys OH– + 20H– COOH + Outside • crystallization or spray drying as -lysine hydrochloride. is catalyzed by three Figure 4 Production of L-lysine with a mutant strain of different aspartokinases. As shown in Figure 5. coli Biomass strains with excellent threonine yield and productivity are now available. enabling high export rates of up to 7 nmol min1 introduced. coli. Figure 3 Structure of the L-lysine exporter and the putative concentration of the fermentation solution. 1 • internal (cytoplasmic) It has a high K value for -lysine (20 mmol l ) at the m L side. The convential 0 route of lysine downstream processing is characterized by: OH– Lys+ Lysine+ NH2 Inside 20H– C+ OH– C+ – • removal of the bacterial cells from the fermentation broth by separation or ultrafiltration. The reason is that E. worldwide. The liquid product contains up to glutamicum. glucose is fed together with ited by L-threonine and its synthesis is repressed by ammonium. L 0 Lysine+ An alternative process consists of biomass separation. One of these isoenzymes is inhib- Corynebacterium glutamicum. in which the entire L-lysine-containing fer- mg(dry weight)1. For the substrate translocation step approximately US$2 per kg. and in nutrients. introduction of auxotrophies. ΔΨ • absorbtion and then collection of lysine in an ion exchange step. E. and whereas reorientation of the carrier involves the mem- with the potential of fermentation technology. The second aspartokinase . coli has much more Glucose effective excretion systems for L-threonine and related amino acids. Although the pathway of L-threonine biosynthesis in Lysine Escherichia coli is much more regulated compared to that in C. This transporter is a genetic and improvements in the L-lysine process should be realized. The L-lysine production curve is shown in Figure 4. A formal products usually present in conventional L-lysine hydro- description of the energetic steps during translocation chloride manufacture are thus avoided in this process. glutamicum. glutamicum.48 Amino Acid Production with sugar ammonium also has to be fed. by fermentation using mutants of coryneform bacteria in version rate relative to sugar used is about 50%. the first reaction in the Time biosynthetic pathway of L-threonine. With the benefits provided the pH gradient and the L-lysine gradient are important. glutamicum. In E. final was manufactured by extraction of protein hydrolyzates or concentration of at least 170 g l1 L-lysine. such as genetic engineering. 50% of a L-lysine base that is stable enough to be marketed. under the optimized culture conditions. L-lysine production strains are able poses. together production strains were developed by empirical classical breeding. the Start glucose feeding phosphorylation of L-aspartate. Until 1986. and the con.

L-serine Therefore. containing the plasmid with the threonine operon. the filtrate is concentrated and kinase the amino acid is isolated by crystallization.10-methylenetetrahydrofolate þ glycine þ H2O Based on the synthesis pathway. provides the activated C-1 compound obtain very high activities of the thrABC-encoding 5. which encodes threonine dehydrogenase. The consequences: first. growth is independent of the addition L-serine has a key position in central metabolism since as of L-isoleucine. Using sugars like glucose as a carbon source. Indeed. sion of 5. as are all the other proteinogenic amino acids. To more importantly. but the price per kg is higher than for inhibited and repressed by L-lysine. second. Currently. Only the regulation by L-threonine and L-isoleucine is shown. The genase Feedback recovery of feed-grade L-threonine is rather simple: Repression Met Homoserine inhibition after the cell mass has been removed from the culture thrB Homoserine broth by ultrafiltration. coli by the originally occurred with methanol-utilizing organisms thrABC operon. and L-cysteine. Threonine biosynthesis feed additive amino acids. and stable high-level expression of thrABC operon. coli strain semialdehyde 1 L-threonine concentrations of more than 80 g l are Homoserine thrA dehydro. derives directly from glycolysis in just three enzymatic chromosomal mutations were introduced to result in an steps (Figure 6). As and homoserine kinase. This mutation has several advantageous much as 8% of the glucose carbon flux is via L-serine. it prevents the tein. at high L-threo. cellular analyses and engineering methods resulted in a breakthrough in microbial L-serine production. but has no other major application. thrC Threonine synthase L-Serine lle Threonine The engineering of an efficient L-serine-producing bacter- Figure 5 Regulation of L-threonine biosynthesis in Escherichia ium is a rather recent example where a combination of coli. A systematic study revealed that removing isoleucine leaky strain. L-threonine has been successfully marketed as a feed Homoserine phosphate additive with a worldwide demand of more than 70 000 tons per year. there is a clear focus on to tetrahydrofolate þ L-serine. like synthesis of L-isoleucine-dependent premature termination of the phospholipids. L-tryptophan. the isoleucine leaky biodegradative reaction catalyzed by SHMT provides gly- mutation has a positive selection effect on all the cells cine required for purine. which in turn can be further . it prevents an excess formation of the reason is that L-serine. protein. however. the thrABC transcription. To prevent the degradation of L-threonine phosphate the gene tdh. the SHMT activities. which is bivalently repressed by L-threonine plus activated by a tetrahydrofolate derivative (THF) to form L-isoleucine. with the interconversion two major targets for the design of L-threonine-overprodu. will be seen below. L-threonine ranks third in produc. which requires L-isoleucine only at bottlenecks in synthesis and preventing L-serine degradation low L-threonine concentrations. The current price is approxi- occurs by the conversion of aspartate--semialdehyde in mately US$40 per kg. and the third one is is therefore smaller. with a price of approximately US$3 per kg. The market isoenzyme is repressed by L-methionine. L-serine is used in infusion solutions. nine concentrations. Moreover. is required for a number of purposes. The thrA gene encodes a bifunctional taking advantage of the serine hydroxymethyltransferase enzyme with aspartokinase and homoserine dehydrogenase (SHMT) reaction.10-methylene-THF. and. The microbial production of L-serine three enzymatic steps that are encoded in E. However. was inactivated. It catalyzes the interconver- acids behind L-lysine and L-glutamic acid. this operon was cloned from a strain in which Aspartate the aspartokinase and homoserine dehydrogenase activ- kinase ities are resistant to L-threonine inhibition and was Aspartyl overexpressed. the SHMT reaction plays a key role in tion volume among the biotechnologically produced amino microbial L-serine production. and dissociation of reactants within the same order of cing strains. and heme synthesis. Lys Aspartate By continuous sugar feeding of such an E. besides being incorporated into pro- undesired by-product L-isoleucine. Additionally. in one of the initial steps of strain development. L-threonine synthesis is regulated L-serine. that is. In the biosynthesis reaction. the prevention of L-isoleucine formation magnitude. obtained with a conversion yield of about 60%. Amino Acid Production 49 Aspartate enzymes. The thr operon is under the control of a single catalyzes the glycine condensation with the C1-unit promoter. this method faced a setback because of by feedback inhibition of the homoserine dehydrogenase the external addition of glycine and low conversion yields. are major issues in the manufacture of L-serine. Furthermore.

C-term of the serA-encoded PGDH represents the the pabAB and pabC genes were deleted. CSL the label revealed that L-serine as an entity is converted to provides some folate to enable restricted growth and pyruvate and a genomic screen revealed furthermore reduced SHMT activity of the strain. and in combinations with serC and serB overexpressed did not overexpressing the three genes of the L-serine synthesis result in significant L-serine accumulation. As men- by L-serine. L-serine to pyruvate. deleted of serine dehydratase the mutant allele serA197 in C. and 5-methyl. obvious that only because of the requirement for purine The study with 13C-labeled L-serine also showed that synthesis and the provision of tRNAfMet for translation glycine was formed from the L-serine added. and serB genes overexpressed. indicating an pathway (pserACB) with the serA gene carrying the dele- intracellur conversion of L-serine. and other activated C. or its intermediate p-aminobenzoate. The 1 units like 10-formyl-THF. a N-formylmethionyl-tRNA and purine synthesis. Therefore. a high L-serine degradative flux via the SHMT not surprising considering that there is no preference of the reaction occurs. As men- To derive from the ‘workhorse’ of amino acid produc. Under industrially relevant conditions Therefore. serine dehydratase contains an [4Fe-4S] cluster involved in THF to serve different demands in metabolism. glutamicum tive to L-serine inhibition but with catalytic activity 13032sdaApabABC pserACB. It is slight transient L-serine accumulation was observed. encoding the domain involved in allosteric control. The medium con- that an sdaA-encoded serine dehydratase is present in tained 35 g l1 solid CSL plus initially 15 g l1 glucose. This is derived from the largely retained (Figure 6). an L-serine producer. with mate lyase catalyzing two steps of THF synthesis. and this was initiation. Therefore. tioned. a number of provide activated C-1 units and this cellular demand can- steps were undertaken. which is allosterically controlled engineers used a trick to deplete SHMT activity. where as activity and growth of the resulting strain were dependent much as 197 amino acyl residues from the C-term were on THF. C. C. SHMT requires THF for functioning. glutamicum either alone or (sdaA) and of the folate synthesis genes (pabABC). converted into 5. likely to be responsible for degradation.10-methenyl. On the right is shown a series of plasmid constructs and their resulting 3-phosphoglycerate dehydrogenase activity in the presence of L-serine. the feedback control of the 3P-glycerate dehydrogenase Therefore SHMT is essential. SHMT the most prominent mutation being serA197. deleted and which resulted in an activity almost insensi. . the metabolic (PGDH) was removed. These were as follows: At first not entirely be bypassed by external metabolite addition. like the pyridoxal-59-phosphate-independent deamination of 5-methyl-THF for L-methionine synthesis. or 10-formyl-THF for then the serA197. SHMT for the forward or backward reaction. tion at its C-term. overexpression of type strain ATCC13032. L-serine conversion catalyzed by SHMT serves to tion. Tracing of reactor based on corn steep liquor (CSL) medium. 5. However. Sequence comparisons revealed that the tioned. serC. 5-formyl-THF. However. a com. When the enzyme was deleted and THF for D-pantothenate synthesis. aminodeoxychorismate synthase and aminodeoxychoris- prehensive set of truncated serA versions was made. Thus the strain eventually selected was C. in the second step. glutamicum. the intracellular fate of the performance of this strain was demonstrated in a 20l 13 L-serine was studied using C-labeled L-serine. glutamicum.10-methenyl-THF.50 Amino Acid Production Figure 6 On the left is shown L-serine synthesis as derived from 3-phosphoglycerate from glycolysis.

decay constant. which is about 60% that niques helped to improve aspartase-containing strains. was about 110 mol l1 h1. Immobilization of the cells in -carrageenan resulted in gen concentration was set to 50% saturation to ensure no remarkably increased operational stability. A column packed with the -carrageenan-immo- the volumetric productivity about 1. L-serine formation occurred from the A plasmid with the aspA gene elevated aspartase forma- beginning. and yields. thus the amination reaction is Polyacrylamide 18 850 120 100 favored. 15 g l1 fructose. Amino Acid Production 51 125 400 350 100 300 DO (% saturation) OUR (mmol/l*h) Growth (OD) Serine (mM) 75 250 200 50 150 100 25 50 0 0 0 25 50 Time (hrs) Figure 7 Performance of an L-serine producer strain in a fermenter showing growth (&). in a 1 m3 column about 100 tons of was reached.1. and further derivatives of this basic strain microbial amino acid production.4 g l1 h1.3. less by- products are formed. and salts. which was present at reactor system. of the wild type. an immobilized cell system Carrageenan 49 400 680 1498 based on E. thus. In this bilized cells produces 200 mmol l1 L-aspartate per hour experiment a final concentration of 345 mmol L-serine per gram of cells.25 h1. As can be seen in Figure 7. coli cells entrapped in a polyacrylamide gel (GA þ HA)b matrix was developed. Aspartase purified from E. and the oxygen uptake rate. the dissolved oxygen. A continuous process enables automation the end of the logarithmic growth of the culture. but significantly higher concentrations can L-aspartate can be produced in 1 month. The equilibrium constant of the deamination Immobilization activity (U/g Half-life productivity reaction catalyzed by the enzyme is 20 mmol l1 at 39  C method cells) (days) (%)a and 10 mmol l1 at 20  C. Carrageenan 37 460 240 397 ment for divalent metal ions. This enzyme catalyzes the reversible immobilized using various methods interconversion between L-aspartate and fumarate plus Aspartase Relative ammonia.1) is used. Compared to be obtained. lized cells has been industrialized by using a fixed-bed OURmax. Furthermore. suggesting a suitable folate supply in the cul. tion in E. thus. the half-life of a Considers the initial activity. coli is a tetramer with a Carrageenan 56 340 70 174 molecular weight of 196 kDa and it has a strong require. Using this system. which can be assumed to contain at least The production of L-aspartate by means of immobi- traces of this vitamin. the aspartase activity could be increased to 120 days. As the isolated enzyme is (GA)b very unstable in solution. inoculation talyst with a half-life of approximately 2 years was of the reactor with cells enabled growth up to a maximum obtained (Table 2). The minimum dissolved oxy. In Enzymatic Production recent years the market for L-aspartic acid has increased L-Aspartic Acid to approximately 30 000 tons per year due to the fact that L-aspartic acid is industrially produced by an enzymatic process in which aspartase (L-aspartate ammonia lyase EC Table 2 Half-life of aspartase in Escherichia coli cells 4. the advantages of this have been developed. In addition. b GA ¼ glutaraldehyde. The and efficient control to achieve high conversion rates and maximal specific productivity was 1. The maximum oxygen uptake rate.45 mmol g1 h1. OUR (^). ture due to CSL. HA ¼ hexamethylene diamine. thus. a bioca- oxygen limitation. and operation period. coli approximately 30-fold. recombinant DNA tech- specific growth rate of 0. DO (&). L-aspartic acid can be easily separated from the reaction mixture by crystallization. the L-serine concentration in the medium (N ). enzymatic production method are higher product concentration and productivity. .

) Handbook of Corynebacterium glutamicum. pp. Taylor Francis Group. Applied Microbiology and Biotechnology ing for the limiting enzymatic steps. In: Eggeling L fermentation plays a key role among the production and Bott M (eds. . Weil B. and Bott M (2006) of the L-lysine secretion carrier opens up an entirely Corynebacterial protein kinase G controls 2-oxoglutarate new field for increasing the overproduction of various dehydrogenase activity via the phosphorylation status of the OdhI L-amino acids. Biosynthesis – Pathways. 259–272. In: Eggeling L and Bott M (eds. 277–304. Leuchtenberger W. pp. and Eggeling L (2001) L-phenylalanine). Eggeling L (2005) Export of amino acids and other solutes. Schultz C. Microbiology Monographs. Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum. Niebisch A. Patek M.) biosynthesis of an amino acid should make it possible to Handbook of Corynebacterium glutamicum. Furthermore. Kabus A. further improvements in microbial processes are New York: Springer Verlag. Winterhalter C. status and prospects. The recent discovery 69: 1–8. Kramer R. London. In: Wendisch VF (ed. New York: CRC Press.) Amino Acid new analytical methods offered by the -omics techniques. Sahm H. Berlin. Bottlenecks in L-amino acid Biotechnological production of amino acids and derivatives: Current synthesis can be removed by amplification of genes cod. a thorough understanding protein. Because of modern pp. microbial 36: 1101–1112. Molecular Microbiology For the synthesis of proteinogenic amino acids. New York: CRC Press. Vrljic M. Raton. methods in the amino acid industry. Boca Raton.52 Amino Acid Production this amino acid is a precursor for the production of the Further Reading dipeptide sweetener aspartame (methyl ester of aspartyl- Bellmann A. The Journal of Biological Chemistry 281: 12300. like DNA chip technology.. Huthmacher K. and Boeck A (2000) Identification of a Future Prospects major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Dassler T. Wittmann C and de Graaf AA (2005) Metabolic flux analysis in of the various elements and mechanisms controlling the Corynebacterium glutamicum. Taylor techniques such as metabolic engineering combined with Francis Group. Boca further influence its production rate in a predictable way. Heidelberg. proteomics. and metabolo.. Regulation and Metabolic Engineering. London. mics. Microbiology 147: 1765–1774. 187–214. Eggeling L (2007) L-serine and glycine. Maier T. and Drauz K (2005) constantly being achieved.

and self-replicating. plants or mammals. aureus strains ABC ATP-binding cassette OM outer membrane ANT aminoglycoside Omp OM proteins nucleotidyltransferases P site peptidyl donor site APH aminoglycoside phosphotransferases PBPs penicillin-binding proteins CSP competence-stimulating peptide qnr quinolone resistance DHFR dihydrofolate reductase QRDR quinolone resistance determining EF-G elongation factor G region LPS Lipopolysaccharide RND resistance-nodule-cell division MATE multidrug and toxic compound SMR small multidrug resistance extrusion VISA strains vancomycin-intermediate S. aureus MDR multidrug resistance strains 53 . Defining Statement Biochemistry of Resistance Introduction Genetics of Resistance Antibiotic Classes Antibiotics Can Act as Pheromones Antibiotic Resistance Biological Cost of Antibiotic Resistance Mechanisms of Resistance Conclusion Acquisition of Resistance Further Reading Glossary them into functional genes by ensuring their correct antibiotic Molecule of microbial origin able to inhibit expression. Abbreviations MFS major facilitator superfamily A site aminocyl receptor site MIC minimal inhibitory concentration AAC aminoglycoside acetyltransferases MRSA methicillin-resistant S. such as antibiotic resistance. Most often double-stranded. Institut Pasteur. A transposon can insert itself into integron DNA element that acquires open-reading nonhomologous DNA. covalently higher concentrations of the antibiotic compared to closed. exit. or originate from information. Antibiotic Resistance B Périchon and P Courvalin. and relocate independently frames embedded in gene cassette units and converts of the general recombination function of the host. France ª 2009 Elsevier Inc. the growth or to kill other microorganisms. integrity. circular. replicon DNA molecule that can replicate chromosome DNA molecule that contains all the autonomously (chromosome or plasmid) genetic information necessary for the life of the resistance When a strain can grow in the presence of bacterium. It plasmid Minichromosome encoding accessory genetic could be synthetic. mutation Any inheritable alteration of DNA. conjugation Unidirectional transfer of genetic transposon (transposable genetic element) DNA information (in this article. a plasmid) involving direct segment able to migrate from replicon to replicon cellular contact between a donor (male) and a recipient (plasmid or chromosome) while retaining its physical (female). other strains of the same species. All rights reserved. microorganisms but not obligatory of microbial origin. Paris. antimicrobial agent Substance active against operon Adjacent genes coordinately expressed. semisynthetic.

acid sequences.g. block the Table 1 Targets of the main classes of antimicrobial drugs Cell wall synthesis Protein synthesis DNA replication Membrane Bacitracin Aminoglycosides Coumarines Polymixins -Lactams Chloramphenicol Quinolones Glycopeptides Fusidic acid Rifampin Fosfomycin Ketolides Sulfonamides Lincosamides Trimethoprim Macrolides Oxazolidinones Streptogramins Tetracyclines . A formylmethionyl-tRNA is in terms of resistance should be in classes rather than in then attached to the peptidyl (P) donor site face of the individualized antibiotics. The 50S subunit is then added. The cell wall protects prokaryotes from the environment Macrolides. Glycopeptides act by binding. by binding to its active site cysteine and thus blocking the formation of muramyl pentapeptide. whereas the related and generally share the same target in the cell and small 30S subunit is composed of proteins and the 16S are thus substrates for the same mechanism of resistance.54 Antibiotic Resistance Defining Statement characterized by the presence of a peptidoglycan located outside the cytoplasmic membrane that is responsible for Resistance of bacteria to antibiotics can be intrinsic or the rigidity of the bacterial cell wall and for the determi- acquired. Acquired resistance results from mutation in a nation of cell shape. aminoglycosides (strep. named 50S and 30S. binding proteins (PBPs).. quinolones). certain aminoglyco. block the transpeptidation events by Resistance of bacteria to antibiotics. tor (A) site. A specific peptidyl transferase mediates a peptide bond between the N-formylmethionine and the adjacent Inhibition of Cell Wall Biosynthesis amino acid. are constituted of two subunit nucleopro- tomycin.. ß-lactams Bacterial ribosomes. an enzyme natural. pentapeptide. the term ‘antibiotic’ is used to designate Fosfomycin inhibits the activity of MurA. Cross- the emergence and more efficient spread of resistance. and gentamicin). has become a major health problem worldwide. implicated in the conversion of the nucleotide diphospho- sides) or entirely synthetic (e. such as penicillins and cephalosporins. also named penicillin- resistance. which is then translocated to the or transformation. which translate the mRNA in amino (penicillins and cephalosporins). The bacterial cell wall is rRNA. Introduction The -lactam antibiotics. These mechanisms can be associated in external face of the cell membrane by a carrier. near the peptidyl transferase center. preventing their incorpora- their interaction with. The large 50S subunit As a consequence. linking by transglycosylases and transpeptidases is responsible for the complete synthesis of the peptidogly- can outside of the cell. such as synthesis of the acquisition of a new genetic information by conjugation muramyl pentapeptide. rRNA. such as erythromycin. kanamycin. members of a given class are closely contains proteins and two rRNA.g. tion into the growing wall. and tetracyclines. in particular multiple binding to the transpeptidases. AUG initiator codon. Antibiotics are defined as secondary metabolites pro. The translation events start with the binding of As will be discussed below. tein particles. and inhibition of. in a noncovalent fash- duced by microorganisms in the environment (generally ion. bind to the 23S and from osmolysis. In this article. which is adjacent to the peptidyl donor site (P site). this implies that the reasoning mRNA to the 30S subunit. Synthesis of the cell wall requires gene located in the host chromosome or from horizontal several steps in the cytoplasm. and The main classes of antibiotics act on four different the adequate aminoacyl-tRNA enters the aminocyl recep- targets (Table 1). but also semisynthetic (e. molecules sugar UDP-N-acetylglucosamine into UDP-muramyl with antibacterial activity. to the C-terminal of D-alanine-D-alanine dipeptide of the soil) active against other microorganisms because of the peptidoglycan precursors. 23S and 5S. a specific target. for example. Antibiotic Classes Inhibition of Protein Biosynthesis Antibiotics are grouped in classes (or families) on the basis of their chemical structure.

and thus stop the elon. As already mentioned. presence of modifying dihydrofolate reductase (DHFR). or (3) absence of the target. lincosa- subunit and block the moving of the tRNA along the ribo. mides. DNA glycosides. of a porin (pore in the external membrane) or by . lactamase (AmpC). glycopeptides. are also intrinsically resistant to low levels of amino- Topoisomerases are essential for cell viability. etc. an essential enzyme for enzymes. Acquired resistance is present only in some strains of the same species or genus. presence of an external membrane in Gram-negative gation of the peptide chain. which leads to loss or decrease in terial species or of the presence of a structural gene. Intrinsic Resistance Intrinsic (or natural) resistance is present in all the bac. recombination. (3) impermeability. These two concomitant events are due Other Targets to a chromosomally encoded vanC gene cluster. Trimethoprim–sulfamethoxazole inhibits the folic Mycobacteria is due to the combination of reduced per- acid metabolism pathway: the first molecule blocks the meability of the bacterial cell wall. inhibit cell wall synthesis in Topoisomerase IV is involved in DNA replication and Gram-positive bacteria by binding to the C-terminal decatenation of the chromosome. both can employ the four major pathways depicted in Figure 1. and transcription. Folic acid is an essential precursor in nucleic acid synth. ification of the target. For example. Resistance to both -lactams and cyclines in esis. This type of resistance could be four major mechanisms of resistance (Figure 1): (1) mod- the result of the physiological characteristics of the bac. There are two major types of resistance to antibiotics: Intrinsic and acquired resistances do not differ in their intrinsic and acquired. and Enterococcus casseliflavus and Enterococcus flavescens are plexes that could block the replication fork. resulting from a particularly low permeability of its outer Lincosamides (lincomycin and clindamycin). D-alanine-D-serine (D-Ala-D-Ser). and monobactams and for an increase in resistance to the penicillins and carbapenems. macrolides. it can be Antibiotic Resistance highly prevalent. Enterococcus Inhibition of DNA Replication spp. (2) production of an enzyme that will inactivate or the target by antibiotics. streptogramins. Enterococcus gallinarum for conformational changes and accumulation of com. in antibiotics. some. in particular by loss for the target. and low affinity for the target (such as PBP and DNA synthesis. D-alanyl-D-alanine (D-Ala-D-Ala) residues of late penta- lones with enzyme-bound DNA complexes is responsible peptide peptidoglycan precursors. bacteria have developed activity of an antibiotic. DNA gyrase). due to inefficient uptake of this class of gyrase is implicated in the control of DNA topology. It delineates the spectrum of On a biochemical point of view. Pseudomonas aeruginosa is a typical organism that exhi- Chloramphenicol binds to the A site and prevents bits a high broad substrate range intrinsic resistance binding by tRNA. membrane (OM) associated with a number of endoge- acting with both the A site and the P site inhibit peptide neous multidrug efflux systems (such as MexAB-OprM bond formation. for which glycopeptides have a low affinity. affinity of the drug for its target or synthesis of a new Natural resistance is often due to (1) inaccessibility of target. Interaction of quino. and by elimination of the D-Ala-D-Ala ending precursors. vanco- DNA replication. Mechanisms of Resistance teria of a given species or genus and could thus be better considered as insensitivity. and MexXY-OprM) and a chromosomally encoded - The ribosome is also the specific target of aminoglyco. teroate synthase. mycin and teicoplanin. sides that act by causing translational errors and by Enterococcus faecium produces an intrinsic low affinity inhibiting translocation. bacilli (such as Escherichia coli) leads to resistance to Tetracyclines bind in the vicinity of the A site of the 30S various drug classes (glycopeptides. by inter. Antibiotic Resistance 55 entrance of the ribosomal tunnel. which impedes the formation of the first peptide bond. PBP 5 responsible for high level resistance to cephalospor- ins. In certain instances. Polymixins increase the permeability of the cell Acquired Antibiotic Resistance membrane.) due to impermeability. (2) low affinity of the antibiotics modify the drug. mechanisms. intrinsically resistant to low levels of glycopeptides by Rifampin is an RNA polymerase inhibitor that hinders synthesis of modified peptidoglycan precursors ending in protein transcription of DNA into mRNA. such as penicillinase production in staphylococci is present in more than 90% of the strains. whereas the second blocks the dihydrop. oxacillin.

Addition PBPs (1a. or assembly represent targets of The macrolide. Competence. As an example. and VanG-type). faecium and Enterococcus faecalis detected in 1986 is due to acquisition of operons encode enzymes producing modified peptido- diminution of its diameter in Gram-negative bacteria. the QRDR (quinolone resistance determining response to a certain population density. Vancomycin resistance in vanco- Alteration or Synthesis of a New Target mycin-intermediate S. Interestingly. impermeability by active efflux of the drug. low Figure 1 Major mechanisms of resistance to antibiotics. antimicrobial agents. 2a. pneumoniae has six erm (erythromycin ribosome methylation) gene. resistance is due to structures. VanB. aureus (VISA strains) is not due to A mechanism frequently used by bacteria to prevent the acquisition of a van gene cluster but due to synthesis of a action of antimicrobial agents is the alteration of specific thicker cell wall that traps vancomycin. Genesis of these stitutes the main mechanism of resistance to these mosaic PBP genes is facilitated by the fact that S. and VanD-type resistance) or in dependent pumps. impermeability by mutation in a porin channel. 2a. or acquisition of new PBPs is prevent protein synthesis. 2x. 1b. topoisomerases. In the absence of mecA. lincosamide. responsible for synthesis of a new PBP. tance to this antibiotic class in Streptococcus pneumoniae is coli) in 23S rRNA by a methyltransferase specified by an mainly due to alteration of PBPs. and 1a that are methylases of the Rmt family (RmtA. pneumoniae were cefotaxime susceptible. Resistance to these molecules is responsible for -lactam resistance. S. The levels of resistance conferred comA gene. In subunits. 2b. aureus strains (MRSA)) is due to acquisition of a gene. production of an or due to modification of PBP 2. the mechanisms is to impede interaction of the antibiotic with base composition mol% GC of the genes in the van its target. These two enzymes. From level resistance could be due to overproduction of PBP 4 top. mecA. in S. Mutations in a specific region of these stimulating peptide (CSP). leading to a targets that have a necessary role in microbial growth. The first clinical isolates of MRSA that have acquired a vanA gene cluster were detected in 2002. alteration of the target. operons suggests that these clusters are composed of genes from various sources (Figure 2). In clinical isolates. which acts as a pheromone. High level resistance to methicillin and all other -lactams in Staphylococcus aureus (methicillin-resistant S. pneumoniae by the Enzyme corresponding portion of the gene from Streptococcus oralis or Streptococcus mitis. with reduced affinity for -lactams. 1b. tion by transformation from related species of streptococci Alteration of the targets of fluoroquinolones type II that are intrinsically less susceptible to -lactams fol. PBP 2a. Enzymatic methylation of 16S rRNA by other low-affinity variants of PBPs 2b. synthesis. despite the fact that the donor strain and the recipient S. and glycan precursors terminating in D-Ala-D-lactate (D-Ala-D- (4) efflux of antibiotics outside of the cells by energy. reduced number of molecules that reach the transglyco- Enzymes involved in several steps of peptidoglycan sylase targets located in the cytoplasmic membrane. topoisomerase IV). and 3) in which point mutations of a methyl group reduces the affinity of the rRNA for could be responsible for -lactam resistance in mutants these three groups of antibiotics that have very different obtained in vitro. DNA gyrase and topoisomerase IV. Acquired resistance to glycopeptides in E. the competence. B. the production region). Alteration of PBPs. pneumo. 2x. counterclockwise. and streptogramin B anti- choice for antibiotics. which is the bacterial DNA synthesis. ArmA confers high level resistance to aminoglycosides. resulting in low biotics act by binding to the 50S ribosomal subunit and thus affinity for -lactams. enzyme inactivating the drug. C. implicated in niae is naturally transformable. The common motif of these various D-Ala-D-Ser (VanE. which is the result of the replacement of a portion of PBP 2x of S.56 Antibiotic Resistance resistance is the mosaic PBP 2x. are composed of two subunits ability to take up DNA from the environment. A typical example of a mosaic PBP-mediated by mutations in the subunits of DNA gyrase or . and 2b). Lac) (VanA. con- lowed by homologous recombination. such as PBPs. and D) or encoded by mosaic genes that result from DNA acquisi. This mosaic PBP 2x is more resistant to cefotaxime. due to the methylation of an adenine residue (A2058 in E. Other mosaic structures responsible for synthesis of low affinity PBP variants have been described (PBP 1a. prevent the fixation of the quinolones on the of CSP is increased by the upregulation of the comC or DNA–enzyme complex. (GyrA and GyrB for DNA gyrase and ParC and ParE for pneumoniae is due to a specific protein. resis. Alteration of three or more PBPs is found in highly resistant isolates.

Within each type. Antibiotic Resistance 57 PR PH vanA vanR vanS vanH vanA vanX vanY vanZ PRB PYB vanB % aa identity vanRB vanSB vanYB vanW vanHB vanB vanXB with vanA 34 23 30 67 76 71 PRD PYD vanD vanRD vanSD vanYD vanHD vanD vanXD % aa identity with vanA 58 42 13 59 69 68 PC vanC % aa identity vanC vanXYC vanT vanRC vanSC with vanC2 71 81 65 91 81 PE vanE % aa identity vanE vanXYE vanTE vanRE vanSE with vanC 53 45 47 60 41 PUG PYG vanG vanUG vanRG vanSG vanYG vanWG vanG vanXYG vanTG Transcriptional regulator Histidine D. qnrA. interact with the  subunit of RNA polymerase. several synthetase. To date. ACP-reductase/-ketoacyl-ACP-synthase. Open arrows represent coding sequences and indicate the direction of transcription. directly. Mutations in the dhfr gene. such as iso-leucyl-tRNA described. . qnrB. notably between the residues are more common than mutations in GyrB or ParE. pneumoniae. three types of qnr genes have been Alteration of other targets. such as rifampin. S. which is respectively. and block transcription initia- the quinolone. and qnrS. and Gram-negative bacteria and topoisomerase IV in Gram. for resistance to mupirocin. protects gyrase and topoisomerase responsible for trimethoprim resistance in IV from quinolone inhibition by binding these enzymes staphylococci. is responsible Antibiotics of the rifamycin family. responsible for rifampin resistance are located positive bacteria.D-dipeptidase % aa identity with vanB 31 16 56 49 46 NA NA with vanD 73 55 23 NA 44 NA NA with vanC 62 40 NA NA 42 41 37 with vanE 55 29 NA NA 41 39 37 Figure 2 Comparison of the van gene cluster. 507 and 534. and isoniazid. including point mutations. fusidic acid. encoding a DHFR. NA. gene. Mutations in the GyrA or ParC subunits in highly conserved regions. which belongs to the penta. Serine regulator kinase Carboxypeptidase Carboxypeptidase racemase D.D. and NADH-enoyl- variants have been reported. insertions.D. The Qnr gene product. elongation factor G (EF-G). The primary target is the DNA gyrase in tion. topoisomerase IV depend on both the bacterial species and encoded by the rpoB gene. Mutations in rpoB have been detected in Another resistance mechanism to fluoroquinolones is Mycobacteria. Mutations. deletions. S. not applicable. Unknown Ligase D. and Neisseria mediated by the plasmid-borne qnr (quinolone resistance) meningitidis. aureus. are peptide repeat family.

In the latter classification. are found in both pathogenic the OM to many molecules. Enterobacter aero- nases on which the -lactamase inhibitor clavulanic acid genes. group 2. whereas MDR efflux pumps are specified by on the aminoglycoside modification. The phosphotrans- pumps. Thus. such as -lactams. group 3. carbapenems. inac- The widespread active export or efflux of antibiotics out- tivate macrolides by cleaving the macrocycle ester. such as by their amino acid sequences or by their response to exposure to this antibiotic. Three genes. This mechanism is thiol-. Other antibiotic enzymatic activity spectrum. Tetracycline-specific efflux pumps. Resistance to chloramphenicol is due to a large variety sion of multidrug transporters is commonly controlled by of chloramphenicol acetyltransferases. pump or due to a mutation in a protein pump that FosB. which are mem- barrier. the small multidrug resis- the bifunctional enzyme AAC(69)-APH(20). and the multidrug and toxic compound fusion product of two genes and possesses an acetyl and a extrusion (MATE) (Table 2). Depending elements. five families of efflux systems tic-inactivating enzymes have been reported. acetyl-. The aminoglycoside multidrug resistance (MDR) phenotype to chemically phosphotransferases (APH) confer a higher level of resis- and structurally unrelated compounds. SMR. have been described: the major facilitator superfamily various aminoglycoside-inactivating enzymes can be present (MFS). more than 50 antibio- the chromosome. drug- tance than aminoglycoside acetyltransferases (AAC) or specific efflux pumps are encoded by mobile genetic aminoglycoside nucleotidyltransferases (ANT). metallo--lactamases. genes code for membrane-associated proteins that reduce are used by several antibiotics. to permeate the OM.58 Antibiotic Resistance Enzymatic Modification chloramphenicol. the latter conferring a (generally from ATP) to a substrate. compounds such as antibiotics. E. and MATE pumps. kinase activity in the same protein. Reduced Uptake of Antibiotics Efflux pump specific for one substrate Gram-negative bacteria possess an OM external to the Tetracyclines inhibit protein synthesis by preventing the peptidoglycan and composed of lipopolysaccharide (LPS) attachment of aminoacyl-tRNA to the ribosomal acceptor and phospholipids. allowing resistance of this ring in penicillins. encoding macrolide 29-phosphotransferases have ary drug transporters. This OM functions as an effective site. chloramphenicol. such as EreA and EreB. present on the chromosome of P. Furthermore. cephalospori- documented in Serratia marcescens. and RND. aminoglycosides. the resistance- in the same host. cephalosporins. some OM proteins Gram-negative and Gram-positive bacteria. This enzyme is respon- Drug efflux systems act in an energy-dependent man- sible for high level resistance in Gram-positive bacteria to all ner by using ATP hydrolysis (ABC) or an ion antiport the aminoglycosides. is enhance the potential of export of this protein. which are widely dis- specific regulatory proteins. The tet efflux (Omp). macrolides. and glycosyltransferases) constitutes a mediated of membrane-based efflux proteins acting as large family of modifying enzymes. penicillinases sensitive to clavulanic acid and extended spectrum -lactamases. efflux mutant is due to overexpression of an endogeneous Fosfomycin is modified by thiol transferases such as FosA. the intracellular drug concentration and thus protect the . Resistance to imipenem in monobactams. except streptomycin and spectinomy- mechanism (MFS. A remarkable example of this coexistence is nodule-cell division (RND). Resistance to these families of antibiotics could be This is a major mechanism of resistance to antibiotics due to the diminution of the porin copy number or such as -lactams. RND. mph(B). the MFS. aeruginosa whereas FosB is As opposed to ABC transporter that usually mediates found in Gram-positive bacteria and. also called porins and acting as aqueous channels. The enzymes can be classified in a number P. SMR. Enterobacter cloacae. resistance to multiple antimicrobial agents. and MATE). FosA. coli. resistances associated with loss of a porin have been four groups have been defined: group 1. which is the tance (SMR). or FosX. and reduction in the size of the pore. are generally responsible for been reported in E. The expres- cin. nucleotidyl-. The OM of P. or fluoroquinolones. To date. found in Gram-negative bacteria. also designed as second- mph(D). on the chro- the export of specific antimicrobial classes. ADP-ribosyl-. mosome of Bacillus subtilis. notably. Antibiotic resistance in an tributed among bacterial pathogens of all genera. and organism to fluoroquinolones. side bacteria limits intracellular accumulation of toxic The transferase group (phospho-. coli and Pseudomonas. other -lactamases weakly sensitive to clavulanic acid. aeruginosa is caused by a loss of the OprD porin in of ways. aeruginosa has a very low permeability to -Lactamases hydrolyze the four-membered -lactam small hydrophilic molecules. has a weak activity. the ATP-binding cassette (ABC). the LPS being responsible for impermeability of bers of the MFS family. and group 4. Generally. Increased Efflux of Antibiotics The macrolide esterases. mph(A). Klebsiella pneumoniae. Efflux pumps have a narrow (such as tetracycline ferases catalyze the transfer of a phosphate group pumps) or a broad specificity.

In Acinetobacter baumannii. fluoroquinolones. Two homologous pumps. MATE. chloramphenicol. The chromosomally-encoded efflux pump EmrAB confers in E. the AcrAB-TolC system is homologous to MexAB-OprM. coli. lipophilic -lactams. and trimethoprim. Substrates of AcrAB-TolC include Efflux pump associated with MDR macrolides. family that confer chloramphenicol resistance. has been detected in adeR or adeS are responsible for the constitutive expres- Mycobacteria. D. subtilis and PmrA of S. coli. Genes coding for QacE and QacE1. baumannii. which is otherwise cryptic in Mycobacterium tuberculosis. initially described in S. NorM (Vibrio parahaemolyticus) An endogenous gene of Listeria monocytogenes. overexpression of AdeABC. aeruginosa also expresses efflux systems of the RND family. membered macrolides. conferring resistance to amino. some of them (such as MexAB-OprM genes. RND. include disinfectants and anti- efflux systems for clinically important antimicrobials. and the dye ethidium hydrophilic quinolones norfloxacin and ciprofloxacin. A fluoroquinolone efflux gene. pneumoniae and Streptococcus pyo. are implicated in fluoroquinolone and encodes a protein of the MFS family and is responsible aminoglycoside resistance. AcrA. The The cmlA genes. small multidrug resistance. erythromycin. fusidic acid. or MexXY-OprM) participate in the intrinsic resistance. VceAB. is responsible for quinolone encode efflux pumps. sible for an increase of the norfloxacin minimal inhibitory an RND tripartite efflux pump. and YdhE (E. A homologous pump. respectively. a chromosomally encoded pro. are implicated in the specific efflux of 14. was found Multidrug and toxic compound extrusion in Vibrio cholerae. rifampin. MFS.and 15. aureus. This septics. As already mentioned. macrolides. the latter being respon. encode exporters of the MFS tance but also accommodates -lactams. detected in Staphylococcus spp. responsible . genes. such as Mycobacterium fortuitum and sion of the AdeABC pump. fluoroquinolones. multidrug and toxic compound extrusion. ribosome. and triclosan. lde. chloramphenicol. and the biocide triclosan. trimethoprim. Small multidrug resistance Resistance-nodule-cell division Substrates of these SMR pumps. is responsible for resis- concentration (MIC). Macrolides also inhibit bacterial growth by binding to ribo. coli resistance to nalidixic acid and to other toxic compounds. ethidium bromide. responsible for macrolide/streptogramin efflux. Most of the tet determinants are found on family can accommodate a broad range of structurally unre- mobile elements. NorA is homologous to Bmr of the Salmonella enterica genome. pneumoniae. tetracycline. and F are also present in resistance in S. tetracycline. aureus The RND family constitutes the most important multidrug (Smr) and E. B. tetra- MFS transporter that confers low level of resistance to the cycline. faecium. coli (EmrE). wild-type A. major facilitator superfamily. are chloramphenicol. Mutations in glycosides and the tetracyclines. Antibiotic Resistance 59 Table 2 Typical substrates of the five classes of antibiotic efflux pumps MFS RND SMR MATE ABC Aminoglycosides Aminoglycosides Aminoglycosides Aminoglycosides Aminoglycosides Chloramphenicol -lactams Chloramphenicol Fluoroquinolones Chloramphenicol Erythromycin Chloramphenicol Erythromycin -lactams Fluoroquinolones Erythromycin Tetracyclines Erythromycin Lincosamides Fluoroquinolones Fluoroquinolones Novobiocin Novobiocin Lincosamides Rifampin Rifampin Macrolides Tetracyclines Tetracyclines Novobiocin Trimethoprim Tetracyclines ABC. specifies an chloramphenicol. resistance-nodule-cell division. coli). In addition to intrinsic resistance by impermeability. Efflux is also implicated in macrolide resistance. The mef P. Tap. detected in S. bromide. The msr(A)/msr(B) and msr(C) genes. trimethoprim. macrolides. named tance to aminoglycosides and decreased susceptibility to qepA and located on a plasmid detected in E. Expression of the adeABC operon is regulated by An efflux pump. B. meropenem. ATP-binding cassette. somes. AcrD and AcrEF also tein of the MFS family. In E. Overproduction of NorA. which are also widespread among MexCD-OprJ system is implicated in fluoroquinolone resis- Gram-negative bacteria. a Gram-negative enteric pathogen. -lactams. tetracy- Major facilitator superfamily cline. novobiocin. SMR. adeRS. and E. MexAB-OprM contributes to resistance to quinolones. lated molecules. a two-component regulatory system. for fluoroquinolone resistance.

one realizes that resistance to other classes of antibiotics is extremely high. a resistant to tetracycline. which can be located either in the of the class are generally resistant to the other members. For racyclines. Gram-negative bacteria that have Gram-positive bacteria. it ing from a bacterial infection. are quinolones. by horizontal transfer. the E. drugs assigned to a Integrons are the most efficient way to achieve co- same class are chemically related. in bacteria that differ by the presence for example. macrolides. Table 3). streptogramins. . This observation stresses that a resistance export of specific antibiotics and have been described in mechanism has no absolute value. the lower the level of resistance. The level of resistance various species. possess the machinery necessary to capture exogeneous eral. Use of a member from the parental cell by a genetic event (following a of a drug class can co-select resistance to another class of mutation or horizontal acquisition of genetic informa. bacteria to other drug classes (first two rows. Biochemical. a recombination site (attI). mechanism. 46% of the strains were susceptible to penicillin G tration of antibiotic (generally expressed as MIC in whereas 54% were resistant to this antibiotic. aeruginosa. cross-resistance implies cross-selection: use of a given antibiotic can select resistance to other members of the Acquisition of Resistance class but not to drugs belonging to other classes. among cularized DNA (gene cassettes). Resistance by a given mechanism will be much lincosamides. the same mechanism will confer to P. although these two drugs have totally biotics belonging to the same class due to a single different structures and targets of action. coli is responsible for resistance to aminoglycosides. Again. G-resistant strains (considered as 100%. On a genetic point of view. If now one considers exclusively the penicillin a genetic alteration relative to its parent (mother cell). norfloxacin. In France in 1999. are composed of genes that However. to penicillin G. Each confers (by cross-resistance) resis- There is a multiplicity of definitions of resistance and tance to a class of antibiotics which. antibiotics with a totally distinct mode of action. Expression of lmrA from Lactococcus lactis in also depends on the degree of susceptibility of the host E. These elements represent a very target of action in the cell. administration Biochemistry of Resistance of the trimethroprim–sulfamethoxazole combination (Bactrim. in other much more often resistant to the other classes of antibio- words. the resistant bacterium (daughter cell) has suffered tics. Similarly. drugs recently developed are more active than old genes: an integrase (intI) that allows recombination of cir- molecules of the same class. and are therefore subject to elegant genetic system for capture and expression of resis- cross-resistance: bacteria that are resistant to one member tance genes. Microbiologic. As we have seen above. can result in a we have already considered the intrinsic and acquired broad spectrum of resistance (MDR). tion). into the genome. which is based on the clinical outcome: compares the rates of resistance of these two groups of success or failure of antibiotic therapy in a patient suffer. respectively. meningitidis. In gen. there are degrees in cross-resistance: the more have been acquired by site-specific recombination. faecalis example. cholerae renders bacteria has a resistance level much higher than N. from other bacteria. Integrons. which resistance. As a result. species exquisitely susceptible to drugs. column 2 in Table 3). among clinical isolates of pneumo- when a strain can tolerate a significantly higher concen. sometimes stabilized by integration information. various mechanisms are associated in the progeny of the bacterium or acquisition of foreign genetic same bacterial host. expression of vcaM of V. They active the drug. For example. chromosome or in plasmids. quinolones. Clinical. ciprofloxacin is much more active than nali- found in Gram-negative and in both Gram-negative and dixic acid. and are located in suffered a mutational event in the target of quinolones. resistance can be acquired by two Co-resistance totally distinct events: Either occurrence of a mutation in the genome leading to vertical inheritance of resistance to the In co-resistance. the antibiotic most prescribed worldwide) has Cross-Resistance 88% chances to co-select a pneumococcus strain resistant Cross-resistance corresponds to resistance to all the anti. tet. For the sake of clarity. the conse- types. efrAB gene is responsible for norfloxacin and ciprofloxacin a species naturally poorly susceptible to antibiotics. cocci. or the absence of a resistance mechanism. pneumoniae (Table 3). bacterium. Genetic resistance is when the daughter cell differs quence of co-resistance is co-selection. This is. and chloramphenicol. If one mg l1). Importantly. in fine. and ciprofloxacin. have thus the same resistance (Figure 3). integrons. For example. only is apparent that the strains resistant to penicillin G are the genetic dimension of resistance is considered.60 Antibiotic Resistance for quaternary ammonium compounds resistance. the type II topoisomerases (DNA gyrase and topoisome- rase IV) become much more resistant to nalidixic acid ATP-binding cassette (that has high MICs) than to ciprofloxacin (that retains Most bacterial ABC drug transporters are implicated in the lower MICs). the case for S. higher if the bacterial species is poorly susceptible.

trimethoprim– sulfamethoxazol (Bactrim). or . is of concern. A mutation in one of glycosides. in which considered as the kidneys of the bacteria since they export most of the antibiotic resistance gene cassettes can be molecules that are toxic. The chromosomal structural genes less frequently. a Another example of this type of resistance is over- gene cassette containing attC inserts by site-specific recom. erythromycin. Tp–Su. This type of resistance is due to a single mechanism excision in an integron. pneumoniae (%) PenG Em Cm Tc Tp-Su PenS (46%) 0 20 14 15 10 PenR (54%) 100 80 38 51 66 EmR 82 100 CmR 77 100 TcR 80 100 Tp-SuR 88 100 Cm. The pumps should be genes have been reported. amino. The emergence of new gene cassettes in class 1 integrons. attI. because they are not only Integrase 3′-conserved segment adjacent. chloramphenicol. such as qnr implicated in resistance to fluoro- R2 Gene cassette quinolones. and streptogra- into the integron contain a single gene and. products of the found. attachment site of the integron. A typical example is the methylation of a specific and a promoter (Pant) that directs transcription of the adenine residue in 50S rRNA that confers high level captured genes. with very different structures. chloramphenicol. lincosamides. in particular. attC. To date. gene for the integrase. In presence of the integron-encoded integrase. Em. expression of efflux pumps that can have very broad bination at the attI site and the gene is transcribed from the substrate ranges. PenG. As in co-resistance. The pumps that are grouped in super- Pant promoter. Site-specific recombination use of any antibiotic that is substrate for one of the Pant intI1 qacEΔ1 sul1 resistance mechanisms will co-select for resistance to all R1 the others. The resistance gene cassettes inserted resistance to macrolides. the genes involved in regulation (activator. trimethoprim. repressor. implicated in the dissemination of antibiotic resistance can select multiresistant bacteria. and antiseptics of the quaternary intI1 qacEΔ1 sul1 ammonium compound family. downstream mins B although these three classes have different from it. outward-oriented promoter for the cassettes. Su. In Gram-negative bacteria. S. resistant. This integrative process or hydrolysis of ATP. a specific attC site. After integration of a gene cassette. have been detected in many Gram-negative and. Pant. The resistance determi- attI nants are tightly linked. Pant intI1 qacEΔ1 sul1 R2 R1 Extended Cross-Resistance Figure 3 Model for site-specific gene cassette integration. lincomycin. sulfonamides. that are increasingly used in household products. ‘extended’ cross-resistance. tance to various drug classes and is thus designated as Horizontal arrows indicate sense of transcription. but co-expressed from the same promoter. five classes of integrons gents. susceptible. and also biocides resistance: the closer of Pant. a class of antibiotics can select for resistance to other drug classes. another families use energy provided by the protonmotive force one can be inserted at the attI site. Int. This accounts for the fact that deter- of the resistance gene. are generally expressed at low level. the higher level of expression such as triclosan. in Gram-positive bacteria. tetracycline. but with differences in genetic and biochemical organization. which is an imperfect inverted chemical structure. There is a clear relationship between the RND pumps can export a large array of antibiotic mole- position of a cassette in the integron and the level of cules. integrase. Tc. Pant fosfomycin. (therefore cross-resistance is dealt) that can confer resis- attachment site of the cassette. Antibiotic Resistance 61 Table 3 Antibiotic resistance in Streptococcus pneumoniae Resistant to (%) S. cellular catabolism. intI1. Gene cassettes for the pumps are positively or negatively regulated and in class 1 integrons confer resistance to -lactams. the is reversible. repeat. erythromycin. R. Class 1 integrons. penicillin G. attC R1 Gene cassette Since the genetic organization of integrons results in Int co-expression of the genes that have been integrated.

often several Transposon of them. a point mutation. In general. A plasmid terium can acquire ‘en bloc’ a multiplicity of resistances. Figure 5 Schematic representation of plasmid conjugation. as described above. Thus. the chromosome is not self- transferable horizontally to other bacteria. Thus. During this process. tion. in turn. the so-called MDR. can carry multiple. a bac- conjugative plasmid coresident in the same cell. After transfer. the complementary strand of the remaining ferred laterally by conjugation or by mobilization. Because of this physical linkage. These mobile genetic elements can be inherited horizontally and vertically. Thus. any gene can be part of a volatile structure as long as it provides intermittent selec- tive advantage to the host and that adequate selective pressure exerts. each bacterium contains a copy of the plasmid (top) and plasmids are self-transferable from cell to cell by conjuga. conjugation. Plasmids and transposons encode functions that are not strictly required for bacterial life but that can provide advantages to the host. Bottom right. They DNA strand in the donor is synthesized while the complementary may carry resistance to several antibiotics.62 Antibiotic Resistance two-component regulatory system) or in the operator will result in overexpression of the pump and leads to resis- tance to its various substrates. The chromosomes are represented in a condensed state. Integrons. Conjugative strand of the incoming DNA is synthesized in the recipient. the smallest genetic event. as a donor. Transposons encode a transposase that allows site-specific insertion and excision. a mechanism that requires physical contact between the donor and the recipient bacterium (Figure 5). in a single genetic event. one strand is transferred from the donor to the recipient. Antibiotic resistance genes are only transiently useful to bacteria and it thus makes sense that they are often transferable and part of mobile genetic elements. resistance determi- nants. and transposons are frequently carried on plasmids. In fact. donor bacterium. selection pressure Figure 4 Schematic representation of the bacterial genome. mobile genetic elements carrying antibiotic resistance genes often encode resistance to heavy metals and detergents. easily up to seven. Mobilizable plasmids can be transferred with the help of a others. bottom left. selection for Transposons resistance to any of them will lead to co-transfer of the Transposons are DNA fragments that can migrate from one replicon to another while retaining their structural integrity (Figure 6). . Chromosomal resistance genes and mutated genes involved in drug resistance are inherited vertically by the next generation of bacteria. Plasmids are extrachromosomal elements that can be trans. Numerous plasmids and transposons carry antibiotic resistance genes. can lead in one step to resistance to a large set of antibiotics. or passively when they are borne by a transferable plasmid. Furthermore. in the case of conjugative transposons of Gram- positive cocci. are found frequently as part of transposons. exerted by biocides may select for antibiotic resistance. The chromosome contains all the genetic information required for the life cycle of the bacteria. They can transfer Plasmid actively. can therefore act. Genetics of Resistance The genome of bacteria is constituted of the chromosome and accessory mobile genetic elements such as plasmids and transposons (Figure 4). After a Plasmids single nick on one of the two complementary DNA strands of the plasmid. recipient bacterium.

of replication is indicated by an arrow) whereas the acceptor (3) transfer frequency of conjugative transposons belonging replicon (lower left) does not. an enterococcal right) contains a copy of the transposon (close bar. The donor replicon (lower frequency of in vitro transfer of Tn916. Antibiotic Resistance 63 promoter regions resulting in transcriptional activation of the regulatory (vanR/vanS) and of the resistance genes. in the same orientation. it has been reported that (1) use of subinhibitory concentrations of penicillins increase the conjugal transfer of plasmid DNA from E. efflux pumps). in the absence of vancomycin. each replicon contains a copy of the element (top) Bacteroides conjugative transposon. faecalis to Bacillus anthracis.. allowing expression of the resistance pathway (synthesis of modified peptidoglycan precursors) and elimination of the normal precursors ending in D-Ala-D-Ala. Resistance to an antibiotic is often inducible by the The frequency of appearance of resistant strains in a bac- antibiotic itself. Thus. The biological cost transduced from the sensor domain to the catalytic domain allows determination of the stability and the potential of VanS. after completion of cycline. aureus and L. and the ability of bacteria to compensate for the fitness cost. and can thus. in which VanS acts as a sensor and mutations occur in genes with essential functions. Selective replication of the to the Tn916/Tn1545 family. some and acquisition of foreign genes. dephosphorylates VanR resulting in arrest of expression of the resistance genes. coli to S. Following recombination and in vivo in the presence of low concentrations of tetra- between the two copies of the element. from E. In this case. and (4) tetracycline also increases the transfer of a transposition. the biological cost of resistance. the drug should be considered terial population depends on several factors such as the as having two types of activities: induction of resistance volume of antibiotic used. in turn.to 100-fold in vitro contains two copies of the element. Acquired resistance to vancomycin is a typical example Acquisition of antibiotic resistance is often associated with a of inducibility. act as a donor. Since antibiotic resistance usually corresponds to a gain of function. and killing of the bacteria that act on distinct targets. Antibiotics Can Act as Pheromones Antibiotics provide selective pressure for resistant bacteria to maintain and disseminate but they can also induce trans- fer of resistance genes. the signal is ments that bear the resistance genes. the growth rate in vitro or in . at the borders of the replicons (middle).g. DNA exchange. The phosphorylated regulator binds to the ceptible and resistant) strains. leading to autophosphorylation of VanS followed reversibility of resistance. In the the replication and maintenance of extrachromosomal ele- presence of vancomycin in the environment. which contain a tetracycline transposon and replicon fusion generate a bireplicon that resistance determinant. It therefore appears that modulation of gene expression probably reflects a good compromise between energy saving and adaptation to a rapidly changing environment. there are two major pathways to low concentrations. several antibiotics can behave like pheromones: they are synthesized by specific cells (such as the Actinomycetes producers) and they act on another cell. For example. on very specific targets to promote antibiotic resistance: mutational events in the chromo. Biological Cost of Antibiotic Resistance latory regions of genes (e. The fitness cost of antibiotic by transfer of the phosphoryl group to the VanR response resistance could be assessed by measuring. monocytogenes. Mutations can occur not only in a structural gene for the target of an antibiotic (as discussed for quinolones) but also in regu. in isogenic (sus- regulator. Expression of the resistance genes of the biological cost because (1) bacteria acquire a new gene or van operon is controlled by a two-component regulatory set of genes responsible for new functions. that is. (2) the resistance system VanS/VanR. the direction conjugative transposon. (2) oxacillin increased the Figure 6 Replicative transposition. Under nonin- ducing conditions. is increased 10. at As already mentioned. VanS acts as a phosphatase. there is an associated biological cost resulting in the loss of fitness of the bacterial host. or (3) of VanR as a transcriptional regulator (Figure 2).

Clinical Microbiological Reviews 20: 79–114. DC: American Society for Microbiology. an acquisition of a second mutation (reverse mutation) and gene epidemics among bacteria (transposons and inte- located in the same (intragenic) or in another (extragenic) grons). resistance-plasmid resistance mutation or loss of the resistant element or (2) epidemics due to the broad host range of conjugation. many in vitro and in observed in clinical isolates. Resistance mutations that Origin. DC: ASM genes encoding tRNAi) partially reduce the fitness cost. and absence of antibiotics. is frequently reduced by a compensated mutants are fixed in the bacterial population. London. White DG. Hughes D and Andersson DI (eds.) (2001) Antibiotic Development and example of a no-cost high level resistance mutation.) (2007) Enzyme-Mediated occur in the fmt or folD gene confer a fitness cost in the Resistance to Antibiotics – Mechanisms. which was already developed drugs. Baron EJ. Some resistant bacteria may have a normal growth Depardieu F. enterica that were Further Reading resistant to a deformylase inhibitor. London: Plenum press. the bacterial reduced uptake.) resistant counterparts. develop (1) strategies to reduce resistance dissemination resistance to several antibiotics. or the growth sites. The methods used to determine the fitness Levy SB (ed. A compensatory evolution could occur to reduce or co-resistance. There are three levels of expo- in an antibiotic-free environment. Cundliffe E. Richmond MH. Jorgensen JH.1 & 2. is a typical Sydney. Advanced Drug Delivery Reviews 57: 1471–1485. Advanced Drug Delivery Reviews (2) involve several classes by extended cross-resistance 57: 1451–1470. suggests that compensation is vivo studies indicate that several pathways will confer.) (2002) Bacterial cost was associated with vancomycin resistance in enter.) (2005) Antibiotics in Laboratory Medicine. and Yolken RH (eds. Murray PR. A specific substitution in the rpsL gene (which Gale EF. Press.) (2005) Antimicrobial Agents. The biological cost associated sion to the susceptible phenotype. resistance to every new antibiotic. mutation. Intragenic mutations in the fmt/folD Prospects for Inhibition. allowing the stabilization of the resistant mosomally located are vertically inherited whereas those bacteria in a natural population. Resistance determinants that are chro- the biological cost.) (2006) Antimicrobial Resistance in Bacteria of Animal targets peptide deformylase. and Courvalin P (2007) suggesting that they have acquired a no-cost resistance Modes and modulations of antibiotic resistance gene expression. under natural conditions.64 Antibiotic Resistance animals. Podglajen I. New York: Taylor & Francis. Piddock LJV (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Taber HW. New York. This stabilization may that are part of mobile genetic elements can be vertically allow resistant strains to compete with susceptible strains and horizontally acquired. Chances of reversi. DC: American Society for genes or extragenic mutations (such as amplification of Microbiology. Washington. resistance genes can easily disseminate gene. compensatory evolution that allows the stabilization of the a reversion to susceptibility is unlikely. vol.) (2005) Frontiers in Intrinsic or acquired resistance to antimicrobial drugs Antimicrobial Resistance. Washington.) encodes ribosomal protein S12). ococci whereas another group found that vancomycin. Bonomo RA and Tolmasky M (eds. Levy. Clinical Microbiological Conclusion Reviews 19: 382–402. Resistance to Antimicrobials. Finally. Washington. Dissemination. Resistance Wright GD (2005) Bacterial resistance to antibiotics: Enzymatic may (1) be limited to one class of antibiotics or degradation and modification. conditions. Once the resistant and with resistance. New York. coli. Similar results were obtained in S. an antibiotic that Aarestrup FM (ed. DC: American Society for Microbiology. Collatz E. Basel: Marcel Decker. nential dissemination of antibiotic resistance: epidemics of lution could be the result of (1) a true reversion of the resistant bacteria among mammals. Thus. Salyers AA. Washington. DC: American Society for Microbiology. Reynolds PE. Lorian V (ed. Pfaller MA. soon or later. London.. Toronto: John Wiley & Sons. New York. The compensatory evo. and McDermott PF (eds. Resistance.) (1992) The Antibiotic Paradox: How Miracle Drugs are cost are also crucial: one study demonstrates that no fitness Destroying the Miracle. Reversion mutations are more common than rever. susceptible enterococcal strains were more fit than their Baltimore. (such as prudent use of antibiotics and increase of surveil- It was observed by competition experiments in lance resistance) and (2) new antibiotics addressing novel Helicobacter pylori that clarythromicin resistance confers a targets and thus escaping from cross-resistance with biological cost in mice. the fitness cost of resistance depends on several Kumar A (2005) Bacterial resistance to antibiotics: Active efflux and factors such as the environmental conditions. . when it exists. Advanced Drug Delivery Reviews 57: 1486–1513. Reduction of this cost. Leclercq R. aureus and Salmonella typhimurium and in rifampin-resistant mutants of E. (2004) Manual of Clinical Microbiology. Bryskier A (ed. and Wax RG (eds. Alekshun MN. resistant bacteria in the population. However. The biological fitness was also partially or totally restored in fusidic acid-resistant mutants of S. could be the result of different mechanisms. mycin resistance in several enteric bacteria. A Tribute to Stuart B. (1972) The Molecular Basis of Antibiotic Action. responsible for strepto. Lambert PA (2005) Bacterial resistance to antibiotics: Modified target species. 5th edn. Washington. Lewis K. a clinically relevant phenomenon. the specific resistance mutation. 8th edn. Inc. It is thus necessary to bility of the resistance are also reduced in case of co. Maryland: Lippincott Williams & Wilkins. and Waring MJ (eds.

) and Introduction to fruit (banana wilt). on the contrary. USA ª 2009 Elsevier Inc. Fungi and mammalian cells fungi associated with disease are considered opportunistic are eukaryotes. the potato Fungi can be unicellular (yeasts) and multicellular or famine. Virginia Commonwealth University Medical Center. and antifungal agents that inhibit synth. and Fludioxonil The Polyoxins and Nikkomycins The Synthetic Pyrimidines The Sordarins The Azoles Dimethomorph and Fluazinam The Allylamines The Phthalimides The Benzylamines. RNA. disease in otherwise healthy individuals. Pneumocandins. Many fungi. corn smut. etc. are important plant and lower animal parasites and can cause damage to crops (wheat rust. chemically induced immunosuppression caused by low nephrotoxicity Damage to the kidney cells. All rights reserved. Defining Statement The Morpholines Introduction The Pyridines The Polyenes The Echinocandins. Other Antifungal Approaches and Dithiocarbamates Further Reading The Benzimidazoles and Methylbenzimidazole Carbamates Glossary in vitro and in vivo Describing or referring to studies emerging fungal infections Fungal infections caused carried out in the test tube and in animals. Abbreviations GVHD graft-versus-host disease AMB amphotericin B HSCT hematopoietic stem cell transplant CSF cerebrospinal fluid MIC mum inhibitory concentration DMPC dimyristoyl phosphatidylcholine NYS nystatin DMPG dimyristoyl phosphatidylglycerol OPC oropharyngeal candidiasis Defining Statement important fungi can exist in each of these morphologic forms and are called dimorphic fungi. system. Fenpiclonil. Historically. Of the estimated Antifungal agents are naturally occurring or synthetically 250 000 fungal species described. or both. molds. Griseofulvin and Papulocandins Cycloheximide The Pradimicins and Benanomycins Pyrrolnitrin. Some medically Ireland to the Americas. Most against yeasts. respectively. opportunistic infections Infections caused by immunocompromised Having a defect in the immune saprophytic fungi or not true parasites. lower animals and plants. was caused by a fungal infection 65 . and orna- mental trees and other plants. pathogens (especially the yeasts) because they live as normal esis of proteins. Antifungal Agents A Espinel-Ingroff. by new or uncommon fungi. which was the reason for the great migration from filamentous (molds) microorganisms. and rarely cause mammalian cells. Thiocarbamates. mycoses and mycotic infections Diseases caused by granulocytopenia/neutropenia Acquired or yeasts or molds. forest (Dutch elm disease). fewer than 150 are produced compounds that have in vitro or in vivo activity known to be etiologic agents of disease in humans. white blood cell counts. and DNA are potentially toxic to flora in humans. VA.

amphotericin B has been are in the last phases of clinical development. Trichosporon beigelii. quantita- patients. creating the high toxicity associated with all polyene amphotericin B and its three lipid formulations. light. and its use in combination with other antifungal agents are based on the type of infection and the status of Amphotericin B the host. which is an important sterol including magnesium. who are at high risk for life-threatening mycoses. or agricultural uses. an internal ester. Scedosporium prolificans. cytopenic host are difficult to diagnose and cause much Amphotericin B replaced 2-hydroxystilbamidine in the mortality. Traces of ergosterol are also recommended during amphotericin B therapy. Amphotericin B is highly protein the triazoles fluconazole. close The polyenes are macrolide molecules that target mem.) administration of 0. detailed data regarding these agents are found in the amphotericin B is effective in the treatment of both references. and hydration status is in the fungal cell membranes. specific. from a soil sam. concentrations of 0. steroids. certain acids. Clinically. and tions with other drugs and applications of the established other emerging fungal pathogens. especially . B and other agents has improved patient care. veterinary. Therefore. under considered as the gold standard antifungal agent for the clinical trials in humans. particularly with certain yeast and mold species. mechanisms of action Malassezia furfur. bicarbonate. Peak serum of 1–3 mg ml1 and trough posaconazole. This drug is also used for the treatment bonds. the number of after the intravenous (i. However. fungal action has been proposed for amphotericin B. fungi and their products glycoside side chain with a primary amino group play an important economic role in the production of alco. Current involved in the overall cell cycle of fungi. Toxicity is the limiting factor during amphotericin B therapy and has been classi- The Polyenes fied as acute or delayed (Table 2). or that have been discontinued management of most systemic and disseminated fungal from additional clinical evaluation. Amphotericin can occur with the administration of electrolytes and other B (the most active molecule) has seven conjugated double concomitant drugs. This article summarizes the most relevant facts such as Candida lusitaniae. which cytosine). Systemic tericins (A and B) were isolated in the 1950s from prophylaxis for patients at high risk for invasive mycoses Streptomyces nodosus. adverse interac.66 Antifungal Agents (potato blight). with a long nificantly increased during the past years. is oxidation-dependent. electrolytes branes containing ergosterol. antibiotics. and so on. infections and opportunistic mycoses. doses. and extends to mammalian cells. safety. mica. pharmacokinetics. Although it pene- pounds have potential use as therapeutic agents.6 mg kg1 fungal diseases caused by both yeasts and molds has sig. Amphotericin B adverse drug interactions ple from Venezuela’s Orinoco River Valley.. tive and qualitative changes in the cell membrane sterols There are more agents for topical treatment as well as have been associated with the development of microbio- agriculture and veterinary use. Two ampho. a free carboxyl group. The fungistatic (inhibition of fungal growth) and fungicidal Because. The drug binds ment and prevention of systemic fungal infections: the to cholesterol. Its half-life of elimination is 24–48 h. Pseudallescheria boydii (Scedosporium apios- and resistance. At the same time. This effect is not 14 antifungal agents are currently licensed for the treat. Candida and Cryptococcus meningitis alone and/or in com- bination with 5-fluorocytosine. empirical antifungal therapy with amphotericin treatment of blastomycosis in the mid-1960s. itraconazole. (Figure 1(a)). duration. hol. Because severe fungal infections in the granulo- Amphotericin B is the most important of the 200 polyenes. and acid pH. especially terminal half-life of up to 15 days. Pores or channels are formed causing osmotic mised patients at high risk for life-threatening mycoses. total dosage. the number of fungal diseases caused by both (lethal) activity of amphotericin B is due to its ability to yeasts and molds has significantly increased during the combine with ergosterol in the cell membranes of suscep- past twenty years. Aspergillus terreus. especially among immunocompro. It is unstable to heat. dimorphic fungi. permum). instability and loss of membrane integrity.1 mg ml1 are usually measured fungin. More trates poorly into the cerebrospinal fluid (CSF). The former com. A second mechanism of anti- the pyrimidine synthesis inhibitor 5-fluorocytosine (flu. A shorter includes yeasts. monitoring of renal function tests.5–1.v. systemic and topical antifungal agents currently licensed The in vitro spectrum of activity of amphotericin B for clinical. regarding the chemical structure. tible fungi. diuresis. the imidazoles miconazole and ketoconazole. among the increased number of immunocompromised Although resistance to amphotericin is rare. recommendations regarding daily dosage. and a of systemic infections in small animals. has also evolved. and the echinocandins caspofungin. and anidulafungin. an aerobic bacterium. Fusarium spp. problem. voriconazole and bound (91–95%). under investigation for the management of severe and resistance to amphotericin B has become an important refractory fungal infections in humans (Table 1). and several agents are logical resistance both in vitro and in vivo. and most of the opportu- description is provided for antifungal compounds that nistic molds. conventional polyene agents. Nephrotoxicity is the most significant delayed adverse effect. Clinically.

b Enilconazole Veterinary Epoxiconazole Agriculture (Continued ) .b AMB lipid complex Systemic mycoses intolerant or refractory to AMB AMB colloidal dispersion Liposomal AMB Liposomal NYS Under investigation Pimaricin Topical keratitisa Phenolic cyclohexane Microtubule aggregation and DNA Griseofulvin Dermatophytic infectionsa inhibition Natural glutarimide Protein synthesis inhibition Cycloheximide Laboratory and agriculture Phenylpyrroles Unknown Fenpiclonil Agriculture Fludioxonil Synthetic pyrimidines Fungal cytosine permease and Flucytosine Systemic (yeasts) in combination with AMBa deaminase Ergosterol inhibition Triarimol Agriculture Fenarimol Anilinopyrimidines Enzyme secretion Pyrimethanil Cyprodinil Azoles Ergosterol biosynthesis inhibition Imidazoles Clotrimazole Topical.Table 1 Antifungal agents.b Nystatin (NYS) Superficial mycosesa. and their use Antifungal class Antifungal target of action Agent Use Polyenes Membranes containing ergosterol Amphotericin B (AMB) Systemic mycosesa. mechanisms of action.b Econazole Isoconazole Oxiconazole Tioconazole Miconazole Topical and veterinaryb Ketoconazole Second-line drug for nonmeningeal systemic mycoses and veterinarya. oral trochea.

aspergillosis. coccidioimycosis. cryptoccosis. long-term prophylaxisa Posaconazole Prophylaxis (invasive Aspergillus and Candida infections). apiospermum and Fusarium infections)a Ravuconazole (BMS-207147. endemic mycoses. salvage therapy (S.Table 1 (Continued) Antifungal class Antifungal target of action Agent Use Fluquinconazole Triticonazole Prochoraz Triazoles Fluconazole Candidiasis. superficial diseases. oropharyngeal candidiasis (OPC) Terconazole Intravaginal Voriconazole Aspergillus and Candida infections. prophylaxisb Itraconazole Candidiasis. ER-30346) Under investigation (phases II and III) Albaconazole (UR-9825) Under investigation (phases I and II) Allylamines Terbinafine Superficial infections Naftifine Topical Benzylamines Butenafine Topical Thiocarbamates Tolnaftate Topical Tolciclate Piritetrade Dithiocarbamates Nonspecific Mancozeb Agriculture Thiram Benzimidazoles and Nuclear division Carbendazim Agriculture methylbenzimidazole carbamates Benomyl Thiophanate Morpholines Ergosterol biosynthesis inhibition Amorolfine Topical Fenpropimorph Agriculture Tridemorph Pyridines Buthiobate Agriculture Pyrifenox .

including candidemia.Echinocandins Fungal (1. other applications for use in humans only. b A human product used in veterinary. commonly used. empirical therapy (febrile neutropenic patients unresponsive to antibacterial therapy). salvage therapy (aspergillosis)a Micafungin Invasive Candida infections. including candidemia Prophylaxis for HSCT patients and liver transplantationa Pradimicins Fungal saccharide Pradamicin FA-2 (BMY None (mannoproteins) 28864) Benanomycins Benanomycin A Under investigation Polyoxins Fungal chitin synthase Polyoxin D None Nikkomycins Nikkomycin Z Under investigation Sordarins Protein synthesis inhibition GM 222712 Under investigation GM 237354 Cinnamic acid Cell wall Dimethomorph Agriculture Oomycete fungicide Oxidative phosphorylation Fluazynam Agriculture Phthalimides Nonspecific Captan Agriculture. . including candidemiaa Caspofungin Invasive Candida infections.3)-glucan synthetase Papulocandins None inhibition Echinocandin B derivative Pneumocandin derivatives Anidulafungin Invasive Candida infections. and antifungals under clinical investigation are listed. see text for other antifungals. Only licensed. horses (dermatophytic infections) Cationic peptides Lipid bilayer of Cecropin None biologimemberanes Indolicidin Synthetic peptides Under investigation Amino acid analogs Amino acid synthesis interference RI 331 Under investigation Azoxybacillins Cispentacin Candida isoleucyl-tRNA synthase Icofungipen Phase II trials inhibitor a Clinical and veterinary use.

(d) ketoconazole. to experimental have been approved for the treatment of invasive fungal animals. However. and a liposomal tericin B.v. despite evidence of nephrotoxi- gastrointestinal (orally) and mucocutaneous candidiasis city reduction. amphotericin B colloidal dispersion. . and the differences in both efficacy and tolerance Amphotericin B lipid formulations among the three formulations. drug interactions. It is an amphoteric tetrane macrolide that has a higher doses of amphotericin B can be safely used. Also. Although it in clinical trials: an amphotercin B lipid complex. the most cost-effective In an attempt to decrease the toxicity and increase the clinical role of these agents as first-line therapies has not efficacy of amphotericin B in patients with deep-seated been elucidated. (e) fluconazole. this antifungal is used mostly for the therapy of amphotericin B.70 Antifungal Agents Figure 1 Chemical structures of some systemic licensed antifungal agents: (a) amphotericin B. several lipid formulations of this antifun- gillosis. a significant improvement in their efficacy (topically). This is not only due to its toxicity after parent. (b) 5-fluorocytosine. and (f) itraconazole. Three similar structure (Figure 2(a)) and identical mechanism of lipid formulations of amphotericin B have been evaluated action to those of conventional amphotericin B. blastomycosis in dogs. not enough information is available regarding their pharmacokinetics. (c) miconazole. when it was isolated from Streptomyces noursei in the early The result is a reduction of human erythrocytes lysis and 1950s. long-term toxi- Lipid Formulations cities. Side effects (especially in cats) and drug gal have been developed since the 1980s. but it is not effective against asper. therapy. fungal infections. compared to conventional amphotericin B has not been eral administration to humans and lower animals but also to demonstrated clearly. an has an in vitro spectrum of activity similar to that of ampho. These interactions are similar to those in humans. It is used for candidiasis in small animals and infections that have failed conventional amphotericin B birds and for otitis caused by Microsporum canis. Although these three formulations its lack of effectiveness when given i. preparations have selective toxicity or affinity for fungal cell membranes and theoretically promote the delivery of Nystatin the drug to the site of infection while avoiding the toxicity Nystatin was the first of the polyenes to be discovered of supramaximal doses of conventional amphotericin B.

V. which contain DMPC and DMPG in a 7:3 ratio. pedal edema I Headache A. K. In the first liposomes. K. M. V Arthralgia. itraconazole. V Transient vision disturbances V (30%) Dizziness I. C. female alopecia K Syndrome of mineralocorticoid excess. V. etc. V. C. evaluations in human subjects are limited. (ambisome). Fl.8:1:0. and Walsh TJ (1998) Clinical pharmacology of systematic antifungal agents: A comprehensive review of agents in clinical use. This (infections by Candida albicans and other Candida spp. cholesterol and amphotericin B in a 2:0. current investigational compounds. P. Streptomyces griseus that is selectively and highly active in vitro against yeasts. An Rash FC. Fl. An (rare) Hepatitis (rare) FC. caspofungin. in a 7:3 liposomal nystatin is significantly superior to that of con- molar ratio (5–10% mole ratio of amphotericin B to ventional nystatin and is well tolerated in experimental lipid).). K. K Diarrhea FC. Liposomal amphotericin B formulation is a stable complex of disk-like structures In the only commercially available liposomal formulation (122 nm in diameter and 4 nm thickness). DC: ASM Press for more detailed information. In: Murray. fluconazole. amphotericin B. FC. V Alopecia (rare) Fl Hypokalemia Fl. thrombocytopenia FC. K. V Anorexia A. (fungal infections caused by Candida spp. (eds. K. . I. I. CV Decreased testosterone synthesis K (I. dimyristoyl phosphatidylcholine (DMPC) Although it has been demonstrated that the efficacy of and dimyristoyl phosphatidylglycerol (DMPG). I. multilamellar liposomes that contained two liposomes. These liposomes contain hydrogenated soy phospatidyl- Liposomal nystatin choline and disteaoryl phosphatidylglycerol stabilized by To protect human erythrocytes from nystatin toxicity. menstrual irregularities. V. C. vomiting A. hypokalemia. flucytosine. multilamellar into large. C. its use was restricted to Amphotericin B colloidal dispersion contains cholesteryl topical applications for the treatment of vaginal candidiasis sulfate and amphotericin B in a 1:1 molar ratio. K. Washington. FC. I. rare) Adrenal insufficiency. It is more toxic for mammalian cells than either Amphotericin B colloidal dispersion amphotericin B or nystatin. P Nausea. An (rare) Decreased renal function (azotemia. thrombophlebitis A Ventricular tachycardia C. Antifungal Agents 71 Table 2 Adverse side effects of the licensed systemic antifungal agents Side effect Drug Fever. V Seizures FL Confusion FC. Anidulafungin. An.) A. K. chills A. FC. acidosis. spherical vesicles (60–70-nm liposomes). FL. FL. Piscitelli SC.). myalgia. I. A. Fl. An Photophobia K. FC Leukopenia. PR et al. FL Anemia A. Advances in Pharmacology 44: 343–500 and Arikan S and Rex JH (2007) Antifungal agents. 9th ed.) Manual of Clinical Microbiology. and putative targets for antifungal drug development. ketoconazole. therefore. amphotericin B was incorporated nystatin has been incorporated into stable. Amphotericin B lipid complex Amphotericin B lipid complex contains a DMPC/DMPG lipid formulation in a 7:3 ratio and a 50% molar ratio of Candicidin amphotericin B to lipid complexes that form ribbon-like Candicidin is a conjugated heptaene complex produced by structures. K. V. An Abdominal pain FC. amphotericin B is incorporated into small unilamellar. V. micafungin. and Apergillus spp. V. An Dyspnea and hypotension (rare) An Reproduced from Groll AH. I. V. This formulation is not commercially available. M Elevation of transaminases FC. but murine models of systemic candidiasis and aspergillosis it led to the development of commercial formulations. FL.4 molar ratio. phospholipids. voriconazole.

This is a glutaramide agent produced by S. cereal seed.72 Antifungal Agents Figure 2 Chemical structures of the most commonly used topical antifungal agents: (a) nystatin. griseus. benzyfuran cyclohexane agent (Figure 2(b)) that binds to RNA. Although cycloheximide had Griseofulvin clinical use in the past. Abdominal adverse side effects natalensis. The therapeutic use of pimaricin is limited to the topical treatment of keratitis (eye infec- tions. It was used in the past as a topical agent. and Fludioxonil potent inhibitor of thymidylate synthetase and interferes with the synthesis of DNA. Fenpiclonil.. and fludioxonil (related to pyrrolnitrin) were the first the clinical use of griseofulvin as an oral agent for treat. It acts as a Pyrrolnitrin. This agent was among the three antifungals that were reported between 1944 and 1947. It also inhibits microtubule Pyrrolnitrin is the fermentation product of Pseudomonas formation and the synthesis of apical hyphal cell wall spp. and other species. therefore. especially in cats. it is frequently used for these infections in small animals. (c) clotrimazole. It is a product of Penicillium janczewski and was the first antifungal agent to be developed as a systemic plant protectant. also in horses) caused by the molds Fusarium spp. Cycloheximide Acremonium spp. terol than for ergosterol and.. of the phenylpyrrols to be introduced as fungicide for ment of dermatophytic infections has become limited. it is highly toxic for mammalian cells. Griseofulvin is a phenolic. (b) griseofulvin. It has a higher binding specificity for choles- have been noted. With the advent of terbinafine and itraconazole. and calves (skin only) as well as Pimaricin is a tetraene polyene produced by Streptomyces for equine sporotrichosis. Fenpiclonil material. it is currently used as a plant fungicide and in the preparation of laboratory media. . Pimaricin However. and (d) terbinafine. horses.

This 5-Fluorocytosine has fungistatic but not fungicidal activ. a side effect of the developed specifically as an anti-Candida agent. Thiabendazole cytosine should not be administered to pregnant women or Thiabendazole was developed as an anthelmintic agent animals. 5-Fluorocytosine should not be used alone for the The basic imidazole structure is a cyclic five-member treatment of any fungal infections. fruits. which is the main active form of the drug. antimicrobial and anticancer drugs. and cyprodinil. It was ketoconazole for cryptococcosis in small animals (very also used in the past in the treatment of superficial yeast toxic for cats) and also for respiratory apergillosis and and dermatophytic infections. cyclosporine. Pyrimethanil. and the other involves the enzyme cytosine The Azoles deaminase. Antifungal Agents 73 The Synthetic Pyrimidines Triarimol. thrombocytopenia. group includes fused ring and N-substituted imidazoles ity mostly against yeasts. Pyrimethanil has activity (without cross- bound permease. Alterations of the genetic The azoles are the largest single source of synthetic anti- regions encoding these enzymes may result in fungal fungal agents. fungi (yeasts and molds). two metabolic sites are responsible for resistance to this compound. which leads to neu. (apples and pears). Thiabendazole has been severe candidiasis in birds. the drug is deaminated to resistance) against Botrytis cinerea (vines. it also interferes gal agents in agriculture. Inside the cell. in the mode of action of 5-fluorocytosine. but it is not inhibit lanosterol demethylase. been used in the past in the treatment of superficial ocytosine. monitoring of the drug concentration in the and two have limited antifungal activity: 1-chlorobenzyl- patient’s serum (serial 2-h levels post-oral administration) 2-methylbenzimidazole and thiabendazole. or pancytopenia (Table 2). Fenarimol. It acts as a competitive antimetabolite not used in medicine but are used extensively as antifun- for uracil in the synthesis of yeast RNA. pyrimethanil. shared in common with a fused benzene ring. Because the drug 1-Chlorobenzyl-2-methylbenzimidazole is administered in combination with amphotericin B. its activity against molds is and the N-substituted triazoles. an enzyme involved in the effective against tumors. but only a preventive effect against Venturia spp. cell lysis. which leads to the inhibition of fluorinated pyrimidine related to 5-fluorouracil and floxur. and ornamental plants) and Venturia spp. The broad spectrum of activity involves the drug or its metabolites. It is an oral. the major therapeutic these compounds is the inhibition of lanosterol demethy- role of 5-fluorocytosine is its use in combination with lase. ring containing three carbon and two nitrogen molecules. which is responsible for the uptake of the drug into the fungal cell. is highly recommended to adjust dosage and maintain serum levels between 40 and 60 g ml1. they are broad spectrum in nature and mostly permeability or synthesizing molecules that compete with fungistatic. The first step is inhibit the secretion of the fungal enzymes that cause plant initiated by the uptake of the drug by a cell membrane. Most of tropenia. At least spp. low-molecular-weight. bacteria. Triarimol and fenarimol are idine (Figure 1(b)). It has latter compound. This drug has been used in combination with and has a limited activity against dermatophytes. One involves the enzyme cytosine permease. and parasites. city associated with 5-fluorocytosine therapy is bone In the fused-ring imidazoles. vegetables. Adverse drug interactions can occur with other yeast and dermatophyte infections. amphotericin B in the treatment of meningitis caused by the yeast Cryptococcus neoformans. The mode of action of inoculum-dependent. synthesis of ergosterol. Clinically. The synergistic antifungal activity of these two agents has been demonstrated in Fused-Ring Imidazoles clinical trials in non-HIV-infected and AIDS patients. Because of its toxic potential. the first azole was discovered in 1944. can induce increased toxicity to 5-fluor. a cytochrome P450 enzyme. used for aspergillosis and penicillosis in dogs. which is responsible for the deamination of the drug to 5-fluorouracil. and other therapeutic agents. The most serious toxi. These activities can be antagonized in vitro by a variety of whereas cyprodinil has systemic activity against Botrytis purines and pyrimidine bases and nucleosides. They that was first developed as an antitumor agent. this biosynthetic pathway. Several enzymes are involved The anilinopyrimidines. and Cyprodinil 5-Fluorocytosine (Flucytosine) Triarimol and fenarimol are pyrimidines with a different The synthetic 5-fluorocytosine is an antifungal metabolite mechanism of action than that of 5-fluorocytosine. these compounds have parasitic activity (anthelmintic) Therefore. 5-fluorouracil. with thymidylate synthetase. As a resistance to this drug by either decreasing the cell wall group. 5-fluoro. a The azole 1-chlorobenzyl-2-methylbenzimidazole was decrease in glomerular filtration rate. . two carbon molecules are marrow suppression (6% of patients).

Miconazole (Figure 1(c)) was the first azole derivative to In the second series. As with clotrimazole. oxiconazole patients. in These series vary in spectrum. Oxiconazole and econazole are less effective than terbinafine and itraconazole in the treatment of ony. its adverse interactions with other and tioconazole drugs. replaced by itraconazole and other triazoles. Bifonazole is seldom utilized as a topical approximately 50% of the patients taking 800 mg or higher agent for superficial infections. due to its Econazole. ketoconazole has been Other frequently used topical imidazoles include econa. The dioxolane series is based on a 1. They occur in Bifonazole is a halogen-free biphenylphenyl methane 10% of the patients receiving a 400 mg dose and in derivative. However. However. isoconazole. Therapeutic failure cation of tioconazole is effective in the management of with ketoconazole has been associated with low serum vulvovaginal candidiasis and as a nail lacquer for fungal levels. Peak plasma concentrations of tic infections. and vomiting (Table 2). 400. sub- stitutions are made at the nonsymmetrical carbon atom Miconazole attached to one nitrogen molecule of the imidazole ring. an administration. and the high rate of relapses. At least dermatophytes) and itraconazole. However. Bifonazole is retained in the concomitant drugs (Table 3). dermatophy. they may slow down the spread of this infection. Enilconazole chomycosis and other infections caused by the This is the azole most widely used in veterinary practice dermatophytes. after systemic highly dependent on the pH of the gastric contents. fungal infections. Ketoconazole requires a variety of fungi (yeasts and molds). and nasal aspergillosis (infused through approximately 2. three series of such compounds have emerged for clinical and agricultural use. and potential uses. Ketoconazole also has been used for a variety of vaginal burning has been associated with intravaginal systemic and superficial fungal infections in cats and dogs. anorexia. efficacy data are not available (veterinary use). 8. and vaginal cream) patients with gastric achlorhydria or treated with antacids for superficial mycoses (nail. oxiconazole. safety and activity. and tioconazole (6.3-dioxolane for dermatophytic infections in humans and other large molecule rather than on the 1-phenethyl molecule. the recommended drugs In this group. and aspergillosis in raptors. Although topical agents do not cure for the intranasal treatment of aspergillosis and . This drug should be caused by the dermatophytes and M. therapy. dermis for a longer time than clotrimazole. In vitro. and saliva. 10-mg oral Ketoconazole pharmocokinetics corresponds to a dual troche). depending on the dose. However. Another limiting factor of ketoconazole therapy is its of activity.74 Antifungal Agents N-Substituted (Mono) Imidazoles onychomycosis as oral drugs do. Clinically is only used as a topical agent cule. It has good series (Figure 1(d)) to be introduced into clinical use and in vitro activity at very low concentrations against a large was the first orally active azole. and skin infections) or H2-receptor antagonists (Table 3). the imidazole ring is intact and substitutions for the treatment of onychomycosis are terbinafine (by are made at one of the two nitrogen molecules. for example. a single appli. monitoring of these levels is recommended in such onychomycosis (nail infections). This drug highly Other intravaginal drugs require 3 to 7-day applications. specific level of antifungal birds.5% vaginal ficial Candida and dermatophyte infections when the latter ointment) (Table 1). Its bioavailability is enzymatic inactivation of this compound. Its limited use is the result of its toxic side numerous and significant adverse interactions with other effects for mammalian cells. CSF. solution. urine. fungal keratitis and pneumonia in horses. scalp. ketoconazole has a broad spectrum of activity compared to that of other azoles. animals. despite its broad spectrum doses. lotion. In noncancer zole (1% cream). tincture. routes of administration. for initial taken with either orange juice or a carbonated beverage. failures. Mild to moderate vulvo. in (1% cream. after corresponding oral doses of 200. Clotrimazole Ketoconazole Clotrimazole is the first member of the triphenylmethane Ketoconazole was the first representative of the dioxolane series of clinical importance (Figure 2(c)). and 20 mg ml1 are measured 1–4 h tubes) in dogs. and/or mild oropharyngeal candidiasis (OPC. and for the intravaginal therapy (single application model with an initial half-life of 1–4 h and a terminal of 500-mg intravaginal tablet) of vulvovaginal candidiasis. furfur. are refractory to griseofulvin therapy. has limited its use to topical applications increase in pH will decrease its absorption. binds to plasma proteins and penetrates poorly into the This drug is also used for candidal stomatitis. adverse side effects. hepatic normal intragastric pH for absorption. isoconazole (1% cream). half-life of 6–10 h. the substitutions are made at a be administered intravenously for the therapy of systemic phenethyl configuration attached to the nitrogen mole. this drug can be effective in the treatment of super- (1% cream and lotion). and 800 mg. In the triphenylmethane series. The most common and dose-dependent adverse effects of Bifonazole nausea.

sulfonylurea Induces potential toxicity of co-compound K Saquinavir. vincristine. mucosal candidiasis (oral. C Rifampin. Although the recom- that is partially water soluble. After multiple doses. benzodiazepines. which have a wider food or gastric pH. triticonazole. are usually measured in healthy subjects after single doses dimefon. of all ages. V Cyclosporine A Nephrotoxicity C. Reduces azole or C plasma concentrations and phenyton K. and esopha- improved resistance to metabolic degradation. the peak plasma as systemic cereal fungicides. rifampin Induces the potential toxicity levels of co-compounds K. Fluconazole is the current drug of choice for Several triazoles are currently licensed for antifungal maintenance therapy of AIDS-associated cryptococcal and systemic therapy and other triazoles are at different levels coccidioidal meningitis. methylprednisone Induces potential toxicity of these compounds K Protein-binding drugs Increases the release of fractions of free drug K. cimetidine Reduces P exposure P Cyclosporine A.) are required for . Other beneficial effects of this substitution are (1) an patients. I.9% in both healthy human subjects and laboratory animals. quinidine. and its absorption is not affected by important agricultural fungicides. I Lovastin. Advances in Pharmacology 44: 345–500 for more detailed information. V. terfenadine. C. Plasma concentrations of 2–7 mg ml1 spectrum of activity than that of the earlier triazoles. phenytoin. and (3) a superior antifungal activity. fluconazole. V Phenytoin. P. formulations of to fungal cell cytochromes than to mammalian cells due fluconazole are available for the treatment of candidemia to the substitution of the imidazole ring by the triazole in nonneutropenic and other nonimmunosuppressed ring. tacrolimus. I. The penetrates well into the CSF and parenchyma of the brain side effects are few. sucralfate. dexamethasone C can be reduced (70 mg of C should be considered) V Omeprazole Reduces omeprazole dose to one half P Phenytoin. carbamazepine. C. fluconazole prophylaxis should be reserved for HIV-infected individuals or AIDS Fluconazole patients. and chronic mucocutaneous candidiasis in patients increased potency. on the route of administration and the drug formulation. I. Fl. nevirapine. The Triazoles Fluconazole does not have in vitro or in vivo activity The triazoles are characterized by a more specific binding against most molds. minimally protein bound. Both oral and i. development of levels are 2. itraconazole. Fl. However. fluquinconazole. posaconazole. and ritonavir compounds Fl. and triticonazole are ity exceeds 90%).d. and Walsh TJ (1998) Clinical pharmacology of systematic antifungal agents: A comprehensive review of agents in clinical use. and putative targets for antifungal drug development. methylprednisolone. voriconazole. and propiconazole. ketoconazole. prochloraz. I Antacids. tacrolimus Increases concomitant drug exposure P Midazolam P Rifabutin Two-way interactions Reproduced from Groll AH. CSF to serum fluconazole concentrations are between 0. Antifungal Agents 75 Table 3 Adverse interactions of the licensed systemic azoles with other drugs during concomitant therapy Azole Concomitant drug Adverse side effect of interaction K. fluquinconazole. (2) an geal). current investigational compounds. I. However. mended dosage of fluconazole for adults is 100–400 mg and excreted largely as an active drug in the urine.d. penicillosis as well as for dermatophytic infections. vaginal. tria. and prochloraz Fluconazole is well absorbed orally (its total bioavailabil- Epoxiconazole. astemizole Fetal arrhythmia K. drug can develop during therapy. isoniazid. It each day (q. V Tacrolimus Tacrolimus can be decreased C Efavirenz. of 100 and 400 mg. rifabutin. Induces potential toxicity co-comgoxin. simvastatin Rhabdomyolysis l Indinavir. cisapride. phenobarbital. chlordiazepoxide. caspofungin. omeprazole. Its pharmacokinetics are linear and independent Epoxiconazole. V. Fl.v. I Nonsedating antihistamines. or for patients with prolonged (2 weeks) and Fluconazole is a relatively small molecule (Figure 1(e)) profound neutropenia (500 cells). Fl. K. since resistance to this of clinical evaluation (Table 1). rifabutin. The resistance to these compounds has been documented. dicyclosporine. and the imidazole. didanosine Reduces azole absorption K.5 times higher than those of single doses. and it has a prolonged half-life (up to 25 h in humans). FL.5 and 0. and the eye.) higher doses (800 mg q. Piscitelli SC. V Warfarin. H2 antagonists.

itraconazole is insoluble in aqueous fluids.o.v. and these effects are usually observed when the patient tions caused by a Candida spp.8 mg ml1. In contrast to the imidazoles and itraconazole. Posaconazole concentrations is recommended during treatment of both exhibits dose-proportional absorption up to 800 mg. itraconazole is soluble only at low pH and is better and an increased potency against most fungi compared to absorbed when the patient is not fasting. Voriconazole (UK-109496) Fluconazole interactions with other concomitant drugs are Voroconazole is a triazole related to fluconazole obtained similar to those reported with other azoles. minimal. The oral solution is better side chain of SCH 51048. distributes widely (Figure 1(f )). fluconazole. In contrast to lation.i. dine and -methylation groups (Figure 3(a)).d.v. itraconazole has been used for the treatment of conazole resistance among Candida krusei (intrinsically aspergillosis. higher doses are recommended and Posaconazole is the product of a modification of the n-alkyl clinical resistance may emerge. itraconazole are commercially available for and multiple doses resulted in a higher (8 times) accumu- the treatment of certain systemic mycoses. are usually obtained after 200-mg dosages aspegillosis. However. and skin rash (1–5%. or anta. is extensively penetrates poorly into the CSF and urine but well into metabolized in the liver (80%). it Voriconazole binds to proteins (65%). and osteomyelitis (caused fluconazole use for the treatment of such infections is by Coccidiodis immitis in large animals). Plasma peak (1. equine sporotrichosis. omeprazole. in vitro and in vivo antifungal activity. Posaconazole has a pro- able clinical response. for more days. muciloginosa. certain isolates of Candida rugosa.)) or after i. The mean half-life of elimination is about 6 h.4 and 1. that exhibit a minimum takes up to 400 mg during several periods of time.i. Voriconazole pharmacokinetics in humans are nonlinear and dosedependent.8–16%). it is nazole (Table 3). non-life-threatening mycoses including unresponsive cases to other azoles.) caused by S. Posaconazole absorption from treatment of HIV-associated oral and esophageal candi. adverse side effects are more frequent (Table 2). see Table 2). Rhodotorula strains. cids. blastomycosis (especially in resistant) and Candida glabrata (15% resistant) isolates. It does not have activity against concomitant H2-receptor antagonists. Due to flu. unchanged in the feces (66%). dependent. these concentrations are also generally well tolerated with only few side effects. As do the other azoles. Its structure is closely related to that of ketoconazole Voriconazole has good oral bioavility. Clinically. hepatic (10–15%). Following single oral Itraconazole doses. especially R. Treatment with itraconazole has longed elimination half-life (20–66 h). this drug should be taken with food and/or Sporothrix schenckii and a significant number of acidic fluids. In inhibitory concentration (MIC) of 8 mg ml1. Therefore. but its use is precluded. has been approved for the primary treatment of invasive respectively. vor- aspergillosis and penicillosis in small animals and birds iconazole acts by inhibiting fungal cytochrome P450- when topical enilconazole is not feasible. for obtained in cancer patients receiving 5 mg kg 1 divided example. divid- superficial and invasive diseases: Drug concentration ing the dose and food or liquid supplements increase 0. apiospermum and Fusarium spp. animals. monitoring of itraconazole plasma are achieved 11–24 h after the actual dose. Fluconazole has been used to treat nasal insoluble in aqueous fluids. the intestinal tract is slow and peak serum concentrations diasis.d. It is fungicidal against some fungi. Similar to ketocona.. the Zygomycetes. and it is highly protein bound (90%). which included a variety of chiral absorbed than the tablet and has become useful for the substituents (Figure 3(b)). peak plasma concentrations were achieved after 2 h Oral and i. The in vitro fungistatic and . is highly protein bound (>98%). No data are available regarding its side effects fluconazole does not exhibit major toxicity side effects or drug interactions in animals.5–4 h) and trough concentra. when the dosage is increased above 1200 mg. ketoconazole as therapy for endemic. It has a large volume distribution (1774 l) and it and 2 mg ml1 by bioassay appear to be critical for favor. but itraconazole has a broader spectrum of into tissues including those of the central nervous system. For reported drug Clinically. it tions between 1 and 2. candidemia or twice daily (b. 14--sterol demethylasemediated synthesis of ergosterol. The drug is 2 days and q. However. dogs). transient visual (10–15%) into two oral solution dosages. For Posaconazole (SCH 56592) more severe mycoses. salvage therapy for other mould infections (capsule) as either single daily dosages (per orally (p. administration (b. itraconazole (200–400 mg/day) supplanted interactions see Table 3. it is not extensively been associated with less adverse and mostly transient metabolized (approximately 14%) and is eliminated side effects (10%) than that with ketoconazole (Table 2). but they are less by replacement of one triazole moiety by fluoropyrimi- frequent than those exhibited by ketoconazole and itraco.76 Antifungal Agents the treatment of severe invasive infections and for infec. erratic in cancer patients or when the patient is taking especially Aspergillus spp. (78–88%) practically unchanged after a single dose.d.5 mg ml1 by high performance liquid chromatography exposure.2 mg ml1 and 0. Voriconazole has an improved in vitro fungistatic activity zole. crytococcosis. Absorption is those of fluconazole.) for and other infections caused by Candida spp. and is found in the urine skin and soft tissues. (2.

dermatophytes. It also has been approved for the treatment of OPC. nausea. altered drug level. . ER-30346) versus-host disease (GVHD) or those with hematologic BMS-207147 is a novel oral thiazole-containing triazole malignancies with prolonged neutropenia from chemother- (Figure 3(c)) with a broad spectrum of activity against the apy. fungicidal activities of posaconazole are similar to those of Investigational Triazoles voriconazole and amphotericin B against yeasts. and crypto- have occurred in 8% of patients (Table 2). with the Posaconazole is generally well tolerated and serious exceptions of Candida tropicalis and C. of the agents against most pathogenic yeasts. it is only used for fluconazole. II–III trials). most opportunistic molds including the and are at earlier stages of development (Table 1). and vomiting) treatment of invasive aspergillosis. (b) posaconazole. ER-30346).8% vaginal creams to be determined in clinical trials in humans (phases and 80 mg vaginal suppositories).4 h) and similar to that of phyte infections. and SDZ 89-485 Oral posaconazole has been approved for prophylaxis of were discontinued from further development due a vari- invasive Aspergillus and Candida infections in patients at high ety of adverse side effects. BAY R 8783. certain phaeoid fungi. Ravuconazole Inducers of the UDP glucuronidation pathway may has a similar or superior in vitro activity compared to those affect posaconazole plasma concentrations (Table 3). than that of itraconazole (1. BAY 3783. Ravuconazole shows good pharmacokinetics in animals that is similar to that of itraconazole. glabrata. The potential use of ravuconazole has yet vulvovaginal candidiasis (0. This indicates Terconazole that it is absorbed at levels comparable to those of itraco- Terconazole was the first triazole marketed for the topical nazole. risk due to being severely immunocompromised hemato- poietic stem cell transplant (HSCT) recipients with graft- Ravuconazole (BMS-207147. and (c) ravuconazole (BMS-207147. SCH 51048. and the Triazoles such as saperconazole (R 66905). rash. SCH 39304. the Other triazoles are currently under clinical investigation dimorphic fungi. It also has adverse effects (most common. candidiasis. coccosis. Antifungal Agents 77 Figure 3 Chemical structures of three triazoles: (a) voriconazole. the half-life ravuconazole (4 h) is longer treatment of vaginal candidiasis and superficial dermato.4 and 0. Currently. However. majority of opportunistic pathogenic fungi. good in vivo antifungal activity in murine models for the increased hepatic enzymes. Zygomycetes.

The benzylamine. tolciclate. UR-9751. bamate fungicides in the mid-1930s. The . UR-9751.2 h in mice to 9. SSY 726. and D 0870 The allylamines act by inhibiting squalene epoxidase. Ferbam. Animal studies suggest that the absorption of this com- pound is almost complete after p.5–2. which results in a decrease in the ergosterol content and continued by its original developers. and other molds. respectively. The in vitro activity an accumulation of squalene..9 h in dogs. nazole for high-dose therapy. dine and increased by rifampin. increased spray intervals to 14 days or more. maneb. also inhibit the synthesis of ergosterol at the level of squalene.78 Antifungal Agents D 0870 The Allylamines Although more in vitro and in vivo studies were con- ducted with D 0870 than with SDZ-89-485. showed good antifungal activity. and albaconazole (UR-9825) ficial dermatophytic infections. It is usually well animals following the administration of single oral doses tolerated at oral doses of 250 and 500 mg per day and the of 10 mg kg1 and the drug was detected in the animals side effects (10%) are gastrointestinal and cutaneous. T-8581 has shown potent in vitro antifungal activity against Candida spp. Candida spp. but its in vitro activity against the yeasts is controversial. The halflife of T-8581 varies in the differ. Thiocarbamates. Methylbenzimidazole Carbamates A great impact on crop protection was evident with the TAK 187. cruzi. Aspergillus spp.. but because activity) of 184 (UR-9746) and 34 mg ml1 (UR-9751) they are only surface-acting materials frequent spray after 8 and 8–24 h. The only fungicide used until the discovery of the dithiocar- pharmacokinetics of these compounds in laboratory ani. and piritetrade. and zineb are has been demonstrated with these compounds against not used as much.14–12 mg ml1) tophytes (oral and topical).7 mg ml1 are detected 1 or 2 h after a single oral dose.. It has this class of antifungals. drug concentrations of 0. this drug was also dis. neoformans. The safety of T-8581 is under evaluation. butenafine. Albaconazole is currently The Benzimidazoles and in phases I–II trials. Their clinical use is limited to the topical treatment of super- UR-9746. These compounds these new triazoles. for the treatment of murine systemic candidiasis and and Dithiocarbamates superior to itraconazole for aspergillosis in rabbits. Terbinafine and Naftifine Therefore. but higher for the common Candida spp. which suggests the Pharmacokinetics and poor activity have limited the use of potential use of this compound as an alternative to fluco. of D 0870 is lower than that of itraconazole against Aspergillus spp. The maximum solubility of T-8581 is superior (41. It follows linear pharmacokinetics over a dose range of 125–750 mg. C. Terbinafine has T-8581 replaced griseofulvin and ketoconazole for the treatment T-8581 is a water-soluble 2-fluorobutanamide triazole of onychomycosis and other infections caused by derma- derivative. and albaconazole are similar fluori.6 mg ml1). High peak concentrations (7. and Aspergillus fumiga- tus. The Bordeaux mixture UR-9746. dosages. evaluation of this compound has been con- tinued by another pharmaceutical company for the Terbinafine (Figure 2(d)) is the most active derivative of treatment of OPC in HIV-infected individuals. and the thiocarbamates. tolnaftate. It has an excellent in vitro activity also shown activity against the parasite Trypanosoma against the dermatophytes and other filamentous fungi. Of those.o. naftifine to topical treatment of dermatophytic infections. It is also effective for the of T-8581 were determined in the sera of laboratory treatment of vulvovaginal candidiasis.8 mg ml1) Naftifine to that of fluconazole (2. The activity of T-8581 is similar to that of fluconazole The Benzylamines. The sera after 24 h. In vitro and in vivo activity applications are required. metabolism of terbinafine may be decreased by cimeti- ent animal models from 3. neoformans. These antifungals lacked detectable toxicity in experimental animal infections.. C. mancozeb mals has demonstrated peak concentrations (biological and thiram are widely used in agriculture. (reaction product of copper sulfate and lime) was the dated triazoles that contain an N-morpholine ring. and KP-103 introduction of the benzimidazoles and other systemic Some in vitro and very little in vivo data are available for (penetrate the plant) fungicides.

The pneumocandins are fermentation products of the mold Zalerion arboricola. is the only with a -hydroxyglutamine instead of the threonine resi- morpholine that has a clinical application for the topical due.8 echinocandins have better in vitro and in vivo antifungal isomerase enzymes in the ergosterol biosynthetic path. The terminal elimination half-life is about (echinocandin B). cinerea and Penicillium expansum have but poor in vivo activity. LY303366. but they possess a hexapeptide core Amorolfine. activity than the papulocandins. Of the three naturally occurring pneumocandins (A–C). an essential dose proportional. since MBC- tiacandin have in vitro activity only against Candida spp. vWF 11899 A–C. this drug was discontinued owing to the The Pyridines incidence of metabolic acidosis associated with its i. Antifungal Agents 79 methylbenzimidazole carbamates (MBCs. tion with N-phenylcarbamate or agents that have a different mode of action. aculeatus (aculeacin A). and Pneumocystis carinii (in rodents). resistant strains of B. and thiophanate) inhibit nuclear division and are The papulocandins A–D. The The morpholines interfere with 14 reductase and 7. carrier. echinulate of 30–50 l. bound in humans (84%). a derivative of fenpropimorph. which leads to an increase in toxic sterols and an ment has resulted in several semisynthetic echinocandins increase in the ergosterol content of the fungal cell.5–2. They act specifically by tions are usually higher than those in plasma in animals. and Other B have certain antifungal activity in vitro and in vivo Morpholines against Candida spp.v. Pharmaceutical develop- way.9 mg ml1 have been measured after single doses of 50–250 mg kg1. The pneumocandins have similar structures to those of Amorolfine the echinocandins. mulundo. doses) are which results in the depletion of glucan. Cilofungin is a biosemisynthetic analog of the naturally occur- ring and toxic (erythrocyteslysis) 4-n-octyloxybenzoyl- echinocandin B. peak levels in plasma (5 or 6 h) of 0. Anidulafungin (VER-002. carbendazim. It has high potency and oral and parental bioavailability. Anidulafungin is moderately protein component of the fungal cell wall. sporiofungin. inhibiting the synthesis of fungal (1. and chae- also systemic agricultural fungicides. but they are nonwater-soluble. and FR 901379. pneumocan- The Morpholines dins. Although it showed good in vitro activity against Candida spp. a branched-chain 14C fatty acid acyl group at the treatment of dermatophytic infections and candidal N-terminus. In The Echinocandins. The Echinocandins and Pneumocandins The echinocandins include echinocandins. fungin sidechain (Figure 4(a)).5–1 h) and a volume distribution occuring metabolites of Aspergillus nidulans var. The Papulocandins benomyl. V-echinocandin) This is another semisynthetic cyclic lipopeptide which Buthiobate and Pyrifenox resulted from an increase of aromatic groups in the cilo- These agents are important agricultural fungicides. A. Pneumocandins.v. Systemic exposures of anidulafungin (post i.3)-glucan synthetase.. but they are important Cilofungin (LY121019) agricultural fungicides. In laboratory animals. and Papularia 40–50 h and its clearance about 1 l/hr. which precluded clinical been isolated. The pyridines are another class of antifungal agents that inhibit lanosterol demethylase. only A and Fenpropimorph. However. L687781. aculeacins. Its phar- macokinetics are linear and is characterized by a short The echinocandins and papulocandins are naturally distribution half-life (0. . Tissue concentra- sphaerosperma (papulocandin). peak levels of 105–1624 ng ml1 are measured and Papulocandins after oral administrations of 100–1000 mg kg1. polyethylene glycol. Tridemorph.and deoxymulundocandin. BU4794F. these compounds should be used in combina- development. Protein binding and side effects have precluded the clin- ical use of these morpholines. proline residue. humans. and variable substituents at the C-terminal vaginitis. with an improved antifungal activity compared to those of the naturally occurring molecules described previously.

80 Antifungal Agents Figure 4 Chemical structures of anidulafungin (VER-002. L693989. Anidulafungin has good in vitro activity against a variety 100 mg daily dose) and other candidal infections (100 mg of yeasts. its MICs for certain observed. neoformans and T. fluconazole. but its fungicidal have been observed with drugs likely to be coadministered activity against some species of Candida is superior to those with anidulafungin. including isolates resistant to itraconazole and loading dose on first day followed by 50 mg daily dose). caspofungin (L 743872 or MK-0991). agents. and molds. V-echinocandin LY303366). and L731373 nia. L733560. Anidulafungin has shown oral efficacy in animal models of systemic candidiasis and pneumocystis pneumo. This compound is not active Laboratory abnormalities in liver functions have been against C. L705589. but no clinically relevant drug–drug interactions molds are higher than those of the azoles. and nikkomycin Z. Anidulafungin has been approved for the treatment of Modification of the original pneumocandin B by phos- candidemia (200 mg loading dose on first day followed by phorylation of the free phenolic hydroxyl group led to the . beigelii.

which results in disruption of the plasma led to the water-soluble semisynthetic molecules membrane and leakage. In humans. Although it was selected for available. Caspofungin is conducted. formulations are derivative of BMY 28864. albicans and C. Further modifications of pneumocandin B teins. Blastomyces bolites of the Actinomycetes. activity in animals against P.5 h and drug concentrations are usually higher BMS 181184 in tissue than in plasma. neoformans. highly protein bound (97%) with a half-life that ranges from 5 to 7. the drug is mostly well tolerated. caspofun. The former com- Micafungin is a semisynthetic derivative of a naturally pounds were discovered during a search for new occurring lipoprotein that was synthesized by a chemical agricultural fungicides and pesticides. carinii. it has in vitro activity the enzyme chitin synthase. beigelii. The Polyoxins and Nikkomycins penic patients unresponsive to antibacterial therapy. L733560. but is inactive of chitin in the fungal cell wall. Its great advantage release. As the other echinocandins. it was not effective in the treatment of sys- HSCT. in vitro and in vivo activity against C. and compared to other new antifungals is its good in vivo rash have been reported (Table 2). tematic candidiasis in mice. fumigatus. immitis (parasitic phase). The transported into the cell via peptide permeases. and L731373. and Histoplasma capsulatum as well as in vitro molecules have also been produced.v. nausea. fever. drug is well tolerated (Table 2). gin (50 mg) has been approved for the treatment of systemic candidiasis and other Candida infections. They act by disrupt. These molecules are against C. fungi. and phylaxis of Candida infections in patients undergoing C. elevation of liver transaminases in humans Candida spp. Both polyoxins modification from a product of the mould Coleophora and nikkomycins are pyrimidine nucleosides that inhibit impedir. caspofungin exhibits favorable dosedependent This compound is either a semisynthetic or biosynthetic linear pharmacokinetics and only i. but several semi-synthetic dermatitidis. activity against C. Clinical trials in humans have not been other semisynthetic pneumocandins. The poor solubility of pradimicin A led to the develop- Caspofungin (MK-0991 or L743872) ment of BMY 28864. albicans. C. and lower activity against the dimorphic led to the discontinuation of this drug. As for anidulafungin and mica- fungin. Antifungal Agents 81 improved. It is protein binding (99%) and plasma concentrations attain a steady state by Polyoxin D day 4 with repeated doses. which leads to the depletion against Candida and Aspergillus species. against C. vomiting. of pradimicin FA-2. Caspofungin has fungistatic and fungicidal further clinical evaluation due its promising in vitro and activities similar to those of anidulafungin against most in vivo data. L705589. refrac- tory invasive aspergillosis or for patients intolerant to other agents and for empiric therapy for febrile neutro. water-soluble pneumocandin B phosphate binding with the saccharide component of mannopro- (L693989). BMY 28864 appears to have good tion of L733560 and was selected for evaluation in clinical in vitro and in vivo activity against most common yeasts trials in humans. immitis. Caspofungin is water soluble. which is a water-soluble derivative Caspofungin (Figure 4(b)) is the product of a modifica. The drug is Benanomycin A not effective for the treatment of disseminated experi- mental infections caused by C. especially Aspergillus spp. It also has fungistatic in vitro activity against some of the other molds. T. As are the and A. neoformas. Nikkomycin Z The Pradimicins and Benanomycins This compound (Figure 4(c)) appears to have both The pradimicins and benanomycins are fungicidal meta. In laboratory This compound has shown the best antifungal activity animals. neoformans. but histamine among the various benanomycins. Micafungin has recently been approved for the treatment of patients with esophageal Although this compound has in vitro antifungal activity invasive (including candidemia) candidiasis and the pro. The polyoxins are produced by Streptomyces cacao and the Micafungin (FK 463) nikkomycins by Streptomyces tendae. and Fusarium spp. these molecules were not Pradimicin A (BMY 28567) and FA-2 (BMY 28864) evaluated in humans. . Clinical trials ing the cell membrane through a calcium-dependent are pending. mild hepatotoxicity. Although studies were conduced in laboratory animals. neoformans.

Clinical trials are pending. Espinel-Ingroff A (1998) Comparison of in vitro activity of the new triazole Natural and Synthetic Cationic Peptides SCH 56592 and the echinocandins MK-0991 (L-743. Clinical Microbiology Reviews 9: 235–272. but it causes skin sensitization in horses. it is not cross-resistant to acids. Clinical Microbiology Reviews fungal activity and when incorporated into liposomes has 12: 40–79.82 Antifungal Agents The Sordarins Synthetic peptides Synthetic peptides have been derived from the natural The natural sordarin GR 135402 is an antifungal fermenta. New York: Indolicidin Cambridge University Press. phenylamides (systemic controllers of Phycomycetes plant infections). It is a Candida isoleucyl-tRNA synthase inhibitor that is currently in phase II trials. Clemons KV and Stevens DA (1997) Efficacies of two novel azole derivatives each containing a morpholine ring. In vitro. C. Milling RJ. GM 222712. Antimicrobial Agents and Chemotherapy 41: 200–203. by the application of specific fungicides. (eds. and Sibley CM (1999) Current and emerging azole antifungal agents. Manual of Clinical Microbiology. The compounds to have in vitro activity against C. cinera as an uncoupler of oxidative phosphorylation. albicans. neoformans. Captan is also Arikan S and Rex JH (2007) Antifungal agents. and Wright K (1995) Control of fungi pathogenic to plants. GM 193663. Cecropin Groll AH. and GM 237354 and A. . Piscitelli SC. Advances in Pharmacology activity varies according to the fungal species being 44: 343–500. European Journal of Clinical Microbiology & Infectious Diseases 8: 352–361. tle. Cationic peptides provide a novel approach to antifungal Journal of Clinical Microbiology 36: 2950–2956. The Phthalimides Further Reading The discovery of captan in 1952 and later of the related Allen DG. Its antifungal for antifungal drug development. Russell PE. Pringle JK. challenged. and Russell NJ (eds. Espinel-Ingroff A and Shadomy S (1989) In vitro and in vivo evaluation of antifungal agents. and cispentacin are amino acid analogs with good in vitro antifungal activity against Dimethomorph and Fluazinam Aspergillus spp. DC: ASM Press.872) and LY303366 against opportunistic filamentous and dimorphic fungi. Darby GK. fumigatus and also show synergistic activity with are synthetic derivatives of GR 135402. Amino Acid Analogs RI 331. current investigational compounds. therapy that warrants further investigation. genase and the biosynthesis of sulfur-containing amino tans on tomatoes and potatoes. Espinel-Ingroff A (1996) History of medical mycology in the United Other Antifungal Approaches States. 222712 and GM 237354 have shown broad-spectrum anti- fungal activity for a variety of yeasts and molds. GM 211676. Washington. et al. They appear tion product of Graphium putredinis.) Fifty Years of Antimicrobials: Past Perspectives and Future Trends. UR-9746 and UR-9751 against systemic murine coccidioidomycosis. the azoxybacillins. and Walsh TJ (1998) Clinical pharmacology of Cecropin is a natural lytic peptide that is not lethal to systemic antifungal agents: A comprehensive review of agents in clinical use. Indolicidin is a tridecapeptide that has good in vitro anti. GM fluconazole in vitro. In: Murray PR. Sheehan DJ. and Burgmann PM (1993) captafol and folpet initiated the proper protection of crops Handbook of Veterinary Drugs. respectively. Hitchcock CA. RI Dimethomorph is a cinnamic acid derivative for use 331 and the azoxybacillins inhibit homoserine dehydro- against Plasmopara viticola on vines and Phytophthora infes. Fluazinam is used in vines and potatoes but Icofungipen (BAY 10-8888 and PLD-118) also acts against B. and putative targets mammalian cells and binds to ergosterol. bactericidal-permeability increasing factor. Philadelphia: Lippincott. 9th ed.) used to treat dermatophytic infections in horses and cat. and the dermatophytes (RI 331 and azox- ybacillins) and also good in vivo activity (cispentacin). In: Hunter PA. activity against experimental aspergillosis in animals. Smith DA. Conlon PD.

which catalyzes the cleavage of a sugar creatinine An end product of energy metabolism found derivative called neuraminic acid. (buffalo hump). The placebo is an agent without the specific effect. placebo An agent used as a ‘control’ in tests of EC50 Concentration of a drug that produces a 50% drugs. the degradation of the hemoglobin from degraded red maintenance therapy Drug treatment given for a long blood cells. for example. for example. in the blood in uniform concentration. but neither sites. observed in some patients receiving antiviral agents for bilirubin A greenish compound formed in the liver from HIV treatment. the concentration of which is activity. one virus can bind to two red cells causing them to group knows which agent it is receiving (the so-called clump (agglutinate). and distributed within the body in a way that preserves monotherapy Treatment with a single drug. Yale University School of Medicine. facial wasting. or made insoluble. lipoatrophy Redistribution/accumulation of body fat antiretroviral agent Any drug used in treating patients including central obesity. 83 . a substitution of valine for is tested on a broad range. which have the property of drug are due to psychological effects or binding to the surface of the red blood cells of some expectations. CT. It is usually given to some patients animal species. chemoprophylaxis Preventive treatment with chemical mutations Changes to the base pair sequence of the agents such as drugs. All rights reserved. which is excreted peptidomimetic A molecule having properties similar by the kidney at a constant rate. in which it target enzyme. drug resistance Decreased susceptibility to antiviral phase III The final stage in testing of a new drug. genetic material of an organism. ‘blind’ design). which designates a specific amino neuraminidase An enzyme. Antiviral Agents E Paintsil and Yung-Chi Cheng. inactivated. components in the linear genetic code in DNA or nephrotoxicity Kidney toxicity. and large population of methionine at residue 184 of the reverse transcriptase patients for comparison to existing treatments and to enzyme of HIV confers resistance to lamivudine (M184V test for rare complications. pharmacokinetic Refers to the rates and efficiency of cytokine One of a variety of proteins which has a uptake. some viruses. in virus yield. messenger RNA. Because there are multiple binding while the test drug is given to others. it is not broken with combination therapies with more than one drug at down. peripheral wasting. Defining Statement Therapeutics for Papillomavirus Introduction Therapeutics for Enteroviral Infections Therapeutics for Herpesvirus Infections Anti-HIV Agents Therapeutics for Respiratory Virus Infections Further Reading Therapeutics for Hepatitis Glossary hepatotoxicity Liver toxicity. time to maintain its effect after the condition has been bioavailability The property of a drug to be absorbed controlled or to prevent recurrence. New Haven. after usually due to changes in the amino acid residues of determination of its safety and effectiveness. body. often elevated in cases of liver damage. mutation. effects of the drug under test and is used to hemagglutinin Specific glycoprotein molecules on the determine to what extent any observed effects of the surface of some viruses. codon A triplet of three consecutive nucleotide nephrolithiasis The presence of kidney stones. Alterations of this rate to those of a peptide or short protein. distribution. For example. the same time. dorsocervical fat enlargement with human immunodeficiency virus (HIV) infection. and ‘cushingoid appearance’ virus to a specific drug. USA ª 2009 Elsevier Inc. contrasted its useful characteristics. are considered an indication of kidney malfunction. and disposition of a drug in the regulatory effect on a cell. present on the surface of acid in the linear sequence of a protein molecule. antiviral resistance The developed resistance of a breast enlargement. alanine aminotransferase An enzyme found in the interferon Any group of glycoproteins with antiviral liver and blood serum. see Table 1 for letter codes of amino acids).

of DNA. hence ‘shingles’) distribution on the trunk. An of viral infections. acid core. resulting from virion A complete virus. usually in a girdle (L. therapeutic index The numerical ratio of the protease An enzyme that catalyzes the cleavage of concentration needed to achieve a desired effect in proteins. T1/2 The time for reduction of some observed quantity. Available . prophylaxis Prevention. Abbreviations HPMPC (S)-1-(3-hydroxy-2-phosphonylmethoxypro- ALT alanine aminotransferase pyl) cytosine CMV cytomegalovirus HPV human papillomavirus CNS central nervous system disease HSE herpes simplex encephalitis CoV coronavirus HSV herpes simplex virus CPK creatinine phosphokinase IFNs interferons CSF cerebrospinal fluid MNR multinucleoside resistance CYP cytochrome P450 NA neuraminidase dATP deoxyadenosine triphosphate NAMs nucleoside-analog-associated mutations dGTP deoxyguanosine triphosphate NK natural killer EBV Epstein–Barr virus PEG polyethylene glycol FDA Food and Drug Administration Pgp P-glycoprotein FEV1 reduced forced expiratory volume RSV respiratory syncytial virus HAART highly active antiretroviral therapy SARS severe acute respiratory syndrome HBeAg hepatitis B e antigen SEM skin. They target stages in the viral life cycle. eye. however. their final. most of host cell that any antiviral drug that interferes even to a the available antiviral agents are effective against only lesser extent with host cell factors may be toxic to the host replicating viruses. a building block goes through to multiply.84 Antiviral Agents prodrug A drug that is given in a form that is inactive teratogenesis Production of fetal abnormalities by and must be metabolized in the body to the active form. active form. including the coat and nucleic infection with varicella zoster virus. depending on the duration and dosage used. thymidine kinase An enzyme that catalyzes the protease inhibitor A substance that inhibits the action transfer of a phosphoryl group from a donor such as of protease enzyme. The development of antiviral agents is ideal antiviral agent should be effective against both not trivial as viral replication is intricately linked with the actively replicating and latent viruses. a virus-specific protease is 50% of the patients and the concentration that needed to cleave some of the virus coat proteins into produces unacceptable toxicity in 50% of the patients. adenosine triphosphate to the sugar (deoxyribose) replication cycle The series of steps that a virus or cell component of the thymidine molecule. viremia The presence of virus in the bloodstream. or mouth HBV hepatitis B virus SPAG small-particle aerosol generator HCV hepatitis C virus TAMs thymidine analog resistance mutations HHV-6 human herpes virus-6 TK thymidine kinases HHV-7 human herpes virus-7 UGT UDP-glucuronosyl transferase HHV-8 human herpes virus-8 VZV varicella zoster virus HIV human immunodeficiency virus Defining Statement Introduction Antiviral agents are drugs approved by Food and Drug Antiviral agents are drugs approved by the Food and Administration (FDA) for the treatment or control of viral Drug Administration (FDA) for the treatment or control infections. In the case of HIV. shingles Eruptive rash. the blood concentration of a drug. by 50%. cingulus. for example. some agent.

and free virus in body fluids. trauma. must be in acyclovir triphosphate concentrations much higher in extremely high in order for the therapy to be acceptable. family: HSV-1. Antiviral agents can be used for viral DNA polymerase. host phenotypic behaviors toward anti. and hence. Acyclovir O translation of mRNA. and antiviral virus (HCV). The goals for treating acute viral acyclovir to its monophosphate metabolite efficiently. respectively. is relatively resistant. 1. hepatitis B virus (HBV). preemptive therapy. Antiviral agents used to treat viral diseases are currently O H2N N N limited. catalyze the phophorylation to acyclovir monophosphate. This article summarizes the most relevant clovir into cellular DNA. In addition. Peak concentrations of approximately 0. viral latency viral DNA synthesis (Figure 1). as illustrated in Figure 1.04 mg ml1). In addition.5–5 h in suppression. The plasma half-life is 2–3 h in older chil- reactivate during periods of stress. Viral encoded thymidine state). both actively replicating and latent viruses. or are 39-hydroxyl group of nucleosides. The elimination . hepatitis C Chemistry. ing other viral DNA replication. budding. synthesis of viral mRNA. higher doses of acyclovir are required in the treatment of VZV infections. human activity papillomavirus (HPV).e. human herpes 15–30%.. The monophosphate is subsequently phosphorylated to decrease the rate of transmission of the virus. Epstein–Barr virus (EBV). present in only herpesvirus-infected cells. thetic acyclic purine nucleoside analogue that lacks the Viruses could stay in the cells as episomal form. and at least half of the available agents are for the OH treatment of human immunodeficiency virus (HIV) infec- tions. which lacks There are eight members of the human herpesviridae a virus-specific TK. cyto- megalovirus (CMV).50 mg ml1). varicella zoster virus (VZV). CMV. The bioavailability of oral formulations of acyclovir is CMV. EBV TK has poor efficiency to utilize Therapeutics for Herpesvirus Infections acyclovir as substrate. the incorporated acyclovir development of resistance to the antiviral agent by the can trap viral DNA polymerase and prevent it from initiat- virus. The others are used for the management of herpes simplex virus (HSV). Two important factors that can required for DNA chain elongation. mechanism of action. In vitro. however. VZV. the growing chain of limit the utility of antiviral drugs are toxicity and the DNA is terminated. Antiviral Agents 85 antiviral agents mainly target stages in the viral life cycle. and human herpes virus-8 (HHV-8). Higher establish latency within the neuronal ganglia of the ner. the goal is to prevent viral sphate inhibits viral DNA synthesis by competing with damage to visceral organs. respiratory syncytial virus (RSV). Acyclovir tripho- For chronic viral infections. infections in immunocompetent patients are to reduce Acyclovir is highly selective for cells engaged in active the severity of the illness and its complications and to viral replication and does not affect noninfected cells.57 mg ml1 are attained after multidose oral administra- The hallmark of the herpesviruses is their ability to tion of 200 or 800 mg of acyclovir. uncoating. maturation of new viral proteins. higher acyclovir concen- trations are required for EBV inhibition. and influenza virus-related Acyclovir [9-(2-hydroxyethoxymethyl) guanine] is a syn- diseases. HSV-infected than in uninfected cells. suppression. and VZV (0. replication of viral RNA and DNA. human herpes virus-6 (HHV-6). HSV-2. viral attach- ment to host cell. An ideal antiviral agent should be effective against kinases (TK). and therefore efficacy deoxyguanosine triphosphate (dGTP) as a substrate for becomes paramount. resulting apeutic index. neonates with normal creatinine clearance. acyclovir is most active pharmacologic and clinical properties of the available against HSV-1 (average EC50 ¼ 0. Acyclovir The target stages in the viral life cycle are. Acyclovir is phosphory- incorporated into host chromosomal DNA without lated to the active triphosphate metabolite that inhibits engaging in active viral replication (i. plasma acyclovir levels are achieved with intravenous vous system or cells of the immune system and administration. Since prophylaxis. (0. The ther- the di-.57 and virus-7 (HHV-7). Varicella virus is much less susceptible to acyclovir than is HSV. and triphosphate by cellular kinases. resulting in little incorporation of acy- an individual. release of N N newly synthesized virus. or immune dren and adults with normal renal function and 2. most of the available antiviral agents are effective against Host cell TK or other kinases cannot phosphorylate only replicating viruses. or ratio of efficacy to toxicity. therefore. or treat- acyclovir triphosphate lacks the 39-hydroxyl group ment of overt disease. HSV-2 antiviral agents.10 mg ml1). The viral polymerase has viral drugs because of either genomic or epigenetic a greater affinity for acyclovir triphosphate than cellular factors could limit the efficacy of an antiviral agent in DNA polymerase.

valacyclo- systemic complications. (b) inhibition of DNA synthesis and chain termination. and convenient daily) is the standard treatment. and Initial and recurrent episodes of genital HSV infection 4 days.86 Antiviral Agents (a) O O O N N N N Herpes TK N Cell kinases N H2N N N H2N N N H2N N N HO O P O P P P O Deoxynucleoside triphosphate pool (b) NH2 N Cytosine O O N P O O NH2 N N Adenine O P N N O O NH2 N N DNApol compex O P N N Adenine O O O Sugar-phosphate N backbone N Acyclovir (guanine) O N N H2N P O O Chain termination Figure 1 The mechanism of action of acyclovir: (a) activation. and time to complete Clinical indications healing (8 vs. safe. virus shedding. pain. with a half-life of approximately 20 h in persons with effective treatment for genital HSV. be suppressed with acyclovir. when initiated within 24 h of onset of can be treated with acyclovir. respectively. 14 days) but is reserved for patients with For most of the clinical indications of acyclovir. Orally administered under ‘Valacyclovir’ and in section Clinical indications acyclovir (200 mg five times daily or 400 mg three times under ‘Penciclovir and Famciclovir’. alternatives. Intravenous acyclo- end-stage renal disease. Oral therapy (200 mg five times vir and famciclovir are as effective. and recurrent episodes can symptoms. Acyclovir is minimally metabolized vir (15 mg kg1 day1 in three divided doses for 5–7 days) and approximately 85% is excreted unchanged in the urine is the most effective treatment for a first episode of genital via renal tubular secretion and glomerular filtration. daily) for 7–10 days shortens the duration of signs and Genital herpes symptoms. . 7. herpes and results in a significant reduction in the median duration of viral shedding. The clinical applications of valacyclovir and Recurrent genital herpes is less severe and resolves famciclovir are detailed in section ‘Clinical indications’ more rapidly than primary infection. of acyclovir is prolonged in individuals with renal dysfunc. respectively. and time to healing by 2. Topical acyclovir is not an tion.

CNS and disseminated disease. and of 0. HSV strains are TK altered and maintain the ability to Varicella phosphorylate the natural substrate. normal host produces some acceleration of the healing of apy of choice and reduces mortality from 70 to 19%. and the number of maximum lesions and 25–30% of patients have no further recurrences while in immunocompetent children. which resides in a latent state in the sensory ganglia Herpes simplex encephalitis following primary varicella (chicken pox) infection. Intravenous acyclovir therapy of herpes zoster in the every 8 h (30 mg kg1 day1) for 10–14 days is the ther. but selec- Varicella or chicken pox. Orally administered acyclovir (200 or VZV will make the use of acyclovir for immunocompe- 400 mg five times daily for 5 days) reduces the time to loss tent children with chickenpox obsolete. mechanism that in young children and is manifested by fever. 43 and 57% of acyclovir recipients. Acyclovir at a dose of 10 mg kg1 age. in whom chicken pox is self-limiting and results Herpes labialis in few complications. 14 days for SEM disease. thymidine. At present. For babies with SEM dis- ease. but infrequent. as evidenced by a reduction of with HIV or transplant recipients. when initiated within 24 h of the onset of the rash reduces vir reduces the frequency of recurrences by up to 80%. is a common.1–0. Oral acyclovir therapy in high doses in immunocompromised patients Herpes zoster with herpes zoster is effective but valaciclovir is superior. Herpes zoster or singles is caused by the reactivation of VZV. 38% of acyclovir recipients returned to zoster-associated pain). with prevalence The disease is more severe in neonates. The recommended visceral complications of herpes zoster in immunocom- treatment for neonatal herpes infection is 20 mg kg1 every promised adults. developed normally. or mouth (SEM) disease. Clinical benefit from apy is not indicated for immunocompromised host with intravenous or oral acyclovir therapy is documented as chicken pox. Occasionally. resistance is mutations in the HSV genome resulting in a respectively. highly contagious tively lose the ability to phosphorylate acyclovir. Acyclovir is the standard therapy at a 8 h of parenteral acyclovir with duration dictated by the dose of 10 mg kg1 (body weight) or 500 mg m2 (body extent of disease. such as those infected improved the outcome. Oral acyclovir therapy recurrent genital herpes. 21 days for surface area) every 8 h for 7–10 days. Chicken pox is generally self-limiting molecules is an alternate. Oral acyclovir (800 mg five times a normal neurologic function. incidence of serious ocular complications such as keratitis gories: skin. especially sporadic fatal encephalitis in the United States. the rash. the duration of fever. The bioavailability of valacyclovir makes it evidenced by a significantly shorter duration of viral an attractive alternative. It is primarily a disease of early of the viral DNA polymerase gene resulting in failure to childhood with 90% of cases occurring in children incorporate the acyclovir triphosphate in progeny DNA 1–14 years of age. mised host substantially reduces morbidity and mortality. of crust by approximately 1 day (7 vs. deficiency or alteration in viral TK activity. Antiviral Agents 87 Oral acyclovir administration effectively suppresses immunocompromised individuals. Daily administration of acyclo. Oral acyclovir ther- quent and severe HSV infections. Furthermore. are afflicted with fre. Oral Neonatal HSV infection acyclovir treatment of zoster ophthalmicus reduces the Neonatal HSV infection is divided into three clinical cate. constitutional symptoms. For babies surviving encephalitis and The most common mechanism for conferring acyclovir disseminated disease. The risk of zoster-associated pain persist- predominantly the causative agent of herpes simplex ing after the healing of the rash correlates with increasing encephalitis (HSE). day) administration results in accelerated healing of the rash and reduction in the severity of acute neuritis. 98% of acyclovir recipients developed normally 2 Resistance years after infection. eye. Intravenous acyclovir therapy significantly system (CNS) disease. Mutation illness caused by VZV. adults. VZV pneumonitis from 45 to <5%. Topical therapy for HSV-1 mouth or lip infections is of the widely use of the varicella vaccine to protect against no clinical benefit. and disseminated (if there is evidence reduces the frequency of cutaneous dissemination and of visceral involvement) HSV disease. importance of acyclovir treatment in otherwise healthy children. Resistance to acyclovir is uncommon. Intravenous acyclovir treatment (500 mg m2 of body Immunocompromised host surface area or 10–12 mg kg1 every 8 h for 7–10 days) Immunocompromised individuals. vesicular rash. and resolution of pain (both acute neuritis and Furthermore. shedding and accelerated lesion healing. central nervous and uveitis.4% and 5–6% in immunocompetent and . 8 days) but does not Acyclovir therapy of chicken pox in immunocompro- alter the duration of pain or time to complete healing. the clinical taking the drug. remains uncertain. mild may result in HSV resistance to acyclovir. and an itchy. HSV-1 is in older adults. Acute HSV infection of the brain is the most common cause of herpes zoster is a painful. debilitating condition.

and is equivalent to plasma affinity for the viral DNA polymerase. respectively. Cidofovir has a Valacyclovir.to 50-fold greater obtained with oral acyclovir. (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) Chemistry. Owing to its unique phosphorylation valacyclovir are similar to that as described for acyclovir. The acyclovir-resistant VZV isolates all had tive for episodic therapy (1 g. and CMV. Acyclovir. cidofovir usually maintains activity against acyclovir. including agitation. once a day). increasing drug activity. is an acyclic phosphonate nucleotide activity analogue of deoxycytidine monophosphate. Cidofovir NH2 Valacyclovir N Valacyclovir HO O N O O O P N N OH O CH3 OH O H2N N N O CH3 NH2 Chemistry. initial episode of genital herpes (1 g. recovered from marrow transplant recipients and AIDS For immunocompromised patients. Adverse effect Acyclovir therapy is associated with few adverse effects. valacyclovir is effec- patients. suppression of recurrent genital herpes (500 mg. as well as ganciclovir. and antiviral cytosine (HPMPC). Valacyclovir is rapidly metabolized into acy. VZV. thereby selectively inhibiting mechanism of action and antiviral activity spectrum of viral replication. A few reports have linked intravenous Adverse effects acyclovir use with CNS disturbances. compared with the levels achieved with doses of intravenous acyclovir. In addition. then serves as a competitive inhibitor of DNA polymerase. three times viral TK for their activity. twice a day for >5 days) and altered or absent viral TK function but remained suscep. The triphosphate metabolite tract. diarrhea. tremors. and headache. or 1 g. The Resistance most common complaints associated with acyclovir therapy The mechanism of resistance to valacyclovir is similar to include nausea. which do not require day. and suppression of recur- frequently than acyclovir-resistant HSV but have been rent genital herpes (1 g or 500 mg. requirements for activation. twice daily for 10 active metabolites with long half-lives (17–48 h). Valacyclovir provides a high bioavail. Valacyclovir has similar side effect profile as acyclovir. mechanism of action. HSV-1 and HSV-2 infections in immunocompetent indivi. Cidofovir is less potent than acyclovir in vitro. and the liver. hallucinations. mutants. is a prodrug of acyclovir (the L-valyl ester of single phosphate group attached therefore it does not require acyclovir). daily for 7–10 days) is superior to acyclovir for the reduc- tion of pain associated with shingles. cidofovir produces duals. HSV-2. Rapid infusions of that of acyclovir. permitting days). twice a day for 1 day) and recurrent genital herpes (1 g or resistant isolates of VZV have been identified much less 500 mg. The cellular DNA polymerase. acyclovir during pregnancy showed that the rate of birth defects did not exceed that expected in the general popu- lation and the pattern of defects did not differ from those Cidofovir in untreated women. twice a day for 3–5 days).88 Antiviral Agents immunocompromised patients. cellular clovir and valine by the enzyme valacyclovir hydrolase kinases sequentially phosphorylate the monophosphate to its (esterase) found in the brush border of the gastrointestinal active triphosphate metabolite. threefold to fivefold higher than that The active form of the drug exhibits a 25. once weekly dosing.and foscarnet-resistant HSV iso- Clinical indications lates. and antiviral activity Cidofovir. once a day).and foscarnet-resistant CMV The antiviral spectrum of valacyclovir encompasses HSV-1. mechanism of action. no crystalline nephropathy has been reported Data on outcomes from pregnant mothers exposed to with its use. episodic therapy for recurrent herpes labialis (2 g. intravenous acyclovir can result in reversible crystalline nephropathy. viral enzymes for conversion to the monophosphate. however. disorientation. ability of acyclovir. twice a tible to vidarabine and foscarnet. Cidofovir has in vitro activity against . cidofovir persists in cells for prolonged periods. and myoclonus. It is effective for treatment of however. Valacyclovir (1 g.

HPV. and hypophosphatemia. HHV-6. and CNS side effects. and adenovirus. OH phonoacetic acid that inhibits all HHVs. is an inorganic pyrophosphate analogue of phos. Unfortunately. headache. foscarnet acts directly on the virus DNA O CH3 polymerase. CNS side effects include headache. mutations in CMV DNA polymer. and antiviral H activity O H 2N N N Foscarnet. Thus. ante. seizures. O CH3 H sphates. rash. cidofovir The cerebrospinal fluid (CSF) concentration of foscarnet is concentrates in kidney cells 100 times greater than in other approximately two-thirds of the plasma level. TK-deficient. diarrhea. painful genital ulcerations. also occurs. These isolates are all DNA polymerase mutants. ase can mediate altered susceptibility. occurs in Foscarnet toxicity includes mainly nephrotoxicity (acute 30–50% of recipients. hypo- calcemia. rash. EBV. with sufficient in immunocompromised patients. may develop isolated or combined hypomagnesemia. nausea. then 5 mg kg1 every other week. . Renal excre- tissues and produces severe proximal convoluted tubule tion is the primary route of clearance of foscarnet with nephrotoxicity when administered systemically. including most ganciclovir-resistant CMV isolate and acyclovir-resistant HO O Valganciclovir HSV and VZV strains. and mucocutaneous acyclovir-resistant (viral TK- dosing regimen is 5 mg kg1 per week during the first deficient) or penciclovir-resistant HSV and VZV infections 2 weeks. fore. hypokalemia. Clinical indications Clinical indication The principal indications are CMV retinitis in AIDS Cidofovir is licensed for treatment of CMV retinitis and has been used to treat acyclovir-resistant HSV infection. acyclovir-resistant her. The patients. and ocular hypotonia. and Foscarnet hallucination. it is administered intravenously. irritability. polyomaviruses. There are anecdotal reports that dividing the VZV in immunocompromised host can be treated with 5 mg kg1 weekly dose into three doses given alternate days foscarnet at dosages lower than that for the management in a week may reduce renal toxicity substantially. there. It inhibits DNA polymerase by blocking the pyrophosphate-binding site and preventing N N cleavage of pyrophosphate from deoxynucleotide tripho. tremor. hydration and coadministration of oral probenecid to prevent Mucocutaneous HSV infections and those caused by nephrotoxicity. Patients rior uveitis. Cidofovir >80% of the dose appearing in the urine. diarrhea. orthopoxviruses. however. The oral bioavailability of foscarnet is about 20%. Adverse effects Nephrotoxicity is the principal adverse event associated Adverse effects with systemic administration of cidofovir. hematologic abnormalities. mechanism of action. NH2 HO pesviruses remain sensitive to foscarnet. develop both in the laboratory and in the clinical setting. CMV. Foscarnet –O O Ganciclovir and Valganciclovir P 3 Na+ O C O– Ganciclovir O– O N N Chemistry. nausea. resulting in complex plasma elimination. of CMV retinitis. which requires activation by a O H2N N N virus-specific TK. and VZV resistant to foscarnet uncommon. Antiviral Agents 89 VZV. Other reported side effects include tubular necrosis and interstitial nephritis). Bone sequestration has limited and variable oral bioavailability (2–26%). Unlike acyclovir. Resistance Resistance The development of resistance with clinical use is Isolates of HSV. fever. metabolic and neutropenia. Other reported side effects include fever. HHV-8. and vomiting.

and thrombocytopenia. an agent affecting DNA synthesis. are less than plasma levels. (1) The alteration of the CMV phosphonotransferase The plasma elimination half-life is 2–4 h for individuals (coded by CMV UL97 gene) reduces intracellular with normal renal function. The phar. doses given for 14–21 days. valganciclovir is convenient to use and may replace The neutropenia is usually reversible with dosage intravenous ganciclovir for initial and maintenance adjustment of ganciclovir. Intracellular ganciclovir triphosphate concentrations are at least tenfold higher in CMV-infected cells than unin. Ganciclovir [9-(1. including CMV pneumonia. therapy with ganciclovir acyclic side chain. requires an induction and maintenance phases. renal function requires adjustment of dosage. additional studies are necessary before merase and inhibits the incorporation of guanosine a recommendation can be made in the use of ganciclovir for triphosphate into viral DNA. the enzyme that catalyzes the initial neonates congenitally infected with CMV. respectively. Because CMV lacks the Gancoclovir has been evaluated in the treatment of gene for TK. In cells infected by HSV-1 or 900 mg once a day for induction and maintenance ther- HSV-2. There Concentrations of ganciclovir in biologic fluids. Resistance is associated with decreased sensitivity up to macokinetics of ganciclovir in neonates is similar to that 20-fold. L-valine ester of ganciclovir. acyclovir because of TK deficiency are also much less Valganciclovir. It is also effective for CMV syndromes. because of the potential toxicity of long-term as a competitive inhibitor of herpes viral DNA poly. most notably as a potent inhibitor of spermatogenesis. Its mechanism of action and spectrum of activity are similar to that of Adverse effects ganciclovir. and (2) mutations in the route of clearance of ganciclovir. serves as sensitive to ganciclovir. ganciclovir has carci- compromised patients. Oral Neutropenia occurs in approximately 24–38% of patients. or withholding of treatment. Thrombocytopenia occurs in 6–19% of patients.and triphosphate infants from hearing deterioration beyond one year of life. As suppression of CMV retinitis in AIDS or other immuno. Ganciclovir has gonadal toxicity in animal models. strains of HSV that are resistant to in adults. In immunocompromised patients receiving prolong The oral bioavailability of ganciclovir is poor (5–7%). mechanism of action. In a phase III phosphorylation of ganciclovir in CMV-infected cells is randomized controlled trial. the first step of phosphorylation to ganciclovir 5 mg kg1 day1 given once daily for 5–7 days per week. Oral valganciclovir can be given in doses The most important side effects of ganciclovir therapy are that result in serum levels that approximate ganciclovir the development of neutropenia. The per dose administered twice a day for 6 weeks) protected final phosphorylation steps to the di. and gastroin- nine] is a nucleoside analogue that differs from acyclovir testinal infection in AIDS and transplant patients. and as active as acyclovir against HSV-1 The induction dose is 10 mg kg1 day1 in two divided and HSV-2 and almost as active against VZV. ganciclovir therapy. serum levels achieved with intravenous ganciclovir. ity against CMV. is by cellular kinases. impaired CMV DNA polymerase (coded by CMV UL54 gene). CMV colitis. Like acy. the prevalence of resistance exceeds 8%. triphosphate into the growing viral chain results in slow- ing and subsequent cessation of DNA chain elongation. The kidney is the major phosphorylation of ganciclovir. and prophylaxis or preemptive nogenic potential. Ganciclovir triphosphate serves However. a finding of unknown significance. Resistance fected cells. therapy. It has 8–20 times greater in vitro activ. Incorporation of ganciclovir congenital CMV infection. and therefore.90 Antiviral Agents Chemistry. and a maintenance dose of clovir. and virus-encoded enzymes.3-dihydroxy-2-propoxymethyl) gua. monophosphate in herpesvirus-infected cells depends on Oral valganciclovir dosage is 900 mg twice a day. It Clinical indications causes an increased incidence of tumors in the preputial Ganciclovir is approved for treatment and chronic gland of male mice. In by having a hydroxymethyl group at the 39 position of the immunocompromised patients. . TK catalyzes the phosphorylation of ganciclovir apy. Occasionally. including are two mechanisms of resistance by CMV to ganciclovir: aqueous humor and CSF. Valganciclovir is orally bioavailable (approximately 60%) and is rapidly con- verted to ganciclovir after absorption. and antiviral treatment of CMV infection in high-risk transplant activity recipients. oral prodrug of ganciclovir. ganciclovir therapy (6 mg kg1 the phosphotransferase encoded by UL97 gene. to ganciclovir monophosphate. treatment.

HSV and VZV isolates resistant to penciclovir have been after which it is oxidized by the liver at the position 6 identified in the laboratory. Penciclovir. diarrhea. like acyclovir. and 1% is approved for episodic therapy of herpes labialis and VZV are similar to those of acyclovir. and VZV. HSV-2-. O every 8 h for 7 days). The elimination T1/2 in healthy subjects is H2N N approximately 2 h. The in vitro recurrent herpes labialis (cold sores). Topical penciclovir inhibitory effects of penciclovir on HSV-1. twice a day for 7 days). The oral bioavail. efficiently than acyclovir in HSV. Both compounds have good activity against HSV-1. and well tolerated in the treatment of HSV infections activity in HIV-infected individuals. famciclovir is as methylene bridge for the ether oxygen in the acyclic effective. . except that therapy of mucocutaneous HSV infections. is relatively inac- Penciclovir tive against CMV and EBV. a accelerates lesion healing and resolution of pain by prodrug of penciclovir with improved bioavailability about 1 day. penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)- 6-deoxyguanine]. a tenfold ciated only with headache. Antiviral Agents 91 Penciclovir and Famciclovir antiviral activity. Compared with valacyclovir. HSV-2. Host cell kinases phosphorylate both penciclovir and acyclovir to a small but comparable extent. Because penciclovir is more stable. Penciclovir is eliminated rapidly and almost unchanged N N by active tubular secretion and glomerular filtration by the OH kidneys. allowing for less frequent dosing. and liver. it has longer testicular toxicity in other rodents. particularly it is not an obligate DNA-chain terminator. safe. HSV-2. Penciclovir is active against O hepatitis B. For immunocompromised patients. suppression of recurrent genital herpes (250 mg. N OH Famciclovir Clinical indications O Famciclovir is available in an oral preparation for treat- N N ment of HSV-1. N episodic treatment of recurrent genital herpes (125 mg.and VZV-infected Adverse effects cells is the major determinant of its antiviral activity. Chemistry. Resistance is attributed to of the purine ring.and VZV-infected cells. and VZV-infected cells after drug was tumorigenic (murine mammary tumors) and causes removal. Famciclovir. It ability of penciclovir is poor (<5%). and antiviral safe. being asso- Penciclovir triphosphate has. Famciclovir is also at least as Penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)] effective as acyclovir for ophthalmic zoster and for a guanine nucleoside analogue is structurally similar to shingles and acute zoster pain in immunocompromised ganciclovir. mechanism of action. ribose part of the molecule. It is available over-the-counter in many (approximately 77%). Therapy with oral famciclovir is well tolerated. Its metabolism and mechan. Penciclovir is phosphorylated more alterations or deficiencies of TK and DNA polymerase. and convenient in the treatment of zoster. Compared with acyclovir. It is well absorbed after oral adminis- tration and is rapidly metabolized to penciclovir by Resistance deacetylation in the gastrointestinal tract. HSV-2. twice a day). is the diacetyl ester of 6-deoxy countries. applied every 2 h during waking hours for 4 days. on average. famciclovir is efficacious for episodic treatment of recur- rent genital herpes (500 mg. differing only be the substitution of a patients. three times a day for 10 days). and for shingles (500 mg. famciclovir is as effective. Penciclovir is available as a 1% cream for topical ism of action are similar to those of acyclovir. O twice a day for 5 days). and nausea. The preferential metabolism in HSV. It is used in O the treatment of the following conditions: initial episodes H2N N of genital herpes (250 mg. Preclinical longer intracellular half-life than acyclovir triphosphate studies of famciclovir indicated that chronic administration in HSV-1-. and VZV infections.

which inhibit both viral nofuranosyl adenine) is active against HSV. and and cellular DNA synthesis. Low levels of drugs can be detected in the aqueous addition. However. Viral DNA synthesis is blocked at lower doses of drug than is host cell DNA synthesis. large doses of vira-A Trifluorothymidine is the most efficacious of these com. vira-A is incorporated into terminal positions of humor. The phosphate derivatives. thus inhibiting elongation. vidar- compound to receive FDA approval in 1963 for treatment abine should be recognized historically as the first of HSV keratitis. HO Trifluorothymidine O Vidarabine F3C NH Vidarabine NH2 N O HO N N O N N HO HO O OH HO Chemistry. Idoxuridine was the first antiviral available as an intravenous formulation. because of poor solubility and toxicity. are cytotoxic to dividing host cells. O photophobia. these nucleo- activity sides are phosphorylated by both viral and cellular kinases Vidarabine (vira-A. and 9-D-arabi- to active triphosphate derivatives. and antiviral activity Idoxuridine (5-iodo-29-deoxyuridine). pounds. Vidarabine is a purine nucleoside analogue that is results in potent antiviral activity but also sufficient host phosphorylated intracellularly to its mono-. mechanism of action. and tri- cytotoxicity to prevent the systemic use of these drugs. VZV. both cellular and viral DNA. unlike acyclovir. di-. adenine arabinoside. Parenteral administration CMV. However. conversion toxicity of these compounds is not significant when applied of vidarabine to its active intracellular derivative does not topically to the eye in the treatment of HSV keratitis. In cornea. . clinical significant resistance has not been O established. antiviral agent licensed in 1977 for systemic treatment. and antiviral analogue. mechanism of action. However. Topically applied dependent DNA polymerases of some DNA viruses idoxuridine or trifluorothymidine will penetrate cells of the approximately 40 times more than those of host cells. I NH Adverse effects N O The ophthalmic preparation of idoxuridine and tri- HO fluorothymidine causes local discomfort. hypersensitivity reactions as well as superficial punctate or epithelial keratopathy. and trifluorothymi- dine (5-trifluoromethyl-29-deoxyuridine) are thymidine Chemistry. Thus. When administered systemically. require viral enzymes at any of the phosphorylation steps. edema of the eyelids. resulting in a relatively Clinical indications selective antiviral effect. and the treatment of choice for HSV keratitis The use of vidarabine was replaced by acyclovir (1 drop of 1% ophthalmic solution instilled in each eye. It is no longer up to nine times a day). and less commonly.92 Antiviral Agents Idoxuridine and Trifluorothymidine Resistance Trifluorothymidine-resistant HSV strains with altered Idoxuridine TK substrate specificity have been selected for in vitro. irritation. Both idoxuridine and trifluorothymidine are effective and The triphosphate derivative competitively inhibits DNA licensed for treatment of HSV keratitis.

However. protease. Therapeutics for Respiratory Virus esis. famciclovir in standard animal models with respect to all cleotide that inhibits CMV replication through an parameters of efficacy. compounds bind to two viral targets simultaneously. NH2 order kinetics with half-life of 62 h. has been discovered. intraocular administration revealed first. a new compound class of helicase–primase Fomivirsen inhibitors. including iritis and vitritis. including inhibitors of ribonucleotide reductase. This class of antisense mechanism. New Prospects for Therapy of Herpesvirus merase resistant mutations. The helicase–primase complex is essential major immediate early region 2 (IE2) of CMV. and DNA polymerase.2 mmol l1. much lower than the 100-fold resistance to acyclovir with similar DNA poly. which for the HSV DNA replication process. with preclinical phar- Chemistry. the helicase and primase subunit of the helicase–primase plementary to a sequence in mRNA transcripts of the enzyme complex. no resistant clinical iso- patients in whom trifluorothymidine cannot be used lates have been reported. BAY 57-1293. No systemic absorption has been observed after intravi- treal administration in humans. namely GCG TTT GCT CTT CTT CTT GCG-39) is com. valacyclovir and Fomivirsen is a 21-nucleotide phosphorothioate oligonu. Resistance These reactions are usually transient or reversible with Resistance to vidarabine is conferred by mutations in the topical corticosteroids treatment. viral DNA polymerase gene. Acyclovir-resistant clinical Infections HSV isolates are always susceptible in vitro to vidarabine. Pharmacokinetic assessment of fomivirsen in humans after intraocular administration is limited.03 and Amantadine and Rimantadine 0. Its oligonucleotide sequence (59. Rimantadine NH2 Clinical indications Fomivirsen is indicated for use in HIV patients with CMV retinitis who are intolerant of or have contraindica- tion to other treatment for CMV retinitis or in whom the disease is recalcitrant to ganciclovir or cidofovir treat- ment. It has activity against cidofovir. mechanism of action.and ganciclovir- resistant strains of CMV. Some Adverse effects of the compounds that have been the focus of drug dis- Ocular toxicity consists of occasional hyperemia and covery in the last decade have been targets of viral increased tearing. Adverse events of fomivirsen are uveitis. with subsequent inhibition of viral replication. In a Amantadine rabbit model. Antiviral Agents 93 Clinical indications Resistance Although trifluorothymidine is the antiviral agent of CMV strains with tenfold decreased susceptibility have choice for the topical treatment of HSV keratitis. fomivirsen inhibits CMV replication in a dose- dependent manner. encoded enzymes. While several classes of compounds are being investi- gated for the treatment of herpesvirus infections. Binding of fomivirsen to the target mRNA results in inhibition of IE2 protein synth. occurring in approximately 25% of patients. The degree of maximal resistance to vidarabine is fourfold. both of low incidence. TK. Recently. Topical vidarabine is superior to idoxuridine in the treatment of HSV ocular Adverse effects infections. with a mean IC50 of between 0. gene expression that are essential for the production of infectious viral particles. for example. Infections In vitro. . Fomivirsen is cleared from the vitreous in rabbits during the course of 7–10 days by a combination of tissue distribution and metabolism. in been selected in vitro. These compounds encodes for several proteins responsible for the viral have no demonstrable activity against either VZV or CMV. vidarabine is a suitable alternative. and antiviral macological profile that outperforms the current standard activity HSV treatment represented by acyclovir.

Although the spectrum of adverse events associated with amantadine and rimantadine are qualitatively similar. CNS side effects. infection and illness in immunocom- secretion. On aver- accompanying the hydrogen flux facilitates the dissocia. and other transmembrane protein that functions as an ion channel systemic complaints. and complexes so that the ribonucleoprotein can enter the 300 mg day1. A. they are less frequent and less severe with rimantadine.g. Amantadine is excreted unchanged significance of isolating resistant strains from the treated in the urine by glomerular filtration and. amantadine and riman- tadine inhibit the acid-mediated dissociation of the matrix protein from the ribonuclear protein complex Resistance within endosomes. caused by type A influenza virus. More severe adverse effects (e. Clinical indications Amantadine is reported to cause side effects in 5–10% of Amantadine and rimantadine are licensed both for the healthy young adults taking the standard adult dose of chemoprophylaxis and treatment of influenza A infections. anxiety. and effects associated with rimantadine. By interfering with the function of the M2 protein. be effective in treatment of influenza A infections in Rimantadine is fourfold to tenfold more active than adults and children if treatment is initiated within 48 h amantadine. The consequences of these drugs are point mutations in the RNA sequence encoding for the the potentiation of acidic pH-induced conformational M2 protein transmembrane domain. These side effects are usually mild and Prophylaxis with either drug prevents approximately cease soon after amantadine is discontinued. Prophylaxis also is indicated if the disorders. vomiting. in comparison with placebo. although 50–60% of infections and 70–90% of clinical illnesses they often disappear with continued use of the drug. influenza vaccine. ing therapy. tubular subject is not clear. The mechanism of action of these drugs of the onset of symptoms. cell nucleus and initiate replication. and antiviral vaccine may be ineffective because the epidemic strain activity differs substantially from the vaccine strain of influenza Amantadine (1-adamantanamine hydrochloride) and riman. Drug therapy results in reduc- relates to the influenza A virus M2 protein. rimantadine has been asso- and children with chronic disorders of the cardiovascular ciated with exacerbations of underlying seizure or pulmonary systems. similar to those in patients infected with drug-sensitive However. fever. being useful only against influenza A infections. Rimantadine is exten- sively metabolized following oral administration. tion of the M2 protein from the ribonucleoprotein Amantadine and rimantadine are given orally at 200.. and for the 2 weeks after vaccination if influenza A tadine (-methyl-1-adamantanemethylamine hydrochloride) already is active in the community. insomnia. appears in the treated subjects within 2–3 days of initiat- lar transport. CNS adverse presumed influenza outbreaks in institutions housing effects associated with rimantadine administration are high-risk persons. however. with impaired creatinine clearance. lightheadedness. 200 mg day1. About 5% of patients but prophylaxis is especially recommended for control of complain of nausea. which occur in 5–33% of patients. The plasma elimination T1/2 is approximately petent people infected with a drug-resistant virus are 12–18 h in individuals with normal renal function. About 25–35% of treated patients shed Absorption of rimantadine is slower compared with resistant strains by the 5th day of treatment. with an elimination T1/2 of 24–36 h. High-risk individuals include adults significantly less. it is used preferentially. are symmetric tricyclic amines with narrow spectrum of Amantadine and rimantadine also have been shown to activity. likely. Approximately 15% of the Adverse effects dose is excreted unchanged in the urine. as well as earlier resumption of for this virus and is activated by pH. . the elimination T1/2 increases in the elderly virus. The drop in pH normal activities. doses exceeding 200 mg daily. respectively. the duration of illness is shortened by about 1 day. This degree of prophy. an integral tion in the duration of viral excretion. hallucinations.94 Antiviral Agents Chemistry. age. The clinical that of amantadine. mental Rimantadine can be given to any unimmunized member of depression and psychosis) are usually associated with the general population who wishes to avoid influenza A. This event occurs early in the viral Resistance to amantadine and rimantadine results from replication cycle. mechanism of action. Resistance typically changes in the viral hemagglutinin during its intracellu. or anorexia. Because of a lower incidence of side confusion. lactic effectiveness approximates that of inactivated are most common and include difficulty in thinking.

4 and 1 days. peak serum concentrations to have decreased infectivity and fitness and therefore less range from 17 to 142 ng ml1 within 2 h of administration likely to be transmitted.3-didehydro-N. cyclohexane-1-carboxylic acid] are sialic acid analogue Inhaled zanamivir. and antiviral respiratory secretions. These drugs also significantly diminish viral replication in Chemistry. no adjustment HO OH in dosing is necessary for renal insufficiency because of the H limited amount of systemically absorbed drug. The lipophilic side efficacy of oseltamivir in preventing culture-proven chain of zanamivir and oseltamivir binds to the influenza influenza is about 90%.5 and 5 h. and provides 29–43% relative reduction in the odds of complications when given within 48 h of onset of symptoms. viral aggregation. Oseltamivir NH2 carboxylate is eliminated unchanged by renal excretion HN through glomerular filtration and tubular secretion. blocking its ability to cleave sialic acid residues. Influenza NA also decreases viral reduces the risk of contracting influenza. virus NA. In general. which are essential for the release of the virus with fever by 84%.3-dideoxy-2.5S)-4-scetamido-5-amino-3-(1-ethylpropoxyl)-1. and spread within 6 weeks during the peak of influenza season significantly the respiratory tract. mechanisms of resistance: mutations in the hemagglutin vir exceeds 1000 ng ml1 in sputum for 6 h after inhalation receptor-binding site. Oseltamivir administered for from infected cells. Systemically absorbed zanamivir is excreted unchanged in Zanamivir the urine. Local respiratory mucosal concentrations of zanami. Zanamivir is available as dry powder activity for inhalation using a breath-activated Diskhaler delivery Zanamivir (4-guanidino-2. 10 mg once daily given for 4 weeks that competitively inhibit influenza virus neuraminidase as seasonal prophylaxis. while oseltamivir is (3R. resis- influenza A and B viruses). The plasma T1/2 is between 2. reduces the likelihood of labora- (NA). Approximately 75% of O orally administered oseltamivir reaches the systemic circu- HN lation in the form of oseltamivir carboxylate. Although serum concentrations of zanamivir increase with decreasing creatinine clearance. mechanism of action. . system. Antiviral Agents 95 Zanamivir and Oseltamivir of a 10 mg dose.4R. HO O Oseltamivir is an ethyl ester prodrug that. Approximately 10% of inhaled tant viruses with mutations in the NA enzyme are thought dose is absorbed systemically. Resistance Zanamivir has poor oral bioavailability and therefore it is Viruses resistant to zanamivir and oseltamivir have been administered by oral inhalation. given at 75 mg twice a day for 5 days. Treatment of O otherwise healthy adults and children with zanamivir and H2N oseltamivir reduces the duration of symptoms by 0. More than 75% of an orally generated after in vitro passage in cell culture. oseltamivir carboxylate. following H hydrolysis by hepatic esterases. and oseltamivir [ethyl ester of >7 years is 10 mg twice daily for 5 days. and mutations in the conserved (i. Serum concentrations of the drug increase in the presence of declining renal function. Clinical inhaled dose of zanamivir is deposited in the oropharynx. influenza virus isolates with reduced susceptibility to approximately 13% of this is distributed to the airways and both NA inhibitors have been reported..e. Influenza virus NA is located on the surface of the tory confirmed influenza (with or without symptoms) by virus and is responsible for cleaving terminal sialic acid 34%. The recommended dose of zanamivir in patients acetylneuraminic acid). There are two lungs. The protective inactivation by respiratory mucous. Zanamivir and oseltamivir are effective against both influenza A and influenza B. and dose adjustment is recom- mended in patients with renal insufficiency. over and above the concentration required to inhibit portions of the NA enzyme active site. and influenza disease residues. O O O Clinical indications Zanamivir and oseltamivir are used for treatment and pre- HN vention of influenza A and B infections. is converted to the active N COOH compound. influenza disease by 67%. The Oseltamivir elimination T1/2 of oseltamivir carboxylate is 6–10 h.

mechanism of action. placebo controlled trials have failed to demonstrate a consistent decrease in need for mechan- ical ventilation. Inhalation of zanamivir has Although respiratory secretions will contain milligram resulted in bronchospasm and reduced forced expiratory quantities of drug. Ribavirin can be administered orally (bioavailability of approximately 40–45%) or intravenously. in individuals with reactive airway diseases or chronic The kidney is the major route of clearance of drug. O NH2 Clinical indications N Respiratory syncytial virus N Ribavirin is licensed for the treatment of carefully N selected. hospitalized infants and young children with HO O severe lower respiratory tract infections caused by RSV. levels may be altered. but there is a of health care workers to ribavirin. Use of aerosolized ribavirin in adults and children with RSV infections reduced the severity of illness and virus HO OH shedding. The most Ribavirin (-methyl-1-adamantane methylamine hydro- common adverse events following aerosol administration chloride) has antiviral activity against a variety of RNA of ribavirin include bronchospasm and malfunction of and DNA viruses. tively. The usual dosage of aeroso- whose mechanisms of action are poorly understood and lized ribavirin is 20 mg ml1 of drug instilled in a small- probably not the same for all viruses. Oral virus. obstructive pulmonary diseases. Aerosol admin. respec- events following administration of oseltamivir have pri. The 59-triphosphate of ribavirin inhibits the formation of the 59-guanylation capping on Lassa fever and hemorrhagic fever the mRNA of vaccinia and Venezuelan equine encephalitis Systemic ribavirin has demonstrated efficacy in the man- viruses. Oral doses of 600 and 1200 mg result however. Zanamivir should be used with caution ml1) can be detected in the plasma. Notably. Aerosol administration of ribavirin results in plasma marily been gastrointestinal with nausea and vomiting levels that are a function of the duration of exposure.96 Antiviral Agents Adverse effects respectively. ribavirin triphosphate concentrates in erythrocytes and Ribavirin persists for a month or longer. Chemistry. the triphosphate is a potent inhibitor of agement of life-threatening infections caused by Lassa viral mRNA (guanine-7) methyltransferase of vaccinia fever and hemorrhagic fever with renal syndrome. however.5 mg volume (FEV1). and antiviral The use of ribavirin for the treatment of RSV infections activity is controversial and remains discretionary.5 mg ml1. inhibit influenza A RNA-dependent RNA polymerase. infections in children. accounting for approximately 40%. . resistance to ribavirin has not been identified as in peak plasma concentrations of 1. Adverse result in 17 and 24 mg ml1 plasma concentrations. Likely. In addition. a clinical problem. Intravenous dosages of 500 and 1000 mg Both NA inhibitors are generally well tolerated. occurring in some patients. its ability to particle aerosol generator (SPAG) and administered for alter nucleotide pools and the packaging of mRNA appears 12–22 h day1 for 3–6 days. duration of stay in intensive care unit.5–3.3 and 2. The capacity of viral mRNA to support protein ribavirin is recommended for prophylaxis against Lassa synthesis is markedly reduced by ribavirin. an enzyme essential for Hepatitis C DNA synthesis. only microgram quantities (0. the persistence of ribavirin in erythrocytes contributes to its hematopoietic Ribavirin toxicity. This process is not virus specific. special containment certain selectivity. inhibition by ribavirin-59-monophosphate of inosine monophosphate dehydrogenase. Resistance istration has become standard for the treatment of RSV Treatment failures with ribavirin occur in some patients. in that infected cells produce more delivery system in an isolation room with negative pres- mRNA than noninfected cells. Ribavirin may fever in exposed contacts. or duration of hospitalization among ribavirin recipients. Ribavirin is a nucleoside analogue ventilator delivery systems. To avoid potential exposure important. This inhibition may have direct effects Oral ribavirin in combination with interferon- (IFN-) on the intracellular level of GMP and other nucleotide is recommended for hepatitis C infection. However. Hepatic metabolism also contributes to the clearance of ribavirin. A major action is the sure is used.

Chemistry. The antiviral effects of IFN ness and caution are warranted and. NA inhibitor administered intramuscularly. Each type of IFN can be produced Ribavirin has been found to be teratogenic and mutagenic through recombinant DNA technology. To expand about 6 days. resultant plasma levels. cardiac arrest. Although peramivir is a unchanged. When given orally rently classified as . researchers various body fluids and metabolism in a number of have been testing a number of investigational agents. and antiviral and concomitant digitalis toxicity. IFNs are active against a wide spectrum of viruses New Prospects for Therapy of Respiratory with RNA viruses being more sensitive than DNA Viruses viruses. IFN is eliminated by inactivation in the antiviral drug arsenal against influenza. Antiviral Agents 97 Adverse effects Therapeutics for Hepatitis Adverse effects attributable to aerosol therapy with ribavirin Interferons of infants with RSV include bronchospasm. The pegylated forms of IFN- (Peg- influenza virus compared with 36% of untreated animals. and antiviral properties. particular concern has mounted of late due lesion). mechanism of action. daily administration. While influenza pandemics have long posed a threat to IFNs are not orally bioavailable and are administered humankind. antineoplastic. hepatitis B and C. Concern has been expressed about the risk to receptors and activates secondary messengers to initiate persons in the room of infants being treated with ribavirin production of multiple proteins. Plasma levels are dose dependent. peaking seasonal or avian influenza because circulating influenza 4–8 h after administration and returning to baseline A viruses as well as the H5N1 strains affecting humans in between 18 and 36 h. Reticulocytosis. particularly females of childbearing age. hypotension. respectively. in general. Eastern Europe. activation of natural killer (NK) cells and other immune cells. Three generally . intramuscularly or subcutaneously (including into a 1957. Absorption of IFN- is more Amantadine and rimantadine are not recommended for than 80% complete after intramuscular or subcutaneous injection. a threat realized to varying extents in 1918. which causes severe acute respiratory syndrome (SARS). inhibition of viral establishment of stringent precautions for administration of protein synthesis. Preclinical stu- IFN- molecule covalently bonded to polyethylene dies in mice and ferrets revealed that the drug could protect glycol (PEG) improves the pharmacokinetic profile of 80% or more of animals exposed to pathogenic H5N1 IFN markedly. the antiviral activity Southeast Asia are resistant to these medications. therefore. Pulmonary function test activity changes after ribavirin therapy in adults with chronic IFNs are glycoprotein cytokines (intercellular messen- obstructive pulmonary disease have been noted. The mechanism of this risk seems to be minimal with limited exposure. organs. . gers) with a complex array of immunomodulating. enza in Southeast Asia. The binding of in preclinical testing. the natural sources of or intravenously. There appears to be some variability in absorption to continued sporadic human cases of H5N1 avian influ. between each of the three classes of IFN and. rash. underscores the need for antiviral drugs for the control of Clinical indications future SARS outbreaks. phocytes. Its use is contraindicated in women IFN to the intact cell membrane is the first step in estab- who are or may become pregnant during exposure to the lishing an antiviral effect. importantly. and conjunctivitis have been asso. and prevention of the viral infection of ribavirin. aware. pneumothorax in ventilated patients. IFN binds to the cellular drug. However. and 1968. fibroblasts. IFN-) offer a more convenient once weekly instead of T-705 is a viral RNA polymerase inhibitor in the preclini. are licensed for the treatment of cal testing stage. IFNs are cur- ciated with the use of ribavirin aerosol. or . Zanamivir peaks at 24 h and then slowly decreases to baseline over and oseltamivir are the only available options. The outbreak in 2003 of a novel coronavirus infection. action is rather complex. The major goals of therapy for hepatitis B are to prevent ome structure have revealed novel potential therapeutic progression of the disease to cirrhosis. and increased cytokine produc- tion. end stage liver targets for antiviral therapy. New insights into the field of Hepatitis B SARS pathogenesis and SARS-coronavirus (CoV) gen. Negligible amounts are excreted in the urine including peramivir and T-705. cells. disease or hepatocellular carcinoma. the include degradation of viral mRNA. transient elevations of serum bilirubin and which. are leukocytes. and Africa. which are pivotal for the aerosol. apnea. Although defense of the cell against viruses. and lym- the occurrence of mild anemia have been reported. The immunomodulating effects of IFN include enhancement of antigen presentation by HLA I and II to the immune system.

liver failure. Genotype and other baseline factors affect the response to PEG-IFN-2a in HBeAg-positive chronic O N hepatitis B. sion of HBV replication to levels less than 1104 copies disorientation. Combination therapy between 0. Genotypes 1 and 4 infec.98 Antiviral Agents accepted indications for treatment are: (1) positive test for modest.0 mg kg1. Therapy for chronic HCV infection is indicated in with sulfur substituted for the 39 carbon atom in the furanose patients with detectable HCV RNA viral load and persis. mechanism of action. per ml (2000 IU ml1) is regarded as a surrogate of treat- ment success with a resultant improvement in serum ALT and hepatic necroinflammatory disease. At higher doses of two times normal. and diarrhea. suppres. been shown to predict a higher likelihood of HBeAg loss in patients with chronic hepatitis B treated with lamivu- Adverse effects dine. occurring in months of therapy. monotherapy or in combination with ribavirin. The triphosphate derivative tory prior to treatment initiation. than other HCV genotypes. biopsy is not manda. attention deficits. sonality changes. S Hepatitis C The aim of therapy for chronic HCV infection is to Chemistry.39-dideoxy. headache. ring [(–) 29. Lamivudine is administered orally at 100 mg day1 Side effects are frequent with IFN (both standard and in the treatment of HBV infections. either as is a competitive inhibitor of the viral reverse transcriptase. and paranoid ideation. with 70% patients becoming . The incidence of lamivudine resis- up to 26% of treated patients.5 h and the plasma T1/2 60% of patients.25 and 8. Lamivudine is eliminated by the is either PEG-IFN-2a or PEG-IFN-2b given alone or kidneys unchanged by both glomerular filtration and in combination with ribavirin. The use of pegylated forms of IFN has become common with the convenience of weekly N dosing. Treatment end points differ in HBeAg IFN. Clearance Lamivudine of serum HBeAg and HBV DNA polymerase occurs with NH2 treatment (30–40%). However. chills.5 mg ml1 are achieved in 1–1. but these symptoms usually Resistance become less severe with repeated treatments. Influenza-like symptoms (i. and malaise) commonly occur. and HBeAg negative disease. however. is reduced with standard IFN therapy. has been The oral bioavailability in adults is >80% for doses used extensively for HCV infections. It is although neutralizing antibodies to recombinant IFNs the first line drug for the treatment of HBeAg and anti- have been reported. as manifested by per- positive. It has significant tently elevated ALT. Lamivudine is phosphorylated to the triphosphate of therapeutic intervention. fibrosis. confusion. The standard treatment of HCV infection is approximately 2–4 h. not clinically relevant. Peak serum concentrations for 40 weeks resulted in sustained responses in more than of 1. tubular excretion. Leukopenia is usually tance is 15–20% per year. Findings of cirrhosis. could be higher. Lamivudine is the (–) enantiomer of a cytidine analogue noma. a marker of repli- cation. though the ideal dose pegylated) administration and are usually dose limiting. and antiviral decrease and ultimately prevent progressive liver damage activity leading to cirrhosis. metabolite by cellular kinases. Increased ALT levels may also occur and (3) alanine aminotransferase (ALT) level greater than as well as nausea. Leukopenia Resistance to lamivudine monotherapy develops within 6 is the most common hematologic abnormality. vomiting. Standard IFN. Elevated serum ALT levels have observation is unknown. The clinical importance of this latter HBe positive disease.e. and dosages should be adapted to crea- tions are associated with lower sustained virologic response tinine clearance. (2) positive hepatitis B e antigen (HBeAg).. 39-thiacytidine]. or even activity in vitro against both HIV-1 and HIV-2 as well as moderate inflammation on liver biopsy support the choice HBV. Lamivudine Hepatitis B DNA polymerase level. Patients with genotypes A and B have a O OH better response in comparison with genotypes C and D patients. and reversible upon discon- HBV DNA. neurotoxicity is encountered. tinuation of therapy. Clinical indications Lamivudine is effective as monotherapy for the treatment Resistance of chronic HBV infection and in combination with other Resistance to administered IFN has not been documented antiretroviral drugs for treatment of HIV infection. or hepatocellular carci. fever.

Lamivudine resistance to HBV is moderate and severe renal impairment. Clearance of adefovir is by renal excretion. The bioavailabil. These mutants have a reduced replication capa.52 vs.g. neutropenia is encountered but at a low in the HBV polymerase B domain (A181V/T) and the D frequency. . N N O These are usually mild. HO sphate metabolites is by cellular kinases. within the catalytic domain (C domain). the spectrum of chronic hepatitis B infection. 6%). 16%). a phosphonate acyclic nucleotide H analogue of adenosine monophosphate. including nausea.4S)-4-hydroxy- ity of adefovir dipivoxil in humans is about 40%. Adverse effects Adefovir Dipivoxil Nephrotoxicity is the major side effect of higher doses of adefovir. Entecavir (2-amino-1. and within the B domain (e.3R. At the city compared with the wild type HBV virus. mechanism of action.and tripho. and antiviral to the lack of a 39 hydroxyl group. The triphosphate HO competes with endogenous deoxyadenosine triphosphate (dATP) in incorporation to the nascent viral DNA result- ing in premature termination of viral DNA synthesis due Chemistry. tion of ALT (48% vs. At the highest doses of 20 mg patients 3 years after adefovir monotherapy. It will be curious to dose. It is also useful for the treatment of lami- Adverse effects vudine-resistant HBV infection. This may be partly because lamivudine does not affect Resistance mitochondrial DNA synthesis and its poor inhibition of Adefovir resistance occurs in approximately 6% of human DNA polymerases. Mutations kg1 day1.. anorexia and diarrhea. M204V or The efficacy of adefovir has been assessed in patients with M204I). The phosphorylation to the di. adefovir resulted resistance is managed by sequential treatment with either in significant improvement when compared with placebo: adefovir or entecavir.g. Lamivudine has an extremely favorable toxicity profile. H2N N N able prodrug of adefovir.9-dihydro-9-[(1S. hepadnaviruses and herpesviruses. Other NH2 dose-related clinical adverse events have been gastroin- testinal events. O O O O O Entecavir Entecavir O Chemistry. Antiviral Agents 99 resistant after 5 years of treatment. improvement in liver histology (53% vs. 0. normaliza- questionable. and antiviral N activity N Adefovir dipivoxil. the advantage of sequen. It has activity against activity HIV. 25%). Adefovir is mono- phosphorylated and is not dependent on initial phosphorylation by viral nucleoside kinases to exert its antiviral effect. and HBeAg seroconversion (12% vs. It causes a proximal convoluted tubule lesion Adefovir dipivoxil characterized by a rise in urea and creatinine. bis(pivaloyloxymethyl)ester of 9-(2- phosphonylmethoxyethyl) adenine. However. domain (N236T) confer resistance to adefovir. Clinical indications which includes the YMDD motif (e. Lamivudine recommended dose of 10 mg once a day.. L180M or HBeAg positive and negative disease and other settings in V173L).55 log copies ml1). is an orally bioavail. It has a 3-(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6- long intracellular half-life of 18 h allowing for a once-daily one). macokinetics is substantially altered in subjects with dence of resistance. monhydrate is a guanosine nucleoside analogue. mechanism of action. reduction tial treatment compared to de novo combination therapy is in HBV DNA (3. intermittent and self-limited O O without the need for concomitant medications or dose N N P interruption. Its phar- know if lamivudine at higher doses will affect the inci. conferred through HBV strains with mutations in the viral polymerase.

with naturally occurring thymidine triphosphate for viral DNA elongation. O OH Studies prior to approval of entecavir for HBV treatment suggested that entecavir did not have anti-HIV activity at clinical relevant concentrations. Telbivudine triphosphate does not recent reports of an anti-HIV activity of entecavir. . Monitoring for long-term toxicity Telbivudine was approved in October 2006 by the FDA is needed. Entecavir resistance requires the following The second phase starts approximately 16–24 h after dosing. the abso- Resistance lute oral bioavailability of telbivudine is not known. Telbivudine is rapidly absorbed after oral dosing with peak plasma concentration achieved within 1–3 h. telbivudine exhibits an apparent single-phase treatment naive is about 1. with T1/2 of 2. M204V/I þ L184G.5 h. recent studies OH have suggested an anti-HIV activity of entecavir at drug concentrations in the low nanomolar range. However. The HBV polymerase binds preferentially to entecavir triphosphate. slower elimination phase was observed with inten- resistant patients after 4 year of switching treatment to sive sampling in healthy volunteers up to 168 h post-dosing. In phase III trials. but not HIV treatment.100 Antiviral Agents Entecavir is efficiently phosphorylated by cellular kinases Telbivudine to the active triphosphate metabolite. dosage reduction of telbivudine and comprised of headache. lamivudine. With chondrial metabolism. for the manage- contrast to other nucleoside analogue. which competes about 15 h. whereas lamivudine triphosphate pre- transaminases or signs of active disease on histological ferentially inhibits the complement DNA synthesis. and entecavir triphosphate does O N not affect human mitochondrial DNA synthesis.2%. virologic rebound decline. 40 h. a presence of a and resistance have been reported in 43% of lamivudine- second. mechanism of action. In Entecavir was approved in March 2005. for treatment of chronic HBV infection. The incorporation of telbivudine into the Clinical indications viral DNA terminates viral DNA chain elongation. Telbivudine is phosphorylated by cellu- half-life of the triphosphate metabolite in vitro studies is lar kinases to the triphosphate metabolite. The effect of entecavir on human cellular polymerase is minimal. In addition. or . In clinical trails . The elimination T1/2 of telbivudine increases with Most adverse events in the phase III studies were mild renal dysfunction. Telbivudine is a competitive The mean elimination T1/2 of entecavir varies from 77 to inhibitor of both HBV viral reverse transcriptase and 149 h in patients with normal function. Over an The prevalence rate of resistance to entecavir in HBV- 8-h period. It is eliminated primarily in the nucleoside with potent and specific activities against HBV urine through glomerular filtration and tubular secretion.e. ente- inhibit human cellular polymerase . such as lamivudine. and gastrointestinal upset. and (3) inhibits DNA- CH3 dependent DNA synthesis (i. tions. cavir monotherapy probably should not be used in telbivudine triphosphate is not a substrate for human DNA individuals with HIV–HBV coinfection who need HBV polymerase and thus will not induce genotoxicity. activity Entecavir is not a substrate of the cytochrome P450 Telbivudine (-L-29-deoxythymidine) is an L-configured (CYP) enzyme system. and antiviral achieving peak plasma concentrations between 0.6–1. The intracellular DNA polymerase. upper respiratory tract infec- is recommended in individuals with renal dysfunction. and other hepadnaviruses. The long plasma T1/2 of telbivudine is consistent with the long intracellular T1/2 (14 h) of its triphosphate in vitro Adverse effects studies. pharyngitis. therefore. responses achieved with Preliminary studies have shown a potent inhibition of entecavir surpassed previously published response rates HBV replication with a safe profile and no effect on mito- for IFN--2b. and adefovir dipivoxil. However. fatigue. cough. entecavir. The most common laboratory abnormality was ALT level greater than five times the Clinical indications upper limit of normal. Entecavir is well absorbed after oral administration Chemistry.5–5 h. S202I. amino acid sequence changes in the reverse transcriptase with a long observed terminal-phase T1/2 of approximately domain of HBV. It affects three-steps in the replication of HBV: (1) prevent the priming of the Telbivudine HBV reverse transcriptase. ment of adult patients with chronic HBV infection who telbivudine preferentially inhibits anticomplement or have active viral replication and/or elevation in liver second-strand DNA. However.. abdominal pain. terminating viral DNA HN synthesis). or M250V. examination. (2) prevent reverse transcribing O of the HBV pregenomic mRNA.

breakdown of muscle tissue. mechanism of action. elvucitabine.6%.02 log10 viral suppression at 34 and 48 weeks. the lack of cytotoxicity. which may be necessary but not sufficient to effect viral control. alamifovir to be licensed for hepatitis B treatment in other countries. with lamivudine-resistant HBV mutants. hence. headache.73 log10 and tance to telbivudine. clevudine was well tolerated without any serious adverse events reported. within 12 months of treatment. in inhibition of viral plus-strand DNA synthesis. and emtricitabine. and  could not utilize the 59-triphosphate of clevu- those treated with lamivudine. antiviral resistance remains a concern with O N O long-term therapy. placebo- recipients of telbivudine had HBV DNA rebound that controlled phase III study in South Korea. telbivudine was found to be superior to lamivudine. Adverse effects In clinical trials. Clevudine Future Prospects O Current antiviral agents either inhibit hepatitis B repli- CH3 HN cation. Adverse effects Resistance Most of the adverse effects of telbivudine reported in In vitro studies suggest that there may be cross-resistance clinical studies were mild to moderate. and antiviral Another challenge is the management of hepatitis B in activity individuals with HIV coinfection. Chemistry. respectively. licensed for the OH treatment of HIV infections. MIV 210. and cough. In HBeAg-negative dine as a substrate and. respec- HBV associated with telbivudine resistance are M204I or tively.02 to 0. B infection in South Korea. but are not yet FDA-approved for this indication. than telbivudine monotherapy. In a randomized. A unique characteristic of clevudine is the slow M204I þ L180I/V. an resistance occurred in the B domain of the polymerase enzyme present in muscle tissue and a marker for the gene. the search for novel agents. and OH treatment strategies with minimal or no resistance and F good long-term safety profile are the focus of ongoing research. Preclinical HBeAg-positive patients a therapeutic response occurred studies revealed that human cellular DNA polymerases . Appropriate combina- Clevudine [1-(2-deoxy-2-fluoro--L-arabinofuranosyl) tion regimens for individuals with coinfections are thymidine] is a nucleoside analogue of the unnatural expected in the near future. fatigue. in 75% of patients treated with telbivudine and 67% of .6%. Moreover. Tenofovir. Clevudine is well absorbed study. The most com. 21. analogue in the pipeline.84 mmol l1. dine once daily for 24 weeks maintained a 3. 8. . patients. -L-FddC. Clevudine was recently approved in South Korea for the There are a number of new nucleoside and nucleotide treatment of hepatitis B after demonstration of potent anti. Clevudine is efficiently HBV treatment on HIV. Long-term toxicity has Clevudine to be closely monitored. also have activity against HBV. Lamivudine. The mechanism of action is mainly serum HBeAg or ALT normalization) after one year. It is likely amdoxovir. Clinical indications Resistance Clevudine is approved for treatment of chronic hepatitis HBeAg-positive. HBeAg-positive patients who received 30 mg of clevu- resistance HBV strains have a high level of cross resis. and HBeAg-negative. after oral administration with estimated long half-life of Using the two drugs in combination was no more effective 44–60 h. rebound of viremia after cessation of treatment. hepatitis B activity in phase II and III clinical trials. phosphorylated by cellular kinases to clevudine-tripho- posite of suppression of HBV DNA and either loss of sphate in target cells. upper respiratory tract infec- tion. valtorcitabine. racivir. abdominal pain. Antiviral Agents 101 with primary end point of therapeutic response (a com. chronic was associated with resistance mutations. or invoke an immune response. the response was 75 and 77% for telbivudine The EC50 of clevudine for HBV inhibition values ranges and lamivudine. The mutations in the RT domain of 2. target treatment of HBV to -L configuration with potent activity against HBV and alter the outcome and take into account the impact of some activity against EBV. In animal studies mon were elevated creatinine phosphokinase (CPK). In the second year of the from 0. . and hepavir B may soon be part of the armamentarium for hepatitis B treatment.

With imiquimod appli- F cation. and 18). Intralesional therapy is painful. IFN treatment is secure FDA approval because of its induction of CYP expensive and there is limited efficacy. Adverse effects include irritation of the adjacent propyl]phenyl]-5[trifluoromethyl]-1. and intralesionally with variable respiratory infections. IFNs have been administered (mostly IFN. treat. and the potential for drug interactions. to reduce the rates of transmission. mechanism of action. including antibody-deficient individuals.2. It acts as a ligand for Toll-like receptor 7 and activates Pleconaril macrophage and dendritic cells to release IFN  and F F other proinflammatory cytokines. particularly. They are more effective if used in combination with ditis. podophyllotoxin. there is no virus-specific effective systemic therapy available. With time. They result in the physical removal of the lesion or the The recently FDA-approved quadrivalent prophylac. Several large con. and systemic effects including fati- gue and influenza-like illness. and at least 20–30% of epithelial cells. its antiviral effect by integrating into a hydrophobic pocket . severe myocar- results. local erosion. 11. The exact mechanism of action is Chemistry. leading to a broad spectrum of human responders will have recurrence. widely used treatments for external genital warts. related pathogens that cause a wide spectrum of human tory properties. and neonates. Pleconaril failed to such as fever and myalgia. In general. N flaking. Imiquimod This is an immunomodulator approved by the FDA for Pleconaril topical treatment of external and perianal genital warts. and paralytic poliomyelitis.102 Antiviral Agents Therapeutics for Papillomavirus Trichloroacetic acid. bone trolled trials have demonstrated inconsistent clinical benefits marrow recipients. antiviral. peutic options have been limited. Therapeutics for Enteroviral Infections Interferon The enteroviruses include nearly 70 serotypes of closely IFNs have antiproliferative. and immunomodula. 16. aseptic meningitis. Future Prospects ment of disease with current therapies has not been shown The current therapies are not targeted antiviral therapies. 3A enzyme activity.5% solution or gel is similar activity in effectiveness to imiquimod but may have more adverse Pleconaril (3-{3. The adverse O effects are. diseases. particularly the interference with oral contraceptives. HPV infection induces the hyperproliferation of response rate is only 60–70%. ranging from benign warts (self-limiting) to malignant neoplasms. thereby inducing a tic vaccine (HPV6/11/16/18) has been shown in clinical bystander immune response. Furthermore. of the use of immune serum globulin and pleconaril systemic therapy is associated with influenza-like symptoms for serious enteroviral infections. systemically.5-dimethyl-4-[[3-methyl-5-isoxazolyl}- effects. There is urgent need to trials to be effective in preventing high-grade vulval and develop specific and effective antiviral agents for HPV vaginal lesion associated with HPV 16 and 18. gradual clearance of warts occurs in about 50% of N patients over an average of 8–10 weeks. application site reactions (irritation. Furthermore.4-oxadiazole) exerts skin. may develop potentially of the use of standard IFN- therapy of condyloma life-threatening enterovirus infections for which thera- acuminatum (caused by HPV) that was refractory to cyto. pruritus. patients. infections. and cryother- apy (with liquid nitrogen or a cryprobe) remain the most HPVs are small DNA viruses with strict epithelial trop. this prophylactic vaccine is expected to reduce the inci- dence of HPV infections. infections due to the vaccine types (HPV6. There are case series destructive therapies. a resin used for many years for topical treatment of warts. Certain either local surgery or podophyllotoxin. from mild nonspecific fever to common upper ) topically. and erosion). ulceration and scarring. illness. and antiviral unknown. induction of nonspecific inflammation. Podophyllotoxin 0. O Podophyllotoxin O N Podophyllotoxin is the main cytotoxic ingredient of podophyllin. but ism. encephalitis.

Long. and darunavir). Combinations of drugs are receptors. there is an urgent need to identify alternative drugs that might be effective. emtricitabine. nelfinavir. and tenofovir). such as vir). tenofovir/ For treatment of enteroviral meningitis. is rela. abacavir/lamivudine. This high level of bioavailability was achieved by the substitution The combination of three or more anti-HIV agents into of trifluoromethyl on the oxadiazole ring that reduces its multidrug regimens. infections may develop chronic meningitis and menin. Immunocompromised host and efavirenz) analogue). Subsequent (2) fusion inhibitors (enfuvirtide). often with a fatal outcome. and (5) protease inhibitors (saquinavir. delavirdine. can efficiently inhibit HIV viral processes. Adverse effects Pleconaril is generally well tolerated. (4) integrase inhibitors (raltegra- Patients with compromised humoral immunity. who contract enteroviral ritonavir. showed a marginal statistical improvement in a clinical score in the pleconaril-treated groups. The most common O adverse events are headache. thus interrupting the infection cycle. two large studies emtricitabine. however. A subsequent small study of 21 infants with proven enteroviral meningitis in Coreceptor Antagonist the United States did not have enough power to show unequivocal benefit with pleconaril. didanosine. and nausea. Antiviral Agents 103 inside the virion. 1 levels. indinavir. There are fixed- dose combinations for zidovudine/lamivudine. none has viral uncoating and blocking viral attachment to host cell reached phase I clinical trial. which is the target of pleconaril. zidovudine/ Enteroviral meningitis lamivudine/abacavir. There are N . The available anti- In a phase I trial of pleconaril for treatment of common cold. (3) reverse transcriptase trials confirmed a modest reduction in length of symptoms inhibitors (nucleoside/nucleotide (zidovudine. Future Prospects N Pleconaril has not been licensed for treatment of enter. Maraviroc Maraviroc Resistance F F Resistance to pleconaril has been reported in some ser- otypes of enteroviruses. diarrhea. lamivudine. for example. HIV drugs are categorized according the step they target there was a significant reduction in rhinorrhea of about within the HIV viral life cycle (Figure 2). This is the start-of-the-art treatment of AIDS or Pleconaril also readily penetrates the blood-brain barrier. fosamprenavir. Drug combinations are. tipranavir. of many anti-HIV agents are available. the mechanism is not well understood. lopinavir. amprenavir. and a reduction inhibitors. goencephalitis. and tenofovir/emtricitabine/efavirenz. and prevents viral replication by inhibiting several investigational compounds. zalcitabine.5 h) after oral dosing. Clinical indications while reducing the likelihood of the development of drug Common cold resistance virus. The viral likely to offer the best chance of cure and protection from capsid structure. enterovirus infections in the future. atazanavir. and non-nucleoside (nevirapine. (1) binding 1. in princi- ple. Pleconaril has broad spectrum and potent activity against enteroviruses and rhinoviruses. HIV-infected individuals. in a severity score as compared to the placebo. those with agammaglobulinemia. tively conserved among the picornaviruses. often termed highly active antiretro- degradation in the liver by enzymes involved in oxidative viral therapy (HAART). N N oviral infections. and minimizing toxicity. NH term use of pleconaril is associated with an increase in menstrual irregularities in women. however.5 days in those on 400 mg three times daily. There are case Fixed-dose combinations and once-daily dosage forms reports of the efficacy of pleconaril in these patients. abacavir. The metabolic stabilization is reflected in the replication to achieve low or undetectable circulatory HIV- drug’s long serum half-life (about 6. stavudine. for common cold in patients treated with pleconaril. coreceptor antagonist (maraviroc). Anti-HIV Agents Pleconaril is 70% bioavailability when given orally. aimed at obtaining synergism between the compounds.

Maraviroc was not active against CXCR4-tropic and multiple antiretroviral agents. The human chemokine receptor CCR5 present on the cell terminal half-life in healthy subjects is 14–18 h. Maraviroc selectively binds to the inhibitors and inducers of these enzymes/transporters.2. Maraviroc is a substrate of CYP3A 1-phenylpropyl}cyclohexanecarboxamide) is the first of and the efflux transporter P-glycoprotein (Pgp). The resistance profile in treatment-naive and treatment- The absolute bioavailability for 100 and 300 mg doses experienced subjects has not been fully characterized. preventing the interaction of HIV-1 gp120 and CCR5 necessary for CCR5-tropic HIV-1 to enter Clinical indications cells. its pharmacokinetics are likely to be modulated by 2007) for HIV treatment. Non-nucleoside analogs Integrase inhibitors Protease inhibitors Figure 2 HIV life cycle showing the stages of intervention of available anti-HIV agents. mechanism of action.1 to 1. dual-tropic viruses (EC50 value >10 mmol l1). dence of viral replication and HIV-1 strains resistant to ture. Chemistry. The anti- viral activity of maraviroc against HIV-2 has not been Resistance evaluated.2.104 Antiviral Agents Binding inhibitors Fusion inhibitors + + Reverse transcriptase inhibitors: 1.4-difluoro-N-{(1S)-3-[exo-3-(3-isopropyl-5. inactive against HIV-1.1]oct-8-yl].4 triazol-4-yl)-8-azabicyclo(3. It inhibits the replication of CCR5-tropic laboratory Maraviroc is approved for use in combination with other strains and primary isolates of HIV-1 in vitro.64 ng ml1) in cell cul. who are treatment-experienced with evi- from 0.25 nmol l1 (0. The mean anti-HIV agents for the treatment of adults with CCR5- EC50 for maraviroc against various strains of HIV-1 ranges tropic HIV-1.05 to 0. membrane. and there- the class of CCR5 coreceptor antagonists licensed (August fore. following serial passage of two dose of 1200 mg administered to uninfected volunteers. CCR5-tropic viruses (CC1/85 and RU570). respectively.5–4 h following single oral been selected in cell culture. Nucleoside/nucleotide analogs 2. The maraviroc Maraviroc is bound (approximately 76%) to human resistant viruses remained CCR5-tropic with no evidence . chrome P450 system to metabolites that are essentially methyl-4H-1. It is principally metabolized by the cyto- Maraviroc (4. HIV- are 23 and 33%. and antiviral activity plasma proteins. Peak plasma concentrations 1 variants with reduced susceptibility to maraviroc have of maraviroc are attained at 0.

and protease inhibitors are susceptible to enfuvirtide in cell culture. Enfuvirtide is catabolized by proteolytic enzymes. toxicity (hepatoxicity) and a statement about the possibi. injection site reactions. virus. was associated with maraviroc resis- tance. In into cells by inhibiting fusion of viral and cellular mem.5%. Eosinophilia is glycoprotein and prevents the conformational changes the primary laboratory abnormality seen with enfuvirtide required for the fusion of viral and cellular membranes. higher rate in patients who received enfuvirtide than in repeat (HR1) in the gp41 subunit of the viral envelope those who did not receive enfuvirtide. failing enfuvirtide-containing regimen. Fusion Inhibitors Enfuvirtide Chemistry. a 3- have been selected in vitro. Lysine Lys K There are no known clinically significant interactions Methionine Met M Phenylalanine Phe F between enfuvirtide and other medications. Alanine Ala A Enfuvirtide is administered twice daily by subcutaneous Arginine Arg R injection. positions 36–38 of the HIV-1 envelope glycoprotein gp41. musculoskeletal symptoms. . A316T and I323V were Resistance shown to be necessary for the maraviroc-resistant pheno- HIV-1 isolates with reduced susceptibility to enfuvirtide type in the HIV-1 isolate CC1/85. QAI (HXB2 resistant isolates showed mutations that resulted in amino positions 315–317). Other symptomatic side effects amide. is the first licensed agent in the class of fusion may include insomnia. In clinical trials. Glutamic acid Glu E Following 90 mg bid dosing of enfuvirtide subcutaneously Glutamine Gln Q in combination with other antiretroviral agents in HIV-1 Glycine Gly G Histidine His H infected subjects. Two amino acid residue substitutions (Table 1. the median Tmax was 4 h (ranged from 4 Isoleucine Ile I to 8 h). Enfuvirtide is used with other anti-HIV agents to treat HIV-1 infection in patients who are treatment-experienced and have detectable viral loads of a change from a CCR5-tropic virus to a CXCR4-using even though they are taking anti-HIV agents. non-nucleoside analogue reverse transcriptase inhibitors. letter codes of amino acids) in the V3-loop region of the HIV-1 envelope glycoprotein (gp160).089 to 107 nmol l1 (0. and in children aged 6 and older. but has no activity against HIV-2. fever.3  15. The product label includes a warning about liver codons encoding gp41 HR1 domain amino acids 36–45. The clinical relevance of these mutations is not acid substitutions at the enfuvirtide binding HR1 domain known. Enfuvirtide interferes with the entry of HIV-1 Several cases of hypersensitivity have been described. and antiviral activity Adverse effects Enfuvirtide. inhibitors. Genotypic analysis of these amino acid residue deletion in the V3 loop. In the RU570 isolate. The absolute bioavailability is 84. and nausea. dizziness.4 to 480 ng ml1). with advanced HIV infection. administration. HIV-1 isolates with reduced susceptibil- Adverse effects ity to enfuvirtide have been recovered from subjects The most common adverse events reported with mara. lates with decreased in susceptibility to enfuvirtide of rash. and diz. abdominal pain. Proline Pro P Serine Ser S Threonine Thr T Clinical indications Tryptophan Trp W Enfuvirtide was approved by the FDA in March 2003 for Tyrosine Tyr Y Valine Val V use in adults. Most of the iso- viroc were cough. greater than fourfold exhibited genotypic changes in the ziness. reverse transcriptase inhibitors. upper respiratory tract infections. bacterial pneumonia was seen at a branes (Figure 2). Single-dose vials contain 108 mg of enfuvirtide Asparagine Asn N Aspartic acid Asp D for the delivery of approximately 90 mg ml1 when recon- Cysteine Cys C stituted. HIV-1 clinical isolates resistant to nucleoside analogue lity of heart attacks. and dual tropic viruses. mechanism of action. It Leucine Leu L is not metabolized by hepatic CYP450 isoenzyme systems. Enfuvirtide binds to the first heptad. phase III studies. Enfuvirtide Amino acid Three-letter code One-letter code is active against R5. headache. Antiviral Agents 105 Table 1 The three-letter and one-letter codes for amino acid The IC50 of enfuvirtide for baseline clinical isolates ranged residues from 0. X4. a linear 36-amino acid synthetic peptide with The most common adverse effects of enfuvirtide are the N-terminus acetylated and the C-terminus is a carbox.

5–1. one 400 mg capsule (delayed-release capsule) once a day. delavirdine. saquinavir. respectively.39-dideoxythymidine) is HO O a pyrimidine analogue with an azido group substituting for the 39 hydroxyl group on the ribose ring. Clinical indications Zidovudine is used in combination with other anti-HIV agents. other retroviruses. through the enzyme uridine diphosphoglucuronosyltrans. It is given most important usage of zidovudine in the last decade as two 100 mg tablets (buffered tablets) twice a day or as has been the peripartum three-part zidovudine regimen. mechanism of action. Didanosine (29. It is first converted to 29.5 h. hypoxanthine and is cleared primarily by the kidneys. Zidovudine penetrates cerebrosp. ritonavir. and other compounds. twice a day). lamivudine. semen. It is administered orally at Clinical indications Didanosine is used in combination 600 mg day1 (300 mg tablet. buffered tablet results in 20–25% bioavailability. occurring in 16 and 24% of the patients. and nosine-59-monophosphate by adenylsuccinate synthetase still is used in combination with other drugs as initial therapy and lyase. Drug is predominately metabolized by the liver inhibitors. including polymyositis. Nucleoside/nucleotide reverse transcriptase inhibitors Zidovudine Adverse effects The predominant adverse effect of Zidovudine zidovudine is myelosuppression. syrup. nevirapine. ml1 with a T1/2 of approximately 1. HIV-1 when zidovudine is combined with didanosine. and antiviral activity Zidovudine inhibits HIV-1 at concentrations of approxi. and antiviral N N activity Zidovudine (39-azido-29. saliva.39-dideoxyinsine-59-monopho- zalcitabine. A 300 mg 59-O--D-glucopyranuronosylthymidine. nous formulations. A ferase to its major inactive metabolite 39-azido-39-deoxy. Peak plasma levels are achieved approximately HIV reverse transcriptase and a chain terminator.5 h after treatment.5–2. kinase and subsequently by creatine kinase or phos- Zidovudine is available in capsule. The triphosphate derivative competitively inhibits HIV reverse transcriptase. Zidovudine is initially phosphorylated by cellular TK and then to its diphosphate by cellular thymidylate kinase. mechanism of action. however.39-dideoxyinosine) is a purine nucleoside mately 0. . as evidenced by O neutropenia and anemia. Oral bioavailability is approximately The triphosphate metabolite is a competitive inhibitor of 65%. and fatigue are common side effects. 39-dideoxyade- drug to be licensed for the treatment of HIV infection. It was the first phosphotransferase and subsequently to 29. Synergy has been demonstrated against Didanosine is activated by intracellular phosphorylation. it inhibits a variety of with inhibitory activity against both HIV-1 and HIV-2. malaise. insomnia. Chemistry. and functions as a chain terminator. spectrum of activity of didanosine is enhanced by syner- inal fluid. Zidovudine has been associated CH3 HN with skeletal and cardiac muscle toxicity. The single with other anti-HIV agents as part of HAART. O N HO O Didanosine Didanosine O N3 N HN Chemistry. sphate by 59nucleotidase and inosine 59-monophosphate indinavir. In addition. It is metabolized to viduals who have altered hepatic function. The elimination oral dose achieves peak plasma concentrations of 0. and intrave. Didanosine is acid labile and has poor solubility.106 Antiviral Agents Reverse Transcriptase Inhibitors which has decreased the incidence of transmission of HIV infection from pregnant women to their infants. Nausea. and breast milk and it crosses the gism with zidovudine and stavudine as well as the protease placenta. It is then converted to diphosphate by adenylate for some patients. it is extended in indi. phoribosyl pyrophosphate synthetase to the triphosphate. The 0. headache.6 mg T1/2 is approximately 1 h.013 mg ml1.

39-didehydro. which is activated by cellular enzymes secretion. antiretroviral regimen. accumulation of body fat (lipoatrophy) have been Other zalcitabine-related side effects include nausea. Neuropathy tends to appear after 3 months of therapy and resolves slowly with Adverse effects Peripheral neuropathy is the major toxic. Antiviral Agents 107 Adverse effects The most significant adverse effect Stavudine associated with didanosine therapy is the development of peripheral neuropathy (30%) and pancreatitis (10%). Pancreatitis can during therapy when stavudine was part of a combination occur. neuropathy and myopathy caused by mitochondrial damage. notably peripheral of approximately 0. Lipoatrophy. The oral bioavailability of stavudine is more than 85%. mechanism of action.03 mmol l1. Inhibition of mitochondrial DNA synthesis is proposed to induce Clinical indications Zalcitabine is used as part of depletion of cellular mitochondrial DNA and it is HAART regimen for HIV-1 infections. with the use of stavudine and other nucleoside reverse transcriptase inhibitors (NRTIs). and antiviral Stavudine penetrates CSF and breast milk. Its mechanism of action is similar to that of zidovudine. However.67 mg kg1 per dose.1 mmol l1. 39-deoxythymidine) is a thymi- HO O dine analogue with significant activity against HIV-1. to its triphosphate derivative. CH3 HN Zalcitabine O N Zalcitabine HO O NH2 N O N Chemistry. The peak plasma concentrations every 12 h). and therefore.39-dideoxycytidine) is a the kidneys unchanged and. mechanism of action. specifically dendritic and monocyte/macrophage cells. and Clinical indications Stavudine is used for HIV infection therefore this offers a theoretical advantage for nondividing in combination with other anti-HIV agents. headache. occurring uncommon. medication discontinuation. the Adverse effects The principal adverse effect of presence of renal insufficiency leads to a prolong plasma stavudine therapy is the development of peripheral T1/2. hepatotoxicity. highly potent inhibitor of HIV-1 replication in vitro.01 to 4. respectively).2 mmol l1 and the T1/2 is approximately 20 min. and cardiomyopathy.75 mg tablet every 8 h). by renal tubular pyrimidine analogue. neuropathy. which range from 0. in part. but does so infrequently. following an oral dose of 0. Thrombocytopenia and regimen that included didanosine.25 mg day1 (one 0. having inhibitory concentrations. Redistribution and neutropenia are uncommon (5% and 10%. The oral bioavailability following zalcitabine adminis- It is administered orally at 80 mg day1 (one 40 mg capsule tration is more than 80%. Zalcitabine inhibits both HIV-1 and HIV-2 at concentrations its use has been limited by delayed toxicity.1 to 0. The development of this complication is related to both dose and duration of therapy. It is excreted by activity Zalcitabine (29. and antiviral activity Stavudine (29. . Chemistry. It is administered ultimately responsible for the delayed toxicity observed orally at 2. The drug is cleared mainly by the kidneys. The enzymes responsible for activation of zalcitabine are cell cycle independent.03 mg kg1 range from 0. Other side effects are ity associated with zalcitabine administration.2 mg ml1 are reached within 1 h of dosing at 0. observed in patients receiving stavudine as part of their vomiting. lactic acidosis and diabetes have been Stavudine observed in patients on antiretroviral regimens O containing didanosine. Stavudine is a cells. Peak plasma concentrations of approximately 1. Fatal and nonfatal pancreatitis have occurred in approximately 35% of individuals.

and antiviral have abacavir hypersensitivity reaction is avoided due to activity Abacavir sulfate. 24-h study period.0 mmol l1). reaction. combination with other anti-HIV agents. is a structural analogue of the purine guanine. and antiviral carbovir triphosphate. and the mean half-life is 0. Lamivudine is Abacavir exhibits potent in vitro antiviral activity against given orally at 300 mg day1 (one 150 mg tablet twice a wild-type HIV-1 (IC50 4. The main route of Abacavir excretion is renal. pancreatitis and carbovir triphosphate was measurable throughout the peripheral neuropathies have been reported. . and 0. difference between the levels of activity of abacavir (IC50 0. to form carbovir monophosphate. mechanism of action. terminates DNA chain elongation. or with zidovudine and 0. reports of fatal reactions caused by repeated (cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1. H2N N N Adverse effects The abacavir hypersensitivity reaction is a potentially fatal syndrome occurring in approximately OH 5% of HIV-infected patients exposed to this nucleoside analogue after a median of 11 days (range: 1–318 days). However.4R)-4-[2-amino-6. or two 300 mg tablets once a day. diphosphate kinase and other enzymes. It is given as a 300 mg tablet twice a day. In pediatric studies. with the highest levels found between 6 and 8 h. the lack of active lamivudine metabolites in the at a dose of 300 mg twice daily as part of a combination mitochondrial compartment of cells. and gastrointestinal or respiratory symptoms. It is also formulated lower than the activity of zidovudine (IC50 in combination with zidovudine. Lamivudine is a guanylate kinase and to the triphosphate by nucleoside competitive inhibitor of the viral reverse transcriptase. neutropenia is encountered but at a ged from <20 to 374 fmol per 106 cells.26 mmol l1) and AZT (IC50 0. The intracellular low frequency. This finding suggests a long half-life for carbovir triphosphate within cells.7 h.7–1. and after Clinical indications Lamivudine is used in incorporation. mechanism of action. Chemistry.040 mmol l1). Cmax is attained after a mean of affinity of lamivudine for human DNA polymerases. levels of intracellular carbovir triphosphate ran- of 20 mg kg1 day1. there is no significant abacavir as fixed-dose combination tablet. Carbovir triphosphate competes with endogenous 29-dGTP for incorporation into the nucleic acid chain. or one 300 mg tablet once a day). A fixed- N dose combination with lamivudine is available (600 mg HN abacavir. but this activity is day.8–1. At the highest doses regimen. Rechallenge with abacavir in individuals presumed to Chemistry. as well as HBV. Abacavir is well absorbed after oral administration Adverse effects Lamivudine has an extremely favorable with a bioavailability between 76 and 96%. to the diphosphate by HIV-1 and HIV-2.108 Antiviral Agents Lamivudine the conversion of abacavir to its active metabolite. The presence of HLA-B5701 has been The phosphorylation pathway of abacavir differs from associated with elevated odds of developing abacavir that of all other nucleoside analogues. This may largely be attributed to the low or multiple doses. is phosphorylation to abacavir activity The chemistry and mechanism of action of monophosphate by adenosine phosphotransferase. plus 300 mg lamivudine). Abacavir Clinical indications Abacavir is used in combination HN with other anti-HIV agents. (1S. which undergoes two Lamivudine has significant activity in vitro against both subsequent phosphorylations.23 mmol l1) against clinical isolates of HIV-1. administration of abacavir following a hypersensitivity methanol. This lamivudine have been described previously in section step is followed by deamination by a cytosolic enzyme ‘Lamivudine’ under ‘Therapeutics for Hepatitis’.5 h. The first step in hypersensitivity reaction. However. rash. Systemic manifestations can include fever. fatigue. After single toxicity profile.

Emtricitabine is eliminated by a is approximately 25%.0003–0. tenofovir is altered in patients with renal impairment. the EC50 ranges from 0. [(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphi- Against HIV-2. 9- for laboratory and clinical isolates of HIV-1 in cell culture. respectively.5 to 2. after peak plasma concentrations occurring at 1–2 h post dose.64 mmol l1 (0. an acyclic nucleoside phosphonate (nucleotide) emtricitabine are similar to that of lamivudine. Emtricitabine is a component of Truvada tenofovir disoproxil nor tenofovir are substrates of (a fixed-dose combination of emtricitabine and tenofovir CYP450 enzymes. Approximately 1% of NH2 patients discontinued participation in clinical trials due F to these events. incorporation into DNA. . The bioavailability of the capsules and oral solution Tenofovir triphosphate inhibits the activity of HIV-1 are 93 and 75%. The high intracellular levels of emtricitabine tripho.4 h. imum serum concentrations are achieved in 1. similar in many ways to lamivudine. respectively. plasma HIV RNA. max- secretion. 39 h. 5-fluoro-1-(2R. The active analogue of adenosine 59-monophosphate. . Tenofovir dis- triphosphate competitively inhibits reverse transcriptase by oproxil fumarate requires initial diester hydrolysis for being incorporated into the viral genome. Tenofovir has antiviral activity in vitro against 200 mg daily. Emtricitabine. The pharmacokinetics of disoproxil fumarate).5S)-[2-(hydroxy. nausea. The IC50 values of tenofovir against HIV-2 range from 1. and antiviral activity in the range of 0.007 to 1. DNA polymerases . has in vitro activity against HIV- 1 that is similar to or approximately fourfold to tenfold more potent than that of lamivudine. which are generally Emtricitabine of mild to moderate severity. nyl]methoxy]propyl]adenine fumarate (1:1). mechanism of action.5 mmol l1. mechanism of action. and antiviral O O O H activity Emtricitabine.2 mmol l1. Tenofovir is primarily excreted by disoproxil fumarate).0013–0. after multiple doses of the drug at a dose of merase . Following oral administration of a combination of glomerular filtration and active tubular single dose of 300 mg to HIV-1 infected patients. and mitochondrial DNA poly- However. by DNA chain termination. by cellular enzymes to form tenofovir triphosphate. emtricitabine.6 sphate achieved are associated with better suppression of to 4. Cmax and AUC values are 296  90 ng ml1 and 2287  685 ng h ml1. Emtricitabine is not an inhibitor of The oral bioavailability of tenofovir in fasting patients human CYP450 enzymes. The mean plasma elimination half-life of emtricitabine Tenofovir triphosphate is a weak inhibitor of mammalian after a single dose is about 8–10 h in HIV-infected patients. and rash. Emtricitabine is rapidly and reverse transcriptase by competing with the natural sub- extensively absorbed following oral administration with strate deoxyadenosine 59-triphosphate and. In vitro studies indicate that neither 200 mg capsule. Antiviral Agents 109 Emtricitabine Adverse effects The most common adverse events are headache. and causing conversion to tenofovir and subsequent phosphorylations termination in DNA chain elongation. H3C HO2C methyl)-1. The oral bioavailabil- Clinical indications Emtricitabine is used with other ity increases when tenofovir is administered after a high- anti-HIV agents. diarrhea. It is administered orally at a once-daily fat meal (40–50% fat). and Atripla (a fixed-dose the kidneys by a combination of glomerular filtration combination of efavirenz. N Tenofovir disoproxil fumarate O N O OH Tenofovir disoproxil fumarate NH2 S N N N O CO2H N O H O P O O O • Chemistry. is a fluorinated O nucleoside analogue of cytosine.9 mmol l1.158 mg ml1) Tenofovir disoproxil fumarate is (a prodrug of tenofovir). the intracellular half-life is approximately HIV-1 with IC50 values ranging from 0. converted to The cellular enzymes involved in the phosphorylation of tenofovir.3-oxathiolan 5-yl]cytosine.0  0. The EC50 for emtricitabine is Chemistry. and tenofovir and active tubular secretion.

It binds to a hydrophobic pocket adjacent to With thymidine-based NRTIs (lamivudine/zidovudine the active site of the reverse transcriptase and causes or stavudine/lamivudine). pancreatitis. and Nevirapine 219Q/E). It is administered orally once daily. acute tubular necrosis. dyspnea. The cytidine analogue select for the M184V mutation (lamivudine. renal failure. However. day). Nevirapine The M184V mutation has been shown to increase is metabolized by liver microsomes to hydroxymethyl- zidovudine susceptibility in the absence or presence of nevirapine. and vir) that lack a thymidine analogue. 300 mg regimens. It is administered orally at The most commonly observed mutations with zidovu. 70R. the following adverse events have been failure. Ser-Arg. The use of Nevirapine has a bioavailability of approximately 65%. emtricitabine in place of lamivudine would presumably Peak serum concentration of 3. the 41–210–215 com- bination is the most prevalent. (2) selection of the key 151M mutation.g. (e. 210W. 210W. and antiviral activity Nevirapine (11-cyclopropyl-5. or Ser-Gly) and generally accom- N N panied by other NAMs. 77L. 41L. Ser-Ser. In addition. H referred to as the 151 complex. Resistance to nucleoside/nucleotide analogue Nucleoside analogue-associated mutations (NAMs) develop by at least three pathways: (1) accumulation of Non-nucleoside reverse transcriptase inhibitors zidovudine or thymidine analogue resistance mutations Nevirapine (TAMs). 67N. early treatment nephrogenic diabetes insipidus. 50% of the patients with M184V also harbor the K65R. the M184V mutation emerges conformational changes that affect replication.. mechanism of action. although clinical data are limited.11-dihydro-4- Resistance patterns with earlier nucleoside methyl-6H-dipyrido[3. tablet. proximal tubulopathy. analogue. consisting of a mutation at codon 69 (typically Ser). 200 mg day1 for the first 14 days (one 200 mg tablet per dine/didanosine or stavudine/didanosine are TAMs. T69 insert) are more prevalent in didanosine- with other anti-HIV agents for the treatment of HIV-1 containing regimens than in lamivudine-containing infection. In triple-nucleoside/nucleotide regimens (tenofovir/ fanconi syndrome. approximately 4 h after a 400 mg oral dose. hepatitis. increased K65R. increased amylase. while the K65R is seen with tenofovir selection pressure. with other anti-HIV agents. 75I. TAMs and multinucleoside resistance (MNR) mutations Single-dose nevirapine is used widely in resource-limited . In clinical isolates. proteinuria. 215Y/F. lamivudine while abacavir appears to favor L74V and abdominal pain. the most common mutation selected for is mild to moderate gastrointestinal events and dizziness. The K65R is the major mutation selected in vitro identified during post-approval use of tenofovir.4]diazepin-6-one) analogue combinations (zidovudine or stavudine with is a reverse transcriptase inhibitor of HIV-1. Resistance patterns with new nucleoside/nucleotide Adverse effects The most common adverse reactions analogue combinations With abacavir/lamivudine as seen in a double-blind comparative controlled study were backbone. lamivudine) The resistance profiles seen with earlier its mechanism of action is different from nucleoside nucleoside analogue combinations are well characterized. 215Y/F and 67N. The M184V Clinical indications Nevirapine is used in combination mutation may increase sensitivity to tenofovir. and 116Y. two TAM N pathways have been observed: 41L.2-b:29. then 400 mg day1 (two daily 200 mg tablets). zidovudine resistance mutations and without regard to which TAM combination is present. M184V/I followed by the L74V mutation at treatment However.110 Antiviral Agents Clinical indications Tenofovir is used in combination (Q151M. Chemistry. renal insufficiency. 70R. liver enzymes. and (3) the 69 insertion N complex. [1. followed by an insertion of two or more amino acids (e. 219Q/E/N/R.. whereas TAMs are slower to arise.39-e]. of these. allergic by tenofovir alone or in combination with abacavir or reaction.4 mg ml1 is achieved yield similar results.g. failure has been associated with a high frequency of M184V. lactic acidosis. hypophosphatemia. fol- O lowed by the mutations 62V. lamivudine/didanosine or tenofovir/lamivudine/abaca- increased creatinine. rapidly. and emtrici- tabine).

1-benzoxazin- N 2-one] is a non-NRTI that can be administered once N daily.25 mmol l1. headache.4 h. resulting in an increase and susceptibility In addition. and human cellular DNA polymerases . and appears rapidly. F F Delavirdine F Cl O Delavirdine N O O H S O HN HN Chemistry. myalgias. and antiviral concentration of efavirenz is approximately 1.4-dihydro-4 (trifluoromethyl)-2H-3. mechanism of action. and diarrhea have also been associated with mutations at codons 181 and 188. . It is infection. Resistance Changes in two sets of amino acid residues Efavirenz (100–110 and 180–190) in the reverse transcriptase gene confer resistance to nevirapine. In addition. The 90–95% inhibitory Chemistry.7– activity Delavirdine (1-[5-methanesulfonamido-1H. mechanism of action. fever. Antiviral Agents 111 settings to prevent mother-to-child transmission of HIV Adverse effects Delavirdine administration is infection. which is associated with blistering. HIV-2 reverse transcriptase. . to >60 mmol l1. Its with other antiretroviral agents for the treatment of HIV-1 mechanism of action is similar to that of nevirapine. 25 nmol l1. Activity is mediated predominately by H O noncompetitive inhibition of HIV-1 reverse transcriptase. and inappropriate behavior have been reported in 1 or 2 patients per 1000. or ulceration (1%). Adverse effects The most common adverse effects Resistance Delavirdine resistance can be generated include the development of a nonpruritic rash in as rapidly both in vitro and in vivo with the codon change many as 50% of patients who received 400 mg day1. Nevirapine monotherapy is associated with resistance most frequently appearing at Efavirenz codon 181. and  are not inhibited by efavirenz. Clinical indications Delavirdine is used in combination Resistance Resistance to efavirenz is caused by with other anti-HIV agents. Delavirdine resistance can be conferred by fatigue. . as seen with other non- administration of drug. Combination therapy has resulted in a 150-fold absorbed rapidly when given orally with bioavailabilty of or greater decrease in HIV-1 RNA levels. Inhibitory concentrations for human DNA desquamation. indol-2-yl-carbonyl]-4-[3-(1-methylethylamino) pyridinyl] piperazine) is a second-generation bis (heteroaryl) Clinical indications Efavirenz is used in combination piperazine licensed for the treatment of HIV infection. associated with a maculopapular rash. identified at 236. vomiting. nucleoside analogue. Delavirdine is metabolized by the liver with an elimination T1/2 of approximately 1. and antiviral N activity Efavirenz [(S)-6-chloro-4-(cyclopropylethynyl)- N 1. Other side effects are less common. It has an Adverse effects The most common adverse events are inhibitory concentration against HIV-1 of approximately skin rash (25%). non-nucleoside analogue. It is administered at 1200 mg mutation in the reverse transcriptase gene as with other day1 (two 200 mg tablets three times a day). moist 0. delusions polymerases are significantly higher. nausea. >60%.

integrase inhibitor with potent in vitro activity against and reduces a three times a day schedule to a twice HIV-1 (IC95 of 33 nmol l1) in the presence of 50% daily or even a once daily regimen.(S) -([N -(2 quinolycarbonyl)-Lasparginyl] Adverse effects amino butyl)-4aS. plasma concentra- tions decrease from Cmax in a biphasic manner. three mutational The clinical efficacy of saquinavir is limited by poor patterns were described (N155H. This allows for decreased dosage. The clinical implications of these find. human serum. Y143R/C). Peak plasma concentrations of In 9 of 41 patients failing raltegravir. Saquinavir (cis-N-tert-butyl-decahydro-2[2(R)-hydroxy- 4 . Saquinavir is boosted with ings are unknown. The pharmacokinetic data for ralte- gravir are supportive of twice daily administration. Saquinavir inhibits serious adverse events was less than 3% across arms. N N N H O H OH O O Clinical indications H2N NH Raltegravir received priority approval from the FDA (October 2007) for treatment of HIV-1 infection in com- bination with other antiretroviral agents in treatment- experienced patients with evidence of HIV-1 replication despite ongoing antiretroviral therapy. capsule) enhances efficacy. twice daily Chemistry. with a Saquinavir half-life of approximately 1 h for the initial () phase and an apparent half-life of approximately 7–12 h for the Saquinavir terminal () phase. mechanism of action.3 . and antiviral activity protease inhibitor class. and. oral bioavailability but improved formulation (soft-gel rarely. It is active against a wide range of wild. detected. They are Raltegravir a potent component of HAART regimens. The combination with ritonavir Raltegravir. Q148K/R/H. a structural analogue of a class of compounds allows the boosted protease inhibitor to maintain pro- with a distinct diketo acid moiety. No lipid HIV-1 and HIV-2 at concentrations of 10 nmol l1 and is abnormalities have been reported so far with raltegravir. Approximately 7–14% of the raltegravir H N dose is excreted unchanged in urine. currently O N O available protease inhibitors are metabolized in the liver O by the cytochrome P450 3A4 (CYP3A4) enzyme system. is a novel HIV-1 longed blood levels. Ritonavir is the most powerful enzyme inhibitor in the Chemistry. Oral bioavailability is approximately 30% with exten- Resistance sive hepatic metabolism. and antiviral activity with or without food.phenyl . peripheral lipodystrophy. mechanism of action. the rate of tidomimetic HIV protease inhibitor. synergistic with other nucleoside analogue as well as selected protease inhibitors. 100 mg twice a day of ritonavir to improve its . with median Tmax values in the fasting state of about 1 h. The dosage of raltegravir is 400 mg administered orally. Raltegravir is absorbed rapidly. Long-term HAART containing protease inhibitors has type and multidrug-resistant HIV-1 clinical isolates and been most strongly associated with syndromes cha- has potent activity against viruses that use CCR5 and/or racterized by dyslipidemia. It is metabolized by hepatic glucuronidation and has no effect O H on CYP3A4. and insulin resistance. no mutations were 35 mg ml1 are obtained following a 600 mg dose.112 Antiviral Agents Integrase Inhibitors Protease Inhibitors Raltegravir Protease inhibitors are used in combination with other anti-HIV agents for treatment of HIV infection. The concept of boosting H H N N involves pharmacokinetic drug interactions. 8aS]-isoquinoline-3[S]-carboxyamide Side effects (mostly mild to moderate) were seen with similar methanesulfonate) is a hydroxyethylamine-derived pep- frequency in the raltegravir and placebo arms. CXCR4 coreceptors for entry. Protease inhi- O OH F bitors are used in combination with ritonavir as the N N N boosting protease inhibitor. while in 32 of 41 patients.

with peak plasma levels of approximately 1. Nelfinavir ability of 60% and achieves peak plasma concentrations of 12 mmol l1 after a 800 mg oral dose.6. indinavir inhibits 90% of HIV isolates.10R. Resistance Chemistry.7. O . 8R. H N N N N O H H S O OH Resistance N Mutations at codon sites 90 and 48 of the protease gene result in approximately a 30-fold decrease in susceptibil- ity to saquinavir. The plasma half-life is O approximately 3 h. At Nelfinavir concentrations of 100 nmol l1. and antiviral activity Ritonavir (10-hydroxy-2-methyl-5[1-methylethyl]-1[2-(1- Indinavir methylethyl)-4-thiazo lyl]-3. Abdominal discomfort. 11R)]) is an HIV protease inhibitor with activity in vitro against HIV-1 laboratory strains (0. mechanism of action. has been reported infrequently.1- codon 82 are the most common. 5 thiazolylmethylester. Adverse effects Although indinavir is well tolerated. diarrhea. O N H Adverse effects Adverse effects include nausea. OH HO H N Resistance Indinavir resistance develops rapidly with monotherapy N and occurs at multiple sites. but all occur at a low frequency.dioxo-8. and antiviral activity Ritonavir has cross-resistance to indinavir. and headache. Antiviral Agents 113 bioavailabilty and efficacy even against saquinavir-resis. Chemistry.12-tetra azatridecan-13-oic-acid. including diarrhea and O O nausea. It is synergistic when administered with N OH OH nucleoside analogue. Oral bioavailability is approximately N H N 80%.8 mmol l1 N after 400 mg administered every 12 h. commonly encoun. dimethylethlaminocarbonyl)-4-(pyridin-3-yl) methylpipera- zin-1-yl]-4[S]-hydroxy-2[R]-phenylmethyl pentanamide} is a peptidomimetic HIV-1 and HIV-2 protease inhibitor. mechanism of action.4. Indinavir is rapidly absorbed with a bioavail. S tered adverse effects include indirect hyperbilirubinemia O (10%) and nephrolithiasis (5%). [5S-(5R.11-bis[phenylmethyl]- Indinavir 2.02–0. Mutations at Indinavir {N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1. Codon 82 is a common mutation in indinavir-resistant HIV isolates. Ritonavir tant HIV strains. Ritonavir Adverse effects S Adverse effects are minimal with no dose-limiting toxi- N cities. The extent of resistance is H directly related to the number of codon changes in the N HIV protease gene.15 mmol l1).

Fosamprenavir Fosamprenavir Resistance Cross-resistance to other protease inhibitors. (tert-butyl-carboxamide methanesulfonic acid salt) is Also. 39S)]-2-[20-hydroxy.7 h. .5 h). another peptidomimetic HIV protease inhibitor.2R)-3-(4-amino-N isobutylbenzenesulfonamido)-1- Adverse effects benzyl-2-hydroxypropylcarbamate. and antiviral activity Adverse effects Nelfinavir [3S-(3R.2R)-3-[[(4-aminophenyl) sulfonyl](isobutyl) S amino]-1 benzyl-2-(phosphonooxy) propylcarbamate mono- O N N calcium salt]. Adverse effects Nelfinavir is well tolerated with mild gastrointestinal complication reported. and I84V. After administration of a single dose of fosamprenavir to HIV-1-infected patients. I47V. and abdominal hydroxyphenyl)pentyl]-decahydroiso-quinoline-3-N.5 and 4 h (median 2. is not common. The drug is metabolized by acid substitutions primarily at positions V32I. mechanism of action. Amprenavir is metabolized in the liver by the cytochrome P450 3A4 (CYP3A4) enzyme system. is an inhibitor of human HIV protease. and antiviral activity plasma elimination half-life of amprenavir is approximately Amprenavir is a hydroxyethylamine sulfonamide peptidomi.2–10 mg ml1 are achieved after dosages of 600–1200 mg. H OH Fosamprenavir is rapidly hydrolyzed to amprenavir by NH2 enzymes in the gut epithelium. as well as mutations in the p7/p1 and p1/p6 Gag and Gag-Pol polyprotein cleavage sites. tration of 10–20 nmol l1. though. Fosamprenavir selects for amprenavir-associated muta- Amprenavir is metabolized in the liver by the cytochrome tions on treatment failure. indinavir. a prodrug of amprenavir [(3S)-tetrahydro- O O O furan-3-yl (1S. mechanism of action. metric with a structure identified as (3S)-tetrahydro-3-furyl N-(1S. diarrhea. particularly saquinavir. vomiting. I50V. events (nausea. hepatic microsomes. It is active at a concen- Side effects profile is similar to that of amprenavir.114 Antiviral Agents Chemistry. pain/discomfort). Nelfinavir is Genotypic analysis of isolates from treatment-naive orally bioavailable at approximately 40%. incidence. Resistance life is 7–10 h. 8aR. M46I/L. or ritonavir. at a much lower P450 3A4 (CYP3A4) enzyme system. and antiviral activity O Fosamprenavir. I54L/M. mechanism of action. 7. which are mild to moderate in severity. CSF concentrations are significant. S O N N H O O NH2 P Amprenavir HO OH Amprenavir Chemistry. The most common adverse events are gastrointestinal 39-phenylthiomethyl-49-aza-59-ox-o-59-(20methyl-39. Inhibitory concentrations of HIV-1 are in the range of Resistance 20–50 nmol l1. 4aR. The plasma half. The oral bioavailability is >70% and peak plasma concentrations of 6. achieving patients failing amprenavir-containing regimens showed peak plasma concentrations of 2 or 3 mg following a mutations in the HIV-1 protease gene resulting in amino 800 mg dose every 24 h. It has anti-HIV-2 activity. the peak concentration occurs between 1. skin rash can occur in patients on amprenavir. The O most frequently demonstrated site of mutation is at O O O codon 30. The Chemistry. 229S.

Tipranavir V82A/C/F/S/T. mechanism of action. Adverse effects mino)-3(S)-hydroxy-5-phenyl-1(S)-benzylpentyl)-3. increased in combination with an A71V mutation.8S. The hyperbilirubinemia is reversible upon discon- of immature.6-dihydro-2-pyrone sulfonamides. The muta- bits the metabolism of lopinavir. the mean EC50 values of lopinavir lipodystrophy in some patients. vir have been selected in vitro and obtained from patients Lopinavir is metabolized by CYP3A.9S. I84V.G48V. the cholesterol and triglycerides. resulting in the production (UGT). O OH Atazanavir is rapidly absorbed with a Tmax of approxi- mately 2. and antiviral activity H O H N N N O Tipranavir [2-Pyridinesulfonamide. It has some activity Resistance against HIV-2 strains. Lopinavir peak plasma concentra. Most common adverse events are nausea.6-dimethylphenoxy)-acetyla.12S)-3. be affected by the presence of three or more of the following nelfinavir. tinuation of atazanavir. I54L/T/V. diarrhea. Atazanavir may cause abnormal lated with ritonavir at 4:1 ratio (Kaletra).5. L24I. However.5 h. Antiviral Agents 115 Lopinavir Chemistry. Chemistry. I47V. N88S. Atazanavir exhibits anti-HIV-1 O O H activity with an EC50 in the absence of human serum of HN N N N O 2–5 nmol l1 against a variety of laboratory and clinical H HIV-1 isolates in vitro. plasma levels of lopinavir. L33F.12-Bis(1. amino acid substitutions in protease at baseline: L10F/I/R/ V. N-[3-[(1R)-1-[(6R)- O N N H H 5. mechanism of action. and M46I.1-dimethylethyl)- Lopinavir 8-hydroxy-4. The most common adverse event in patients is the asymp- methyl-2(S)-(2-oxo(1. In the presence electrocardiogram findings. K20M/N/R.3-diazaperhydroinyl)butanamin)] tomatic elevations in indirect (unconjugated) bilirubin is an inhibitor of the HIV protease. against HIV-1 laboratory strains ranges from 65 to 289 nmol l1 (0. and saquinavir). A71V. viral isolates with the I50L mutation are phenotypically Resistance resistant to atazanavir but show in vitro susceptibility to Virologic response to lopinavir/ritonavir has been shown to other protease inhibitors (amprenavir. and antiviral activity Lopinavir [N-(4(S)-(2-(2. and lipodystrophy. .10.6. and antiviral activity Atazanavir [(3S. noninfectious viral particles. ritonavir. treated with atazanavir or atazanavir/ritonavir. thereby increasing the tions associated with resistance to atazanavir are I50L. Atazanavir-resistant clinical isolates from treatment-naive harbored the I50L mutation Adverse effects (after an average of 50 weeks of atazanavir therapy). mechanism of action. prevents cleavage related to inhibition of UDP-glucuronosyl transferase of the Gag-Pol polyprotein. Tipranavir F F Atazanavir N F Atazanavir O H N O S O O N OH O OH Chemistry. The mean elimination half-life of atazanavir in healthy volun- teers and HIV-infected adult patients is approximately 7 h. and I84V. and ritonavir inhi. lopinavir. often. Atazanavir is metabolized in the liver by the cytochrome P450 3A4 (CYP3A4) enzyme system.13-pentaazatetradecanedioic acid dimethyl ester.11-dioxo-9 (phenylmethyl)-6-[[4-(2-pyridi- nyl)phenyl]methyl]-2. HIV-1 isolates with a decreased susceptibility to atazana- tion occurs approximately 4 h after administration. sulfate (1:1)] is an azapeptide inhibi- tor of HIV-1 protease.04–0. and of 50% human serum. increased serum glucose.18 mg ml1).6-dihydro-4-hydroxy-2-oxo-6-(2-phenylethyl)-6-pro- O O pyl-2H-pyran-3-yl]propyl]phenyl]-5-(trifluoromethyl)] is a nonpeptidic HIV protease inhibitor belonging to the class of 4-hydroxy-5. indinavir. M36I. It is coformu.

lipodystrophy. G73. and hepatitis. A71T. Tipranavir is predominantly metabo. vailability of a single 600 mg dose of darunavir alone and Response rates are reduced if five or more protease inhibitor. adult patients. increased cholesterol. Darunavir is primarily not given concomitant enfuvirtide with tipranavir/ritonavir. phase IIb trial. with EC50 ranging Darunavir. K45I. L10F. to darunavir and harbored three to six of the following indinavir.3-b]furan-3-yl ester monoethanolate. regardless of causality or frequency. so does the number of possible effective . coadministered with 100 mg ritonavir. daily. K55Q. lopinavir. increased cholesterol. after coadministration with 100 mg ritonavir twice daily associated mutations are present at baseline and patients are was 37 and 82%. Darunavir. V77I. Other side effects are increased tri- HIV-1 isolates that were 87-fold resistant to tipranavir glycerides. R41E/S/T. M36I. surgery or other medical conditions. at furo[2. Tipranavir-resistant viruses selected for in vitro have K70E. with other anti-HIV agents. respectively. were diarrhea. were selected in vitro by 9 months and contained 10 increased glucose. Resistance and nasopharyngitis. coadministered with 100 mg ritonavir twice treatment of HIV-1 infected adult who are highly treat. atazanavir. coadministered with 200 mg of ritonavir. atazanavir. and increased liver enzyme levels. or who are Adverse effects receiving medications known to increase the risk of The most common treatment-emergent adverse events bleeding such as antiplatelet agents or anticoagulants. V82L. was absorbed following oral administration with a ment-experienced with evidence of viral replication. I84V. such as those with HIV-1 strains resistant Tipranavir/ritonavir should be used with caution in to more than one protease inhibitor. the amino acid substitution V32I devel- navir. and increased triglycerides. The absolute oral bioa- have HIV-1 strains resistant to multiple protease inhibitors. Ritonavir inhibits CYP3A. and antiviral activity HIV-1 and clinical isolates in vitro. nelfinavir. lopinavir. V32I. indinavir. used in combination with other anti-HIV agents for the Darunavir. protease mutations that developed in the following order: L33F. T74S. As the number of effective drugs increases. It is an steady state following a dose of 500/200 mg twice daily inhibitor of the HIV protease. L33. and oped on darunavir/ritonavir (600/100 mg twice a day) in ritonavir but remain sensitive to saquinavir.2 to 8.2R)-3-[[(4-aminophenyl)- The effective mean elimination half-life of tipranavir/ sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenyl- ritonavir in healthy volunteers and HIV-infected adult methyl) propyl]-carbamic acid (3R. has from 0. nelfinavir. against laboratory strains and clinical isolates of HIV-1 lized by the CYP 3A4 enzyme system. I13V. patients who may be at risk of increased bleeding from trauma.8 and 6.5–4 h.to 21-fold decreased susceptibility 90% of HIV-1 isolates resistant to amprenavir.116 Antiviral Agents Tipranavir inhibits the replication of laboratory strains of Chemistry. amino acid substitutions S37N/D.7–5. metabolized by CYP3A. Resistance A71V.0 h. lipodystrophy. In clinical trials tipranavir Darunavir-resistant virus derived in cell culture from had less than fourfold decreased susceptibility against wild-type HIV had 6. in the form of darunavir ethanolate. and I54V/T. and laboratory strains of HIV-2 with median EC50 values Tipranavir. of HIV infection in antiretroviral treatment-experienced cranial hemorrhage with the use of tipranavir/ritonavir. or I85V in the protease. is ranging from 1. In decreased susceptibility to the protease inhibitors ampre. Darunavir exhibits activity with a light meal.5 nmol l1 (0. and L89. nausea. Adverse events include rash.07 mmol l1 (18–42 ng ml1). (>10%) reported in the de novo subjects. I47. greater than 30% of virologic failure isolates and substi- tutions at amino acid position I54 developed in greater than 20% of virologic failure isolates. thereby increasing the plasma concentrations of daruna- Adverse effects vir when given in combination. ritonavir.0 ng ml1). Other substitutions Darunavir that developed in 10–20% of darunavir/ritonavir virolo- Darunavir gic failure isolates occurred at amino acid positions I15. or Tmax of approximately 2.6aR)-hexahydro- patients is approximately 4. or saquinavir.03 to 0. the following chemical name: [(1S. is indicated for the treatment There have been reports of both fatal and nonfatal intra. headache. mechanism of action. respectively.3aS. H O O O O O H S • C H OH Future Prospects in HIV Therapeutics H H O N N 2 5 H H OH The simplification of HAART regimens has been a high NH2 priority for many years.

Journal of discovered 29. Baba M. (2002) New helicase-primase new NRTIs in phase II clinical trials are MIV-310. The New England Journal of substituted thymidine analogues with potent antiviral Medicine 340: 1255–1268. inhibitor as drug candidates for the treatment of herpes simplex SPD754. Breuer J. a novel thymidine compounds discovered as anti-HIV agents targeted at analog. resistance profile when compared to other thymidine Jefferson TO. but as a consequence of by Public Health Service grant AI-38204 from NIAID. and L-d4FC. 16(1): 17–23. the previous edition. Gut 57(1): 105–124. Seminars in Pediatric Infectious Diseases 13(1): 12–21. Nature Medicine 8(4): 392–398. combinatorial chemistry. Soong SJ. and Rivetti D analogues and maintains activity against multidrug resis. inhibitor of HIV-1 replication and is much less inhibitory Desmond RA. Leung CH. Dutschman GE. and to mitochondrial DNA synthesis and cell growth in cell Whitley RJ (2006) Enteroviral meningitis: Natural history and outcome cultures than its progenitor stavudine. New drug discovery strategies attempt at circumvent- ing the current drug resistant problem by focusing on either novel targets or new compounds capable of sup. Antiviral Chemistry & Chemotherapy 16(4): 217–221. Balfour HH. Antimicrobial Agents and Chemotherapy 50(7): 2409–2414. Cheng YC. McMahon MA. et al.. et al. Cochrane Database of Systematic Reviews. Jilek BS. Johnson RW. structurally related to stavudine. No. phase I and II clinical trials anticipated in 2008. infections will be available with the advent of modern and Whitley RJ and Kimberlin DW (2005) Herpes simplex: Encephalisits children and adolescents. Antiviral Agents 117 regimens. 2. reduced toxicity and shows a unique activity profile against drug- resistant mutants. Jr. et al. There are several nucleoside analogues in pre. There is increasing number of 29. Not only is their Work performed and reported by the authors was supported medication burden simplified. In: Lederberg J. Rong H. It also has a unique Dworkin RH. Journal of Clinical Virology 30: 115–133. Further Reading pressing HIV strains that are resistant to current Arvin AM (2002) Antiviral therapy for varicella and herpes zoster. San Diego: It is anticipated that new and effective treatments for viral Academic Press. improved adherence to therapy. Clinical Infectious Diseases 44: S1–S26. and exerts persistent antiviral activity even after B. virtually any step in the replicative cycle of the virus Tanaka H. metabolite accumulates in cells much longer than stavu. they should experience Portion of the current article were reproduced from better control of HIV and thus reduced morbidity. The triphosphate of pleconaril therapy. Dusheiko G and Antonakopoulos N (2008) Current treatment of hepatitis dine. and Huang DB (2005) Advances in antiviral therapy. Other Kleymann G. . Accortt NA.39-didehydro-39-deoxy-49-ethynylthymi. The trend toward fixed-dose combinations and Acknowledgments once-daily dosage forms of many antiretroviral drugs has provided welcome relief to patients. Fischer R. Clinical Virology 34(S1): S147–S150. and Li Y (2005) New targets and activity and less cytotoxic. it appears to have potent entecavir – effects on HIV-1 replication and resistance. 2nd edn.) Encyclopedia of Microbiology. Betz UAK. The New antiviral activity in treatment-experienced patients with England Journal of Medicine 356: 2614–2621. written by Richard Whitley. removal of drug from culture. It is currently in preclinical studies with in healthy adults. and computer-aided design of Wu JJ. Kumamoto H. Pang KR. (2007) The HBV drug in phase II trials is TMC125. Demicheli V. Art. compounds with greater specificity targeting on viral life Dermatologic Clinics 23: 313–322. vol. Summary Whitley RJ (2000) Antiviral agents. dine. (eds. (1999) Antivirals (non-AIDS). An example is the recently inhibitors of HBV replication to combat drug resistance. Notably are the novel 49. (2007) Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of are treatment naive.39-didehydro-39-deoxy-49-ethynylthymidine. Antimicrobial Agents and Chemotherapy 51(11): 3870–3879. cycle.: CD001265. Jones M. clinical and clinical studies. Seminars in Pediatric Infectious Diseases improved technology. (2006) Neuraminidase inhibitors for preventing and treating influenza tant HIV strains. et al. A new non-nucleoside analogue disease. Ying CX. based on molecular biology. Talley L. et al. Di Pietrantonj C. Brennan TP. inhibitors. Haraguchii K. Villano SA. is a more potent De Clercq E (2004) Antiviral drugs in current clinical use. Issue 3. and Cheng YC (2005) 49-Ethynylstavudine (49-Ed4t) has potent anti-HIV-1 activity with and novel targets in development. (2007) Recommendations for the management of herpes zoster. resistance mutations to this drug class or in patients who Paintsil E.

phylogeny Inference of evolutionary relationships elongate. or highly saline environments. simple organic and inorganic materials. orthologues Genes or proteins with identical or very DNA replication Process of copying parental strands similar functions in different organisms inferred from of DNA to daughter strands. Baltimore. usually with an optimum hyperthermophiles. with nuclei and includes all eukaryotes (plants. between 0 and 20  C. Includes many but not all Archaea. using DNA polymerases phylogenetic or experimental studies. including all members of euryarchaeota A phylogenetic group. usually containing domains. MD. membrane-bounded nucleus. of both the Archaea and Bacteria. prime. or kingdom. which grow in Archaea One of three phylogenetic groups. or nucleotide or amino acid sequence differences among domains. accessory proteins such as initiation and termination haloarchaea Halophilic microorganisms belonging to factors. control the process of protein folding and turnover in the methanogens Anaerobic microorganisms that use cell. of life at the highest level that possesses cells orthologues. Defining Statement Archaeal Genomics Introduction Biotechnological Applications of Archaea Historical Recognition Conclusion Archaeal Ecology and Environmental Biology Further Reading Novel Molecular and Genetic Characteristics of Archaea Glossary halophiles Salt-loving organisms. at the highest level. domains. Bacterial cells are metagenomics Large-scale sequencing of DNA prokaryotic and lack nuclei. extremophiles Microorganisms that grow and flourish thermophiles Heat-loving organisms that grow in environments inhospitable for the growth of most optimally at elevated temperatures. and terminate the process. prokaryotes Microorganisms that do not possess a fungi. like the Archaea. usually defined by the Bacteria One of three phylogenetic groups. the archaeal domain. above kingdom (or phylum). Archaeal saturation. middle temperature range. isolates. of hydrogen. temperature optima above 50  C. and some thermophiles and optimally at low temperatures. and other accessory proteins that initiate. All rights reserved. environment. or kingdom. animals. crenarchaeota A phylogenetic group. or ambient temperature on Earth’s surface (20–42  C). and in isolated or cloned from microbial communities in the contrast to Eucarya. among microorganisms based on the extent of Eucarya One of three phylogenetic groups. such as acetate. cells are morphologically prokaryotic cells and lack mesophiles Organisms that grow optimally in the nuclei. and the Archaea characterized by many hyperthermophilic generate methane as the primary end product. the Archaea characterized by all halophiles and psychrophiles Cold-loving organisms that grow methanogens. and carbon dioxide for their metabolism. Archaea (overview) S DasSarma. and protozoa). usually with other organisms. USA ª 2009 Elsevier Inc. University of Maryland Biotechnology Institute. of life (along with the Bacteria and Eucarya) at salt concentrations in excess of sea salinity up to the highest level. J A Coker. which may be analyzed for genes and chaperones Molecular machines that assist and genomes using computational analysis. and P DasSarma. 118 . hyperthermophiles genomics An approach to studying organisms by grow optimally above 80  C. determining the complete sequence of a genome transcription The process of synthesizing messenger followed by computational and functional analysis of the RNA from DNA using RNA polymerase and other encoded genes.

They also have unique features. cold. Stadtman and followed the devel- 0. Ehrenberg in 1838. Although Archaea are tional century. consisting of cells methanogens from complex microbial communities was bounded by a single lipid membrane and lacking a finally accomplished in the mid-twentieth century nucleus. A second major group of Archaea. C. hot. Ichi. and into proteins using the protein synthesis machinery. like their vannielii. and a few have phototrophic capabilities and 1868. which contain branched chain isopre. Archaea of the microscope. Archaea dominate many extreme environments and are widespread in many common environments. Some of the earliest named methanogenic translation) that are simplified versions of their eucaryal species were Methanobacterium formicicum. or anaerobic trenches kilometers below the ocean surface. Barker. Archaea have been detected in the mammalian gastroin. Woese and coworkers in 1977. A role for microbes in others are heterotrophs and grow on complex organic methanogenesis was first described by A. Eucarya. Archaeal genomes are K. with anaerobic methanogenic species were recognized as a coherent group in the tree of life giving rise to marsh gas (combustible air). The Archaea are prokaryotic microorganisms that are mem. acidic. Methanococcus counterparts. and T. However. transcription. H. transcription. but their classification as Archaea required an addi- can use light energy for growth. After the advent of microscopes in the fifteenth cen- testinal tract. including thermostable DNA polymerases of the third domain of life. Abbreviations PCR polymerase chain reaction ANME anaerobic methane-oxidizing Archaea rRNA ribosomal RNA GINS Go. Methanogenic discovered until the late twentieth century. Many Archaea are chemoauto. Woese and coworkers in the latter third of the twentieth century (Figure 1). Archaea have archaeal halophiles producing red and pink hues in been detected in nearly all environments examined using hypersaline ponds used to harvest salt from the sea. lar to nucleated eucaryal cells. R. described as using small ribosomal RNA (rRNA) sequence compari. Archaea have many useful qualities that have been translated into applications in Archaea are prokaryotic microorganisms that are members biotechnology. through the work of microbiologists. other factors. a scientific term originally introduced trophs and can grow on simple inorganic chemicals. the halophilic noid units in fatty chains linked to a glycerol-1-phosphate Archaea (or haloarchaea). most well-characterized archaeal species. were identified as agents of food . alkaline. Their information Historical Recognition transfer systems (DNA replication. and San TBP TATA-binding protein MCM minichromosome maintenance TFB transcription factor IIB PCNA proliferating cell nuclear antigen tRNA transfer RNA Defining Statement head group via ether linkages. Many microorganisms studied by early microbiologists were Archaea. Nii. Béchamp in materials. by C. The isolation of pure species of prokaryotic in their morphology. but their fundamental distinction from common Bacteria escaped notice until the pioneering Introduction phylogenetic work of C. distinct from be called Archaea were evident well before the invention the other two domains – Bacteria and Eucarya. known as ‘bacteria’. By contrast. Schnellen. including 16S described in ancient texts. and trans- lation) are simplified versions of their eucaryal counterparts. membrane lipids. prokaryotic microorganisms became collectively been identified thus far.5–5. A. and Methanosarcina barkeri. were not where they are sometimes dominant. aminoacyl–tRNA synthetases. culture-independent molecular techniques. near hydrothermal marine vents located in that are very salty. metabolic activities of microorganisms that would later bers of the third branch (or domain) of life. R. early as the time of the Roman empire. Archaea (overview) 119 translation The process of decoding messenger RNA including ribosomes. some of which grow optimally above Archaea have been cultured from extreme environments 100  C. and methodology. distinct from Bacteria and for polymerase chain reaction (PCR). includ- ing the mammalian gastrointestinal tract. and salt-loving sons by C. some of their molecular characteristics are simi. but no archaeal species causing disease has tury. G.75 Mbp circles and include genes for information opment of specialized anaerobic microbiological transfer machineries (DNA replication. hyperthermophilic rRNA sequencing.

Crenarchaeota Protreobacteria positive Halobacterium Ciliates Thermoproteus Methanococcus Chloroplast Pyrodictium Thermoplasma Thermococcus s yru Cyanobacteria Marine nop Crenarchaeota Pyro. also shared with some thermophilic species. and certain diverse microorganisms. M. center of the protein synthesis machinery. In the early branched isoprenoids (Figure 2). Woese and his students intensively Studies of membrane lipids in the 1960s by M. and Halobacterium salinarum. F. respectively. However. the combination of which gives these microorganisms became possible to recognize Archaea as a distinct phy- the ability to grow phototrophically. though widely employed at 1968. when salting was widely used for preserving fish ether bonds linking hydrocarbon side chains with methyl- and meats before the advent of refrigeration. (a) Typical diphytanyl glycerol diether phospholipid without a head group. tha Flagellates lobus Me Flavobacteria Trichomonads Microsporidia Thermophiles Diplomonads Figure 1 An evolutionary tree. suggesting a Taxonomy of these halophilic Archaea continues to be common relationship between these metabolically controversial. two kingdoms (Euryarchaeota and Crenarchaeota) and representative genera are shown.120 Archaea (overview) Bacteria Archaea Eukarya Green Animals nonsulfur Mitochondria Slime molds Euryarchaeota Fungi Methanosarcina Plants Gram. Stoeckenius discovered that these Halobacterium present. W. and the green portions highlight the isoprenoid hydrocarbon chains. process common to all known free-living organisms. With the advent of molecular biology in the 1970s. termed purple membrane. logenetic group. Subsequently. family. or to propose novel evolutionary region of the membrane. In eries of lipid chemistry. Many relationships among microorganisms. Halobacterium cutirubrum. especially since the order. were not originally used for taxonomic or phy- species contain a light-driven proton pump in a specialized logenetic grouping. as opposed to the twentieth century. the red portions highlight the glycerol-1-phosphate backbone (stereoisomer used in Bacteria and Eucarya). spoilage. (b) Dibiphytanyl glycerol tetraether lipid common to thermophiles. it tion. . carrying out a philes and methanogens were of unusual composition. these unusual characteristics of membrane lipids were ium). Kates studied small rRNAs. Petter. it became clear that were named Halobacterium halobium (Halobacterium salinar. have the ability to produce buoyant gas vesicles for flota. In both panels. haloarchaeal isolates were named glycerol-3-phosphate head group and typical ester bond Bacillus halobius ruber and Bacterium halobium by H. and subsequent isolates most other organisms. Klebahn linkages to straight-chain acyl group fatty acids found in and H. Within domain Archaea. the blue portions highlight the ether bonds. They found glycerol-1-phosphate as the head group and Woese reasoned that since all known organisms contain (a) (b) O– O– O– O– O P O P O O O O H2 O C H2 C CH C CH H2 H2 HC C C O C O H2 H2 O OH Figure 2 Characteristic archaeal lipids. the surprising discov- genera include the term bacterium rather than archaeum. emphasizing the three-domain view of life. molecules that form the catalytic and coworkers showed that those present in many halo.

growing in the 55–80  C temperature range. Using RNA fingerprinting (catalogs of K. Jannasch and M. Many ideal molecular chronometer to infer deep evolutionary new thermophilic species were identified by W. for example. Deep Lake. including Halobacterium sp. . Yellowstone National Park. especially after the discovery by RNA oligonucleotides generated with sequence-specific H. organisms to have their genomes completely sequenced. Archaea (overview) 121 small rRNAs (either 16S or 18S). and Eucarya – to emphasize Methanocaldococcus (formerly Methanococcus) jannaschii and a the existence of three fundamentally different types of handful of other microorganisms became the first free-living organisms in the tree of life (Figure 1). and others. like the Godzilla Vent in the Mid-Atlantic Ridge. (c) Wetlands and marshes. J. are a rich source of hyperthermophiles. in the north arm of Great Salt Lake. 100  C) became known through both culture-dependent O.. and in 1990 Woese. and as their central The last two decades of the twentieth century brought functions are evolving slowly. Mottl of microbial communities nucleases). (a) (b) (c) (d) (e) (f) Figure 3 Archaeal habitats. Photo courtesy of S DasSarma. California. Stetter. Photo courtesy of Australian Antarctic Data Centre. Halorubrum lacusprofundi. (d) Thermophilic mats radiating from Grand Prismatic Spring. and M. in cases of high-pressure environments. This classification was confirmed by sequence ana. onments (named ‘archaebacteria’ or ancient bacteria) on the hyperthermophilic species (growing best above 80  C or other. (b) Antarctic lakes. Photo courtesy of W Whitman. (f) Archaeal acidophilic biofilms seen at the Richmond Mine at Iron Mountain. in the Vestfold Hills where the psychrophile. with the common bacterial species grouping the original thermophilic Archaea available for molecular together on the one hand (dubbed ‘eubacteria’ or true phylogenetic analysis included only species of Thermoplasma bacteria) and uncommon species inhabiting diverse envir. For example. (e) Hydrothermal vents called ‘black smokers’ in deep-sea marine trenches. (a) Bloom of haloarchaea. Zillig. While yotic species. Wheelis proposed the new names – and culture-independent techniques. Photo courtesy of Yellowstone National Park. relationships. and Sulfolobus. Kandler. they would serve as the about exciting developments in archaeal research. Photo courtesy of J Banfield. was isolated. Utah is visible as red brine. O. where methanogens are found and studied. domain Archaea. near deep-sea hydrothermal vents (Figure 3). a deep division was discovered among prokar. Photo courtesy of D Kelley. even above lysis of rRNAs and their genes. Bacteria.

with Euryarchaeota in blue and Crenarchaeota in orange. but they generally reflect differing evolu.2 THSC1 Sulfolobales MarBenthGpC YNPFFA Crenarchaeota MarBenthGpB/DHVC1 Figure 4 An unrooted tree of the Archaea emphasizing 16S rRNA sequences obtained from culture-independent methods. The major phyla are bolded. Modified from Schleper C. However. sequences has recently led to the proposal of many new mostable polymerases that improved the PCR method. Nature Reviews Microbiology 3: 479–488.1A pOWA133 AAG Korarchaeota Nanoarchaeum equitans Thermoproteales SAGMCG-1 Desulfurococcales FFS Group I. progenote near the root of the tree of life. Jurgens G. which have nuclei surrounded by membrane and DHVE6 Euryarchaeota DHVE3 PENDANT-33 Halobacteriales MarB Group III enth Methanomicrobiales SAGMA-S/T SA1 GpE Group II ANME-1-GBa WH Rice ANME-1B CA VADIN cluster1 ANME-1-AT Thermoplasmata ANME-1A SAGMA1 DHVE1 Methanosaetaceae Methanobacteriales ANME-2A/B ANME-2C Archaeoglobales ARC1 Methanococcales ANME-3/ B Methanosarcina Methanococcoides DHVE4 Thermococcales Methanolobus/ Methanohalophylus Methanopyrus Group I. indicating environmental and metagenomic studies has greatly that further detailed studies are necessary to shed light on increased our current knowledge of the archaeal domain. ‘B’ indicates the position of bacterial outgroups. collec. the evolutionary unity of the Archaea and their A great deal of interest has recently focused on the nature distinction from the Bacteria and Eucarya are now firmly of the earliest evolutionary events giving rise to the three established. this hypoth- have largely confirmed the results of 16S rRNA-based esis is not universally accepted. with a common tions of all ribosomal proteins) as well as comprehensive sets ancestor to both Archaea and Eucarya diverging later to of predicted proteins from complete genome sequencing form these two domains (Figure 1). However. Korarchaeota. Most phylogenetic studies are consistent doms Crenarchaeota and Euryarchaeota (Figure 1). . domains of life. Many with Bacteria branching earliest from the ancestral cell or studies using orthologous proteins and genes (e. some of A combination of genome sequencing together with these branches are poorly resolved (Figure 4)..g. investigators have even proposed that the more complex tionary rates and uncertain gene histories. Named phyla and dark triangles refer to the cultured and acronyms refer to the many noncultured Archaea.1B Group I. Relationships between individual have been proposed to account for these groups. Some species do vary. as is the subdivision of Archaea into the king. and Nanoarchaeota. and Jonuscheit M (2005) Genomic studies of uncultivated archaea. several Pyrococcus species yielded novel ther. As a result. phyla. including lateral Eucarya. the true diversity of existent archaeal phyla and taxa. and many different scenarios phylogenetic analysis.1C Group I. including two additional kingdoms of Archaea.122 Archaea (overview) helping to confirm the three-domain view of life. increasing the value of Archaea for biotechnology. At about or horizontal gene transfers. Dotted lines refer to uncertainties in phylogenetic positions. The great diversity of the same time.

As a whole. group 1. Novel 16S have led to the realization that a much greater diversity of rRNA genes of terrestrial hot spring Crenarchaeota includes Archaea exists than was previously known (Figure 4). over the past 30 in these environments include marine group I. The importance of these Archaea has been case is for the population of Cenarchaeum symbiosum cells in explored through the use of 16S rRNA gene libraries and symbiotic association with the marine sponge. However. ecosystems and harbor a species richness that is 20-fold ques. it has become clear that the major. Soil environments are the Earth’s most diverse extreme environments using culture-dependent techni. Besides using growth on traditional pure cultures. diversity are made more difficult to obtain in these environ- organisms that thrive in extreme habitats (Figure 3) because ments due to their remoteness. some investigators have used geological sampling data isms quite challenging. Estimates of archaeal diver- archaeal 16S rRNA gene sequences through sequencing of sity in thermal spring environments vary in number. Culture-independent methods have estimated that up to 5% of the microbial community in soils are Archaea. and Nanoarchaeota. However. through culture- Archaeal Ecology and Environmental independent techniques. symbiotic. of microbial mats of cold methane seeps found in the world’s teristics of the common ancestor as a cellular organism with oceans. numerous novel strains have been Biology identified. However. The used as a good approximation. Sulfolobales. Sufficient quantities of the DNA of this psychrophilic approaches are not precise tools showing a full representation species were obtained for the reconstruction of a representa- of the microbial community in an environment. the marine years a combination of culture-dependent and culture. they can be tive complete genome by metagenomic sequencing. making an in-depth study of these organ. and novel genera of Thermococcales. Such studies have shown that assembled genome sequence suggested that C. benthic groups. for example in the plethora of microenvironments that dominate vent communities. the uncultured try and recreate the environment in laboratory media. with few exceptions from the environment in which the organism was obtained. Archaeoglobales. scientists have nearly all of the originally cultivated archaeal microorgan. Axinella mex- other culture-independent methods. Novel archaeal 16S rRNA sequences have been identified This method results in cell exposure to nearly natural nutri- from a variety of microbial habitats. Therefore. predominately anaerobic universal common ancestor of all life on Earth diverged to methane-oxidizing Archaea (ANME). Archaeal cells or their 16S rRNA genes have also a cytoplasmic membrane and a sophisticated translation been detected in freshwater environments and comprise up apparatus are however generally accepted today. and Korarchaeota (Figure 4). evolving from a protocellular ‘RNA world’. or whole community culture ity of known groups of Archaea are still not available as are vigorous and ongoing. or thermophilic).1A. in soils include members of the group I. A most interesting soil types. It has also been shown degree of uncertainty still exists with respect to how the last that certain Euryarchaeota. Typical Archaea found methanogenic. Thermal springs have also isms are widespread. through but cells remain sequestered to stimulate growth. a marine groups I–III (see Figure 4). with Global Distribution of Archaea the Crenarchaeota alone accounting for up to 3% in some Although Archaea were originally discovered inhabiting niches. Archaea (overview) 123 many genes interrupted by introns. ques.1B and ANME. However. In fact. ANME. it has been estimated that the Earth’s branches of prokaryotes are hypothesized to have evolved oceans sustain 1028 archaeal cells. symbiosum Archaea account for up to 60% of prokaryotic cells in certain may have ammonia oxidizing capability. archaeal cells commonly make up a substantial fluorescent-labeled probes (fluorescent in situ hybridization) fraction of the prokaryotic cell population. independent techniques has shown that archaeal microorgan. and can be detected in most common been a rich source for novel Archaea.e. marine Archaea include members of group I. A few archaeal strains have been cultivated from hydro- thermal vent communities. Although these icana. to 5% of the prokaryotic population. Archaea commonly found through recent studies using culture-independent techni. Examples of uncultured subsequently by loss of the nuclear membrane and introns. comprise up to 50% form the three separate primary lineages. . to (i. hydrothermal microenvironments.. environmental samples and probing archaeal cells using however. Korarchaeota and Nanoarchaeota). media. group II. including fresh and salt ent environments and has been successful for the enrichment water. their global distribution has been shown primarily higher than that of the ocean. hydrothermal vents and thermal springs. With over 8000 archaeal 16S rRNA gene sequences Attempts to bring novel noncultured archaeal microor- deposited in databases. the Archaea have been recognized as micro. detection of resisted laboratory cultivation. and diverse of some previously nonculturable species. Others Archaea have all been shown to be members of the two have used semipermeable chambers in which nutrients flow kingdoms Crenarchaeota or Euryarchaeota (Figure 4). predicted that Archaea do comprise a majority of cells in isms were found to be extremophilic (halophilic. and and additional simplification of their genomes. most of which have and nonextreme environments as well. Both sediments. ganisms into axenic. are the more ancient pelagic marine environments and up to 80% in some marine group. Predicted charac. Estimates of microbial Traditionally.

a diverse and Sampling the surrounding environment for possible increasing number of genera (28 at present) have been primary sources of energy can help corroborate their described (Table 1). This is often carried out by measuring from numerous environments of varying salinity and gen- the concentrations of molecules that are already known erally dominate over Bacteria and a few Eucarya at the to serve as primary energy sources (various carbon and highest salinity extremes. Table 1 Taxonomy of Archaea Euryarchaeotaa Archaeoglobales Archaeoglobaceae Archaeoglobus Ferroglobus Geoglobus Halobacteriales Halobacteriaceae Haladaptatus Halalkalicoccus Haloalcalophilium Haloarcula Halobacterium Halobaculum Halobiforma Halococcus Haloferax Halogeometricum Halomicrobium Halopiger Haloplanus Haloquadratum Halorhabdus Halorubrum Halosarcina Halosimplex Halostagnicola Haloterrigena Halovivax Natrialba Natrinema Natronobacterium Natronococcus Natronolimnobius Natronomonas Natronorubrum Methanobacteriales Methanobacteriaceae Methanobacterium Methanobrevibacter Methanosphaera Methanothermobacter Methanothermaceae Methanothermus Methanococcales Methanocaldococcaceae Methanocaldococcus Methanotorris Methanococcaceae Methanococcus Methanothermococcus Methanomicrobiales Methanocorpusculaceae Methanocorpusculum Methanomicrobiaceae Methanoculleus Methanofollis Methanogenium Methanolacinia Methanomicrobium Methanoplanus (Continued ) . Haloarchaea have been isolated ecological roles. however. etc.). Haloarchaea predominate in nitrogen compounds. family (Halobacteriaceae). hydrogen.124 Archaea (overview) With the advent of extremely high-throughput Halophilic Archaea metagenomic sequencing and single-cell genome- All salt-loving halophilic Archaea (also called haloarch- sequencing methodologies. it may be possible to per- aea) belong to the kingdom Euryarchaeota and have form complete genome sequencing of many more been classified into a single order (Halobacteriales) and archaeal organisms and model their roles in ecology.

Archaea (overview) 125 Table 1 (Continued) Methanospirillaceae Methanospirillum Unclassified Methanoregula Methanocalculus Methanolinea Methanopyrales Methanopyraceae Methanopyrus Methanosarcinales Methanosaetaceae Methanosaeta Methanotrix Methanosarcinaceae Methanimicrococcus Methanococcoides Methanohalobium Methanohalophilus Methanolobus Methanomethylovorans Methanosalsum Methanosarcina Thermococcales Thermococcaceae Palaeococcus Pyrococcus Thermococcus Thermoplasmatales Ferroplasmaceae Ferroplasma Picrophilaceae Picrophilus Thermoplasmataceae Thermoplasma Unclassified Thermogymnomonas Unclassified Aciduliprofundum Methanosphaerula Crenarchaeotaa Caldisphaerales Caldisphaeraceae Caldisphaera Cenarchaeales Cenarchaeaceae Cenarchaeum Desulfurococcales Desulfurococcaceae Acidilobus Acidococcus Aeropyrum Desulfurococcus Fervidococcus Ignicoccus Staphylothermus Stetteria Sulfophobococcus Thermodiscus Thermofermentum Thermosphaera Pyrodictiaceae Geogemma Hyperthermus Pyrodictium Pyrolobus Unclassified Caldococcus Ignisphaera Nitrosopumilales Nitrosopumilaceae Nitrosopumilus Sulfolobales Sulfolobaceae Acidianus Metallosphaera Stygiolobus Sulfolobus Sulfurisphaera Sulfurococcus Thermoproteales Thermofilaceae Thermofilum Thermoproteaceae Caldivirga Pyrobaculum Thermocladium Thermoproteus Vulcanisaeta Nanoarchaeotaa Nanoarchaeum Korarchaeotaa Korarchaeum a Major phyla (kingdoms) are listed in bold. and third column. . second column. families. genera. with subphyla following. First column contains orders.

halorhodopsin. protein. Haloarcula. dopsins. which contains Certain species of haloarchaea are able to grow anaerobi- an unusually high concentration of magnesium. nitrogen oxide. and in some cases nitrate. and cally via the fermentation of arginine. Halogeometricum. and Lake higher Eucarya. The accumulation of and hydrocarbons. some as old as 200 million years Some haloarchaea. Halococcus. Traditionally. can also mediate phototactic responses. produce a light-driven pro- isolates. reduces osmotic stress to the Photophosphorylation Oxidative phosphorylation (aerobic and anaerobic) H+ H+ H+ Cl– O2 DMSO. Halobacterium. as well as natural reduced by high salinity). in Halobacterium acts as a light- Haloarchaea are able to perform aerobic and anaerobic driven chloride pump (Figure 5). mainly KCl. microbial composition of the Dead Sea. All genera of haloarchaea are able to use effects of high salt concentrations through a process of amino or organic acids as a carbon source. which is sources utilized by haloarchaea include sugars. Species of Halobiforma. the north arm of Great Salt Lake in the mically linked to the protein bacterio-opsin. for example. known as sensory rho- from less salty environments such as coastal oceans. where isolates of Halobacterium. A third class of retinal sources such as fish sauces and animal hides. The membrane potential and proton-motive force are also used to drive many metabolic processes. respiration. and soils. Other carbon selective uptake of salts known as ‘salting in’. which is western United States (separated from the south arm by a similar to the photopigment in the visual systems of railroad causeway). in their cell membrane Haloferax. The true age of isolates from (Figure 5). The purple color is due to for haloarchaea are other neutral and alkaline hypersaline light absorption by the chromophore retinal that is che- lakes. anaerobic fermentation (substrate-level phosphorylation). swimming toward beneficial green light and away from such as Halobacterium. have yielded haloarchaeal Halobacterium. Halomicrobium. Natronococcus. and Haloterrigena have been isolated bacteriorhodopsin in Halobacterium. high levels of bacteriorhodop- ancient salt deposits is quite controversial. Under conditions with sufficiently high Magadi in the Rift Valley of Africa. halophilic Archaea. fumarate. Haloarchaea have the capability to grow by aerobic or anaerobic respiration (oxidative phosphorylation). and Halorubrum. and/or photophosphorylation (using light-driven pumps in the membrane and lemon-shaped gas vesicles for flotation). used by few nonarchaeal organisms. phically for a period of time. ton pump. (from the Permian period). such as Haloarcula. cells may of Haloarcula. shallow have low oxygen concentrations (oxygen solubility is ponds for isolating salts from the sea. for example. since some sin produced in response to limiting oxygen and high metabolic activity occurring in the entrapped state cannot light intensity form a two-dimensional crystalline lattice be strictly ruled out. where species levels of bacteriorhodopsin and light intensity. Lake Assal in Djibouti. such as sodium ion extrusion. and Halorubrum. trimethyla- a square-shaped species) are typically detected. were isolated from salted protein damaging blue and UV light. Another typical environmental niche called the purple membrane. and flagellar (extracellular line) rotation. most are facultative aerobes Haloarchaea have been shown to resist the denaturing (Figure 5). In some strains. Since their natural environments often salts internally. amino acid uptake. Halorubrum. and Natronomonas have use the proton-motive force generated to grow phototro- been isolated.126 Archaea (overview) environments such as artificial crystallizer ponds. bacteriorhodopsin. marshes. Terminal electron acceptors during Haloarcula. and Haloquadratum (including anaerobic growth include dimethylsulfoxide. TMAO ATP ADP + Pi Ornithine H+ Citrulline Arginine Carbamoyl CO2 + NH4+ phosphate Substrate-level phosphorylation Na+ H+ Na+ AA Figure 5 Physiology of haloarchaea. however. glycerol. grow anaerobically. ancient salt deposits. . Retinal proteins similar to Halogeometricum. many haloarchaea are able to solar salterns. The mine.

and Methanosaeta.g. which would result have been found in three main types of ecosystems. Methano. uptake systems and sodium–proton antiporters Some bacteria in these communities also produce hydro- (Figure 5). often also hot (e. As a result. these products to generate methane. and methionine. Methanospirillaceae. where typical methanogens include spe- sequences. peat bogs.. in sediment. solfataras. Methanococcales. like the uptake of amino acids. . via degrade biopolymers into alcohols and fatty acids. but they form a broad group comprising 5 environment is to exist as part of syntropic communities orders (Methanobacteriales. includ- therefore these nutrients are available only for a relatively ing both light repair (photolyase) and dark repair short time. Archaea (overview) 127 cell membrane but creates an intracellular milieu that is The substrates used to generate methane can be harsh and challenging for biological macromolecules. Genomic analysis has also shown that the proteins Another type of methanogenic environment exists of haloarchaea are highly acidic. and Metha. Methanosarcina. In fact. all methanogens are (Table 1). These environments are Methanogenic Archaea are microorganisms that are cap. In these environments. rumen fluid and have revealed that surface negative charges facilitate the digestive tracts) where species of Methanobacterium. for Methanococcaceae. may dominate or compete with other anaerobic nothermaceae) and 31 genera (Table 1). or consortia with Bacteria. Methanocorpusculaceae. condition-tolerant vegetative cells to have been found In the third type of environment. communities of resident bacteria concentration and relatively low NaCl concentration. the substrates for thus far are both haloarchaea. morph into a left-handed form. and structural studies inside multicellular organisms (i. and methanogens natural environment. carbon dioxide type (hydrogeno- Halobacterium. aggregation. Methanogens titive in sulfate-rich marine environments since sulfate serve an important role in the global carbon cycle. the gens grow faster and predominate over those that utilize most radiation-resistant strain as well as the most space acetate in these environments. Within the anoxic zones. which periods of increased salinity due to evaporation and accounts for two-thirds of the methane generated in resulting decline and decomposition of less halophilic these environments. 10 ity of methanogens to oxygen. for example. most common methanogenic environments are fresh- tion (via salting out) of most nonhaloarchaeal proteins. like the The main adaptive strategy for methanogens in the haloarchaea. and the both membrane potential and ATP-driven potassium fatty acids subsequently into acetate and carbon dioxide. pleting the conversion of organic carbon into methane However. rice fields. such as sulfate-reducing Bacteria for hydrogen. The arranged into three groups: methyl or other one-carbon internal salt concentration of most halophilic species. formation of a hydration shell. such as methanol. even in these environments. has been measured to be as high as the trophic).e. These organisms Bacteria. methanogens gas. geothermal gases (e. The sodium-motive force is important for gen as a metabolic end product. and Methanosarcinales).. most reducing zones within the various habitats. Z-DNA. methanogenesis are not of biological but geological origin. Haloarchaeal cells maintain high internal KCl In these environments. they inhabit the families (Methanobacteriaceae. Methanogens then utilize metabolic activities. water sediments. and decreasing aggregation and precipitation. alternating GC sewage digesters. The most widely which generally are present in high concentrations during used substrate for methanogenesis is acetate. carbon dioxide and hydrogen gas) are the sub- Methanogenic Archaea strates used by methanogens. members of the kingdom Euryarchaeota. and Methanimicrococcus are common. the sulfate-reducing Bacteria. and well as a greenhouse gas that has been implicated many other diverse species have been isolated in global warming. and hot springs) able of producing methane gas. meaning that the products may still utilize one-carbon substrates.g. the hydrogenotrophic methano- (nucleotide excision repair) systems. The in desolvation. Methanocaldococcaceae. increasing their solubility Methanobrevibacter.. called cies of Methanobacterium. and Some DNA sequences. reducers are able to outcompete them for hydrogen. denaturation. species. Hydrogenotrophic methanogens are usually less compe- tively inhabit strictly anoxic environments. up to 5 M salts. which are not used by as the substrates for methanogenesis. Methano. This process is syntropic. geysers. of metabolic activities of other microorganisms are used methylamines. example. and precipita. Due to the extreme sensitiv- microbiales. like type (methylotrophic). com. methanogens Methanosarcinaceae. Methanosaetaceae. Taxonomically. Methanothermobacter. the digestive systems solar illumination of many hypersaline environments has of animals actively absorb intermediates from the break- also resulted in development of tolerance to radiation for down of complex organics produced by bacteria and haloarchaea via active DNA repair mechanisms. are extremely sensitive to oxygen and therefore selec. a potential fuel source as and species of Methanothermus. Methanopyraceae. and acetoclastic (Figure 6). Methanopyrales. the strictly anaerobic zone of microbial mats microbiaceae. swamps. The high Unlike the external environments.

The most diverse group of cultured Archaea are thermo- Shallow vents are sites of hydrothermal activity within a philic. and oxygen). Overview of the three pathways responsible for the biogenesis of methane. The red arrow indicates the ability of some Archaea to reverse the hydrogenotrophic pathway to produce CO2. Sulfolobus. Pyrodictiaceae. and lactate) and acceptors (sulfate. which can then move in the forward direction to produce methane. iron (II). The blue arrow indicates that CO. Ignicoccus. most thermophilic . 5 families (Archaeoglobaceae. New Zealand. while between seawater and magma. data about their microorganisms have been isolated from solfataras. These organisms are capable of using a wide Desulfurococcus. and hydrothermal vents. Cenachaeales. Geoglobus.128 Archaea (overview) Methylotrophic Hydrogenotrophic Acetoclastic One carbon compounds 6e– CO2 Acetate 2e– 2e– ATP Methyl-CoM Formyl-methanofuran Acetate-Pi Formyl. Thermofilum. Pyrobaculum. Isolates of the former king- such as Palaeococcus. Stetteria have been isolated. In these sites. the Sulfolobales. and 32 genera (Table 1). which is later converted into CO2. Pyrobaculum. Desulfurococcales. and diversity of molecules as electron donors (sulfur. genera and Crenarchaeota kingdoms. Pyrococcus. and Although many species of thermophiles and Thermoproteaceae). iron (III). and Thermoproteales). springs from sites in Iceland. These hyperthermophiles have been isolated. tetrahydrosarcinapterin tetrahydrosarcinapterin 2e– 2e– Methyl- Methylene. (Table 2). Thermoproteus have been found in solfataric fields and hot hydrogen gas. Picophilaceae. Pyrodictium. Hydrothermal vent commu- nities can be divided into two groups: shallow and deep. Archaeoglobus. and 10 genera (Table 1). Sulfolobaceae. tetrahydrosarcinapterin tetrahydrosarcinapterin 2e– Methyl. hot metabolic capabilities and habitats are still accumulating springs. Thermophilic Archaea Russia. Archaeoglobus. 7 families last of which has been proposed to constitute a new archaeal (Caldisphaeraceae. Japan. is a by-product of the acetoclastic pathway. These communities often contain (Caldisphaerales. Thermococcales. Thermofilaceae. Cenachaeaceae. Italy. and Thermoplasmatales). Acetate-CoA tetrahydrosarcinapterin Methylene- Methenyl. Thermococcaceae. and found near subsurface volcanoes and at the boundary Thermoplasmataceae). However. Desulfurococcaceae. and dom consist of 3 orders (Archaeoglobales. members of which occupy both the Euryarchaeota few 100 m below the ocean surface. nitrate. and the United States. usually kilometers beneath those of the latter kingdom consist of 5 orders the ocean surface. Methyl-CoM tetrahydrosarcinapterin Methyl- 2e– Coenzyme M 2e– CH4 Figure 6 Pathways of methanogenesis. Deep vent communities are Ferroplasmaceae. kingdom. and Nanoarchaeum.

it is pos. Ferroglobus 4H2 þ HNO3 ! NH4OH þ 2H2O Pyrolobus 4H2 þ CO2 ! CH4 þ 2H2O Methanopyrus. Methanocaldococcus 6H2O þ NO3– þ 2FeCO3 ! NO2– þ H2O þ 2 Ferroglobus Fe(OH)3 þ HCO 3 2S0 þ 3O2 ! H2S þ CO2 Sulfolobus. They have a greater content of charged residues and intrahelical charge pairs forming Other Extremophilic Archaea salt bridges than mesophiles. though by increasing the number of saturated fatty acids in their not yet axenically cultured. all of which were isolated from Thermophiles have improved membrane lipid stability Antarctic lakes (Figure 3). a large majority of thermophilic isolates are increase to levels experienced by hyperthermophiles. Sulfolobus Acetate þ 8Fe(III) þ 4H2O ! 2HCO3 þ 8Fe(II) þ 9Hþ Ferroglobus. Thermofilum. Acidianus 2S0 þ 3O2 þ H2O ! 2H2SO4 Acidianus. thermophilic proteins. Methanogenium frigidum. supercoils and protects it against thermal denaturation. bacterial. symbiosum. Stygiolobus. Thermophilic Archaea contain three the thermophilic Archaea. Geoglobus H2 þ 6FeO(OH) ! 2Fe3O4 þ 5H2O Pyrobaculum Arsenate þ H2 ! arsenite þ H2O Pyrobaculum environments have very low concentrations of oxygen. C. Acidianus. Desulfurococcus. Some thermophiles have a high internal components to prevent denaturation and degradation. capable of growth at low temperatures. Thermodiscus. Hsp60 (or thermo. Only three cultured some family). Aeropyrum H2 þ ½O2 ! H2O Sulfolobus. Methanococcus. and other cellular into chromatin. Ignicoccus. Thermophilic proteins also appear to be smaller. Pyrobaculum. bilayer. Hsp70. They species of psychrophilic Archaea – Methanococcoides burtonii. and Halorubrum lacusprofundi – have vering unfolded proteins to chaperones. The upper limit for retain stability. Another method eucaryal. but monolayer membranes philes are chemoautotrophic. Pyrolobus Organic compounds þ H2SO4 ! H2S þ CO2 Archaeoglobus Organic compounds þ S0 ! H2S þ CO2 Thermoproteus. and relationships rather than stability factors. The extreme temperatures at which thermophilic and Another means of stabilizing DNA is the employment of especially hyperthermophilic Archaea thrive require the DNA-binding proteins and compaction of the genome adaptation of proteins. has been maintained in the . like haloarchaea. Hyperthermophiles also use novel dibiphytanyl and hyperthermophilic habitats (found at depths of sev. Pyrolobus. bilayers may become unstable. Acidianus. which These microorganisms have incorporated subtle changes may help prevent chemical damage that can occur at high in amino acid sequences that are important for stabilizing temperatures. Thermofilum 4H2 þ SO2 4 ! H2S þ 4H2O Archaeoglobus H2 þ HNO3 ! HNO2 þ H2O Pyrobaculum. and compared to denatured proteins. glycerol tetraether components. Pyrobaculum. archaeal. which assists in refolding by deli. concentration of potassium ions. Metallosphaera. As temperatures surprisingly. Stetteria. lipids. Thermococcus. In order to increase the stability of DNA. Archaea (overview) 129 Table 2 Thermophilic metabolic reactions Metabolic reaction Organism Organic compounds þ O2 ! H2O þ CO2 Sulfolobus. Methanothermus. lipid anaerobes and all hyperthermophiles and many thermo. hyperthermophilic life has been extended in recent hyperthermophilic microorganisms possess a novel years to increasingly higher temperatures (current max. Metallosphaera. Thermoplasma Organic compounds ! CO2 þ H2 Pyrococcus Aromatic compound þ Fe(III) ! HCO3 Ferroglobus þ Fe(II) þ H2O þ Hþ H2 þ S0 ! H2S Pyrodictium. there are very few well- families of heat shock proteins. which Psychrophilic Archaea may also result in increased stability. also contain prefoldin. Sulfurisphaera. In addition. Thermoproteus. which introduces positive DNA imum 121  C) under high pressures (>120 MPa). Relatively used to improve the stability of proteins of thermophiles little is known about most psychrophilic organisms that is through the action of chaperones. which help to refold grow and thrive in these environments. Pyrococcus Organic compounds ! CO2 þ fatty acids Staphylothermus. enzyme. However. been studied in detail. and in some cases more basic. reverse gyrase. Desulfurococcus. Not mation of a lipid monolayer (Figure 2). Over 80% of the Earth’s biosphere is at or below 4  C and sible that these differences may reflect evolutionary harbors a wide variety of species. DNA. Metallosphaera. characterized psychrophilic Archaea. Sulfolobus 2FeS2 þ 7O2 þ 2H2O ! 2FeSO4 þ 2H2SO4 Acidianus. which results in the for- eral kilometers) are completely devoid of sunlight. and small heat shock proteins (sHsp).

the membrane of some acidophiles have an extremely low respectively. and microorganisms in these environments. Studies have shown that an abundance of acidic and basic amino acids. It has been suggested that the tae). archaeal viruses have also led to a hypothesis that DNA replication may have evolved independently in multiple Acidophilic and alkaliphilic Archaea domains. while at neutral Much of the current knowledge of archaeal molecular pH they are unable to assemble into liposomal structures. thrive at the extremes of pH. some methanogenic species were also found to be capable Methanosalsum) have also been isolated. bacterial aspects of the molecular that alkaliphiles could not survive at elevated tempera. These findings led to comparative ana- as sodium chloride. and spherical) have been found. and thermophiles negative charges in the cell wall repel hydroxide ions in (e. specialized cloning and expression vectors . In many cases. and the proteins of halophiles and thrive in pH extremes while keeping an internal pH close some thermophiles were found to be highly charged with to neutral is not well understood. which include diverse species representing all three domains of life. few genes effects. do not structure are responsible for cold activity. Thermococcus) have been isolated. although the present minate in the environment. acidic springs) as well as man-made Characteristics of Archaea sites (acid mine drainage. The first is to have acidic thermolithotrophicus. and Thermoplasma. Since the initial discovery of archaeal viruses with head– Like thermophiles.130 Archaea (overview) laboratory and studied through cocultivation with its mar. The number and There is still uncertainty on the precise mechanisms of diversity of these novel forms indicate that they predo- adaptation of psychrophilic proteins. and other biomolecules for unique shapes (fusiform..g. Methanogenic alkaliphiles (e. soda lakes) and man-made from DNA ! RNA ! protein) is the same in all the sites. linear. Other methanogens.. internally. Typical genera of the acidophilic Archaea include Sulfolobus. In addition. bottle-shaped. except for the head–tail variety. biology of Archaea were also noted. and Natronomonas are typical species involved in DNA replication. and Sulfolobus shiba- polymers in their cell wall. and by the combined action of the sodium/proton antiporter Sulfolobus solfataricus and Thermococcus kodakarensis) have system. recently several alkaliphilic thermophile of a nuclear membrane and coupling of transcription and species (e. including the absence tures.. which help to maintain physiological pH processes through gene knockout and replacement systems. The process of information transfer (known as the central Ferroplasma. in depth using genetic and biochemical analysis. Psychrophiles show significant homology to nonarchaeal varieties or to also employ ‘antifreeze’ proteins that inhibit formation of proteins of recognizable function. Interestingly. such as lysis of many of the highly conserved macromolecular Haloarcula. All cultured archaeal ice crystals within the cell to mitigate their damaging viruses have double-stranded DNA. proton permeability at acidic pH (<4. biology is based on genome sequencing and bioinformatic This suggests that loss of membrane integrity may be the approaches..0).e. modifications to their example. Methanococcus maripaludis and Methanosarcina acetivor- the external alkaline environment. low temperature). Therefore. selenocys- How acidophilic and alkaliphilic Archaea are able to teine and pyrrolysine). Lipids of psychrophiles also incorporate more are shared between archaeal gene families though there is unsaturation allowing them to remain fluid at lower some evidence of sharing within families.g. haloarchaea.. in Archaea these processes are evaporitic conditions at these lakes lead to high concen. For tially at low pH. transcription. NRC-1 and Haloferax volcanii. many new archaeal viruses with novel and adapted their proteins. Acidic environments have been studied all around the world and consist of Novel Molecular and Genetic natural (solfataras. bioleaching reactors). activity at an extreme temperature (i. Natronococcus. the translation apparatus of Vulcano Island. potassium uptake. The genes of sequenced view is that multiple subtle changes in overall protein archaeal viruses. however. However. however. For alkaliphiles. Studies of temperatures.g. Pyrococcus furiosus. psychrophilic Archaea have also tail phenotype. These organ. Methanococcus external high-pH environment. Italy. Alkaline environments dogma of molecular biology. with information flowing also consist of natural (e. In addition. Soda lakes have stable pH values at or above 10. replication have been conducted in detail in certain metha- isms have developed two levels of defense from the nogenic and thermophilic Archaea (e. and ATPase-driven proton also provided genetic systems for the study of fundamental expulsion..g.. The second defense is ans. more closely related to those of Eucarya than they are to trations of sodium carbonate and usually other salts. Archaeal Viruses ine sponge host. haloarchaea. biochemical studies of transcription and DNA membrane and cell wall are also important. such those of Bacteria. Acidophilic and alkaliphilic microorganisms. Halobacterium sp.g. The three domains. droplet-shaped. lipids. such as on translation.g. It was thought translation. of incorporating nonstandard amino acids (e. a few systems have been studied reason why these organisms are able to grow preferen.

The AAAþ domain also proteins. bacterial and eucaryal systems. symbio. Archaea (overview) 131 and novel selectable markers (e. Through recently solved cocrystal structure thermal denaturation. mevinolin. while the Euryarchaeota C-terminal portion consisting of a type IA topoisomerase contain essential PolB and PolD family polymerases. a Euryarchaeota-specific Reverse gyrase is a multidomain protein. and to those in Bacteria. with circular chromosomes an AT-rich region called the duplex unwinding element. proliferating cell nuclear antigen (PCNA). which are typically inverted. of the DNA. This is in RNA primers are created. sum). These sites – found in intergenic regions – contain The genomes of Archaea are similar in size and structure large sequence repeats. Halobacterium sp. These organisms encode 1–6 histone proteins in their Ichi. demonstrated using the genetic system of the halophilic Among hyperthermophilic Archaea (as well as Euryarchaeote. Nii. and while they do not strand breakage. and neomycin-resistance) were developed to DNA replication in Archaea contains elements of both the aid in analysis. ducted using biochemical approaches using both philic archaeal genomes. Thermophilic and especially hyperthermophilic Orc/Cdc6 gene and serve as a binding site for the result- species also require the genome to be protected from ing protein. with eucarya-like replica- tion proteins acting to replicate a bacteria-like genome. simvastatin. It adds positive supercoils by a combination of However. but are more specific to of the DNA helix leads to recruitment of the minichro- certain phyla. This novel topoisomerase to this rule. the genes for most of these replication factors have been tion. the sliding clamp loader replica- contrast to the well-conserved four histone proteins in tion factor complex opens and then loads the sliding clamp. NRC-1. The difference between archaeal and eucaryal DNA. GINS (Go.. which dimerize for stability. solfatar- proteins to maintain reversible compaction. all of which are abundant in Crenarchaeota and some thermophilic and involved in DNA replication and/or repair in Archaea. and Alba proteins. by up to 35 . and interacts with another protein complex. This local opening have been found in Archaea.5–5. five. which are uniquely archaeal. the crenarchaeote. and PolD. C. Alba proteins are ligase I and uracil DNA glycosylase. Once the and sometimes hexamers in the presence of DNA. Archaea have evolved chromatin analysis. Alba is subject to acetyla.g. hyperthermophilic Bacteria). The archaeal chromatin proteins are of two the recognition helix into the major groove and wing into main types. one. The PolB polymerases are DNA primer- increases the rigidity of DNA and stabilizes double. the eucarya-like Aeropyrum pernix and S. genomes. Archaea initiate replication at one (e. inserting transcription. PCNA has to access many regions of their genome simultaneously as also been shown to interact with the flap endonuclease – opposed to differentiated eucaryal cells that require access both of which are eucarya-type proteins – as well as DNA to more limited regions of their genomes. C. Pyrococcus abyssi) Genomic Architecture or multiple sites (S. directed DNA polymerases that do not require PCNA stranded DNA at high temperatures. and form tetramers which links MCM with the archaeal primase. contacts the minor groove. Eucarya that form octomers and nucleosomes in association or. they do require it . domain. onto the with DNA. with an PolD family enzyme. Alba dimerizes for stability and Crenarchaeota and Euryarchaeota. Reverse gyrase has for efficient synthesis but do require it for strand displa- also been shown to bind to internal nicks as well as the cement. solfataricus) in their circular chromo- some.g. which is icus Orc/Cdc6 proteins were found to bind the DNA essential for genomic function in DNA replication and replication site via their winged helix domain. stabilizing them by reducing the rate of for DNA binding and extension. PolB family enzymes. DNA Replication puromycin. The Crenarchaeota possess only N-terminal portion containing a helicase domain and a PolB-type DNA polymerases.. which decreases its affinity for DNA binding. symbiosum. require PCNA for strand displacement. 0. and three in Japanese). which results in unwinding In addition. several other families of chromatin proteins and kinking.75 Mbp in size requiring significant (1000-fold) These chromosomal DNA replication origins (origin compaction for packaging into the small prokaryotic binding or ORB) are usually but not always near an cells.. is an exception actions of the two domains. two. and the corresponding While most studies of DNA replication have been con- genes are present in one or two copies within most thermo. hyperthermophilic Euryarchaeota. as in Eucarya. PCNA tethers the DNA polymerase to DNA and histones may reflect the need for prokaryotic archaeal cells increases the length of DNA chains produced. the thermal denaturation There are two classes of DNA polymerase involved in of genomic DNA is inhibited by the introduction of DNA replication in Archaea: PolB.g. mosome maintenance (MCM) complex. histones similar to the ubiquitous eucaryal the minor groove (Figure 7). similar to the eucaryal positive supercoils using the reverse gyrase enzyme. Histone proteins are mainly found in the Euryarchaeota The MCM complex continues the unwinding of DNA and in a few early branching Crenarchaeota (e. and San. The PolD polymerases prefer a primed template ends of DNA. the essential nature of binds to DNA and possibly RNA.

and synthesized by the archaeal primase are usually removed can bypass UV photoproducts. and has been shown to be genetically responsible for nucleotide exci- DNA Repair sion repair in at least one species. NRC-1. Sulfolobus tokodaii. encode a type I RNaseH as well. such as the damage recognition DNA. However. the type II RNaseH and flap endonu. clease act cooperatively to process Okazaki fragments on Many species with Y family polymerases also contain the lagging strand. Once the primers are removed. eucarya-type that these strictly anaerobic Archaea are also exposed to single-strand binding (replication protein A) proteins UV radiation under some circumstances. while PolD acts as the lagging strand ing unrepaired DNA lesions.132 Archaea (overview) (a) DNA (b) + AAA domain TBP DNA ORC/CDC6 Winged-helix protein domain TFB Figure 7 Structure of DNA replication and transcription initiation proteins bound to DNA. for efficient DNA synthesis. not all Archaea contain all of these ase when the nucleotide is encountered in the template repair proteins and others. ADP and Mg2þ in the structure are colored pink respectively. suggesting ment. imidine dimers and abasic sites. radiation such as the Sulfolobales and Halobacteriales. An important property of archaeal eucaryal enzymes. The bacterial excision repair machinery (UvrABC) is present in certain mesophilic Euryarchaeota. some diverse Archaea. Moreover. Dpo4 in polymerases may act on either side of the replication S.or cies have no exposure to UV or visible light. The RNA primers member of the Y family of error-prone polymerases. Fen1 is involved Archaeal mismatch repair machinery differs from the in Okazaki fragment processing in DNA replication. and XPB and XPD ‘read ahead’ function for uracil. and seal nicks in the DNA. NRC-1. (a) Model structure of an Orc/Cdc6 protein bound to an origin DNA. It has been hypothesized that low mutation frequency in some species suggests the in the Euryarchaeota. XPF-ERCC1 and Fen-1 replicative B family DNA polymerases (PolB family) is a (a homologue of XPG) nucleases. which stalls the polymer. Deep-sea spe- bind the DNA and DNA ligases (either ATP. some of these enzymes are involved deamination is more frequent at high temperatures. This type of repair is most important for the proteins XPA and XPC found in Eucarya. such as cyclobutane pyr- by a type II RNaseH. are absent thermophilic Archaea since uracil resulting from cytosine altogether. Green strands correspond to the transcriptional promoter DNA. Cdc/Orc6 protein is colored according to secondary structure (-helices are orange and -sheets yellow). lack these enzymes. Dpo4 (a DinB homologue in Bacteria) is a fork (leading or lagging strand). while type I RNaseH removes the last the UV photoproduct repair enzyme photolyase. and translesion bypass polymerases in Archaea indicates that Pyrobaculum aerophilum. The distribution of such as Halobacterium sp. Halobacterium sp. some Archaea encode a DNA polymerase. PolB acts as the leading strand existence of an efficient mechanism. and common bacterial MutS/MutL pathway. PolB family lesion bypass DNA polymerase. Methanosarcinales also contain these enzymes. for example. helicases. possibly homologues of the eucaryal equivalents. however. Archaea have DNA repair proteins (like their replication Thermophilic Archaea likely use a different pathway for counterparts) that are usually more similar to their nucleotide excision repair. DNA strands are green. solfataricus. For repair of remain- DNA polymerase. although the XPB and XPD are involved in transcription initiation. (b) Model structure of the TBP–TFB–DNA complex. In they are generally found in organisms exposed to UV such organisms. in other cellular processes. . for example. ribonucleotide of the RNA primer of the Okazaki frag. In the Crenarchaeota. and therefore NADþ-dependent).

furiosus enzyme to identify com. Since no TATA-interacting hydrogen.. 23S. may ment of RNA polymerase. activity at most eucaryal RNA polymerase II promoters. and the for example. Studies copies of the corresponding genes. and they generally contain to the subunits of the P. Some known about the processes of transcript elongation and transcripts have also been shown to contain polyadenine termination in Archaea. with the B subunit split and bacterial character. tion sequence was located 39 to the UGA codon. Archaea also use the contain introns. respectively) have been compared resemble operons of Bacteria. frequently organized into transcription units or operons. but all of its ribosomal proteins into B9 and B0. and subunits of hydrogenase catalyzing the utilization of tion factor genes are essential. and it may be inserted cotran- combinations (e. bound to the promoter. multiplicity of M. while the TFB protein Two unusual amino acids present in methanogenic binds a B-recognition element immediately upstream of Archaea are selenocysteine and pyrrolysine. Interestingly. Some Archaea (e. haloarchaea sence of the amino acid. although not the host of ever.. methyltransferase enzyme. incorporation of glutamine and asparagine proceeds other eucaryal transcription initiation factors. different temperatures). Selenocysteine proteins contain two imperfectly repeated sequences from is inserted at certain opal (UGA) stop codons through a internal gene duplications. maripaludis showed that the inser- of transcription factor genes is common in some Archaea. multiple transcrip. although their genes are with the exception of TFS. in the methylamine likely contain a novel regulatory system involving recogni. fewer copies of the three rRNA genes – 16S. Archaea (overview) 133 Transcription known to transcribe through DNA-binding proteins with- out the aid of elongation factors. their genomes for use of the standard genetic code. or  factors. Consistent with this hypothesis. transcription is started by recruit. for their response to heat shock). Archaeal RNA polymerase is tails at their 39 ends. which possesses ment of tRNA (transfer RNA). both the TBP and TFB neither appears to be uniquely archaeal. pyrrolysine. slationally using a specialized amber suppression tRNA. like those of the haloarchaeal bacterio-opsin gene. and aminoacyl–tRNA synthetase genes in eucarya-like TATA-binding protein (TBP) and transcrip. likely reflecting their relatively slow growth rate com- The promoter structure and transcription initiation fac. Recent studies of M. while methanogens and The translation system of Archaea has hybrid eucaryal haloarchaea have 12 subunits. compared to only 4 subunits in the single bacteria-type RNA polymerase. TBP binds to the TATA box upstream using GatABC amidotransferase. . consistent with about 25 bp upstream of the transcription start point. as found in eucaryal transcripts. Crystal structures of RNA polymerase have eucaryal homologues.. The RNA polymerase of Pyrococcus and the Crenarchaeota have 11 subunits Translation (RpoBA9A99DE9FLHNKP). and 5S – mon structural motifs. tors of Archaea are also eucaryal in nature. This is dis. tion as well as factor-mediated termination. mutagenesis studies have The existence of a coupled transcription/translation shown that while some genes have the requirement of a mechanism as commonly observed in bacterial species canonical TATA box. tinctly eucaryal features. The position of pyrrolysine is tion of different promoters by specific TBP–TFB at an amber (UAG) codon. like recent acquisition or divergent functions (e.g. kodakarensis. barkeri showed the pre- proteins have been found in the Archaea. a fraction of which may 10 and 35 recognition elements. enzymes with 11 or 12 subunits. However. The ribosomal protein genes from both Bacteria and Eucarya (Escherichia coli and of Archaea are organized into multigene clusters that Saccharomyces cerevisiae. there are no known archaeal homologues of eucaryal Pol as found in Bacteria.g. Instead. pared to well-characterized Bacteria. the haloarchaea. relatively little is be capped in vitro using the viral capping enzyme. T. Archaea contain a full comple- tinct from the bipartite bacterial promoter. others use promoters that deviate has been observed in at least one archaeal species. Archaeal Haloarcula marismortui) contain multiple rRNA operons promoters have an AT-rich region (TATA box) located that differ by 3% or more in sequence. by modification of the amino acids on the charged tRNAs found in Bacteria. of the transcriptional start site. and nine TFB genes have been found in the genome. Selenocysteine has been found in proteins including Genetic analysis in haloarchaea showed that although a those involved in reduction of carbon dioxide to methane fraction of these genes may be deleted. where up to six TBP genes decoding event is more similar to Eucarya. although the TATA box. Archaeal transcripts are not thought to be Once the TBP and TFB transcription factors are modified with 59-end caps. from the consensus. The genomes of most archaeal context-dependent suppressor tRNA (Sec-tRNASec) and species sequenced to date have shown just one or two directed by a selenocysteine insertion sequence. although primary transcripts. However. and lacks most Pol II elongation factors. jannaschii and M. Similarly with termination. similar to the eucaryal The transcriptional machinery in Archaea also has dis- Pol III enzyme. Transcription in Archaea is carried II termination factors. how- tion factor IIB (TFB) (Figure 7).g. archaeal transcription out using a simplified version of the eucaryal RNA poly- termination suggests sequence-directed intrinsic termina- merase II-like system.

the twin.1 Coccus Yes Anaerobic Mesophilic Methanopyrus kandleri AV19 1.3 Coccus Yes Aerobic Hyperthermophilic Archaeoglobus fulgidus DSM 2.0 57.6 Irregular Yes Anaerobic Hyperthermophilic 4304 coccus Cenarchaeum symbiosum A 2. Yes Aerobic Mesophilic Moderate disks Haloquadratum walsbyi DSM 3.2 47. Archaeal Genomics and proteolytic processing. methylation.2 46. are distinctly archaeal.9 36.5 Coccus Yes Anaerobic Hyperthermophilic Korarchaeum cryptofilum 1.7 62. In addition.5 62. such as hypusina. redox proteins.3 Irregular Yes Anaerobic Mesophilic coccus M.134 Archaea (overview) Further.3 Irregular Yes Anaerobic Hyperthermophilic Moderate DSM 2661 coccus Methanococcoides burtonii DSM 2.6 40. Temp.1 Coccus No Aerobic Thermophilic Methanobrevibacter smithii ATCC 1.7 53.7 33.6 49. Dozens of archaeal genome sequences have been completed some.7 Irregular Yes Anaerobic Hyperthermophilic Moderate 5456 coccus Ignicoccus hospitalis KIN4/I 1.0 65.1 43. proteins.and O-linked modifica- tion. NRC-1 2. Yes Aerobic Mesophilic Extreme 43049 disks Halobacterium sp. such as the methylation of methyl-coenzyme M and are available in public databases (Table 3). range req. including both N. especially among haloarchaea.7 56.1 Rod No Microaerophilic Hyperthermophilic Ferroplasma acidarmanus fer1 1.7 31.2 48. For protein secretion.1 Pleomorphic. tion and thiolation. lipid modification.9 Square Yes Facultative Mesophilic Extreme 16790 Hyperthermus butylicus DSM 1. and a few archaeal general secretory (Sec) machinery is a hybrid of from thus far unclassified organisms.3 61.0 Coccus No Anaerobic Mesophilic labreanum Z Methanoculleus marisnigri JR1 2.6 65.0 Rod No Anaerobic Mesophilic 35061 Methanocaldococcus jannaschii 1. Sizes of the sequenced eucaryal and bacterial systems. These include trast to Bacteria. where it is mainly used for secretion of acetylation.0 Ultrathin No Anaerobic Thermophile filament Metallosphaera sedula DSM 5348 2.0 Irregular Yes Anaerobic Mesophilic coccus M.75 Mbp for Table 3 Table of Archaea with completely sequenced genomes Salinity Organism Size GC Shape Motility Oxygen req. Nanoarchaeum equitans.5 Mbp for the arginine (Tat) protein export is also extensively used in smallest.6 30. to over 5. maripaludis C6 1.3 56. glycosylation. disulfide bond formation.0 Irregular Yes Anaerobic Mesophilic Nankai-3 coccus Methanococcus maripaludis C5 1.8 50.9 31. and in con- have been found in archaeal proteins. phosphorylation. Nearly reductase and the unique lipid moieties of haloarchaeal three-quarters of the genomes are from Euryarchaeota.7 Rod No Facultative Psychrophilic Caldivirga maquilingensis IC-167 2. amino acid modification.3 Irregular Yes Anaerobic Mesophilic coccus Methanocorpusculum 1.9 Rod Yes Facultative Mesophilic Extreme Haloferax volcanii DS2 4. archaeal genomes ranges from less than 0.2 Irregular Yes Anaerobic Mesophilic coccus M.5 Pleomorphic.8 33.1 Rod Yes Anaerobic Hyperthermophilic Moderate (Continued ) . Aeropyrum pernix K1 1. the with one-quarter being from Crenarchaeota.5 Rod No Anaerobic Mesophilic Haloarcula marismortui ATCC 4.8 33. Many of these modifications are shared among the three domains of life.7 33.7 31. a wide range of posttranslational modifications some Archaea. maripaludis C7 1.1 Irregular Yes Anaerobic Mesophilic coccus Methanococcus vannielii SB 1. maripaludis S2 1. however.8 Irregular Yes Anaerobic Psychrophilic Moderate 6242 coccus Methanococcus aeolicus 1.

2 Rod No Aerobic Mesophilic Picrophilus torridus DSM 9790 1.7 Coccus No Aerobic Thermophilic 639 Sulfolobus solfataricus P2 3.6 Sphere No Anaerobic Hyperthermophilic Natronomonas pharaonis DSM 2. while mesophilic methanogens and halophiles mophile. range req. Methanothermobacter thermoautotrophicus (originally the three-domain view of life and also revealed complex- Methanobacterium thermoautotrophicum).7 Coccus No Anaerobic Hyperthermophilic Sulfolobus acidocaldarius DSM 2.5 45.1 41.7 63.5 Irregular No Anaerobic Mesophilic coccus Methanosphaera stadtmanae 1.9 53. trophic hyperthermophilic methanogen from a deep-sea These initial genome sequences reinforced the validity of trench.2 51. an aerobic halophilic so far. an autotrophic ity in gene histories as a result of lateral gene transfers and thermophilic methanogen from an anaerobic sewage unequal evolutionary rates. an aerobic hyperther- methanogens.0 Irregular Yes Anaerobic Hyperthermophilic KOD1 coccus Thermofilum pendens Hrk 5 1.9 40.6 35.7 41.2 36.8 Coccus No Aerobic Hyperthermophilic Thermococcus kodakarensis 2.7 Irregular No Anaerobic Mesophilic coccus Methanosarcina barkeri Fusaro 4.6 Sphere No Anaerobic Mesophilic DSM 3091 Methanospirillum hungatei JF-1 3.5 36. Temp.8 49.2 54.8 Irregular No Aerobic Hyperthermophilic coccus Sulfolobus tokodaii 7 2.2 Coccus No Anaerobic Mesophilic Methanosarcina mazei Go1 4.6 39. Thermophiles represent the largest group of sludge digester. Relatively few acidophi.9 Irregular Yes Anaerobic Hyperthermophilic coccus Staphylothermus marinus F1 1.8 27.8 49.9 Pleomorphic Yes Facultative Thermophilic Noncultured methanogenic 3. Halobacterium sp.6 Coccus No Anaerobic Thermophilic Methanosarcina acetivorans C2A 5.5 Cylinder.6 Rod Yes Anaerobic Thermophilic 4184 Pyrococcus abyssi GE5 1.6 34.8 44.6 Mesophilic archaeon RC-I M. .7 32.2 Rod Yes Facultative Hyperthermophilic 11548 Pyrobaculum islandicum DSM 1.2 Curved Yes Anaerobic Mesophilic Methanothermobacter 1. a hyperthermophile number of hydrothermal vent species and high-temperature from a hydrothermal vent.8 57.5 Rod No Anaerobic Mesophilic Methanosaeta thermophila PT 1.1 Rod Yes Anaerobic Hyperthermophilic 13514 Pyrobaculum calidifontis JCM 2.8 Coccus Yes Anaerobic Hyperthermophilic Pyrococcus horikoshii OT3 1.0 Coccus No Aerobic Thermophilic Pyrobaculum aerophilum IM2 2.6 46.5 54. (originally Methanococcus) jannaschii. Pyrococcus horikoshii.0 Pleomorphic Yes Facultative Thermophilic 1728 Thermoplasma volcanium GSS1 1.1 52. archaeon probably isolated from salt used in food preser- Among the first archaeal genomes to be sequenced vation.7 42. which metabolizes sulfur and were M.0 35. A. NRC-1. Thermoplasma acidophilum. acetivorans. and psychrophilic Archaea are represented pile. grows at high temperature and under acidic conditions. No Anaerobic Thermophilic thermautotrophicus  H irregular rod Nanoarchaeum equitans Kin4-M 0.6 Rod No Anaerobic Hyperthermophilic Thermoplasma acidophilum DSM 1.9 39. including a thermophile.0 57. solfataricus. Methanoregula boonei 6A8 2.5 31. an auto. an acidophilic and constitute the next largest groups. Archaea (overview) 135 Table 3 (Continued) Salinity Organism Size GC Shape Motility Oxygen req. slightly thermophilic heterotroph from a coal refuse lic.4 Rod Yes Facultative Hyperthermophilic Pyrobaculum arsenaticum DSM 2. alkaliphilic.1 Rod Yes Aerobic Mesophile Moderate 2160 Nitrosopumilus maritimus SCM1 1. Archaeoglobus fulgidus.1 55.7 Irregular Yes Anaerobic Hyperthermophilic coccus Pyrococcus furiosus DSM 3638 1. a sulfur-metabolizing Archaea with completely sequenced genomes. and S. pernix.

fulgidus DSM 4304. Pyrococcus horikoshii duction. Most of the proteins encoded in the genome. fulgidus genes of M. jannaschii involved in transcription. jannaschii genome. jannaschii. The second archaeon to have a complete genome almost 60%. and translation PCR amplification from genomic DNA. have addressed the high radiation resistance of Pyrococcus gun sequencing approach involving hybridization to yield species and a genetic system has been reported for the multiple sequence ladders in a single experiment. thermautotrophicus were also found to be predicted genes were identified. with 20% Methanothermobacter thermautotrophicus related to functional genes and 22% to conserved sequences with unknown function. with a pI of about 8. A number of postgenomic studies sequenced using a novel ‘multiplex’ whole-genome shot. Eucarya than in Bacteria. a thermophilic was found to be basic. The majority of genes. pernix K1. yielded -ketoglutarate dehydrogenase.136 Archaea (overview) Methanocaldococcus jannaschii a 2. It was only the fourth ences. jannaschii. was found to be a circle of 1. 28% were Aeropyrum pernix related to sequences with unknown functions. which uncharacterized yet conserved proteins. produced a complete 1. Archaeal diversity the time of publication. indicating that there was a low degree of mal vent. The genome con- About a quarter of the genes encoded functionally tained 1738 predicted protein-coding sequences. an autotrophic to be clearly eucaryal and several of the biosynthetic archaeon. None of the A. fulgidus lacking methanogenic pathways. jannaschii. The genome proteins that are removed autocatalytically with conco- sequence was assembled using a physical map and verified mitant religation of flanking sequences. reading frames present. A. The average protein sequence was for M.66 Mbp circular chro- pathways for nucleotides. The genome also encoded 14 related hyperthermophilic Pyrococcus species. were reported earlier). furiosus species. three tRNA genes with introns. microbial genome of any kind to be determined (only and coding for environmental sensors.67 Mbp circular genome. regulatory and Haemophilus influenzae. for M. However. was functionally . by long PCR products amplified from the genomic DNA. related to the sequences with unknown function. acid cycle were present. As in the case of M. amino acids. which are insertions within OT3. were found to be new. and replication were more similar to those found in introns were found. The and one protein gene with an intein. jannaschii DSM2661 before. cell division.74 Mbp. ome sequence determined. horikoshii proteins containing 18 inteins. mapping along the entire length of the genome using most DNA metabolism. remaining gene products were novel and did not show any significant similarity to known sequences in the data- bases when reported. transcription. all but one of the genes for the tricarboxylic The first sulfur-metabolizing organism to have its gen. indicating a methanogen (named M. thermautotrophicus H. transla- genes contained inteins. was found to have a 1. Like the two methanogenic Archaea sequenced The first archaeal genome.75 Mbp circular chromosome was to high temperature. and metabolism were more similar The complete genome sequence of the first of three closely to those found in Bacteria. only 38% of its genes could be was however supported by the finding of about 25% new assigned a cellular role with high confidence. Of the 1855 open and two tRNA genes contained introns. the genomes displayed the expected differ- shotgun sequencing and assembly. the majority of genes coding for energy pro. About 54% were most similar to other completed genome sequence. A total of 2694 proteins for M. and 27% The first strictly aerobic hyperthermophile to have a were entirely new. As expected for an aerobic Archaeoglobus fulgidus organism. The 1. extrachromosomal circles by whole-genome random However. Mycoplasma genitalium. about 46% of the predicted pro- teins could be assigned putative functions based on similarities to previously sequenced genomes. 25% of which were more similar to eucaryal sequences. and transport functions required for sulfur metabolism. A. At which were shared with M. confirming the special evolu- tionary position of Archaea in the evolutionary tree. and cofactors mosome sequence and two (58 and 16 kb) were similar to orthologues in the M. the information processing systems were found (originally Methanococcus jannaschii). thermoautotrophicum at the time of possible means of adaptation of hyperthermophilic proteins sequencing). but only 19% had significant matches to the Crenarchaeota isolated from a coastal solfataric ther- M. Four interrupted related to genes with putative function and about 20% genes were identified. The single exception. two-thirds of were identified as likely genes by statistical analysis. P. conservation among orthologous genes in the two The structure of this genome was verified by restriction sequenced methanogens. one of related P. a member of Archaea.18 Mbp circular genome containing 2436 predicted genes. although five tRNA genes with tion. Eleven genes contained inteins the few accomplished in this manner. A total of 2061 putative genes were found. Synechocystis sp. with A.

56 Mbp circular chromosome. indicating that contain a 2. Its DNA replication. As a result of its ease of intracellular salinity. for DNA replication and cell cycling studies. Analysis of the 1509 predicted genes showed typical archaeal characteristics as well as a rela- tively large fraction of bacterial genes (about 10%) likely Nanoarchaeum equitans acquired through lateral gene transfers. and 12 and 2. The genome was found to be GC-rich core cellular functions. The aerobic crenarchaeote S. appears to be highly plastic. is the smallest sequenced to date. cated that N. acidophilum were also present. Methods have been predicted proteins. and transcription systems were found to The genome of an acidophile that is also a slightly ther. may be a member of the Euryarchaeota. with 700922). M. as well as evidence of integrase-mediated insertion events. A substantial The genome of the hyperthermophilic obligate symbiont. representing an entirely new archaeal Halobacterium sp. Phylogenetic analysis has indi- didates for laterally transferred genes. kandleri developed for facile cultivation and genetic manipulation proteins show an unusually high content of negatively of this aerobic mesophile. transcription. kandleri encode 2977 predicted proteins. including those for DNA replication and repair. it has become a popular using 16S rRNA suggested that it belonged to a very model among haloarchaea and for classroom teaching.99 Mbp genome on a single chromosome and archaeal methanogens may be monophyletic. including construction of gene charged amino acids. kandleri AV19. suggesting that it representing 12 families. M. NRC-1 kingdom (Nanoarchaeota). . deep branch. NRC-1 (ATCC N. some of which may represent inte- DSM 1728 (growing best at 59  C and pH 2). The genome was (up to 15%) of genes coded for proteins with similarity to a variety of Bacteria. and translation. an adaptation to its relatively high knockouts and replacements. kan- dleri consistently groups with other archaeal methanogens.69 Mbp circle coding 1692 fraction of laterally transferred genes. and The genome of M. number of genes similar to the phylogenetically distant N. harboring a dynamic 2. the DNA replication. with over 200 diverse trans- Fourteen introns were also discovered in its tRNA genes. posable IS elements present. respectively. genes for many pNRC200. equitans may be a very early branching archaeal lineage. 191 kb. equitans Kin4-M. active sodium– Methanopyrus kandleri proton antiporter and potassium uptake systems. equitans has one of the most compact genomes. and appears to have rela- with Bacteria and Eucarya. Ignicoccus. which coinhabits the narchaeote. for Sulfolobus species and used as model systems. 40% of which appear to was found to lack many proteins involved in signaling and be archaea-specific.57 Mbp genome with a 95% of its genome coding and specifying 550 putative 2. kandleri was found to share Sulfolobus solfataricus organization of genes involved in methanogenesis with M. pNRC100. including lipid. which was thought to sophisticated photosensory and signal transduction path- be a deeply branching archaeon close to the root of the ways. and transcription. where genomic DNA served as template and 29-modified Phylogenetic studies showed that a substantial fraction oligonucleotides were used for priming. 365 kb. In either case. genome comparisons indicated that M.01 Mbp circular chromosome and two related large proteins. As in other Archaea. was grated plasmids. was sequenced employing a novel method systems resembled more complex eucaryal organisms. The Halobacterium NRC-1 gen- obtains many of its biomolecules from Ignicoccus. some researchers have reported contradictory results. and translation tree of life. solfataricus P2 was found to jannaschii and M. Archaea (overview) 137 replaced by a 2-oxoacid:ferredoxin oxidoreductase. 36% of which were unrelated to any previously reported.49 Mbp. A genetic system has been developed sequenced and yielded a 1.3% shared exclusively regulation of gene expression. M. phile was that of Halobacterium sp. also indicating can. However. S. be similar to Eucarya and multiple replication origins mophilic member of the Euryachaeota. However. circle of only 0. T. extrachromosomal DNA circles. Like those of halophiles. suggesting that it The first complete genome sequence of an extreme halo. Although phylogenetic analysis laboratory culturing and study. cell cycle. are lacking.9%) and contained 91 transposable IS elements. type proteases and chaperones have been of interest in this archaeon. In addition. ome coded 2630 predicted proteins. and (65. a same environments. Analysis of the genome sequence showed the presence of pathways for uptake and utilization of amino acids. nucleotide biosyntheses. that grows in coculture with a cre- Crencharchaeote. especially The lack of a rigid cell wall and the presence of eucarya. The genome tively few genes acquired via lateral transfer. solfataricus. thermautotrophicus. were identified. amino acid. indicating that it contains a high found to be a GC-rich (61%) 1. DNA repair and recombi- Thermoplasma acidophilum nation.

with Methanococcus maripaludis higher fidelity (proofreading exonuclease activity) and pro- ducing larger products (greater processivity). kandleri. kodakaraensis. the latter being useful for However. maripa. and the inteins present in M. medi- knockout system has been developed for postgenomic cal. Based on genome analysis. the yielded a 1. PCR enzymes are stantial number of genes (about 30%) were absent from only one of a group of extremozymes from Archaea that Pyrococcus and unique to T. including hydrogenases and eight including M. the process using archaeal enzymes. Although the term ‘extremozymes’ is used philic Euryarchaeote that cohabits environments with for enzymes from both archaeal and bacterial extremo- Pyrococcus. and may influence both the efficiency Biotechnological Applications of Archaea of caloric uptake and nutritional health of the host. kodakaraensis KOD1 is a sulfur-reducing hyperthermo. Of the 1722 predicted (from Thermococcus litoralis).66 Mbp circular genome. However. furiosus). cheese production) and in detergent applica- branching unclassified Archaea that may represent an tions. has been completely sequenced and genes and 45 tRNA genes. analysis of this hyperthermophile and it has become a popular model for postgenomic studies among thermophiles. A facile genetic system sequencing. Complete pathways for methanogen.g. jan. cryptofilum OPF8 is a member of a large group of deep. Archaeal proteases are also in use for baking. thermautotrophicus and M. which the organism adhesin-like proteins.138 Archaea (overview) Methanobrevibacter smithii this is the result of horizontal gene transfer or evidence of a common ancestor. The genome lacks complete pathways for those found in the gut mucosa.09 Mbp T. Thermophile and psychrophile enzymes fill a entirely new archaeal kingdom (Korarchaeota). The development of PCR in the could be annotated. and (4) competition for nitrogenous nutrient pools. Industrial and Agriculture Applications Korarchaeum cryptofilum In industrial settings. (2) regulated expression of de novo synthesis of several cofactors. Since Archaea are often found in extreme environments Thermococcus kodakaraensis and are evolutionarily distinct from Bacteria and Eucarya. dairy (e. led to the A popular genetic model among methanogens M. Similar organisms have also been identified in human Biotechnology has long been one of the most important dental caries. an enzyme that logue could be identified.5% were unique. driving forces behind studies of archaeal microorganisms. A facile gene have contributed to the development of chemical. enzymes identified in methanogens and thermophiles. jannaschii.59 Mbp chromosome containing 1617 protein-encoding ATCC 35061. . is initiation mechanism. M. and genomic biotechnology. and ligase from Thermococcus.. an energy-saving of crenarchaeal and euryarchaeal genes and it is unclear if method. 44% could be assigned a function. variety of different needs. the K. and Vent a 1. archaeal extremozymes have played an important genome revealed 2306 putative genes. cryptofilum genome appears to be a hybrid laundry washing at lower temperatures. The presence of transposable genetic 1980s using DNA polymerase from the thermophilic bac- elements similar to Pyrococcus species suggested transfer of terium Thermus aquaticus was followed by enhancement of genes between the two related genera. has been developed for this organism. half of which role in biotechnology. Other useful enzymes for mole- protein-coding genes. also in use for facilitating strand separation in DNA naschii homologues were absent. hydrogenotrophic methanogen with (from Pyrococcus woesei). they serve as an excellent source of novel enzymes and T. Comparative and organism is expected to be an obligate anaerobe and grow functional genomics of this strain indicates that it persists heterotrophically using peptide and amino acid degrada- in the gut by (1) production of surface glycans resembling tion pathways. Pfu (from P. Its genome consists of a single The genome of the human gut methanogen. Annotation of the 2. archaeal lipases are used both in K. B DNA topoisomerase V from M. fermentation products produced by saccharolytic bac- teria. a sub. cular biology applications include a thermostable DNA 48% were conserved but had unknown functions. No Orc gene homo.85 Mbp circular genome. smithii affects digestion of dietary polysaccharides. Extremozymes The need for more PCR thermostable enzymes. kodakaraensis philes. smithii 1. (3) consumption of a variety of probably scavenges from its environment. identification of archaeal extremozymes such as Pwo ludis is a mesophilic. biomolecules. implying an unusual replication relaxes both negatively and positively supercoiled DNA. esis were identified. and restriction–modification 7. A group selenocysteine-containing proteins. The presence of M.

for example. culture-inde- example. Proceedings of the National Academy oped for improving the stability of vaccines. and Archaea have been used to develop novel biomedical Fleischmann EM (eds. and Jonuscheit M (2005) Genomic studies of uncultivated Archaea. With the limited availability of fossil fuels. Woese CR. Physiology gical contactor. Patents have been issued for Woese CR (2000) Interpreting the universal phylogenetic tree. and methane is extracted at the top. baking. no archaeal patho- development of optical storage devices with the ability gens have been confirmed to date.and color-sensitive photoelectric widespread occurrence of archaeal species worldwide in devices. paper manufacturing. Journal of Molecular Evolution 46: 1–17. association with the human gastrointestinal tract. Potentially. The find- fish sauce (Nam Pla). Douady CJ. Totowa. bacteriorhodopsin in purple membrane.) 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Nature Reviews Microbiology 3: 479–488. while trehaloses may be used in food prepara. Kandler O. 2nd edn. structure of archaeal ribosomes is being used for under. include pendent techniques have extended our appreciation of the holography and light. Read T. NRC-1 in vaccine States of America 97: 8392–8396. Boone DR. However. of alternative fuels. physiology. the study of Archaea has provided the foundation Archaea have been identified as candidates for production for conceptualizing early cellular evolution on planet Earth. Haloarchaea (along with halophilic Bacteria) have Although Archaea are the most recently described group of long been used in the fermentation processes involved microorganisms from a modern molecular phylogenetic in the production of fermented foods. Science 229: 717–725. Since then. The of Sciences of the United States of America 74: 5088–5090. as well as an and Molecular Biology. standing the basis of antibiotic action and computer-aided and Eucarya. for importance to ecological processes. researchers are attempting to harness this ability to pro. Thermophilic alcohol Howland JL (2000) The Surprising Archaea: Discovering Another Domain of Life. around sand grains at high temperatures. the United States of America 87: 4576–4579. Robb FT. many diverse Archaea have tion and as preservatives. Oxford UK: Blackwell Publishing. of oil and gas production by breaking down guar gum Norwich. Investigations have also rapidly expanded our knowledge and understanding of their molecular biology.

sulfate. When CO2 dioxide (CO2). nitrate. which can be light or chemical energy). hydrogen. energy cannot be obtained by oxidation of an organic carbon substrate (formate and methanol oxidation and reassimilation of carbon dioxide by some aerobes can be Autotrophic Modes of Life viewed as an exception of autotrophy). energy is provided by photosynth- sustain life. carboxylation Is the addition of CO2 or bicarbonate to primary production Is the formation of organic another carbon-containing molecule. occurs in aerobic as 140 . in microorganisms) for Heterotrophic organisms (and this includes humans) gen. Primary production (using the maximum gain of reducing equivalents. Defining Statement Assessment of Distribution of the Different Pathways Autotrophic Modes of Life Regulation Conversion of Inorganic Carbon to Cell Carbon Further Reading Mechanisms for CO2 Assimilation Glossary CO2 fixation Describes the conversion of CO2 (a gas) to autotrophy Self-feeding. the source of reducing power is provided by inorganic compounds The biosynthesis of organic carbon starting from inorganic such as water.5-bisphosphate carboxylase/ dehydrogenase oxygenase Defining Statement transferred to a terminal electron acceptor such as oxygen (or also to nitrate. Because CO2 is the sole source of carbon represents quantitatively the most important biosynthetic for autotrophic organisms. to CO2 this process then requires: reducing equivalents and autotrophic organisms are essential for life and autotrophy input of energy. Abbreviations NMR nuclear magnetic resonance CbbR Calvin–Benson–Bassham cycle regulator PEP phosphoenolpyruvate CFI carboxylating factor for isocitrate RuBisCO Ribulose-1. Instead. reducing equivalents and process in the overall carbon cycle. autotrophy is the ability of an an organic compound containing carbon–carbon bonds organism to synthesize all cell carbon constituents as well as the assimilation of this CO2 fixation product. Autotrophic CO2 Metabolism B E Alber. but also growth factors added to the medium). Therefore. therefore. or sulfate. Therefore. every single carbon in the cell would inorganic carbon species Used in autotrophy are become labeled (except the ones derived from essential carbon dioxide (CO2). energy conservation. Columbus. bicarbonate (HCO 3 ). resulting in a compounds from inorganic carbon species using light or carbon–carbon bond. Likewise. USA ª 2009 Elsevier Inc. Autotrophy is the ability of an organism to assimilation is viewed as the reversal of carbon oxidation synthesize all cell carbon from CO2 alone. The complete oxidation of organic carbon esis or by reduction of oxidized inorganic compounds such compounds to CO2 by heterotrophic organisms allows as oxygen. reduced sulfur compounds or carbon species by autotrophic organisms is a prerequisite to ammonium. CO2 assimilation Describes the biosynthesis of cell carbon constituents (biomass) starting from carbon dioxide or bicarbonate. if an heterotrophy Organic compounds are utilized as autotrophic organism was to be grown in the presence carbon sources. exclusively from inorganic carbon. carbon monoxide (CO) or cyanide (CN). etc. OH. CO2 fixation by autotrophic organ- erally oxidize their carbon sources completely to carbon isms refills the organic carbon pool. chemically derived energy. All rights reserved. of labeled CO2. Ohio State University.

The CO2 is the same for any given organism. Ribulose-1. the CO2 assimilation mechanism used by a given organism. the Thiobacillus neapolitanus). In addition to When considering the biosynthesis of all carbon compounds carboxysomes. The further conver- sion of acetyl-CoA to C3. that CO2 assimilation in Eukarya (quantitatively most important in green plants) is of microbial origin. RuBisCO has a rather slow turn- Conversion of Inorganic Carbon to Cell over even at saturating levels of CO2. Currently. the reductive citric acid cycle (Arnon–Buchanan the different carboxylating enzymes are specific for either cycle). The conversion of pyruvate to the C4-com. there are at least assimilation sequences are known presently: the reductive five such systems known in cyanobacteria. are able to plete) reductive citric acid cycle. way). and the 3-hydroxypropionate/4-hydroxybuty- These aspects of CO2 metabolism have not been addressed rate cycle). did heterotrophic life been proposed. Furthermore. because the function of these protein microcompartments were first average oxidation level of any cell is close to zero (equal suggested for Halothiobacillus neapolitanus (formerly: to oxidation state of formaldehyde. Carboxysomes provide a microenvironment for RuBisCO with locally elevated CO2 The general equation for cell carbon biosynthesis from (and most likely decreased oxygen) concentrations. In addition. which covers the structural and functional aspects of the carboxysome in more detail. reductive citric uncatalyzed interconversion of CO2 =HCO3 – rate-limiting. Similar cellular inclusions were found in used for CO2 assimilation (see below). and Eukarya. hypotheses. Autotrophic CO2 Metabolism 141 well as in anaerobic environments. atmosphere is only about 10 mmol l1. That is. processes evolve after the establishment of autotrophy pound oxaloacetate proceeds either by direct carboxylation or did heterotrophic life forms capable of metabolism of (pyruvate carboxylase) or via carboxylation of phosphoe.or C4-compounds is not discussed Evolutionary Aspect in detail in this chapter. cycle). Independent of pentose phosphate cycle (Calvin–Bassham–Benson cycle). CH2O). compartments and Synthesis of Central Precursor Metabolites assemblages’. and the majority of the cellular RuBisCO enzyme. an aerobic thiosulfate oxidizer and amount of ATP required is dependent on the mechanism obligate autotroph. low-molecular compounds present in the ‘primeval soup’ nolpyruvate (PEP) catalyzed by either PEP carboxylase or precede autotrophy? The speculations. Carboxysomes con- trophic CO2 fixation follows the general reaction scheme: sist of several different proteins. Five CO2 autotrophic CO2 metabolism. However. but in each case the reductive carboxylation of acetyl-CoA to pyruvate. among them specific shell proteins. there are various CO2/bicarbonate transpor- from CO2. oxaloacetate. The rapid interconversion of bicarbo- 3-hydroxypropionate/4-hydroxybutyrate cycle. it is sufficient to understand the synthesis of ters involved in the CO2 concentration mechanism that central precursor metabolites. 2- bicarbonate across membranes adds a fascinating aspect to oxoglutarate. The reader is referred to ‘Intracellular structures of prokaryotes: Inclusions. this enzyme also Carbon catalyses an apparent wasteful competing oxygenation reac- Overall Equation tion in the presence of oxygen. Bacteria. pyruvate (3-hydroxypropionate/malyl-CoA in most autotrophs. The question as to whether autotrophy is a primitive trait uvate synthase (pyruvate:ferredoxin oxidoreductase).5-bispho- sphate carboxylase/oxygenase (RuBisCO). and the CO2 or bicarbonate. how- ever. It is important to remember. Therefore. as the CO2 Concentrating Mechanisms chloroplasts from such organisms are thought to have The concentration of dissolved CO2 at pH7 under an air arisen from a cyanobacterial endosymbiont. acid cycle. . and PEP carboxykinase. or 3-phosphoglycerate (reductive pentose phosphate cycle) from either CO2 or bicarbonate. or 3-phosphoglycerate. The rather recently recog- central carbon metabolism from which building blocks for nized protein-facilitated transport of CO2 and/or polymers are made and include acetyl-CoA. the 3-hydroxypropionate/malyl-CoA cycle. The C5-compound 2-oxoglutarate is presentations of the various controversial views on this formed from acetyl-CoA and oxaloacetate by enzymes of subject will not be part of this overview. Archaea. has has been controversial. thrive autotrophically. catalyzed by pyr. the major CO2 fixation enzyme. the reductive acetyl-CoA pathway (Wood–Ljungdahl path- CO2 or bicarbonate has to first enter the cell. has Km values for CO2 that range from 10 to 300 mmol l1. Members of all three the oxidative citric acid cycle or via a (complete or incom- domains of life. These are intermediates of deliver bicarbonate into the cell. auto- oxygenic phototrophic cyanobacteria. pyruvate. carbonic anhydrase (catalyzing the interconversion CO2 þ 4H þ nATP ! ½CH2 O þ H2 O þ nADP þ nPi of bicarbonate and CO2). These nate and CO2 catalyzed by carbonic anhydrases is at issue as autotrophic pathways account for the net synthesis of long as carbon flux rates are high enough to make the acetyl-CoA (reductive acetyl-CoA pathway.

does not requires additional CO2 to be fixed by RuBisCO to com- include a regeneration step. known. albeit one of the fastest enzymes RuBisCO.5-bisphosphate 5× carboxylase/oxygenase