Advances in Microbial Physiology

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ISBN: 978-0-12-387661-4
ISSN: 0065-2911
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11 12 13 14 10 9 8 7 6 5 4 3 2 1
Contributors to Volume 59
MUKTAK AKLUJKAR, Department of Microbiology and Environmental Bio-

technology Center, University of Massachusetts, Amherst, Massachusetts,
USA
LESLEY A.H. BOWMAN, Department of Molecular Biology and

Biotechnology, The University of Sheffield, Sheffield, United Kingdom
JESSICA E. BUTLER, Department of Microbiology and Environmental Bio-

USA
KELLY A. FLANAGAN, Department of Microbiology and Environmental

Biotechnology Center, University of Massachusetts, Amherst,
Massachusetts, USA
ASHLEY E. FRANKS, Department of Microbiology and Environmental Bio-

USA
JON M. FUKUTO, Department of Chemistry, Sonoma State University,

Rohnert Park, California, USA
LUDOVIC GILOTEAUX, Department of Microbiology and Environmental

Massachusetts, USA
J.S. HALLINAN, School of Computing Science, Newcastle University,

Newcastle, United Kingdom
DAWN E. HOLMES, Department of Microbiology and Environmental Bio-

USA
viii CONTRIBUTORS TO VOLUME 59
K. JAMES, School of Computing Science, Newcastle University, Newcastle,

United Kingdom
DEREK R. LOVLEY, Department of Microbiology and Environmental Bio-

USA
NIKHIL S. MALVANKAR, Department of Microbiology and Environmental

Massachusetts, USA
SAMANTHA MCLEAN, Department of Molecular Biology and Biotechnology,

The University of Sheffield, Sheffield, United Kingdom
KELLY P. NEVIN, Department of Microbiology and Environmental Biotech-

nology Center, University of Massachusetts, Amherst, Massachusetts, USA
ROBERTO ORELLANA, Department of Microbiology and Environmental

Massachusetts, USA
ROBERT K. POOLE, Department of Molecular Biology and Biotechnology,

The University of Sheffield, Sheffield, United Kingdom
CARLA RISSO, Department of Microbiology and Environmental Biotechnol-

ogy Center, University of Massachusetts, Amherst, Massachusetts, USA
AMELIA-ELENA ROTARU, Department of Microbiology and Environmental

Massachusetts, USA
PRAVIN M. SHRESTHA, Department of Microbiology and Environmental

Massachusetts, USA
TOSHIYUKI UEKI, Department of Microbiology and Environmental Biotech-

nology Center, University of Massachusetts, Amherst, Massachusetts, USA
A. WIPAT, School of Computing Science, Newcastle University, Newcastle,

United Kingdom, and Institute of Cell and Molecular Biosciences,
Newcastle University, Newcastle, United Kingdom
TIAN ZHANG, Department of Microbiology and Environmental Biotechnol-

ogy Center, University of Massachusetts, Amherst, Massachusetts, USA
Geobacter: The Microbe Electric’s
Physiology, Ecology, and Practical
Applications
Derek R. Lovley, Toshiyuki Ueki, Tian Zhang, Nikhil S.
Malvankar, Pravin M. Shrestha, Kelly A. Flanagan, Muktak
Aklujkar, Jessica E. Butler, Ludovic Giloteaux, Amelia-Elena
Rotaru, Dawn E. Holmes, Ashley E. Franks, Roberto Orellana,
Carla Risso and Kelly P. Nevin
Department of Microbiology and Environmental Biotechnology Center, University of

Massachusetts, Amherst, Massachusetts, USA
ABSTRACT
Geobacter species specialize in making electrical contacts with extracellular

electron acceptors and other organisms. This permits Geobacter species to
fill important niches in a diversity of anaerobic environments. Geobacter
species appear to be the primary agents for coupling the oxidation of
organic compounds to the reduction of insoluble Fe(III) and Mn(IV)
oxides in many soils and sediments, a process of global biogeochemical
significance. Some Geobacter species can anaerobically oxidize aromatic
hydrocarbons and play an important role in aromatic hydrocarbon removal
from contaminated aquifers. The ability of Geobacter species to reductively
precipitate uranium and related contaminants has led to the development
of bioremediation strategies for contaminated environments. Geobacter
species produce higher current densities than any other known organism in
microbial fuel cells and are common colonizers of electrodes harvesting
electricity from organic wastes and aquatic sediments. Direct interspecies
electron exchange between Geobacter species and syntrophic partners
ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 59 Copyright # 2011 by Elsevier Ltd.

ISSN: 0065-2911 All rights reserved
DOI: 10.1016/B978-0-12-387661-4.00004-5
2 DEREK R. LOVLEY ET AL.
appears to be an important process in anaerobic wastewater digesters.

Functional and comparative genomic studies have begun to reveal
important aspects of Geobacter physiology and regulation, but much
remains unexplored. Quantifying key gene transcripts and proteins of
subsurface Geobacter communities has proven to be a powerful approach
to diagnose the in situ physiological status of Geobacter species during
groundwater bioremediation. The growth and activity of Geobacter species
in the subsurface and their biogeochemical impact under different
environmental conditions can be predicted with a systems biology
approach in which genome-scale metabolic models are coupled with
appropriate physical/chemical models. The proficiency of Geobacter
species in transferring electrons to insoluble minerals, electrodes, and
possibly other microorganisms can be attributed to their unique “microbial
nanowires,” pili that conduct electrons along their length with metallic-like
conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter
species do not contribute to this long-range electron transport, but
cytochromes are important for making the terminal electrical connections
with Fe(III) oxides and electrodes and also function as capacitors, storing
charge to permit continued respiration when extracellular electron
acceptors are temporarily unavailable. The high conductivity of Geobacter
pili and biofilms and the ability of biofilms to function as supercapacitors
are novel properties that might contribute to the field of bioelectronics.
The study of Geobacter species has revealed a remarkable number of
microbial physiological properties that had not previously been described
in any microorganism. Further investigation of these environmentally
relevant and physiologically unique organisms is warranted.
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Distribution and Abundance of Geobacter Species . . . . . . . . . . . . . . . . . 6
3. Brief Description of Geobacter Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4. Phylogeny and Genomic Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
5. Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.1. Fe(III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.2. Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.3. Other Extracellular Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . 19
5.4. Other Microorganisms—Syntrophy . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Electron Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes, Humics,
Fe(II), U(IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
6.2. Aromatic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
7. Extracellular Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
7.1. Microbial Nanowires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
7.2. Cytochromes and Multicopper Proteins . . . . . . . . . . . . . . . . . . . . . . 30
GEOBACTER PHYSIOLOGY AND ECOLOGY 3
7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide . . . . . . . 34

7.4. Model for Extracellular Electron Transfer to Electrodes . . . . . . . . . 37
7.5. Extracellular Electron Transfer in Syntrophy . . . . . . . . . . . . . . . . . . 40
7.6. Model for Extracellular Electron Transfer to Other Extracellular
Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7.7. Capacitor Role of Cytochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
8. Regulation of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.1. Sigma Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
8.2. Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
8.3. Two-Component Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.4. Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.5. Nucleotide-Based Second Messenger . . . . . . . . . . . . . . . . . . . . . . . 50
8.6. Summary Statement on Regulation . . . . . . . . . . . . . . . . . . . . . . . . . 51
9. Environmental Systems Biology of Geobacter . . . . . . . . . . . . . . . . . . . . . 52
9.1. Environmental Transcriptomics and Proteomics . . . . . . . . . . . . . . . 52
9.2. BUGS (Bottom-Up Genome-Scale) Modeling . . . . . . . . . . . . . . . . . 54
10. Biogeochemical Impacts of Geobacter Species . . . . . . . . . . . . . . . . . . . . 56
11. Practical Applications of Geobacter Species . . . . . . . . . . . . . . . . . . . . . . 57
11.1. Bioremediation: Natural Attenuation and Engineered . . . . . . . . . . . 57
11.2. Producing Methane from Organic Wastes and
Hydrocarbon Deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
11.3. Microbial Fuel Cells, Electrosynthesis, and Bioelectronics . . . . . . 61
12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1. INTRODUCTION
Geobacter species represent a rare example of a genus of microorganisms

that are abundant and play an important biogeochemical role in a diversity
of natural environments, yet are easily cultured and can be genetically
manipulated for physiological studies. Although there are other Fe(III)-
reducing microorganisms that have been studied in more detail, it is clear
that Geobacter species are generally the predominant Fe(III)-reducing
microorganisms in many soils and sediments in which Fe(III) reduction is
an important process. Physiological studies with Geobacter species have
revealed a number of novel microbial properties that have an important
impact on the geochemistry of some anaerobic soils and sediments and,
in some instances, have practical applications.
As detailed in subsequent sections, the following microbial processes
were first identified in studies with Geobacter species: (1) oxidation of
organic compounds to carbon dioxide with Fe(III) or Mn(IV) as the elec-
tron acceptor, (2) conservation of energy from organic matter oxidation
coupled to Fe(III) or Mn(IV) reduction, (3) production of extracellular
magnetite from microbial Fe(III) reduction, (4) anaerobic oxidation of an

aromatic hydrocarbon in pure culture, (5) microbial reduction of U(VI),
(6) microbial reduction of Co(III), (7) utilization of humic substances as
an electron acceptor for microbial respiration, (8) oxidation of organic
compounds to carbon dioxide with an electrode serving as an electron
acceptor, (9) conservation of energy from the oxidation of organic com-
pounds coupled to electron transfer to an electrode, (10) the potential
for an electrode to serve as an electron donor to support microbial respira-
tion, (11) use of cytochromes as capacitors to permit respiration in the
absence of exogenous electron acceptors, (12) extracellular electron trans-
fer via microbial nanowires, (13) organic metallic-like long-range conduc-
tion of electrons along a protein filament, (14) production of conductive
biofilms with conductivities comparable to that of synthetic polymers,
and (15) the potential for interaction with syntrophic partners via a direct
electron transfer (Fig. 1).
The reduction of Fe(III), and to a lesser extent Mn(IV), by Geobacter
species can play an important role in carbon cycling in water-saturated
soils and aquatic sediments and further influences the geochemistry of
these environments through the release of dissolved Fe(II) and Mn(II) as
Dominance in Current Metallic-like

Genus Geobacter petroleum- conduction in pili
GS-15 reported established production
contaminated and biofilms
aquifer Microbial
Anaerobic nanowires
Anaerobic
aromatic Electrode-driven
benzene
hydrocarbon Humics Genetic dechlorination
degradation
degradation reduction System G. sulfurreducens
genome
1985 1990 1995 2000 2005 2010 2015
U(VI) Humics as Electrode-

reduction donor driven Interspecies
respiration electron
Role of transfer
Reductive Importance in
Additional novel G. sulfurreducens Motility, dechlorination methanogenic
properties of described chemotaxis,
GS-15 reported aggregates
and pili
Figure 1 Time line of important discoveries associated with Geobacter species.

well as trace metals, metalloids, and phosphate that adsorb onto Fe(III)
and Mn(IV) oxides. In fact, the studies that led to the discovery of the first
Geobacter species were initially designed to better understand the flux of
phosphate from aquatic sediments that contributes to algal blooms.
Geobacter reduction of U(VI) and radionuclides can have an important
influence on the migration of these compounds and is considered to be a
potential tool for mitigating environmental contamination. Geobacter spe-
cies play an important role in degrading a diversity of organic contaminants
in groundwater, both under natural attenuation and engineered bioremedi-
ation strategies. The ability of Geobacter species to exchange electrons
with electrodes has inspired several new strategies for bioenergy and
bioremediation. A recent surprise is the realization that Geobacter
species are important syntrophic microorganisms, forming partnerships
with methanogenic microorganisms, under conditions where they can
significantly contribute to the conversion of organic wastes, or hydrocarbon
deposits, to methane. The production of Geobacter-based materials
with novel electronic properties is a newly emerging field of study.
The number of publications on Geobacter species is relatively small but
continues to grow (Fig. 2) as does awareness of the environmental
relevance of these organisms and their potential practical applications.
The purpose of this review is to provide a broad overview of what has been
learned about Geobacter species since they were discovered 25 years ago.
Due to time and space constraints, not every publication mentioning
Geobacter species could be reviewed.
Geobacter items published
140 4500
Geobacter items cited
4000
120
3500
100
each year
each year
3000
80 2500
60 2000
1500
40
1000
20 500
0 0
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
1995
1997
1999
2001
2003
2005
2007
2009
Figure 2 Publications and citations each year with Geobacter as a topic

according to data from the Thomson Reuters ISI Web of Knowledge.
2. DISTRIBUTION AND ABUNDANCE OF GEOBACTER

SPECIES
The hallmark physiological capability of Geobacter species is their ability

to couple the oxidation of organic compounds to the reduction of Fe
(III), which allows Geobacter species to fill key niches in the anaerobic
microbial food chain of sedimentary environments such as aquatic
sediments, wetlands, rice paddies, and subsurface environments in which
Fe(III) reduction is an important terminal electron-accepting process
(Lovley, 1987, 1991, 1993, 1995, 2000b). For example, molecular analysis
of the metabolically active microorganisms in Fe(III)-reducing rice paddy
soils revealed that Geobacter species accounted for 85% of the
microorganisms consuming acetate, the key intermediate in anaerobic deg-
radation of organic matter (Hori et al., 2010). Other factors such as the
“remarkably low” maintenance energy requirement of Geobacter species
may also be an important factor in their success in subsurface
environments (Lin et al., 2009).
Molecular analyses, which avoid cultivation bias, have generally found
that Geobacter species are the most abundant Fe(III)-reducing
microorganisms in environments in which Fe(III) reduction is actively tak-
ing place (see, e.g., Anderson et al., 2003; Cummings et al., 2003; Holmes
et al., 2007; Hori et al., 2010; Islam et al., 2004a; Kerkhof et al., 2011;
Rooney-Varga et al., 1999; Röling et al., 2001; Snoeyenbos-West et al.,
2000; Stein et al., 2001; Vrionis et al., 2005). Pure culture isolates have been
recovered from a diversity of environments (Table 1). Further, molecular
(Fig. 3) and/or enrichment studies have detected Geobacter in diverse
environments such as aquifers contaminated with petroleum (Alfreider
and Vogt, 2007; Anderson et al., 1998; Botton et al., 2007; Coates et al.,
1996; Holmes et al., 2007; Prakash et al., 2010; Rooney-Varga et al., 1999;
Salminen et al., 2006; Snoeyenbos-West et al., 2000; Van Stempvoort
et al., 2009; Winderl et al., 2007, 2008); groundwater contaminated with
landfill leachate (Kuntze et al., 2011; Lin et al., 2005, 2007; Röling et al.,
2001; Staats et al., 2011); environments contaminated with organic acids
(Azizian et al., 2010; Stults et al., 2001); contaminated soils and aquatic
sediments (Blothe et al., 2008; Cummings et al., 2000; Haller et al., 2011;
Halm et al., 2009; Manickam et al., 2010); uranium-contaminated subsur-
face sediments amended with organics to promote metal reduction (Akob
et al., 2008; Amos et al., 2007; Anderson et al., 2003; Baldwin et al., 2008;
Brodie et al., 2006; Burkhardt et al., 2010, 2011; Callister et al., 2010;
Cardenas et al., 2008, 2010; Chandler et al., 2010; Chang et al., 2005;
t0005 Table 1 Geobacter species available in pure culture listed in the order in which the species were described.
Genome Electron donors Other Optimal

size and oxidized with Fe Fe forms electron growth
Name Source informationa (III)b reducedc acceptorsa,d temperature Referencese
Geobacter Aquatic 4,011,182 bp Ac, Bz, Bzef, PCIO, Fe Mn(IV), 30 Lovley et al.
metallireducens sediments GC%—59.5 BtOH, Buty, Bzo, (III)-Cit Tc(VII)*, (1987,
Aklujkar BzOH, p-Cr, U(VI), 1993),
et al. (2009) EtOH, p-HBz, AQDS, Lovley and
p-HBzo, humics, Phillips
p-HBzOH, IsoB, Nitrate (1988a,b)
IsoV, Ph, Prop,
PrOH, Pyr,
Tol, Val
Geobacter Contaminated 3,814,139 bp Ac, H2 PCIO, Fe Tc(VII)*, 35 Caccavo
sulfurreducens ditch GC%—60.9 (III)-Cit, Co(III), et al. (1994),
Strain Fe(III)-P U(VI), Lin et al.
PCA— AQDS, S , (2004)
Méthé et al. Fum, Mal,
(2003) O2
Strain
KN400—
Nagarajan
et al. (2010)
“Geobacter Contaminated Ac, EtOH, For, PCIO, Fe Mn(IV), 30 Coates et al.
humireducens” wetland H2, Lac (III)-Cit AQDS S , (1998)
(strain JW3) nitrate,
Fum,
Geobacter Deep Ac, EtOH For, PCIO, Fe- Mn(IV), 25 Coates et al.
chapellei subsurface Lac NTA AQDS, (2001)
Fum
Geobacter Aquatic Ac, Buty, EtOH, PCIO, Fe AQDS 30 Coates et al.
grbiciae sediments For, Prop, Pyr (III)-Cit (2001)
(continued)
Table 1 (continued)

Geobacter Contaminated Ac, Buty, Bzo, PCIO, Fe AQDS, 30 Coates et al.

hydrogenophilus aquifer EtOH, For, H2, (III)-Cit Fum (2001)
Prop, Pyr, Suc
Geobacter Freshwater Ac, BtOH, Buty, PCIO Mn(IV), 30 Straub and
bremensis ditch Bzo, EtOH, For, So, Fum, Buchholz-
Fum, H2, Lac, Mal Cleven
Mal, Prop, PrOH, (2001)
Pyr, Succ
Geobacter Freshwater Ac, EtOH, For, PCIO, Mn(IV), 30 Straub and
pelophilus ditch Fum, H2, Mal, Akaganeite So, Fum, Buchholz-
Prop, PrOH, Pyr, Mal Cleven
Succ (2001)
Geobacter Fe(III)- 4,615,150 bp Ac, Bzo, BtOH, Fe(III)-Cit, AQDS, 30 Nevin et al.
bemidjiensis reducing GC%—60.3 Buty, EtOH, Fe(III)- Fum, Mal, (2005)
subsurface Aklujkar Fum, H2, IsoB, NTA, Fe Mn(IV)
sediment et al., 2010 Lac, Mal, Prop, (III)-P,
Pyr, Succ, Val PCIO
Geobacter Acetate- Ac, BtOH, EtOH, Fe(III)-Cit, AQDS, 17–30 Nevin et al.
psychrophilus impacted For, Lac, Mal, Fe(III)- Electrode, (2005)
aquifer Pyr, Succ NTA, Fe Fum, Mal,
sediment (III)-P, Mn(IV)
PCIO
Geobacter Freshwater Ac, Bze, Bzo, Fe(III)-Cit, PCE, TCE, 35 Sung et al.
lovleyi sediment Buty, Cit, EtOH, PCIO nitrate, (2006)
For, Glu, Lac, Fum, Mal,
MeOH, Prop, So, U(VI),
Succ, Tol, YE, Mn(IV)
Geobacter Sedimentary Ac, Buty, BtOH, Fe(III)-Cit, AQDS, 30 Shelobolina
pickeringii kaolin strata EtOH, MeOH, Fe(III)- Mal, Fum, et al.
Glyc, Lac, Pyr, NTA, Fe Mn(IV), (2007a,b)
Succ, Val (III)-P, S , U(VI)*
PCIO
Geobacter Sedimentary Ac, Buty, BtOH, Fe(III)-Cit, Nitrate, 30 Shelobolina
argillaceus kaolin strata EtOH Glyc, Lac, Fe(III)- Mn(IV), et al.
Pyr, Val NTA, Fe S , U(VI)* (2007b)
(III)-P,
PCIO
Geobacter Subsurface soil Ac, Act, H2 Fe(III)- Mal, Fum, 30 Nevin et al.
thiogenes NTA nitrate, So, (2007),De
TCA Wever et al.
(2000)
Geobacter Uranium- 5,136,364 bp Ac, EtOH, Lac, Fe(III)- AQDS, 32 Shelobolina
uraniireducens contaminated GC%—54.3 Pyr NTA, Fe Fum, Mal, et al. (2008)
subsurface (III)-P, Mn(IV),
sediment PCIO, U(VI)*
smectite
Geobacter Tar-oil- Ac, Buty, Bz, Bzo, Fe(III)-Cit, Fum 25–32 Kunapuli
toluenoxydans contaminated BzOH, For, m-Cr, PCIO et al. (2010)
sediment Prop, Pyr, Ph,
p-Cr, Tol
Geobacter Heavy metal- 4,304,501 bp Ac, Buty, For Fe(III)-Cit, Fum, Mal, 30 Prakash
daltonii and GC%— Bzog, Tolg PCIO So, U(VI) et al. (2010)
hydrocarbon- 53.5%
contaminated
shallow
subsurface
sediment
“Geobacter Uranium- 5,277,406 bp Ac, Act, Asp, PCIO, AQDS 22–25 Holmes
andersonii” contaminated GC%—61.2 EtOH, For, Fum, subsurface et al.
strain M18 subsurface Glt, Lac, Mal, Pyr, sediment (2011c)
sediment Succ, YE
“Geobacter Uranium- 4,745,806 bp Ac, Act, Ala, Bzo, PCIO, AQDS, 22–25 Holmes
remediiphilus” contaminated GC%—60.5 EtOH, For, Glt, subsurface Mn(IV) et al. (2011c)
strain M21 subsurface Lac, Pyr, Ser, sediment
sediment Succ, Xyl, YE
“Geobacter Acetate- Ac, Act, Asp, Bzo PCIO, AQDS, 22–25 Holmes
aquiferi” impacted Cit, Cys, EtOH, subsurface Mn(IV) et al. (2011c)
strain Ply1 aquifer For, Fum, Lac, sediment
sediment Mal, Pyr, Xyl, YE
(continued)
Table 1 (continued)

“Geobacter Acetate- Ac, Act, Bzo, PCIO, 25–30 Holmes

plymouthensis” impacted Butyr, Fum, Lact, subsurface et al. (2011c)
strain Ply4 aquifer Mal, Pep, Prop, sediment
sediment Pyr, Succ
a
Links for genome information: Geobacter metallireducens—http://www.ncbi.nlm.nih.gov/genome?
Db¼genome&Cmd¼ShowDetailView&TermToSearch¼18912Geobacter sulfurreducens strain PCA—http://www.ncbi.nlm.nih.gov/genome?
Db¼genome&Cmd¼ShowDetailView&TermToSearch¼379Geobacter sulfurreducens strain KN400—http://www.ncbi.nlm.nih.gov/nuccore/
CP002031.1Geobacter bemidjiensis—http://www.ncbi.nlm.nih.gov/genome?Db¼genome&Cmd¼ShowDetailView&TermToSearch¼22805Geobacter
lovleyi—http://www.ncbi.nlm.nih.gov/genome?Db¼genome&Cmd¼ShowDetailView&TermToSearch¼22459Geobacter uraniireducens—http://www.
ncbi.nlm.nih.gov/genome?Db¼genome&Cmd¼ShowDetailView&TermToSearch¼20999Geobacter daltonii—http://www.ncbi.nlm.nih.gov/genome?
Db¼genome&Cmd¼ShowDetailView&TermToSearch¼23754Geobacter andersonii—http://www.ncbi.nlm.nih.gov/genome?
Db¼genome&Cmd¼ShowDetailView&TermToSearch¼26944Geobacter remediiphilus—http://www.ncbi.nlm.nih.gov/genome?
Db¼genome&Cmd¼ShowDetailView&TermToSearch¼24728.
b
Abbreviations for electron donors and acceptors: acetate (Ac), acetoin (Act), alanine (Ala), anthraquinone-2,6-disulfonic acid (AQDS), aspartic acid
(Asp), benzaldehyde (Bz), benzene (Bze), benzoate(Bzo), benzylalcohol (BzOH), butanol (BtOH), butyrate(Buty), citrate (Cit), p-cresol (p-Cr),
m-cresol (m-Cr), cysteine (Cys), elemental sulfur (So), ethanol (EtOH), formate (For), fumarate (Fum), glucose (Glu), glutamic acid (Glt), glycerol
(Glyc), p-hydroxybenzoate (p-HB), p-hydroxybenzaldehyde (p-HBz), p-hydroxybenzylalcohol (p-HBzOH), hydrogen (H2), isobutyrate (IsoB),
isovalerate (IsoV), lactate(Lac), malate (Mal), methanol (MeOH), manganese oxide (Mn(IV)), peptone (Pep), phenol (Ph), propanol (PrOH),
propionate (Prop), pyruvate (Pyr), serine (Ser), succinate (Succ), tetrachloroethylene (PCE), trichloroethylene (TCE), toluene (Tol), trichloroacetic
acid (TCA), valerate (Val), xylose (Xyl), yeast extract (YE).
c
Fe(III) forms: Poorly crystalline iron oxide (PCIO), ferric citrate (Fe(III)-cit), ferric nitrilotriacetic acid (Fe(III)-NTA), ferric pyrophosphate
(Fe(III)-P)
d,*
Organism has the ability to reduce the metal but not determined whether energy to support growth is conserved from reduction of this metal.
e
Reference in which the capacity to grow via Fe(III) reduction is described, followed by references with other physiological traits.
f
Zhang et al. (2011).
g
Electron donors utilized with fumarate as electron acceptor only.
Figure 3 Neighbor-joining tree showing the phylogenetic relationship within

the genus Geobacter based on 16S rRNA gene sequences. The clone sequences
having > 98% 16S rRNA gene sequence identities were grouped into a single clus-
ter. Cultured representatives (black), including isolates whose genomes are fully
sequenced (red) are shown in the figure. Isolation source and the reference for
both pure culture isolates (blue) and representatives environmental clone
sequences (black) are also shown at the right side of the tree. The sequences
assigned as unpublished in the NCBI and SILVA databases are presented with
their accession number. All sequences (> 1300 bases) were obtained from the
SILVA SSU_106 Ref database (Pruesse et al., 2007) and manually aligned in
ARB program (Ludwig et al., 2004) before the phylogenetic tree construction.
The scale bar represents 10% sequence divergence.
Holmes et al., 2002; Hwang et al., 2009; Istok et al., 2004; Kerkhof et al.,
2011; Michalsen et al., 2007; Mohanty et al., 2008; North et al., 2004; Pea-
cock et al., 2004; Scala et al., 2006; Wan et al., 2005; Wilkins et al., 2007,
2009; Williams et al., 2011; Xu et al., 2010); subsurface environments with
high arsenic concentrations (Héry et al., 2008; Islam et al., 2004a; Lear
et al., 2007; Weldon and MacRae, 2006); environments contaminated with
chlorinated compounds or dechlorinating enrichment cultures (Amos et al.,
2007; Bedard et al., 2007; Imfeld et al., 2010; Kim et al., 2010; Macbeth
et al., 2004; Sorensen et al., 2010; Sung et al., 2006; Yoshida et al., 2005);
wetland and aquatic sediments (Brofft et al., 2002; Cifuentes et al., 2000;
Coates et al., 1998; Costello and Schmidt, 2006; Costello et al., 2009; Martins
et al., 2011; Musat et al., 2010; Roden et al., 2006, 2008; Stein et al., 2001;
Straub et al., 1998); freshwater seeps (Blothe and Roden, 2009; Bruun
et al., 2010; den Camp et al., 2008); acidic springs, peat, or sediments
(Adams et al., 2007; Kusel et al., 2008, 2010; Percent et al., 2008); pristine
aquifers (Flynn et al., 2008; Holmes et al., 2007); rice paddy or other soils
(Cahyani et al., 2008; Conrad et al., 2007; Friedrich et al., 2004; Hansel
et al., 2008; Hiraishi et al., 2005; Hori et al., 2010; Ishii et al., 2009; Noll
et al., 2005; Scheid et al., 2004; Zhu et al., 2009); soil rhizosphere (Fernando
et al., 2008); mangrove sediments (Zhang et al., 2008); a 1700-year-old
wooden spear shaft (Helms et al., 2004); iron-rich snow (Kojima et al.,
2009); clay wall material (Kitajima et al., 2008); dental unit water supply sys-
tems (Singh et al., 2003); methanogenic digesters (Cervantes et al., 2003,
2004; Morita et al., 2011; Riviere et al., 2009; Tsushima et al., 2010; Werner
et al., 2011); and the deep subsurface (Coates et al., 1996, 2001; Kovacik
et al., 2006; Shimizu et al., 2006). In many of these studies, it was concluded
that Geobacter species had an important role in influencing the soil/sedi-
ment/groundwater biogeochemistry and/or promoting bioremediation.
Another environment in which Geobacter species or closely related
Desulfuromonas, Geopsychrobacter, and Pelobacter species are often
abundant is on the surface of electrodes harvesting electricity from organic
matter in wastewater or systems initiated with wastewater inocula
(Aelterman et al., 2008; Borole et al., 2009; Butler et al., 2010a; Call
et al., 2009; Chang et al., 2008; Choo et al., 2006; Cusick et al., 2010; Freguia
et al., 2010; Ishii et al., 2008; Jung and Regan, 2007, 2011; Kiely et al., 2011a,b;
Kim et al., 2007, 2008b; Lee et al., 2003, 2008, 2009; Li et al., 2010; Liu et al.,
2008; Luo et al., 2010; Parameswaran et al., 2010; Shimoyama et al., 2009;
Torres et al., 2009a; Xing et al., 2009), as well as sediments (Bond et al., 2002;
De Schamphelaire et al., 2010; Holmes et al., 2004a,d; Kato et al., 2010; Liu
et al., 2007; Reimers et al., 2006; Tender et al., 2002; White et al., 2009; Williams
et al., 2010).
3. BRIEF DESCRIPTION OF GEOBACTER SPECIES
A significant number of pure culture isolates of Geobacter species are

available (Table 1; Fig. 3). All Geobacter isolates are Gram-negative rods
that are capable of oxidizing acetate with the reduction of Fe(III). Other
commonly conserved features include the ability to reduce Mn(IV),
U(VI), elemental sulfur, and humic substances or the humic substance ana-
log anthraquinone-2,6-disulfonate (AQDS). Many isolates have the ability
to use other small molecular weight organic acids, ethanol, or hydrogen as
an electron donor (Table 1).
The two most heavily studied Geobacter species have been G. meta-
llireducens and G. sulfurreducens. G. metallireducens was the first Geobacter
species recovered in pure culture (Lovley and Phillips, 1988a; Lovley et al.,
1987, 1993a). It was with this isolate that many of the novel physiological
attributes listed in Section 1 were discovered. The recent development of a
genetic system for G. metallireducens (Tremblay et al., 2011a) is likely to
refocus attention on this organism to elucidate the physiology of important
novel properties, such as anaerobic benzene degradation.
Geobacter sulfurreducens was the first Geobacter species for which met-
hods for genetic manipulation were developed (Aklujkar and Lovley, 2010;
Coppi et al., 2001; Kim et al., 2005; Lloyd et al., 2003; Park and Kim, 2011;
Rollefson et al., 2009; Ueki and Lovley, 2010a), and therefore it has served
as the Geobacter of choice for functional genomic studies designed to
understand Geobacter metabolism, gene regulation, and extracellular elec-
tron transfer. It was the first Geobacter species found to use hydrogen as
an electron donor, or to grow with elemental sulfur as an electron accep-
tor. The originally isolated strain was referred to as strain PCA (Caccavo
et al., 1994). A commonly used strain of G. sulfurreducens derived from
strain PCA is frequently referred to as strain DL-1 (Coppi et al., 2001)
because this culture was maintained for many transfers in the laboratory
and may have accumulated a significant number of mutations that were
not present in the originally isolated PCA strain. For example, the DL-1
strain only poorly reduces Fe(III) oxide unless it is adapted for growth
on Fe(III) oxide for long periods of time. The capacity for effective Fe
(III) oxide reduction was recovered via adaptive evolution (Tremblay
et al., 2011b).
Another valuable strain of G. sulfurreducens is strain KN400, which was
recovered in a study designed to adaptively evolve G. sulfurreducens for
growth on electrodes (Yi et al., 2009). Although the KN400 and DL-1
strains have an identical 16S rRNA gene sequence, they have some
important physiological differences. In addition to producing more current

than DL-1 (Yi et al., 2009), KN400 also reduces Fe(III) oxides much faster
(Flannagan et al., 2011). One reason for this may be greater expression of
pili in KN400, which, as discussed below, is thought to be a major conduit
for electron transfer to Fe(III) oxide. Further, strain KN400 is motile,
whereas strain DL-1 is not. This can be attributed to interruption of the
gene for the master regulator for flagella gene expression, FrgM, in DL-1
(Ueki et al., 2011). Motility is important in Fe(III) oxide reduction, as
described below, and flagella could play a role in biofilm formation on
electrodes.
Some Geobacter isolates have been isolated in studies focused on novel
physiological properties such as the ability to use aromatic compounds
(G. toluenoxydans; Kunapuli et al., 2010) or reduction of Fe(III) in clays
(G. pickeringii, G. argillaceus; Shelobolina et al., 2007b). G. lovleyi (Sung
et al., 2006) is the only Geobacter species that has been shown to reduc-
tively dechlorinate the chlorinated solvents tetrachloroethylene (PCE)
and trichloroethylene (TCE) that are common groundwater contaminants
and 16S rRNA gene sequences closely related to the pure culture have
been recovered in dechlorinating enrichment cultures (Daprato et al.,
2007; Dennis et al., 2003; Duhamel and Edwards, 2006, 2007; Duhamel
et al., 2004) as well as subsurface environments contaminated with
chlorinated solvents (Amos et al., 2007; Kim et al., 2010; Macbeth et al.,
2004; Sorensen et al., 2010; Sung et al., 2006). G. (formerly Trichlorobacter)
thiogenes is the only other known dechlorinating Geobacter, reducing
trichloroacetic acid (De Wever et al., 2000; Nevin et al., 2007).
One of the goals of isolating pure cultures of Geobacter species is to
obtain isolates that are representative of the Geobacter species that pre-
dominate in environments of interest. Therefore, the isolates Geobacter
uraniireducens (Shelobolina et al., 2008), Geobacter andersonii (Holmes
et al., 2011c), and Geobacter remediiphilus (Holmes et al., 2011c) are of spe-
cial interest because their 16S rRNA gene sequences match 16S rRNA gene
sequences that were found to predominate during active Fe(III) reduction in
the uranium-contaminated aquifer in Rifle, CO when it is amended with
acetate. These isolates may be particularly useful for elucidating the physiol-
ogy of Geobacter species in such systems.
The isolates from the Rifle, CO site are in a phylogenetic clade, known as
subsurface clade I, which as discussed in the next section, is a phylogenetically
coherent group of Geobacter species that have been found to predominate
in a diversity of aquifers in which Fe(III) reduction is important
(Holmes et al., 2007). Other isolates that are also in the clade include
G. bemidjiensis, G. humireducens, G. bremensis, G. daltonii, and
G. plymouthensis. Some of these isolates were recovered from surface

sediments (Table 1).
Subsurface clade II of the Geobacter species includes some subsurface
isolates such as G. chapellei, which was isolated from a deep subsurface
aquifer in which Fe(III) reduction was important (Lovley et al., 1990).
G. psychrophilus and Geobacter aquiferi were isolated from the same ace-
tate-impacted aquifer from which the subsurface clade I isolate
G. plymouthensis was derived (Table 1).
4. PHYLOGENY AND GENOMIC RESOURCES
Geobacter species are in the family Geobacteraceae, which is within the

domain Bacteria, phylum Proteobacteria, class Deltaproteobacteria, and
order Desulfuromonadales. The order Desulfuromonadales branches phy-
logenetically between the orders Syntrophobacterales and Desulfarculales.
The Geobacteraceae family can be further divided into three distinct
clusters: Geobacter, Desulfuromonas, and Desulfuromusa (Holmes et al.,
2004b). The genera Malonomonas and Geopsychrobacter fall within the
Desulfuromusa cluster, Geothermobacter and Geoalkalibacter fall within
the Desulfuromonas cluster, and Pelobacter species are scattered through-
out all three clusters (Fig. 4).
Comparative genomics suggest that the last common ancestor of the
Geobacteraceae was an acetate-oxidizing, respiratory species capable of extra-
cellular electron transfer, and that specialization for fermentative/syntrophic
growth in Pelobacter species evolved at least twice (Butler et al., 2009).
Pelobacter species have lost numerous genes, including most of the c-type
cytochromes, important in extracellular electron transfer, while gaining
unique genes for fermentative and syntrophic growth (Butler et al., 2009;
Haveman et al., 2006). P. carbinolicus may have lost multiheme c-type cyto-
chrome genes and other genes with multiple closely spaced histidine codons,
including the 14-subunit NADH dehydrogenase complex, due to an autoim-
mune response of its CRISPR locus against the histidyl-tRNA synthetase gene
(Aklujkar and Lovley, 2010). Initial studies suggested that Pelobacter
species could reduce Fe(III) (Lovley et al., 1995); further investigation with
P. carbinolicus revealed that Fe(III) reduction was indirect through a sulfur
shuttle (Haveman et al., 2008). P. carbinolicus can grow as a syntroph (Schink,
1992) but does this via interspecies hydrogen transfer rather than the direct
electron transfer that has been documented with Geobacter species (Summers
and Lovley, 2011). Early on it was proposed that P. propionicus should be
f0020 Figure 4 Maximum-likelihood tree showing the phylogenetic relationship
between the members of the family Geobacteraceae within the class
Deltaproteobacteria using 16S rRNA gene (> 1300 bp). The numbers at the branch
points are tree puzzle support values. Only values greater than 50 are shown. For
further methodological details, see the legend to Fig. 3.
placed in the genus Geobacter (Lonergan et al., 1996), but its significantly dif-
ferent evolutionary trajectory and physiology might warrant a separate genus
designation.
Geobacter species can be grouped (Fig. 3) into three distinct clades:
“subsurface clade 1,” “subsurface clade 2,” and the “G. metallireducens
clade” (Holmes et al., 2007). Molecular studies have shown that most of
the Geobacter species that predominate in Fe(III)-reducing subsurface
environments fall into “subsurface clade 1” or “subsurface clade 2”
(Holmes et al., 2007).
Nine Geobacter genomes and two Pelobacter genomes have been
completely sequenced, including two strains of G. sulfurreducens (Table 1).
Descriptions and comparisons of these genomes are available (Aklujkar
et al., 2009, 2010; Butler et al., 2009, 2010b) as are in silico metabolic
models based on these genomes (Mahadevan and Lovley, 2008;
Mahadevan et al., 2006, 2011; Scheibe et al., 2009; Segura et al., 2008; Sun
et al., 2009, 2010; Yang et al., 2010) and other computational analyses
(Krushkal et al., 2007, 2011; Mahadevan et al., 2006; Qu et al., 2009; Tran
et al., 2008; Yan et al., 2007). Also datasets of numerous genome-scale tran-
scriptional and proteomic analyses that may be a useful resource are avail-
able (Ahrendt et al., 2007; Butler et al., 2007; Conlon et al., 2009; Ding
et al., 2008; Franks et al., 2010b; Holmes et al., 2006, 2009; Khare et al.,
2006; Kim et al., 2008a; Krushkal et al., 2007; Leang et al., 2009; Methe
et al., 2005; Nevin et al., 2009; Nunez et al., 2006; Postier et al., 2008; Qiu
et al., 2010; Strycharz et al., 2011a; Yan et al., 2006).
5. ELECTRON ACCEPTORS
Geobacter species can use a diversity of electron acceptors to support anaer-

obic growth (Table 1), and there is evidence that G. sulfurreducens can grow
via oxygen reduction at low oxygen tensions (Lin et al., 2004). Soluble elec-
tron acceptors that can be reduced intracellularly include nitrate, fumarate,
and chlorinated compounds (Table 1). Biochemical studies have identified
protein fractions with nitrate- and nitrite-reductase activity (Murillo et al.,
1999; Naik et al., 1993; Senko and Stolz, 2001) in G. metallireducens, and
the fumarate reductase of G. sulfurreducens, which has the dual role of act-
ing as succinate dehydrogenase, was identified with a gene deletion
approach (Butler et al., 2006). Separate subsections discussing major extra-
cellular electron acceptors follow.
5.1. Fe(III)
At the circumneutral pH in most environments in which Geobacter thrive, Fe

(III) is highly insoluble. In subsurface sediments in which Geobacter species
were active, poorly crystalline Fe(III) hydroxides and structural Fe(III) of
phyllosilicates were the Fe(III) sources in the clay fraction that were reduced
(Shelobolina et al., 2004). Fe(III) forms that mimic these are the best insoluble
Fe(III) sources for cultivating Geobacter species. Crystalline Fe(III) oxides,
which microorganisms do not appear to significantly reduce in natural
sediments (Phillips et al., 1993), are poor electron acceptors for the cultivation
of Geobacter species. Initial attempts to enrich for acetate-oxidizing, Fe(III)-
reducing microorganisms with crystalline Fe(III) forms only yielded meth-
ane-producing enrichment cultures. It was the adoption of poorly crystalline
Fe(III) oxide, synthesized by neutralizing Fe(III) chloride solutions, that led
to successful enrichment and isolation of G. metallireducens. As outlined in
the references in Table 1, subsequent Geobacter species have been enriched
and isolated on a diversity of electron acceptors. However, Fe(III) that either
comes from the environment of interest (Shelobolina et al., 2007b), or closely
resembles that Fe(III), may be most likely to lead to the isolation of the most
environmentally relevant strains.
Reducing the particle size of Fe(III) oxides may accelerate rates of
Fe(III) reduction (Bosch et al., 2010), but whether Geobacter species can
be conveniently cultivated on nanoscale Fe(III) oxides has not yet been
determined. G. metallireducens could use the insoluble Fe(III) complex
Prussian Blue (Fe4[Fe(CN)6]3), an environmental contaminant, as an elec-
tron acceptor to support growth (Jahn et al., 2006).
Geobacter species can be conveniently grown with soluble chelated,
Fe(III) forms (Lovley, 2000b), but whole genome gene expression (Holmes
et al., 2011b) and proteomic (Ding et al., 2008) studies have suggested that
cells grown with soluble Fe(III) have significantly different physiologies than
cells grown on Fe(III) oxide. Soluble Fe(III) appears to be reduced at the
outer cell surface (Coppi et al., 2007), but as discussed below, the number
of proteins that G. sulfurreducens requires for reducing chelated Fe(III) is
significantly less than for Fe(III) oxide reduction.
5.2. Electrodes
After Fe(III) reduction, electron transfer to electrodes is probably the

most studied form of respiration in Geobacter species (Lovley, 2006a,b,
2008b, 2011a; Lovley and Nevin, 2011). All Geobacter species that have
been evaluated have the capacity for electron transfer to electrodes, which,
as discussed in subsequent sections, may have several practical
applications. Geobacter and closely related Desulfuromonas and Geo-
psychrobacter species were the first microorganisms found to conserve
energy to support growth by coupling the oxidation of organic matter with
electron transfer to electrodes and appear to be most effective
microorganisms in carrying out this form of respiration (Bond et al.,
2002; Holmes et al., 2004d; Ieropoulos et al., 2005; Ren et al., 2007). Geo-
bacter or closely related species are frequently the most abundant
microorganisms that colonize electrodes from mixed species inocula or nat-
ural communities, especially under strict anaerobic conditions (see many
references in Section 2). Frequently, the Geobacter species enriched are
most closely related to G. sulfurreducens, which is consistent with the
finding that G. sulfurreducens strains produce the highest current densities
of any known pure cultures and accomplish this with very high (> 90%)
coulombic efficiencies (Nevin et al., 2008; Yi et al., 2009). This is expected
to give them a competitive advantage in colonizing electrodes.
A wide diversity of conductive materials are appropriate electrode
surfaces for Geobacter species. Solid graphite is the most commonly
employed (Bond and Lovley, 2003), but other graphite/carbon materials
(Adachi et al., 2008; Srikanth et al., 2008; Ieropoulos et al., 2010; Selembo
et al., 2010), carbon cloth (Nevin et al., 2008), gold (Richter et al., 2008),
steel (Dumas et al., 2008a), platinum (Marsili et al., 2008; Yi et al., 2009),
and a diversity of conductive polymers (K.P. Nevin, unpublished data)
are also effective.
5.3. Other Extracellular Electron Acceptors
Mn(IV) oxides are typically much less abundant than Fe(III) oxides in soils
and sediments and are more susceptible to abiotic reduction (Lovley and
Phillips, 1988b). There has been much less focus on Mn(IV) reduction than
Fe(III) in Geobacter studies, but many of the aspects of mechanisms for Fe
(III) oxide reduction described below are likely to apply to Mn(IV) reduction.
Geobacter species can reduce a wide diversity of metal ions. It is possible
to grow Geobacter species on some of these electron acceptors when they
are provided in high concentrations in laboratory cultures, but all are gen-
erally found in low abundance in natural environments and would not sup-
port significant amounts of growth. Therefore, it is unlikely that Geobacter
species have evolved specific mechanisms to conserve energy to support
growth with these metal ions as electron acceptors. It seems more likely
that the ability of Geobacter species to reduce metal ions results from the
generalized ability to move electrons to the outer cell surface and onto
redox-carriers that can nonspecifically transfer electrons to a wide variety
of redox-active species, as described in subsequent sections. The environ-
mental significance of the reduction of these metals is that, in general,
reduction decreases solubility, and hence mobility. This can have impor-
tant geochemical consequences in natural environments, such as aiding in
ore deposit formation and, as described in subsequent sections, can be an
effective bioremediation tool.
For example, the discovery that Geobacter species can reduce soluble
U(VI) to the less soluble U(IV) provided a microbial model for U(VI)
reduction in sedimentary environments where U(VI) reduction had previ-
ously been considered to be an abiotic phenomenon and suggested a strat-
egy for removing uranium from contaminated waters (Lovley et al., 1991).
Some Geobacter species can grow with U(VI) as the sole electron acceptor
(Lovley et al., 1991; Sanford et al., 2007), but even in contaminated
environments, U(VI) availability is much less than that of Fe(III), which
supports most of the Geobacter growth (Finneran et al., 2002a).
G. sulfurreducens can use Co(III)-EDTA as an electron acceptor to
support growth (Caccavo et al., 1994). 60Co(III) chelated with EDTA is
a contaminant of nuclear operations and is highly mobile, whereas the
Co(II) produced from microbial reduction is much less mobile (Gorby
et al., 1998).
G. metallireducens conserved energy to support growth from the reduc-
tion of soluble V(V) to less soluble V(IV) with acetate as the electron
donor (Ortiz-Bernad et al., 2004b). In groundwater in which V(V) was a
co-contaminant with U(VI), stimulating the growth of Geobacter effec-
tively removed V(V). However, abiotic reduction of V(V) with Fe(II) pro-
duced from Fe(III) oxide reduction may have also contributed to V(V)
removal (Ortiz-Bernad et al., 2004a).
In a similar manner, Geobacter species may enzymatically reduce
Tc(VII), but abiotic reduction by Fe(II), produced by Geobacter species
or other organisms, is a more likely reaction in soils and sediments (Lloyd
and Macaskie, 1996; Lloyd et al., 2000). G. metallireducens appeared to
reduce Cr(VI) (Lovley et al., 1993a), but this is another species that
Fe(II) can readily reduce abiotically. Other contaminant radionuclides that
Geobacter species can reduce include Np(V) (Lloyd et al., 2000) and Pu
(IV) (Boukhalfa et al., 2007; Rusin et al., 1994).
Geobacter species can reduce Ag(I) precipitating Ag(0) (Law et al.,
2008; Lovley et al., 1993a) and Hg(II) reduction has also been reported
(Lovley et al., 1993a; Wiatrowski et al., 2006). Although a diversity of other
Fe(III)-reducing microorganisms reduced Au(III), the Geobacter species

tested did not (Kashefi et al., 2001).
Some Geobacter species have the ability to reduce elemental sulfur
(Table 1). Elemental sulfur is expected to be abundant in sediments near
the interface of the zones of Fe(III) reduction and sulfate reduction
because sulfide produced in the sulfate reduction zone and entering a zone
containing Fe(III) will abiotically reduce the Fe(III) with the formation of
elemental sulfur. G. metallireducens (Kaden et al., 2002) and possibly other
Geobacter species may be sensitive to sulfide, therefore the reported
inability of some Geobacter species to grow with elemental sulfur as the
electron acceptor may be related to sulfide sensitivity or inappropriate cul-
ture conditions. As discussed below, the mechanisms for elemental sulfur
reduction in G. sulfurreducens are expected to be rather nonspecific, and
it is expected that all Geobacter species should be capable of transferring
electrons to sulfur in a similar manner.
Humic substances are a heterogeneous class of complex organic com-
pounds that can be the most abundant form of organic matter in some soils
and sediments. The structure of humic substances is poorly understood,
but it is known that they contain quinone moieties that are potential elec-
tron acceptors for Geobacter species and some other microorganisms
(Coates et al., 1998; Jiang and Kappler, 2008; Klapper et al., 2002; Lovley
and Blunt-Harris, 1999; Lovley et al., 1996a, 1998; Roden et al., 2010; Scott
et al., 1998; Wolf et al., 2009). If Fe(III) is also available, the hydroquinone
moieties produced when the quinones are reduced can abiotically reduce
Fe(III) to Fe(II), regenerating the quinone state. In this manner, the humic
substances or humic substance analogs, such as AQDS, function as elec-
tron shuttles between the Geobacter species and the Fe(III). Providing
humic electron shuttles can accelerate the reduction of Fe(III) oxide and
make it feasible for Geobacter species to reduce some Fe(III) forms, such
as crystalline Fe(III) oxides, that are otherwise only poorly reduced
(Lovley et al., 1996a, 1998).
5.4. Other Microorganisms—Syntrophy
In the absence of alternative electron acceptors, Geobacter species can

transfer electrons to syntrophic partners. For example, early studies
demonstrated that most Geobacter species could produce hydrogen from
a variety of organic electron donors (Cord-Ruwisch et al., 1998), suggesting
that these Geobacter species might form syntrophic associations with the
well-established principle of interspecies hydrogen transfer (McInerney
et al., 2009; Stams and Plugge, 2009) in which one microorganism disposes
of electrons via the production of hydrogen gas and the partner organism
consumes the hydrogen with electron transfer to an electron acceptor the
other partner cannot utilize.
Acetate-oxidizing cocultures could be established with G. sulfurreducens
and either Wolinella succinogenes or Desulfovibrio vulgaris the first time
that rapid anaerobic syntrophic oxidation of acetate at moderate
temperatures was demonstrated (Cord-Ruwisch et al., 1998). However,
growth yields of G. sulfurreducens in the coculture with W. succinogenes
were higher than expected if G. sulfurreducens was disposing of electrons
via hydrogen production. This initially led to the suggestion that a soluble
c-type cytochrome released in the medium served as an electron shuttle
between the two species (Cord-Ruwisch et al., 1998), followed by the sug-
gestion that cysteine added to the medium was the electron shuttle (Kaden
et al., 2002). A third alternative, currently under investigation, is direct
interspecies electron transfer as described below.
Cell suspensions of G. metallireducens and W. succinogenes oxidized
toluene with the reduction of fumarate (Meckenstock, 1999). The electron
carrier between the two organisms was not investigated, but the poor
capacity for G. metallireducens to produce hydrogen would suggest
that an alternative to interspecies hydrogen transfer might have been
necessary.
The ability of Geobacter species to form syntrophic interactions was fur-
ther investigated in cocultures of G. metallireducens and G. sulfurreducens
(Summers et al., 2010). Multiple lines of evidence suggested that as the
coculture adapted for rapid syntrophic growth, electrons were directly
exchanged between the two species via electrically conductive connections
that will be described later. As detailed later, there is increasing evidence
that direct interspecies electron transfer may be an important feature of
Geobacter in anaerobic environments (Lovley, 2011a,c, Morita et al., 2011).
Another possibility in natural environments is that environmental
components may aid in interspecies electron transfer. For example, in the
reduced state, humic substances and other organics that have quinone/
hydroquinone moieties can serve as electron donors to support anaerobic
respiration (Lovley et al., 1999). G. metallireducens was able to transfer
electrons derived from acetate oxidation much more rapidly to
W. succinogenes in the presence of the humics analog AQDS, which served
as an electron acceptor for acetate oxidation by G. metallireducens, and the
reduced AQDS served as an electron donor for W. succinogenes (Lovley
et al., 1999).
6. ELECTRON DONORS
6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes,

Humics, Fe(II), U(IV)
The universal ability of all Geobacter species to oxidize acetate with Fe

(III) serving as the sole electron-acceptor points to their key ecological/
biogeochemical role in soils and sediments. Acetate is the key extracellular
intermediate in the anaerobic degradation of organic matter (Lovley and
Chapelle, 1995). Although there are some Fe(III)-reducing
microorganisms that can completely oxidize fermentable organic com-
pounds, such as sugars and amino acids (Lovley et al., 2004), they do not
appear to be competitive with fermentative microorganisms. For example,
in sediments in which Fe(III) reduction was the predominant terminal elec-
tron-accepting process, glucose was metabolized to acetate and other
minor fermentation acids (Lovley and Phillips, 1989). Therefore, minerali-
zation of organic matter can only take place if there are Fe(III) reducers
capable of coupling the oxidation of acetate to the reduction of Fe(III).
Further, acetate is a common additive to stimulate the activity of Geo-
bacter species for in situ bioremediation of uranium-contaminated ground-
water. Therefore, understanding acetate metabolism is key for
understanding the ecology of Geobacter species.
Acetate is oxidized via the TCA cycle in Geobacter species (Champine
and Goodwin, 1991; Champine et al., 2000; Mikoulinskaia et al., 1999).
As recently reviewed in detail (Mahadevan et al., 2011), studies on acetate
transporters (Risso et al., 2008a) as well as iterative genome-scale meta-
bolic modeling and experimental studies (Mahadevan et al., 2006; Risso
et al., 2008b; Segura et al., 2008; Tang et al., 2007) have identified addi-
tional features of the pathways for acetate metabolism that will not be
repeated here.
Even though the Geobacter species whose genomes have been
sequenced were isolated from geographically diverse environments, there
is high conservation of acetate metabolism genes including the genes
encoding acetate transporters and the eight enzymes of acetate oxidation
via the TCA cycle (citrate synthase, aconitase, isocitrate dehydrogenase,
2-oxoglutarate:ferredoxin oxidoreductase, succinyl:acetate CoA-transferase,
succinate dehydrogenase/fumarate reductase, fumarase, and malate dehy-
drogenase) (Butler et al., 2010b). All the genomes encode a 14-subunit
NADH dehydrogenase complex, except G. lovleyi encodes a 12-subunit
NADH dehydrogenase complex. All except G. metallireducens encode the
NAD-dependent ferredoxin:NADP oxidoreductase (Nfn) complex, and all

encode a putative NADPH:menaquinone oxidoreductase (Sfr) complex
(Coppi et al., 2007). The genes that encode ATP synthase are divided into
two conserved operons in every Geobacter genome (Butler et al., 2010b).
In addition to acetate, other short-chain fatty acids, alcohols, and hydro-
gen serve as electron donors for some Geobacter species (Table 1). In
G. sulfurreducens, the enzyme required for hydrogen oxidation has been
identified as a four-subunit NiFe hydrogenase (Coppi et al., 2004). How-
ever, this enzyme is only conserved in G. sulfurreducens,
G. uraniireducens, G. bemidjiensis, “G. andersonii,” “G. remediiphilus,”
and G. lovleyi (Butler et al., 2010b). In addition to the respiratory hydrog-
enase, there are multiple other cytoplasmic and membrane-bound hydro-
genases found in Geobacter species, all quite distinct from the well-
studied hydrogenases in related Desulfovibrio species (Coppi, 2005). There
is a hydrogenase specific to Geobacter that predominate in the subsurface
(Butler et al., 2010b).
Genome analysis suggests that G. metallireducens metabolizes propio-
nate via the 2-methylcitrate pathway (Aklujkar et al., 2009; Bond et al.,
2005), whereas G. bemidjiensis is predicted to metabolize propionate via
the methylmalonyl-CoA pathway (Aklujkar et al., 2010). Activation of
butyrate and valerate with a kinase and a transferase to form butyryl-
CoA and valeryl-CoA, respectively, has been predicted from genome
sequences (Aklujkar et al., 2009, 2010), but the expression of butyrate
kinase did not increase in G. metallireducens fed butyrate (Peters et al.,
2007a). G. bemidjiensis also possesses a long-chain fatty acyl-CoA dehy-
drogenase, fadE, which is absent from the genome of G. metallireducens,
suggesting that G. bemidjiensis has the potential for long-chain fatty acid
metabolism. Long-chain fatty acid degradation was documented in the
closely related Desulfuromonas palmitatis (Coates et al., 1995).
G. metallireducens and G. sulfurreducens could reduce nitrate and fuma-
rate, respectively, with the reduced humic substance analog AQDS
(Lovley et al., 1998). G. metallireducens can oxidize Fe(II) and U(IV) with
nitrate as an electron acceptor (Finneran et al., 2002b; Weber et al., 2006).
However, the capacity to conserve energy to support growth with these
electron donors has not been demonstrated.
Electrodes poised at a low potential can serve as an electron donor for
Geobacter species. Electrode-dependent reduction of nitrate (Gregory
and Lovley, 2005), fumarate (Gregory et al., 2004), U(VI) (Gregory and
Lovley, 2005), and chlorinated compounds (Strycharz et al., 2008) has been
documented, as has the reduction of protons to produce hydrogen gas
(Geelhoed and Stams, 2011). Initial studies have suggested that the route
for electron transfer from electrodes to Geobacter species may be different

from electron flow in the opposite direction for current production (Dumas
et al., 2008b; Strycharz et al., 2011a).
6.2. Aromatic Compounds
As detailed later, Geobacter species are often abundant within zones of

petroleum-contaminated aquifers in which aromatic hydrocarbons are
being removed, and it has been suggested that they can play an important
role in converting hydrocarbons in systems designed for recovery of hydro-
carbon deposits as methane. G. metallireducens was the first Geobacter
species found to degrade aromatic compounds and the first microorganism
of any kind in pure culture found to degrade an aromatic hydrocarbon
(Lovley and Lonergan, 1990; Lovley et al., 1989). Of particular interest is
the ability of G. metallireducens to anaerobically degrade benzene (Zhang
et al., 2011). Other than the hyperthermophile Ferroglobus placidus
(Holmes et al., 2011d), G. metallireducens is the only organism in pure cul-
ture that has been reported to be capable of degrading benzene through
defined anaerobic pathways.
A number of other Geobacter species, isolated from a diversity of con-
taminated environments, are capable of degrading aromatic compounds
(Table 1). Geopsychrobacter electrodiphilus, which is closely related to
Geobacter species, also metabolizes aromatic compounds (Holmes et al.,
2004d). All Geobacter species that are capable of degrading aromatic com-
pounds degrade benzoate. Only four (G. metallireducens, G. grbiciae,
G. toluenoxydans, and G. daltonii) are capable of metabolizing toluene.
The mechanisms for the degradation of aromatic compounds in Geo-
bacter species have primarily been investigated in G. metallireducens. Ben-
zoyl-CoA is a central intermediate in the metabolism of all monoaromatic
substrates (Fig. 5). Benzoyl-CoA reductase catalyzes the dearomatization
of benzoyl-CoA to cyclohexan-1,5-diene-1-carboxyl-CoA via a Birch-type
reduction (Boll, 2005b; Boll and Fuchs, 1995; Boll et al., 2000; Kung
et al., 2009; Peters et al., 2007b). G. metallireducens has a class II ben-
zoyl-CoA reductase, which until recently (Holmes et al., 2011d) was
thought to be found in all anaerobes degrading benzoate (Löffler et al.,
2011). Whereas class I benzoyl-CoA reductases couple the hydrolysis of
two ATP with the transfer of two electrons from reduced ferredoxin to
benzoyl-CoA (Boll, 2005a), the class II enzymes, which were initially dis-
covered in G. metallireducens (Kung et al., 2009), do not have an ATP
requirement (Löffler et al., 2011). In order to overcome the high redox
Figure 5 Aromatic hydrocarbon oxidation pathways in Geobacter species.

Enzymes of the aromatics degradation pathways indicated: benzylsuccinate synthase
(BssABCD), benzylsuccinate CoA-transferase (BbsEF), benzylsuccinyl-CoA dehy-
drogenase (BbsG), phenylitaconyl-CoA hydratase (BbsH), 2-[hydroxy(phenyl)
methyl]-succinyl-CoA dehydrogenase (BbsCD), benzoylsuccinyl-CoA thiolase
(BbsAB), alcohol dehydrogenase (adh), 4-hydroxybenzaldehyde dehydrogenase
(PcmO), benzoate-CoA ligase (BamY), phenylphosphate synthase (PpsABC),
phenylphosphate carboxylase (PpcBD—these are enzymes distinct from the PpcB
and PpcD cytochromes), p-cresol methylhydroxylase (PcmIJ), 4-hydroxybenzoyl-CoA
reductase (PcmRST), benzoyl-CoA reductase (BAmBCDEFGI), cyclohexadienoyl-
CoA hydratase (BamR), hydroxyenoyl-CoA dehydrogenase (BamQ), and oxoenoyl-
CoA hydrolase (BamA).
barrier associated with ring reduction, class II benzoyl-CoA reductases

may be driven by either membrane potential or electron bifurcation (Kung
et al., 2010).
A cluster of eight benzoate-induced genes (Bam BCDEFGHI) is
thought to code for the class II benzoyl-CoA reductase (Kung et al.,
2009). BamBC subunits are sufficient to catalyze the reductive
dearomatization of benzoyl-CoA in vitro (Kung et al., 2010). BamB
contains the tungstopterin active site and an Fe–S cluster, whereas BamC
contains three more Fe–S clusters (Kung et al., 2009). BamDEFGHI are
thought to participate in an uncharacterized electron activation process.
Proteome analysis revealed that BamFG was found exclusively in the
membrane fraction (Heintz et al., 2009). Therefore, it was proposed
that the benzoyl-CoA reductase complex is membrane associated. Activity
of class II benzoyl-CoA reductase was also detected in extracts of
G. bemidjiensis and several other obligate anaerobes (Löffler et al., 2011).
Additional reactions (Fig. 5) complete the conversion of benzoyl-CoA to 3-

hydroxypimelyl-CoA (Wischgoll et al., 2005), which is then oxidized to ace-
tyl-CoA and CO2 via beta-oxidation. This process includes the oxidative
decarboxylation of glutaryl-CoA to crotonyl-CoA by the decarboxylating
glutaryl-CoA dehydrogenase of G. metallireducens (Wischgoll et al., 2009).
The toluene degradation pathway of G. metallireducens (Fig. 5) appears
to be similar to the one found in the denitrifier Aromatoleum aromaticum
(Butler et al., 2007; Rabus, 2005; Rabus et al., 2005). The benzylsuccinate
synthase adds toluene as a radical to fumarate to form benzylsuccinate.
Benzylsuccinate is then activated to benzylsuccinyl-CoA, which is
metabolized to benzoyl-CoA (Rabus, 2005; Rabus et al., 2005). Proteomic
and functional genomics studies have suggested that the p-cresol degrada-
tion pathway in G. metallireducens is similar to the one found in Pseudo-
monas putida (Butler et al., 2007; Cronin et al., 1999; Peters et al., 2007a).
PcmO, a 4-hydroxybenzaldehyde dehydrogenase, could be responsible
for the conversion of 4-hydoxybenzaldehyde to 4-hydroxybenzoate in the
p-cresol pathway and for the conversion of benzaldehyde to benzoate in
the benzyl alcohol pathway (Butler et al., 2007). G. metallireducens
expressed more PcmO when grown on p-cresol compared to acetate or
benzoate (Peters et al., 2007a). No experimental evidence suggests that
PcmO is involved in benzyl alcohol oxidation.
Phenol degradation in Geobacteraceae is believed to be similar to that in the
facultative anaerobic denitrifier Thaurea aromatica, which was initially used to
describe the phenol pathway (Butler et al., 2007). Genes coding for
phenylphosphate synthase and carboxylase were upregulated when
G. metallireducens was grown on phenol compared to acetate and benzoate
(Schleinitz et al., 2009). Out of four phenylphosphate carboxylase subunits of
T. aromatica, only homolog genes coding for two subunits were found in the
G. metallireducens and Geobacter daltonii genomes. Despite the absence of
the other genes, phenylphosphate carboxylase activity was still detected
(Schleinitz et al., 2009). Further studies are necessary to understand the bio-
chemistry of phenylphosphate carboxylation in Geobacteraceae. It was
suggested that the benzoate-CoA ligase might also catalyze the conversion
of 4-hydroxybenzoate to 4-hydroxybenzoyl-CoA (Butler et al., 2007). How-
ever, biochemical studies demonstrated that the 4-hydroxybenzoate-CoA
ligase activity in G. metallireducens is found in another enzyme (Peters et al.,
2007a; Wischgoll et al., 2005).
Most of the aromatic degradation genes are found on a 300-kb genomic
island in G. metallireducens (Butler et al., 2007). This region contains 244
genes, which code for, among other things, the benzoyl-CoA reductase,
the benzoate-CoA ligase, phenol and p-cresol degradation pathways, the
cyclohexadienoyl-CoA hydratase, the hydroxyenoyl-CoA dehydrogenase,

the oxoenoyl-CoA hydrolase, and fatty acid oxidation enzymes (Butler
et al., 2007). Identical transposons and repetitive sequences found in this
region indicate that possible horizontal transfer events might be responsi-
ble for the acquisition of those genes (Butler et al., 2007). Most of those
244 genes have no ortholog in Geobacteraceae species that are unable to
degrade aromatic compounds (Butler et al., 2007). Genes coding for the
toluene degradation pathway are found on two operons located in another
genomic island of 167 kb (Butler et al., 2007).
Little is known about the regulatory network associated with aromatic
degradation in Geobacteraceae. Recently a G. metallireducens two-
component system formed by the histidine kinase BamV and the response
regulator BamW was found to regulate the transcription of bamY, the gene
coding for the benzoate-CoA ligase (Juárez et al., 2010). BamVW caused
an increase in the transcription of bamY after exposure to benzoate,
4-hydroxybenzoate, or p-cresol. A transcriptional regulator, which in the
presence of acetate represses the expression of bamA, and possibly other
genes whose expression is induced during growth on benzoate, has been
identified in G. bemidjiensis (Ueki, 2011). Genes for phenol or p-cresol deg-
radation are also transcribed when benzoate is the sole electron source
(Peters et al., 2007a; Schleinitz et al., 2009). However, accumulation of the
corresponding proteins was observed only during growth on phenol or
p-cresol, but not with benzoate (Peters et al., 2007a; Schleinitz et al., 2009).
Therefore, the existence of posttranscriptional regulation mechanism(s)
was proposed to explain these observations.
Many aspects of aromatic degradation pathways are still unknown
and need to be characterized in order to further comprehend the role of
Geobacter species in contaminated environments. These studies should be
accelerated by the recent development of methods for genetic manipulation
of G. metallireducens.
7. EXTRACELLULAR ELECTRON TRANSFER
Effective extracellular electron transfer is one of the hallmark physiologi-

cal features of Geobacter species. The capacity to exchange electrons with
its extracellular environment defines the unique ecological niche of
Geobacter species and is an important feature of the many practical
applications of this genus. Extracellular electron transfer in Geobacter
species is accomplished through unique mechanisms that have yet to be

described in any other organism.
7.1. Microbial Nanowires
One of the most surprising discoveries in the study of extracellular electron

transfer in Geobacter species has been the finding that G. sulfurreducens,
and presumably other Geobacter species, produces pili that are electrically
conductive (Malvankar et al., 2011b; Reguera et al., 2005). Initial
indications that pili were important in extracellular electron transfer came
from the observation that G. metallireducens expressed pili when grown on
Fe(III) or Mn(IV) oxides, but not when grown with soluble, chelated
Fe(III) as the electron acceptor (Childers et al., 2002). Studies on pili in
G. sulfurreducens have demonstrated that this organism can produce
pilin-like filaments from several different proteins, but the most abundant
filaments are those comprising PilA (Klimes et al., 2010).
Deletion of the gene for PilA, the structural pilin protein, inhibited Fe(III)
oxide reduction (Reguera et al., 2005). Conducting atomic force microscopy
demonstrated that the pili were conductive across their diameter (Reguera
et al., 2005). The atomic force microscopy revealed that there were other
proteins associated with the pili, but they acted as insulators. Therefore, it
was proposed that a method for electron transfer to Fe(III) oxide was long-
range electron transport along the pilin filaments. Further, although electron
hopping between cytochromes is the accepted method for biological electron
transfer over distance, it was suggested that cytochromes did not mediate
the electron transport along the pili (Reguera et al., 2005). This concept was
seriously questioned (Shi et al., 2007) because there was no known mechanism
for electron transfer along protein filaments.
However, subsequent studies have provided a mechanism. The pili of
G. sulfurreducens possess metallic-like conductivity comparable to syn-
thetic conducting polymers, such as the organic metal polyaniline
(Malvankar et al., 2011b). When pilin preparations were spotted on a
two-electrode system, they formed a network that conducted electrons
between the two electrodes. Preparations from a pilA deletion mutant
had conductivities comparable to the buffer control. Treating the pilin
preparation to denature any cytochromes that might have remained
associated with the pili had no impact on conductivity. Upon cooling from
room temperature, the pilin conductivity increased exponentially, a hall-
mark of quasi-one-dimensional organic metals. The temperature response
would not have been observed if electron hopping between cytochromes
was responsible for the electron transfer. Studies on the impact of pH

changes on conductivity and X-ray diffraction analysis of purified pilin pre-
parations suggested that p–p interchain stacking between aromatic
moieties of pilin amino acids may confer the metallic-like conductivity.
This hypothesis is currently under investigation.
The possibility of electron transport along a protein filament without the
involvement of cytochromes is a paradigm shift in biology. The metallic-like
mechanism for electron transport along the pili of G. sulfurreducens under
in vivo conditions is fundamentally different than the conductivity proposed
for filaments of other microorganism such as Shewanella oneidensis,
which was only demonstrated in fixed preparations and was reported to
be dependent on the presence of cytochromes (Gorby et al., 2006).
7.2. Cytochromes and Multicopper Proteins
One of the most striking features of Geobacter species is their abundant

c-type cytochromes and the large diversity of cytochromes encoded in Geo-
bacter genomes (Butler et al., 2010b; Ding et al., 2006; Méthé et al., 2003).
With the exception of G. lovleyi, Geobacter species possess ca. 100 c-type
cytochrome genes per genome (Butler et al., 2010b). There are nine famil-
ies of well-conserved c-type cytochromes, four of which are encoded
together and may constitute a quinone:ferricytochrome c oxidoreductase.
However, most of the cytochromes are poorly conserved among the Geo-
bacter species and some cytochrome families have only been found in a sin-
gle species of Geobacter (Butler et al., 2010b). This, coupled with the fact
that the function of c-type cytochromes has only been significantly studied
in G. sulfurreducens, makes it difficult to make broad generalizations about
cytochrome function in Geobacter species.
One family of c-type cytochromes that is well conserved is the PpcA
family of triheme periplasmic cytochromes. These are among the most
abundant c-type cytochromes in Geobacter species and were first studied
biochemically in the closely related Desulfuromonas acetoxidans (Banci
et al., 1996; Bruschi et al., 1997; Czjzek et al., 2001) and G. metallireducens
(Afkar and Fukumori, 1999; Champine et al., 2000) and then with more
detailed functional studies in G. sulfurreducens.
PpcA purified from G. sulfurreducens contained the expected three
hemes with a molecular weight of 9.6 kDa and a midpoint potential of
169.5 mV (Lloyd et al., 2003). Although PpcA is related to the earlier
studied cytochrome in D. acetoxidans, its redox properties are distinct
(Pessanha et al., 2006). Purified PpcA reduced Fe(III) and other metals,
but its periplasmic location makes direct reduction of Fe(III) unlikely

(Lloyd et al., 2003). The heme groups of PpcA are oriented in parallel or
perpendicular to each other (Morgado et al., 2010b), an arrangement
expected to facilitate rapid electron transfer within and between proteins
(Mowat and Chapman, 2005). Deletion of ppcA did not impact fumarate
reduction but did impact reduction of the extracellular electron-acceptors
Fe(III), AQDS, and U(VI) with acetate as the electron donor. However,
with hydrogen as the electron donor, reduction of extracellular electron
acceptors in the mutant and wild type were comparable.
There are four homologs of PpcA in G. sulfurreducens, designated
PpcB-PpcE. The function of these homologs appears to be different. Sur-
prisingly, deletions of ppcB, ppcC, or ppcE increased rates of Fe(III)
reduction (Shelobolina et al., 2007a). Whereas PpcA appears to be
expressed constitutively, PpcD expression is enhanced during growth on
Fe(III) oxide (Ding et al., 2008). Only PpcB is downregulated in cultures
grown on soluble iron (Ding et al., 2008). Structural and thermodynamic
characterizations such as the organization, redox potential, and oxidation
of hemes further suggest different functions of the homologues (Morgado
et al., 2008, 2010a; Pokkuluri et al., 2010). Only PpcA and PpcD can couple
e/Hþ translocation across the inner membrane (Morgado et al., 2010a),
and only PpcC displays polymerization and redox-dependent conforma-
tional changes (Morgado et al., 2007). Much more research into the func-
tion of these periplasmic cytochromes and their interaction with inner
and outer membrane components is required.
Early studies on G. sulfurreducens found significant Fe(III) reductase
activity in membrane fractions, which involved cytochromes (Gaspard et al.,
1998; Magnuson et al., 2000). One of these cytochromes was purified
(Magnuson et al., 2001) and was most likely the subsequently described
OmcB (Leang et al., 2003). This cytochrome has a molecular weight of
89 kDa, 12 hemes, and gross midpoint potential of 190 mV with some
hemes appearing to have much more negative potentials (Magnuson et al.,
2001). The purified protein was capable of reducing Fe(III) oxide and che-
lated Fe(III). OmcB is embedded in the outer membrane, with a portion of
the molecule exposed to the outer surface (Qian et al., 2007). Deleting the
gene for OmcB inhibited reduction of Fe(III) citrate and Fe(III) oxide
(Leang et al., 2003). Deletion mutants adapted to growth on Fe(III) citrate,
but not Fe(III) oxide (Leang et al., 2005). The presence of multiple RpoS-
dependent promoters upstream of upregulated cytochromes in the Fe(III)
citrate-adapted mutant suggests that an activated RpoS response permitted
G. sulfurreducens to compensate for the loss of OmcB (Krushkal et al., 2009).
OmcB is encoded downstream from another cytochrome, designated

Orf2, in an operon with a third protein of unknown function (Leang and
Lovley, 2005). This operon has been duplicated, and the other copy is imme-
diately downstream. In all the other Geobacter species genomes, there is at
least one operon with similarity to this one, and in several, there are tandem
repeats of the operon as well (Butler et al., 2010b). While the Orf2 cyto-
chrome is well conserved across all species, the sequence of the gene in the
omcB position in the operon varies substantially. In all cases, this gene
encodes a multiheme cytochrome, but some of the sequences are very diver-
gent and cannot be called omcB homologs. Thus, while the operon is con-
served and even duplicated, and the sequence of the Orf2 cytochromes is
well conserved, there may be less pressure for the large outer membrane
cytochrome to maintain a specific sequence.
Whereas OmcB is embedded in the outer membrane, several of the G.
sulfurreducens c-type cytochromes are fully exposed on the outer cell surface.
OmcS is a six-heme c-type cytochrome with a molecular weight of 47 kDa
(Qian et al., 2011). Its midpoint redox potential is 212 mV, more negative
than that of the periplasmic c-type cytochromes. However, the available evi-
dence suggests that individual hemes span a wide range of potentials. The gene
for OmcS is the most upregulated gene during growth on Fe(III) oxide versus
growth on Fe(III) citrate (Holmes et al., 2011b) and this is reflected in the pro-
teome (Ding et al., 2008) and in initial studies that detected omcS transcripts in
cells grown on Fe(III) oxide, but not Fe(III) citrate (Mehta et al., 2005). It is
also highly expressed under some conditions during growth on electrodes
(Holmes et al., 2006) and in cocultures of G. sulfurreducens and G. meta-
llireducens (Summers et al., 2010). Purified OmcS reduced a diversity of poten-
tial extracellular electron acceptors for G. sulfurreducens, including Fe(III)
oxide, U(VI), and humics, and also bound Fe(III) oxide (Qian et al., 2011).
OmcS is specifically associated with the pili of G. sulfurreducens (Leang
et al., 2010) and is required for growth on Fe(III) oxide, but not Fe(III) citrate
(Mehta et al., 2005).
OmcE is another c-type cytochrome found on the outer cell surface, but
its specific localization has yet to be pinpointed. It also has not been
purified but is predicted to have a molecular weight of 32 kDa and four
hemes (Mehta et al., 2005). Expression patterns of OmcE (Ding et al.,
2008; Holmes et al., 2006; Kim et al., 2008a; Nevin et al., 2009), as well as
gene deletions studies (Mehta et al., 2005), suggest that OmcE plays a role
in extracellular electron transfer in wild-type cells, but cells can adapt to
the loss of OmcE.
In contrast to OmcE and OmcS, OmcZ is not required for the reduction
of insoluble Fe(III). However, of all G. sulfurreducens cytochromes studied
to date, only OmcZ is absolutely necessary for high-density current pro-

duction (Nevin et al., 2009). In its mature extracellular form, OmcZ has a
molecular weight of 30 kDa, with eight hemes, including an unusual
CX14CH motif (Inoue et al., 2010). Its midpoint potential is 220 mV,
but as with other multiheme cytochromes individual hemes cover a wide
range of potentials. The purified protein can reduce a range of typical sol-
uble extracellular electron acceptors, and Mn(IV) oxides, but only poorly
reduced Fe(III) oxide. This corresponds with increased expression of
OmcZ during growth on Mn(IV) oxide, but not Fe(III) oxide, versus
growth on Fe(III) citrate (Holmes et al., 2011b). The poor solubility of
OmcZ in water might help maintain it within the extracellular matrix
(Inoue et al., 2010). OmcZ is specifically localized at the biofilm–anode
interface in high-current density biofilms (Inoue et al., 2011). It does not
associate with filaments and its expression patterns suggest that its natural
function may be to promote the reduction of extracellular soluble electron
acceptors.
The cytochrome encoded by gene GSU1334 is homologous to OmcZ
and a deletion mutant exhibited defects in Fe(III) oxide and U(VI) reduc-
tion (Shelobolina et al., 2007a). However, caution in interpreting such
phenotypes is warranted without additional study. For example, deleting
genes for several cytochromes predicted to be on the outer surface
inhibited Fe(III) reduction, but this could be attributed to the lack of
proper expression or localization of OmcB or other outer-surface
cytochromes in these mutants (Kim et al., 2005, 2006, 2008a). In a similar
manner, deletion of the gene for MacA, a cytochrome of interest because
it is more highly expressed during growth on Fe(III), inhibited Fe(III)
reduction (Butler et al., 2004), but deletion of macA was also associated
with a lack of OmcB (Kim and Lovley, 2008).
Another cytochrome of interest is PgcA, which is upregulated during
growth on Fe(III) oxide (Ding et al., 2008; Holmes et al., 2011b). Selection
for enhanced growth of G. sulfurreducens on Fe(III) oxide selected for
mutations that increased expression of PgcA (Tremblay et al., 2011b).
Further, PgcA is a member of one of the cytochrome families conserved
across several Geobacter species (Butler et al., 2010b). Further study of
this cytochrome is underway.
The putative multicopper protein, OmpB, which is localized to the outer
surface of G. sulfurreducens (Qian et al., 2007), also appears to be involved
in the reduction of Fe(III) oxide but is not required for the reduction of
soluble Fe(III) (Mehta et al., 2006). The pseudo-pilin OxpG is required
for OmpB export. The OmpB homolog, OmpC, is also important for opti-
mal Fe(III) oxide reduction but has not been experimentally localized and
has different expression patterns than OmpB (Holmes et al., 2008).

Homologs with four copper-binding sites, two at the N-terminus and two
at the C-terminus, are found in all of the Geobacter genomes, though the
protein size ranges from ca. 800 to 1700 aa (Butler et al., 2010b). Phyloge-
netically, the omp genes form two distinct clades, the B-type and the C-
type, and not all genomes contain both types (Holmes et al., 2008). No
homologs were found in the two Pelobacter genomes. Various potential
roles for OmpB and OmpC have been suggested (Holmes et al., 2008;
Mehta et al., 2006), but purification and characterization of the proteins
are required to better evaluate these possibilities.
The many other underexplored cytochromes and other putative redox-
active proteins in G. sulfurreducens warrant further study, as do proteins likely
to be involved in cytochrome export (Afkar et al., 2005), and the cytochromes
in other Geobacter species. For example, G. uraniireducens increases the tran-
scriptional expression of several cytochromes when cultured in anoxic
sediments versus growth on soluble electron acceptors (Holmes et al., 2009).
Additional structural studies will provide important insights into their function
(Londer et al., 2002, 2006a,b; Morgado et al., 2007, 2009; Pessanha et al., 2004,
2006; Pokkuluri et al., 2004, 2008, 2009, 2011).
Development of genetic systems for Geobacter species other than
G. sulfurreducens will also aid in functional analysis, as will the approach
of determining which cytochrome functions can be completed in mutants
of G. sulfurreducens with cytochrome gene sequences from other
Geobacter species (Yun et al., 2011a).
7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide
Several models have been advanced for how Geobacter species transfer
electrons to insoluble Fe(III) oxides. A miscalibrated spectrophotometer
in initial studies with G. metallireducens (Gorby and Lovley, 1991) resulted
in the mistaken suggestion that b-type cytochrome(s) were important in
extracellular electron transfer, but subsequent studies demonstrated a role
for c-type cytochromes in the reduction of Fe(III) and other metals
(Lovley et al., 1993a). An early model for Fe(III) oxide reduction by
Geobacter sulfurreducens suggested that it released a low-molecular-weight
c-type cytochrome, which acted as an electron shuttle between cells and
Fe(III) oxide (Seeliger et al., 1998). However, this concept was refuted in
a number of studies, including studies in the laboratory, which initially
developed the electron shuttling concept (Lloyd et al., 1999; Nevin and
Lovley, 2000; Straub and Schink, 2003).
Evidence consistent with the need for direct contact is the lack of Fe(III)
reduction when cells are separated from Fe(III) oxide contained within
microporous alginate beads (Nevin and Lovley, 2000) or agar (Straub
and Schink, 2003). This was observed with G. metallireducens (Nevin
and Lovley, 2000) as well as G. sulfurreducens, G. bremensis, and
G. pelophilus (Straub and Schink, 2003). In contrast, Shewanella (Nevin
and Lovley, 2002b) and Geothrix (Nevin and Lovley, 2002a) species, and
Fe(III)-reducing enrichment cultures (Straub and Schink, 2003), produced
shuttles that permitted reduction of Fe(III) oxide at a distance. Further, G.
metallireducens also did not appear to produce chelators that could solubi-
lize Fe(III), whereas Shewanella (Nevin and Lovley, 2002b) and Geothrix
(Nevin and Lovley, 2002a) species did solubilize Fe(III) under similar
conditions.
Although some of the components that appear to be involved in elec-
tron transfer to Fe(III) oxides have been identified, the understanding of
how these, and potentially other components, fit together is far from com-
plete. As noted above, OmcS is likely to have an important role in Fe(III)
oxide reduction because (1) OmcS expression is highly upregulated during
growth on Fe(III) oxide (Ding et al., 2008; Holmes et al., 2011b; Mehta
et al., 2005); (2) gene deletion studies indicate that the OmcS is required
for Fe(III) oxide reduction (Mehta et al., 2005); (3) OmcS is specially
associated with pili (Leang et al., 2010), which, as described above, are
electrically conductive and are required for Fe(III) oxide reduction; and
(4) purified OmcS can transfer electrons to Fe(III) oxide and may bind
Fe(III) (Qian et al., 2011). The simplest explanation for these observations
is that electrons that are transported along the pili are transferred to Fe
(III) oxide via OmcS. There is no obvious route for electrons to get to
OmcS other than the pili and the lack of Fe(III) reduction in the absence
of OmcS suggests that electrons cannot be directly transferred from the pili
to Fe(III) oxide.
There is little information on how electrons are transferred to the pili.
This could conceivably take place in the periplasm, or even the inner mem-
brane, but the requirement for OmcB, which is located in the outer mem-
brane, suggests that electron transfer near the outer surface of the cell is
more likely. The fact that OmcB is embedded in the outer membrane
suggests that it might be difficult for OmcB and pili to associate closely
enough for electron transfer between the two. The need to mediate elec-
tron transfer from OmcB to the pili at the outer cell surface may explain
why other potentially redox-active outer-surface components, such as
other c-type cytochromes and the putative multicopper proteins OmpB
and OmpC, are important in Fe(III) oxide reduction.
The role of other outer-surface cytochromes in Fe(III) oxide reduction is

also not completely understood. OmcE can be an abundant c-type cyto-
chrome under some growth conditions, but cells can eventually overcome
deletion of omcE and reduce Fe(III) oxide (Mehta et al., 2005). It has been
proposed that OmcZ localized in an extracellular matrix could be important
in Fe(III) oxide reduction (Rollefson et al., 2011), but this is not consistent
with several observations including (1) OmcZ is not required for Fe(III)
oxide reduction (Nevin et al., 2009), (2) low expression of omcZ in cells
growing on Fe(III) oxide (Holmes et al., 2011b), and (3) purified OmcZ only
poorly reduces Fe(III) oxide (Inoue et al., 2010).
If OmcB is the conduit for electrons out of the cell and toward pili, then
the next question is what is the electron donor for OmcB? Periplasmic
cytochromes are potential sources, ferrying electrons from the inner
membrane to the outer membrane. As noted above, a number of periplasmic
c-type cytochromes have been identified in G. sulfurreducens, but no electron
transfer link between these cytochromes and OmcB, or any other electron
acceptor, has been documented.
Diagrams for how the electrons may flow to Fe(III) oxide from
G. sulfurreducens are available (Lovley, 2011c), but clearly we are still at
the hypothesis stage and more research on electron transfer out of the cell
is warranted. Novel strategies for elucidating important components are
likely to be helpful. For example, adaptive evolution for improved Fe(III)
oxide reduction in G. sulfurreducens provided further evidence for the
importance of pili in Fe(III) oxide reduction as well as identifying an addi-
tional c-type cytochrome that may be involved (Tremblay et al., 2011b).
Studies on species other than G. sulfurreducens are also warranted to
look for commonalities that are general features of electron transfer to
Fe(III) oxides in all Geobacter species. For example, unique PilA
sequences are conserved in Geobacter species (Reguera et al., 2005) and
recent gene deletion studies have demonstrated that PilA is required for
Fe(III) oxide reduction in G. metallireducens (Tremblay et al., 2011a).
In contrast, outer-surface cytochromes’ sequences are poorly conserved in
Geobacter species (Butler et al., 2010b), suggesting that there is less specific-
ity in cytochrome requirements. However, there is still an opportunity to
look for commonality in mechanisms. For example, if electrons cannot be
directly transferred from pili to Fe(III) oxides, then it would be expected
that G. metallireducens, which does not have an OmcS homolog (Butler
et al., 2010b), would possess another cytochrome, which like OmcS, is
associated with pili and necessary for Fe(III) oxide reduction.
Additional research is also required on the early steps of electron trans-
fer across the inner membrane and to the electron carriers responsible for
the terminal steps in electron transfer to Fe(III) and other extracellular

electron acceptors. Although possible electron carriers can be identified
from genome sequences, experimental studies are required before defini-
tive models can be developed. One of the key features of extracellular
electron transfer in Geobacter species is the poor energy yields available
from this mode of respiration in comparison with the reduction of soluble
electron acceptors within the cell (Esteve-Nunez et al., 2004, 2005;
Mahadevan et al., 2006). This can be attributed, at least in part, to the fact
that intracellular reduction of electron acceptors consumes protons along
with electrons, but when electrons are transferred out of the cell, this pro-
ton sink is lost, requiring export of protons that does not contribute to the
development of a proton-motive force across the inner membrane
(Mahadevan et al., 2006, 2011).
7.4. Model for Extracellular Electron Transfer to Electrodes
Like Fe(III) oxide, electrodes represent an insoluble, extracellular electron

acceptor. Initial studies with G. sulfurreducens suggested that it did not
produce electron shuttles in order to promote electron transfer to
electrodes (Bond and Lovley, 2003) and electrochemical studies supported
this conclusion (Busalmen et al., 2008, 2010; Marsili et al., 2008, 2010;
Marsili et al.; Richter et al., 2009). This is consistent with the similar con-
cept of direct electron transfer to Fe(III) oxide.
However, there are major differences between the electrodes and Fe
(III) oxide because electrodes function as stable long-term electron
acceptors, whereas once Fe(III) is reduced in one location cells need to
find additional sources of Fe(III). The stability of the electrode as an elec-
tron acceptor makes it possible for Geobacter to produce thick (> 50 mm)
biofilms on electrodes (Franks, 2010; Franks et al., 2009; Nevin et al.,
2009; Reguera et al., 2006), which are not formed during growth on Fe(III)
oxide. Thus, the necessity to transfer electrons through a biofilm may
require different electron transport strategies and may place different
selective pressures on cells.
Fashioning one coherent model for electron transfer from
G. sulfurreducens to electrodes that can accommodate all the data avail-
able in the literature is difficult. There is substantial confusion in the liter-
ature because models generated from preliminary data are often ruled out
as more data becomes available. For example, early studies in our labora-
tory investigated electron transfer in systems producing relatively low
amounts of current in which most of the cells were closely associated with
the anode surface. Under those conditions, OmcS was highly expressed
and was essential for current production (Holmes et al., 2006). In contrast,
in subsequent studies with systems producing much more current, OmcS
was not highly expressed and cells adapted to produce current comparable
to that of wild type when OmcS was deleted (Nevin et al., 2009). Rather,
OmcZ was highly expressed in the high-current density biofilms. OmcZ
and OmcS do not appear to have equivalent functions, based on their dif-
ferent localization and other factors, and it is generally the case that when
OmcS is highly expressed OmcZ expression is low and vice versa. The
geometry of the electrode material may also influence gene expression
patterns, and presumably electron transfer pathways, with expression
patterns on graphite fiber electrodes resembling more closely the expression
in the early low-current density biofilms on planar graphite surfaces (K.P.
Nevin et al., unpublished data). Therefore, instead of attempting to develop
one universal model for electron transfer to electrodes, we have focused on
electron transfer in thick (> 50 mm) electrode biofilms, which produce
high-current densities, because a major goal is to understand the production
of high-current densities in order to further optimize current output.
An initial observation in the development of higher current densities was
that the increase in current was proportional to the increase in biomass on
the anode, suggesting that cells at great distance from the anode were con-
tributing to current production (Reguera et al., 2006). Subsequent studies
have confirmed the high metabolic activity of such cells (Franks et al.,
2010b). The finding that deleting pilA prevented high-current densities led
to the hypothesis that networks of pili in the G. sulfurreducens biofilms con-
ferred conductivity on the biofilm and a route for electrons released from
cells at distance to be transported to the electrode (Reguera et al., 2006).
Consistent with this concept, modeling studies indicated that the high-
current density in microbial fuel cells would be feasible only if Geobacter
biofilms were assumed to be electrically conductive (Marcus et al., 2007;
Torres et al., 2008, 2009b). However, the suggestion that biofilms of Geo-
bacter species could be conductive contrasted with previous studies, which
had demonstrated that the biofilms of bacteria act as insulators (Dheilly
et al., 2008; Herbert-Guillou et al., 1999; Muñoz-Berbel et al., 2006).
Measurement of the conductance of viable G. sulfurreducens biofilms with a
novel two-electrode system revealed that the biofilms that had been grown
with an electrode as the electron acceptor had remarkable conductivity,
comparable to that of synthetic organic conducting polymers, such as poly-
aniline and polyacetylene (Malvankar et al., 2011b). In contrast, biofilms
grown in the same system, but with fumarate as the electron acceptor, had
low conductivity. The biofilms of Escherichia coli and Pseudomonas
aeruginosa were not conductive. Evaluation of strains of G. sulfurreducens

with different biofilm conductivities demonstrated a strong correlation
between the abundance of PilA in the biofilm and conductivity, suggesting that
the conductivity was related to the extent of pilin production.
The temperature dependence of biofilm conductivity was similar to that
of pilin preparations, demonstrating a metallic-like conduction mechanism,
which was further confirmed with electrochemical gating studies
(Malvankar et al., 2011b). These results suggested that the biofilm conduc-
tivity was related to the metallic-like conductivity of the pilin network. None
of these results support the concept of electron hopping through biofilms via
c-type cytochromes. Further, denaturing the c-type cytochromes in the bio-
films had no impact on conductance and there was no correlation between
conductance and cytochrome content of the biofilms. These results suggest
that the novel metallic-like conductivity in G. sulfurreducens can be
attributed to the surprising metallic-like conductivity of its pilin networks.
Consistent with the apparent importance of pili in conduction of
electrons through G. sulfurreducens biofilms, the gene for PilA is among
the most highly upregulated genes in current-producing biofilms (Nevin
et al., 2009). Selective pressure for enhanced current production yielded a
strain of G. sulfurreducens that produced more pili (Yi et al., 2009).
Deletion of pilA significantly inhibited current production, with only cells
near the electrode surface remaining metabolically active (Reguera et al.,
2006). Although the pilin constructed of PilA may have a structural role
in biofilm formation under some conditions (Reguera et al., 2007), the pilA
deletion mutant readily formed thick biofilms on the graphite electrode
material if fumarate was provided as an alternative electron acceptor
(Nevin et al., 2009).
The concept of electron transport through G. sulfurreducens biofilms via
conductive pilin networks contrasts with many studies that have suggested
that more traditional electron transfer via cytochromes moves electrons
through the biofilms. Biofilms of wild-type G. sulfurreducens growing on
electrodes are visibly red, due to the cytochrome abundance. Many studies
have provided evidence that cytochromes are oxidized and reduced in G.
sulfurreducens biofilms in electrical contact with electrodes (Esteve-Núñez
et al., 2011; Fricke et al., 2008; Jain et al., 2011; Liu et al., 2010b, 2011;
Marsili et al., 2008, 2010; Millo et al., 2011; Richter et al., 2009; Srikanth
et al., 2008; Strycharz et al., 2011b), but the interpretation that this
represents electron transfer through the biofilm by electron hopping via
c-type cytochromes in analogy with redox hydrogels (Heller, 2006; Richter
et al., 2009) is not consistent with the studies (Malvankar et al., 2011b) on
biofilm conductance.
The likely explanation for this apparent discrepancy is that the electro-
chemical analyses only probed the biofilm-electrode interface and not the
entire biofilm (Dumas et al., 2008a; Franks et al., 2010a). The cytochromes
at the interface may function as an electrochemical gate, promoting
electron transfer to the electrode surface (Dumas et al., 2008a).
A likely candidate for a cytochrome functioning as an electrochemical
gate is the outer-surface c-type cytochrome OmcZ. The OmcZ gene is one
of the most highly upregulated genes in current-producing cells, and if omcZ
is deleted, the cells produce low levels of current (Nevin et al., 2009). There is
much higher resistance for electron transfer to electrodes in cells lacking
OmcZ, which was originally interpreted as OmcZ conferring conductivity
throughout the biofilm (Richter et al., 2009). However, this cannot be correct
as the conductance of biofilms of a strain with lower abundance of OmcZ was
higher than those of wild type (Malvankar et al., 2011b). Further, cells
throughout the biofilm express omcZ (Franks et al., 2011). OmcZ
accumulates at the biofilm-electrode interface, consistent with the electro-
chemical gate hypothesis (Inoue et al., 2011).
The reason that OmcZ or other cytochromes might be required to facili-
tate current production is that a significant energy barrier might exist across
the biofilm-electrode interface similar to a semiconductor–metal interface
(Lange and Mirsky, 2008). The wide range of reduction potentials ( 420
to 60 mV) of the multiple hemes in OmcZ (Inoue et al., 2010) might help
overcome this energy barrier in a manner similar to electrochemical gating
in molecular electronics (Vanmaekelbergh et al., 2007).
7.5. Extracellular Electron Transfer in Syntrophy
The finding that key elements of extracellular electron exchange in

G. sulfurreducens were required for effective electron exchange between
G. metallireducens and G. sulfurreducens and the finding that the
aggregates were electrically conductive (Summers et al., 2010) suggest that
components of these cells can form an electrically conductive matrix which
permits direct electron exchange between the partners (Lovley, 2011a,c).
OmcS was very abundant in the aggregates, which was attributed to a
mutation in a regulatory gene of G. sulfurreducens that was selected
for during adaption for ethanol metabolism. Introducing a strain of
G. sulfurreducens with the regulator inactivated hastened adaption for eth-
anol metabolism, whereas deleting the gene for OmcS or PilA prevented
aggregate formation and ethanol metabolism. These results suggest that
electron transfer through OmcS and the pili of G. sulfurreducens is an

important part of the interspecies electron exchange.
Which components of G. metallireducens are important for the electron
exchange are not known, but this question should now be addressable
because of the recent development of methods for genetically man-
ipulating G. metallireducens (Tremblay et al., 2011b). The study of direct
electron transfer from electrodes to cells (Strycharz et al., 2011a) may help
elucidate the electron-receiving component of the model.
It is important to recognize that aggregation of the two syntrophic
partners may not be a prerequisite for direct interspecies electron transfer.
In a manner similar to the mechanisms proposed for Fe(III) oxide reduc-
tion, Geobacter species could establish temporary contact with a recipient
cell, offload electrons, and then move on.
7.6. Model for Extracellular Electron Transfer to Other

Extracellular Electron Acceptors
The display of multiple low-potential c-type cytochromes on the outer sur-

face of Geobacter species confers the capacity to reduce a wide diversity of
soluble electron acceptors at the outer cell surface. Reduction of these
electron acceptors may be rather nonspecific. For example, deleting the
genes for individual outer-surface cytochromes only partially inhibited
the ability of G. sulfurreducens to reduce humic substances and AQDS.
Only when the genes for OmcB, OmcE, OmcS, OmcT, and OmcZ were
deleted in the same strain was humic substance and AQDS reduction
eliminated (Voordeckers et al., 2010).
Although the final product of U(VI) reduction is U(IV), the initial
reduction of U(VI) may be a one electron transfer followed by dispropor-
tionation of U(V) to U(VI) and U(IV) (Renshaw et al., 2005). Initially it
was considered that U(VI) might be reduced in the periplasm (Lloyd
et al., 2002), but the accumulation of uranium in the periplasm that was a
main line of evidence for periplasmic reduction was later found to be an
artifact (Shelobolina et al., 2007a). Systematic deletion of the genes for
the most abundant outer-surface c-type cytochromes in a study comparable
to one on reduction of humic substances has indicated that the site of
reduction is the outer surface of the cell (R. Orellana, unpublished data).
Purified OmcZ (Inoue et al., 2010) and OmcS (Qian et al., 2011) reduce
U(VI), and it is likely that many low-potential c-type cytochromes will
be capable of U(VI) reduction (Lovley et al., 1993b). It seems likely that
the other metallic ions that Geobacter species can reduce may also be
reduced in a similar nonspecific manner.
In vitro studies with the abundant periplasmic c-type cytochrome of the
closely related Desulfuromonas acetoxidans demonstrated that this cyto-
chrome could reduce elemental sulfur in vitro (Pereira et al., 1997) and
periplasmic reduction of sulfur has been a model. However, systematic
reduction of the outer-surface c-type cytochromes of G. sulfurreducens
has suggested that elemental sulfur is also reduced at the outer cell surface
(S. Dar, unpublished results).
7.7. Capacitor Role of Cytochromes
In addition to their role in extracellular electron transfer discussed above,

and possible environmental sensing discussed later, the abundant c-type
cytochromes of Geobacter species may have the additional role of function-
ing as capacitors for electron storage under some environmental conditions
(Esteve-Nunez et al., 2008; Lovley, 2008a). The sheer abundance of c-type
cytochromes in cells growing under electron-acceptor-limiting conditions
or in biofilms producing electrical current makes the cultures/biofilms visi-
bly red. The capacity to store electrons in periplasmic and outer-surface
cytochromes may be beneficial for Geobacter species because Fe(III)
sources are heterogeneously dispersed and there are likely to be periods
when Geobacter species are not in direct contact with Fe(III) oxides. In
fact, when Geobacter species are most actively growing during in situ ura-
nium bioremediation they are highly planktonic and thus periodically out
of contact with Fe(III) (Anderson et al., 2003; Dar et al., 2011; Kerkhof
et al., 2011). Cytochromes positioned beyond the inner membrane, in the
oxidized state, can accept electrons from inner membrane electron carriers,
permitting continued respiration even when Fe(III) is not available.
Energy conservation under such conditions is expected to be similar to
when Fe(III) is available because conservation of energy results from elec-
tron transfer components in the inner membrane. Subsequent steps in elec-
tron transport to extracellular electron acceptors do not conserve
additional energy for the microorganism; they are just necessary in order
to provide electron acceptors for electron transfer across the inner mem-
brane. Once cells contact Fe(III), the electrons stored in the cytochromes
can be discharged.
Another observation consistent with this concept is that G. meta-
llireducens specifically expresses flagella when grown on insoluble Fe(III)
or Mn(IV) oxides, but not when grown on soluble Fe(III) citrate (Childers
et al., 2002). Deleting a gene for flagella production inhibited Fe(III) oxide
reduction in G. metallireducens, but not the reduction of Fe(III) citrate
(Tremblay et al., 2011a). Changing the sequence of a gene for a master
regulator of flagella to confer motility in the otherwise nonmotile
G. sulfurreducens enhanced Fe(III) oxide reduction (Ueki et al., 2011).
The negative impact on Fe(III) reduction of making G. metallireducens
nonmotile was stronger when the cells were grown with sediment Fe(III)
oxides as the electron acceptor than in cultures with synthetic Fe(III)
oxides, consistent with the greater dispersal of Fe(III) oxides in sediments
(Tremblay et al., 2011a). Chemotaxis might guide Geobacter species to
Fe(III) and Mn(IV) oxide sources (Childers et al., 2002).
Studies with current-producing biofilms of G. sulfurreducens have con-
firmed this inference of c-type cytochromes conferring capacitance
(Malvankar et al., 2011a). The biofilms had capacitance comparable to that
of synthetic supercapacitors. Multiple lines of evidence demonstrated that
the biofilm capacitance could be attributed to the c-type cytochromes. As
discussed below, this novel form of capacitance may be a useful contribu-
tion to the field of bioelectronics.
8. REGULATION OF METABOLISM
In order to understand how Geobacter species function in diverse

environments, and how they are likely to change their metabolism in response
to changes in environmental conditions, it is important to understand
how gene expression is regulated. The elucidation of regulatory networks in
Geobacter species is in its infancy, but some progress has been made.
8.1. Sigma Factors
Sigma factors play a key role in the regulation of gene expression in

response to changing environments, and bacteria typically employ multiple
sigma factors to optimize their responses. Each species of sigma factors
recognizes specific promoter elements of a certain set of genes and initiates
their transcription. The genome of G. sulfurreducens encodes homologs of
RpoD (s70), RpoS (sS, s38), RpoH (sH, s32), RpoN (sN, s54), RpoE
(sE, s24), and FliA (RpoF, sF, s28) found in E. coli and many other bacteria
(Méthé et al., 2003). In other bacteria, RpoD is generally the major sigma
factor for most housekeeping genes (Ishihama, 2000). Alternative sigma
factors are required for stress response genes. For example, RpoS is
required for general stress response genes including stationary-phase genes.
RpoH is necessary for heat-shock genes. RpoN is involved in transcription
of genes for nitrogen deficiency and some other stresses. FliA participates in
regulation of flagella and chemotaxis genes. RpoE represents
extracytoplasmic function (ECF) sigma factors, which are involved in regula-
tion of genes for outer membrane or periplasmic proteins.
The RpoD homolog appears to be the major sigma factor in G.
sulfurreducens as transcriptomic analysis has demonstrated that a large
number of genes were transcribed by RNA polymerase containing RpoD
(Qiu et al., 2010). It is likely that G. sulfurreducens RpoD recognizes pro-
moter elements similar to those of other bacterial RpoD homologs (Qiu
et al., 2010; Yan et al., 2006). However, genetic and biochemical studies
for RpoD have not been conducted.
The RpoS homolog is the stationary-phase sigma factor in G.
sulfurreducens (Nunez et al., 2004). It is also involved in response to oxygen
exposure and in growth with oxygen as the electron acceptor. An rpoS-dele-
tion mutant exhibited less viability at the stationary phase than the wild-type
strain and slower recovery after exposure to oxygen. The mutant was also
defective in reduction of insoluble Fe(III) oxide but not of soluble Fe(III)
citrate. Transcriptome and proteome analyses revealed genes in a variety
of cellular functions under the control of G. sulfurreducens RpoS, which
include oxidative stress and nutrient limitation response genes and genes
for c-type cytochromes (Nunez et al., 2006). Among the c-type cytochromes
is MacA, which is known to be critical for Fe(III) reduction (Butler et al.,
2004). It appears that promoters recognized by RpoS in G. sulfurreducens
are similar to those recognized by RpoD, as found in other bacteria (Yan
et al., 2006). However, genes regulated by RpoS in G. sulfurreducens are
diversified from those in other bacteria (Santos-Zavaleta et al., 2011). These
studies suggest that the RpoS homolog plays important roles in
G. sulfurreducens under conditions that Geobacter species typically encoun-
ter in subsurface environments.
RpoH is the heat-shock sigma factor in G. sulfurreducens (Ueki and Lovley,
2007). Expression of the rpoH gene was induced by heat shock from 30 to
42 C and appears to be controlled by RpoH itself as well as the HrcA/CIRCE
system. HrcA is known to be a repressor for heat-shock genes by binding the
CIRCE (Controlling Inverted Repeat of Chaperon Expression) element in
other bacteria (Schulz and Schumann, 1996; Zuber and Schumann, 1994).
An rpoH-deletion mutant of G. sulfurreducens was unable to grow at 42 C,
whereas the wild-type strain could. The expression of heat-shock response
genes decreased dramatically in the rpoH-deletion mutant.
In contrast to most other bacteria for which RpoN is dispensable under

some conditions, the RpoN homolog appears to be a vital sigma factor in
G. sulfurreducens (Leang et al., 2009). Transcriptome analysis demonstrated
that RpoN regulates a large number of genes involved in a wide range of cel-
lular functions including those encoding enzymes for ammonia assimilation,
which are predicted to be essential under all growth conditions in
G. sulfurreducens. RpoN also regulates genes that were shown to be important
for growth in subsurface environments or electricity production in microbial
fuel cells, such as flagella biosynthesis, pili biosynthesis, and c-type
cytochromes. Promoter elements recognized by the G. sulfurreducens RpoN
are highly similar to those recognized by other bacterial RpoN homologs.
Transcription initiation by RNA polymerase containing RpoN requires an
enhancer-binding protein, and the G. sulfurreducens genome encodes more
putative transcription factors in the enhancer-binding protein family than most
bacteria (Karlin et al., 2006; Méthé et al., 2003). Thus, the RpoN homolog is a
global regulator controlling a complex transcriptional network modulating
physiological responses in G. sulfurreducens.
The FliA homolog appears to regulate flagella and chemotaxis genes in
G. sulfurreducens as analysis of the G. sulfurreducens genome identified
sequences similar to those recognized by other bacterial FliA homologs
(Leang et al., 2009; Tran et al., 2008). As noted elsewhere, flagellar motility
and chemotaxis are likely to be important for growth in subsurface
environments. However, the function of the FliA homolog has not been
experimentally studied in Geobacter species.
The RpoE homolog has not been characterized in Geobacter species.
RpoE homologs or ECF sigma factors have been shown to control a vari-
ety of cellular functions in other bacteria. Amino acid sequences of ECF
sigma factors appear to be more diverse than those of other families of
sigma factors (e.g., RpoD, RpoS, RpoH, RpoN, and FliA). Therefore, it
is difficult to predict the function of the RpoE homologs in Geobacter spe-
cies solely by their sequences. Whereas homologs of RpoD, RpoS, RpoH,
RpoN, and FliA are highly conserved among Geobacter species whose
genome sequences are available, RpoE homologs are not, suggesting that
RpoE might be involved in regulation of species-specific features.
8.2. Transcription Factors
Transcription factors generally regulate genes for more specific cellular

functions than sigma factors and further fine-tune gene regulation in
response to environmental and physiological changes. Transcription
factors include an activator and a repressor, which promote or inhibit tran-

scription by RNA polymerase, respectively. Some transcription factors can
function as both an activator and a repressor. The G. sulfurreducens genome
encodes 151 putative transcription factors (Méthé et al., 2003). Transcription
factors classified as a response regulator on the basis of sequence similarity
are described in the section on two-component systems below.
The novel transcriptional repressor, HgtR, is induced in G.
sulfurreducens when hydrogen is available as an electron donor and
represses expression of citrate synthase and other genes encoding enzymes
involved in central metabolism (Ueki and Lovley, 2010a). HgtR also
regulates a gene encoding a putative transcription factor in the GntR fam-
ily. Target genes of this GntR homolog in Geobacter species have not been
identified. A transcription factor in the RpoN-dependent enhancer-binding
protein family appears to regulate expression of hgtR, which has sequences
highly similar to the RpoN recognition consensus sequences in its pro-
moter region. Further, a gene encoding an enhancer-binding protein is
located upstream of the hgtR homologs in Geobacter species. These
enhancer-binding protein homologs contain a domain similar to the C-ter-
minal domain of the iron-only hydrogenase large subunit at the N-termi-
nus. It is possible that these enhancer-binding protein homologs sense
environmental and/or intracellular hydrogen and activate the hgtR
homologs. Therefore, it is likely that a novel regulatory cascade mediated
by multiple transcription factors’ genes controls expression of central
metabolism genes in Geobacter species.
Studies on the adaption of G. sulfurreducens to grow on lactate revealed
a transcriptional regulator that regulates expression of the genes for succi-
nyl-CoA synthetase (Summers et al., 2011), a TCA cycle enzyme that is
required for growth on lactate, but not acetate (Galushko and Schink,
2000; Segura et al., 2008). The G. sulfurreducens gene GSU0514, which is
a homolog of the IclR transcription factor, encodes a transcriptional
repressor for the succinyl-CoA synthetase subunit genes sucC and sucD
(Summers et al., 2011). Mutations in GSU0514 were selected for during
adaption for enhanced growth on lactate, which enhanced expression of
sucC and sucD and promoted lactate metabolism (Summers et al., 2011).
Another adaptive evolution study identified the transcription factor,
GSU1771, which controls the expression of genes that are important for
Fe(III) oxide reduction (Tremblay et al., 2011b). GSU1771 is a homolog
of Streptomyces antibiotic regulatory protein (SARP) (Wietzorrek and
Bibb, 1997). Adaption of G. sulfurreducens for more rapid growth on Fe
(III) oxide yielded strains that accumulated a mutation that interrupted
the GSU1771 gene. Inactivation of GSU1771 in the wild-type strain
enhanced the ability to reduce Fe(III) oxide and increased the expression of
the gene for PilA, the structural protein for the electrically conductive pili.
Although dissimilatory metal reduction by Geobacter species has been
extensively studied, effects of the availability of metals for assimilatory
purposes on growth and activity of Geobacter species have gained less
attention. For instance, Fe(II) appears to play a critical physiological role
in Geobacter species as they contain an unusually large number of iron-sul-
fur proteins such as c-type cytochromes, which have been shown to be
essential for dissimilatory metal reduction. The Fe(II)-dependent tran-
scription factor, Fur, is an important regulator for Fe(II) influx in other
bacteria (Escolar et al., 1999) and all available Geobacter genomes contain
a cluster consisting of homologs of fur, as well as feoB, which encodes an
iron uptake protein and ideR, another Fe(II)-dependent transcription fac-
tor (O'Neil et al., 2008). In chemostat cultures, the expression of the fur-
feoB-ideR cluster decreased as Fe(II) concentrations increased, suggesting
that transcript abundance could serve as an indication of limitation of iron
for assimilation. Monitoring transcript abundance of the Geobacter species
in groundwater surprisingly revealed that iron availability might be limiting
under some bioremediation conditions (O'Neil et al., 2008). Analyses of
Geobacter genomes identified sequences in feoB and other genes that are
similar to other bacterial Fur-binding sites, suggesting that Fur controls
feoB in Geobacter species.
A number of biological processes in energy generation, nitrogen assimi-
lation, and detoxification require nickel-dependent enzymes such as
hydrogenase, carbon monoxide dehydrogenase, and urease (Mulrooney
and Hausinger, 2003; Zhang et al., 2009). The Ni(II)-dependent transcrip-
tion factor NikR is known to regulate nickel transporters in other bacteria
(Chivers and Sauer, 1999; Wang et al., 2009). The G. uraniireducens NikR
homolog was shown to bind the promoter regions of two different genes,
nik(MN)1 and nik(MN)2, which encode ABC-type transporters (Benanti
and Chivers, 2010). The DNA-binding mode of the G. uraniireducens
NikR homolog was distinct from other members of the NikR family.
Geobacter species are likely to encounter oxygen intrusions in subsur-
face environments, particularly at the oxic/anoxic interface where Fe(III)
sources are abundant. Thus, the ability of Geobacter species to tolerate
exposure to low concentrations of oxygen and even grow with oxygen as
the terminal electron acceptor may be a critical factor to survival in subsur-
face environments (Lin et al., 2004; Mouser et al., 2009a). Many bacterial
cells are equipped with oxidative responsive systems, which are regulated
by RecA and LexA (Butala et al., 2009; Cox, 2007). LexA is a transcription
factor controlling genes in the SOS system. The G. sulfurreducens genome
encodes two independent LexA homologs, which appear to be

autoregulated (Jara et al., 2003). Unlike other bacterial LexA, G.
sulfurreducens LexA homologs may not control recA and other genes
known to be involved in the SOS system because sequences similar to G.
sulfurreducens LexA-binding sites located in the G. sulfurreducens lexA
genes are absent from these SOS system genes in G. sulfurreducens. Thus,
G. sulfurreducens may employ unique regulatory mechanisms in oxidative
responsive systems.
Transcriptional regulators control the expression of genes involved in
the degradation of aromatic compounds. BgeR is a transcriptional repres-
sor that regulates genes for the metabolism of aromatic compounds as well
as another transcription factor involved in aromatic metabolism in
G. bemidjiensis (Ueki, 2011). BgeR belongs to the Rrf2 family, but the sim-
ilarity is limited to the N-terminal region, which is likely a DNA-binding
domain. Its C-terminal region does not show similarity to known proteins
except for BgeR homologs in other Geobacter species. It is likely that
genes for aromatic compound degradation are controlled by regulatory
cascades consisting of multiple transcription factors in Geobacter species.
This is a topic that warrants further study because of its potentially
important role in bioremediation.
8.3. Two-Component Systems
Geobacter species have one of the highest IQs of bacteria, which is a mea-
sure of the adaptive potential of an organism on the basis of the total num-
ber of signaling proteins including the two-component system encoded in a
given genome (Galperin, 2005). The genomes of Geobacter species encode
an unusually large number of genes for the two-component signaling
proteins (Aklujkar et al., 2009, 2010; Méthé et al., 2003). The two-component
system typically consists of a sensor histidine kinase, which senses environ-
mental signals, and a response regulator, which generally influences the gene
expression necessary for the adaptation (Egger et al., 1997). The two compo-
nents are often colocalized in the same operon in other bacteria (Mizuno,
1997), but this is often not the case in Geobacter species. Most of the puta-
tive sensor domains of the two-component systems in Geobacter species
are unique or show similarity to uncharacterized systems in other bacteria.
Some of the sensor domains have a c-type heme-binding motif (Londer
et al., 2006b; Pokkuluri et al., 2008) and may participate in redox control
of complex biological processes in Geobacter species.
An important response regulator is PilR, which is an RpoN-dependent

enhancer-binding protein that regulates expression of pilA, the gene for
the structural pilin protein (Juarez et al., 2009). The histidine kinase PilS
appears to regulate PilR because pilS is immediately upstream of pilR.
Transcriptomic and bioinformatic approaches identified a number of genes
including those for c-type cytochromes, such as OmcB and OmcS, under
the control of PilR (Juarez et al., 2009; Krushkal et al., 2010). Adaptive evo-
lution for syntrophic growth of G. metallireducens and G. sulfurreducens
selected for a strain of G. sulfurreducens with a mutation in pilR, which
enhanced production of the c-type cytochrome OmcS (Summers et al., 2010).
Novel regulatory cascades consisting of two two-component systems reg-
ulate nitrogen-fixation gene expression in G. sulfurreducens (Ueki and
Lovley, 2010b). GnfM, a member of the enhancer-binding protein family,
contains a receiver domain at the N-terminus and thus is also a response
regulator of a two-component system in which the activity of GnfM
appears to be regulated by the histidine kinase GnfL. The GnfM gene
seems to be essential for growth, probably because it regulates the expres-
sion of genes involved in nitrogen metabolism that are important even
when ammonium is present (Leang et al., 2009). In addition to the nitrogen
metabolism genes, the GnfL/GnfM system activates genes encoding regu-
latory proteins of a two-component system during nitrogen fixation that
comprises the histidine kinase GnfK and the response regulator GnfR,
which has an RNA-binding domain. GnfK modulates the activity of GnfR
by phosphorylation. Phosphorylated GnfR exhibits RNA-binding activity.
The GnfK/GnfR system regulates by transcription antitermination the
expression of a subset of the nitrogen-fixation genes, such as nifH, nifEN,
nifX, glnK, and amtB, whose transcription is activated by the GnfL/GnfM
system and whose promoter region contains transcription termination
signals. The GnfK/GnfR system plays a critical role in the nitrogen-fixation
gene regulation as deletion mutants of gnfK or gnfR are defective in
growth dependent on nitrogen fixation.
Expression of the gene bamY, which encodes benzoate-CoA ligase, is
induced during growth of G. metallireducens on benzoate (Butler et al.,
2007; Wischgoll et al., 2005). The bamV and bamW genes encoding a puta-
tive histidine kinase and a putative response regulator, respectively, are
located in the vicinity of the bamY gene (Wischgoll et al., 2005). Aromatic
compounds induced the expression of the bamV gene (Butler et al., 2007;
Wischgoll et al., 2005). The bamY gene contains an RpoN-dependent
promoter and the response regulator BamW is also an enhancer-binding
protein (Juárez et al., 2010). Addition of benzoate, p-cresol, or
p-hydroxybenzoate to cultures of E. coli heterologously expressing bamV
and bamW induced expression of a b-galactosidase gene fused to the bamY

promoter, demonstrating the role of this two-component system in
controlling aromatics metabolism (Juárez et al., 2010).
FgrM, which is a member of the enhancer-binding protein family as well
as a response regulator, is the master transcriptional regulator for flagellar
gene expression in Geobacter species (Ueki et al., 2011). FgrM interacts
with RpoN to control transcription of a number of flagella genes, including
the gene for FliA, which controls expression of some flagella-related genes,
including chemotaxis genes. Thus, it appears likely that the expression of
flagella-related genes is controlled in a cascade manner.
8.4. Chemotaxis
Unlike well-characterized motility systems such as those found in E. coli and

Bacillus subtilis, both of which have a single chemotaxis system, Geobacter
species contain multiple chemotaxis systems or homologs of the chemotaxis
system (Tran et al., 2008). One of the chemotaxis(-like) systems, designated
the a-group, is unique to Geobacter species and is predicted to be involved
in flagellar motility (Ueki et al., 2011). A second chemotaxis-like system,
designated the b-group and found in d-proteobacteria, is involved in the regu-
lation of the expression of extracellular proteins, such as the c-type
cytochromes, OmcS and OmcZ (Tran et al., 2011). Proteins of both the a
and b-groups were abundant in groundwater during acetate-stimulated in situ
uranium bioremediation (Wilkins et al., 2009).
Geobacter species appear to have an unusually large number of chemo-
receptor (MCP) genes (Aklujkar et al., 2009, 2010; Méthé et al., 2003; Tran
et al., 2008). Several MCP genes are located in proximity to other chemo-
taxis genes on the genome in Geobacter species but most are scattered on
the genome. Some are predicted to play a role in chemotaxis (Ueki et al.,
2011). It is possible that other MCP genes are involved in signal transduc-
tion pathways mediated by chemotaxis-like systems.
8.5. Nucleotide-Based Second Messenger
Stringent response, originally observed during amino acid starvation in

E. coli, is affected by (p)ppGpp, which is known to act as a global regulator
in physiological adaptation to a variety of environmental changes
(Braeken et al., 2006; Potrykus and Cashel, 2008). In G. sulfurreducens,
ppGpp and ppGp were produced in response to nutrient limitations and
ppGpp accumulated as the result of oxygen exposure (DiDonato et al.,

2006). The production of ppGpp in G. sulfurreducens was dependent on
the rel gene encoding a homolog of the bifunctional RelA/SpoT protein,
which has both (p)ppGpp synthetase and hydrolase activity. Deleting rel
affected expression of genes involved in protein synthesis, stress responses,
and electron transport systems, and enhanced growth with fumarate as the
electron acceptor, but increased oxygen sensitivity and diminished the
capacity for Fe(III) reduction. Bioinformatic analysis suggested that genes
influenced by the Rel/ppGpp signaling system are also controlled by Fur
and RpoS (Krushkal et al., 2007).
Riboswitches, noncoding RNA elements found in the untranslated region
of mRNA, are known to sense and bind cellular metabolites to control gene
expression (Lioliou et al., 2010; Waters and Storz, 2009). Geobacter species
possess riboswitches termed GEMM (genes related to the environment,
membranes and motility) (Weinberg et al., 2007), which have been shown to
sense c-di-GMP in other bacteria (Sudarsan et al., 2008). G. uraniireducens
has the largest number of c-di-GMP riboswitch homologs among bacteria
whose genomes have been sequenced (Weinberg et al., 2007). In G.
sulfurreducens, genes known to be differentially regulated during metal reduc-
tion and electricity production, such as omcS and omcT, were found to contain
a c-di-GMP riboswitch signature in their noncoding region of mRNA
(Weinberg et al., 2007). The 50 untranslated region of omcS mRNA is critical
for omcS expression (B-C. Kim et al., unpublished data).
Another c-type cytochrome whose expression appears to be controlled
with a GEMM riboswitch is PgcA, which contains a GEMM riboswitch
sequence between the predicted RpoD-dependent promoter and the start
codon (Tremblay et al., 2011b). An increase in c-di-GMP in E. coli results
in the upregulation of lacZ under the control of the pgcA-associated GEMM
riboswitch (B-C. Kim et al., unpublished data). Adaptive evolution of G.
sulfurreducens for improved growth on Fe(III) oxide selected for strains that
had either a single base-pair change or a one-nucleotide insertion in the
GEMM riboswitch of the pgcA gene. Introduction of either of the GEMM
riboswitch mutations in the pgcA gene into the wild-type strain increased
the abundance of pgcA transcripts, consistent with increased expression of
pgcA in the adapted strains.
8.6. Summary Statement on Regulation
The abundance of regulatory genes and novel sensing capabilities found in

Geobacter species suggest that they are highly attuned to their
environment and have evolved to be able to sense a wide diversity of envi-

ronmental cues. For the most part, even the regulatory systems that have
already been studied so far have only been examined in a rather prelimi-
nary manner and there are many other regulatory systems that have yet
to be investigated. A better understanding of these systems will greatly
aid the development of models to predict the activity of Geobacter species
under different environment conditions.
9. ENVIRONMENTAL SYSTEMS BIOLOGY OF GEOBACTER
The availability of pure cultures of Geobacter species closely related to

those that are abundant in Fe(III)-reducing environments has made it
possible to take a systems approach to the study of Geobacter ecology in
subsurface environments. For example, quantifying key gene transcripts
or proteins can provide a diagnosis of the in situ physiological status of
Geobacter species, providing insights into metabolic patterns that are likely
to be much different than when the microorganisms were grown under
nutritionally replete conditions in the laboratory. Even estimating how fast
microorganisms are metabolizing in natural environments can be difficult,
especially in subsurface environments which are difficult to sample.
Understanding in situ physiological status is key for bioremediation,
making it possible to rationally design strategies to modify the in situ
activity of Geobacter species (Lovley, 2003; Lovley et al., 2008).
9.1. Environmental Transcriptomics and Proteomics
Several strategies were investigated to elucidate the rate of activity of Geo-

bacter species in the subsurface. One successful approach was based on
monitoring gene transcript abundance (Holmes et al., 2005; Williams
et al., 2011) or protein abundance (Wilkins et al., 2011; Yun et al., 2011b)
of the key TCA cycle enzyme citrate synthase. The citrate synthase
sequences of Geobacter species are more closely aligned with those of
eukaryotes, rather than other prokaryotes (Bond et al., 2005; Méthé
et al., 2003), simplifying the task of designing PCR primers to specifically
amplify citrate synthase sequences of Geobacter species. Chemostat studies
with G. sulfurreducens demonstrated a direct correlation between rates of
acetate metabolism and transcript abundance for citrate synthase (Holmes
et al., 2005). Subsequent field studies in which the in situ levels of
Geobacter citrate synthase gene transcripts were monitored demonstrated

that as acetate availability in the groundwater was artificially manipulated,
the metabolism of the in situ Geobacter community responded accordingly,
increasing expression of citrate synthase when acetate concentrations were
elevated and repressing expression when acetate levels dropped (Holmes
et al., 2005; Williams et al., 2011). A similar metabolic response was noted
when citrate synthase protein levels were quantified with an antibody-
based approach (Yun et al., 2011b).
Attempts to monitor bulk rates of respiration by quantifying the tran-
script abundance of genes specifically involved in electron transfer pro-
cesses was less successful (Chin et al., 2004), but recent studies with
sulfate reducers have demonstrated that important insights into per-cell
rates of metabolism might be obtained with such an approach (Miletto
et al., 2011; Villanueva et al., 2008), suggesting that it might be productive
to revisit this approach in Geobacter species.
Recent studies demonstrated that the abundance of a predominant
c-type cytochrome in groundwater correlated well with the activity of Geo-
bacter species and the effectiveness of uranium bioremediation (Yun et al.,
2011b). Subsequent functional analysis suggested that this cytochrome
might function similarly to the OmcS of G. sulfurreducens.
As important as it is to understand rates of metabolism, it is equally
important to understand the factors that control those rates. Quantifying
key gene transcripts or proteins has been shown to be a useful tool for
diagnosing which nutrients or stresses might be limiting the growth of the
subsurface Geobacter community. For example, measuring transcript abun-
dance (Holmes et al., 2004c; Mouser et al., 2009b) or protein abundance
(Yun et al., 2011b) of the nitrogen-fixation protein NifD can indicate
whether Geobacter species in the subsurface are limited for ammonium
and need to fix atmospheric nitrogen. This information can guide bioreme-
diation because it may be beneficial for cells to be ammonium-limited
during uranium bioremediation, but for bioremediation of hydrocarbon-
contaminated groundwater ammonium limitation is likely to slow contami-
nant removal.
In a similar manner, molecular analysis of the in situ physiological status
of the subsurface Geobacter community has provided insights into phos-
phate limitation (N'Guessan et al., 2009), acetate availability (Elifantz
et al., 2011), iron limitation (O'Neil et al., 2008), and oxidative stress during
uranium bioremediation (Mouser et al., 2009a). Monitoring transcript
abundance for a ribosomal protein made it feasible to estimate growth
rates of Geobacter species during uranium bioremediation and is expected
to be an important tool in evaluating the proposed slow growth of
microorganisms in undisturbed subsurface environments (Holmes et al.,

2011a). The increased expression of a key Geobacter enzyme in the degrada-
tion of aromatic hydrocarbons in response to hydrocarbon contamination in
groundwater was documented with an antibody-based approach (Yun et al.,
2011b). Continued analysis of Geobacter communities with high-throughput
proteomics (Callister et al., 2010; Wilkins et al., 2009) and broad transcriptomic
approaches (Holmes et al., 2009) is expected to identify other key gene tran-
scripts and proteins that will serve as diagnostic tools for better understanding
the ecology of Geobacter species during bioremediation and in undisturbed
soils and sediments.
9.2. BUGS (Bottom-Up Genome-Scale) Modeling
A major goal in microbial ecology is to be able to not only describe the

distribution of microorganisms and their activity, but to predict microbial
distributions and interactions with other microorganisms and the environ-
ment under a diversity of environmental conditions. One strategy for this
is to couple genome-scale metabolic models with the appropriate models
that can describe physical/chemical conditions and their changes in
response to predicted microbial activity (Lovley, 2003; Lovley et al., 2008;
Mahadevan et al., 2011; Zhao et al., 2010).
We have termed this modeling approach bottom-up genome-scale
modeling, abbreviated BUGS modeling, to differentiate it from the
increasingly popular top-down approach of beginning with a global analy-
sis of genes, gene transcripts, and proteins in environments of interest. The
two approaches are complementary. An advantage of the top-down com-
munity wide approach is that it can rapidly provide a “parts list,” an
accounting of the diversity of genes and proteins and their relative abun-
dance. However, this is a highly descriptive approach and it is difficult to
make predictions about the response of microorganisms to changes in envi-
ronmental conditions from such lists. In BUGS modeling, genome-scale
metabolic models are made for the microorganisms that predominate in
an environment of interest and their interaction with each other and their
environment is modeled. BUGS modeling is a slower, iterative process, but
in the end provides a knowledge base, based on first principles of microbial
physiology, that should have broad applicability and predictive power.
Subsurface environments in which Geobacter species predominate have
proven to be good test cases for the BUGS modeling concept. The addition
of acetate to groundwater to stimulate dissimilatory metal reduction results
in a bloom of Geobacter species, which are the primary microorganisms
influencing subsurface biogeochemistry during this period. Further, Geo-

bacter is one of the rare examples where isolates of the species that pre-
dominate in the environment of interest are available in pure culture. In
these still early days of the annotation of microbial genomes, the study of
relevant pure cultures is necessary because many important physiological
features, as well as the function of many genes, cannot be ascertained
solely from genomic sequences.
The details of the generation of genome-scale models for environmental
studies have recently been reviewed (Mahadevan et al., 2011) and will not
be repeated here. The initial genome-scale modeling of G. sulfurreducens
proved to be an important driver for hypothesis-driven research and rev-
ealed important metabolic features (Mahadevan et al., 2006, 2011; Yang
et al., 2010). For example, the mechanisms for acetate uptake, catabolic
and anabolic utilization, as well as energy conservation during reduction
of internal (fumarate) or external (Fe(III)) electron acceptors were
elucidated in an iterative process of laboratory experimentation and in sil-
ico modeling. The genome-scale model of G. sulfurreducens was an impor-
tant tool for analyzing the incorporation of carbon into amino acids, and
revealed that isoleucine was synthesized via the citramalate pathway
(Risso et al., 2008b). Continued development of genome-scale metabolic
models in other Geobacter species (Sun et al., 2009) and closely related
organisms (Sun et al., 2010) is identifying commonalities in metabolic
strategies as well as adding new metabolic modules, such as the pathways
for the degradation of aromatic compounds found in G. metallireducens
and other Geobacter species.
To date, BUGS modeling of the biogeochemical impacts of Geobacter
species has been applied to relatively simple subsurface environments.
In initial studies, the genome-scale metabolic model of G. sulfurreducens
was coupled with reactive transport models to determine geochemical
changes when acetate was added to groundwater to stimulate in situ ura-
nium bioremediation (Scheibe et al., 2009). Initial results were encouraging.
The predominance of Geobacter species during acetate-amended ura-
nium bioremediation is rare and some of the most important ecological
interactions in soils and sediments are those between different
microorganisms. Therefore, in order to expand the BUGS modeling con-
cept, the interactions between Rhodoferax and Geobacter species in the
subsurface were modeled. Like Geobacter species, Rhodoferax fer-
rireducens is an acetate-oxidizing Fe(III) reducer (Finneran et al., 2003).
Rhodoferax has a higher growth yield from acetate, but slower growth rate
than Geobacter and Rhodoferax cannot fix nitrogen whereas Geobacter
can. First, a genome-scale model of R. ferrireducens was generated (Risso
et al., 2009). Then the genome-scale models of G. sulfurreducens and R.

ferrireducens were used to model their growth under different environmen-
tal conditions (Zhuang et al., 2010). The modeling predicted that, as has
been observed experimentally (Mouser et al., 2009b), Geobacter and
Rhodoferax species are likely to coexist in subsurface environments in
which the slow degradation of organic matter deposited with the sediments
drives microbial metabolism. Where ammonium concentrations are rela-
tively high, Rhodoferax will predominate. However, the modeling pre-
dicted that when acetate is added to the groundwater to promote the
growth of Fe(III) reducers, Geobacter outgrows Rhodoferax because of
its much faster growth rate. This prediction is consistent with what is
observed during bioremediation. The BUGS modeling approach is now
being expanded to evaluate the competition between Geobacter species
and sulfate-reducing Desulfobacter species, which compete with Geobacter
species for acetate. Over time more complex communities, potentially first
grown in the laboratory (Miller et al., 2010) and then studied in the field,
can be modeled.
At some point, it may be possible to greatly accelerate the BUGS
modeling process by building models from genomes sequenced directly
from the environment. However, as of now, gene annotation and the abil-
ity to predict even simple physiological properties, such as growth rate,
from the genome sequence are not sufficiently advanced.
10. BIOGEOCHEMICAL IMPACTS OF GEOBACTER SPECIES
Previous reviews have detailed many of the substantial geochemical

impacts that Geobacter species can have on anaerobic soils and sediments
(Lovley, 1991, 1993, 1995, 2000a,b), and these topics will not be covered in
detail here. Important geochemical changes that take place in Fe(III)- and
Mn(IV)-reducing environments in which Geobacter species are abundant
can include the production of magnetite, siderite, and other Fe(II) and
Mn(II) minerals; the release of iron, trace metals, metalloids, and phos-
phate into pore waters; other changes that influence the pH and ionic
strength of pore waters; and changes in soil porosity as the result of reduc-
tion of Fe(III) in clays. The degradation of organic carbon in soils and
sediments coupled to the reduction of Fe(III) and Mn(IV) can contribute
significantly to anaerobic organic matter degradation with the release of
carbon dioxide. Any organic matter degraded in this manner results in less
reduction of sulfate and less production of methane. Ions of metals and
metalloids are natural constituents of soils and sediments, and as noted in

other sections, the ability of Geobacter species to reduce these can influ-
ence their geochemical fate.
In some instances, the activity of Geobacter species in subsurface
environments can have deleterious impacts on groundwater quality. For
example, undesirably high concentrations of Fe(II) and Mn(II) as the
result of microbial reduction of Fe(III) and Mn(IV) are common
(Anderson and Lovley, 1997; Chapelle and Lovley, 1992; Lovley, 1997).
Geobacteraceae are abundant in groundwaters with high arsenic con-
centrations in which Fe(III) reduction has been implicated in the release
of arsenic (Héry et al., 2010; Islam et al., 2004a; Smedley and Kinniburgh,
2002; Weldon and MacRae, 2006). Possibilities for arsenic fluxes from
sediments to groundwater include release of arsenic adsorbed onto Fe(III)
oxide and/or the reduction of As(V) to As(III), which is more soluble.
For example, Geobacter species are thought to play a key role in the mobili-
zation of arsenic from West Bengal sediments (Héry et al., 2008; Islam et al.,
2004a,b, 2005a,b) where arsenic release takes place after Fe(III) reduction,
rather than occurring simultaneously (Islam et al., 2004b). It has been
proposed that Geobacter species related to G. uraniireducens and G. lovleyi
may be the primary catalysts for As(V) reduction (Héry et al., 2010) but
As(V) reduction in these species has not yet been documented.
Some Geobacter species can methylate mercury and their activity may
be an important source of this environmental toxin in iron-rich freshwater
sediments (Fleming et al., 2006). Pure cultures capable of mercury methyl-
ation include Geobacter strain CLFeRB (Fleming et al., 2006) as well as
G. hydrogenophilus, G. metallireducens, and G. sulfurreducens and the
closely related Desulfuromonas palmitatis (Kerin et al., 2006). Environ-
mental conditions that control the extent to which Geobacter species
methylate mercury are beginning to be examined (Schaefer and Morel,
2009; Schaefer et al., 2011) and warrant further study.
11. PRACTICAL APPLICATIONS OF GEOBACTER SPECIES
11.1. Bioremediation: Natural Attenuation and Engineered
11.1.1. Aromatic Hydrocarbons
Geobacter species are often important components of the microbial commu-

nity in aquifers polluted with petroleum or landfill leachate (Alfreider and
Vogt, 2007; Botton et al., 2007; Holmes et al., 2007; Lin et al., 2005, 2007;
Röling et al., 2001; Rooney-Varga et al., 1999; Staats et al., 2011; Van
Stempvoort et al., 2009; Winderl et al., 2007, 2008) which can be attributed,
at least in part, to the ability described above of some Geobacter species to
degrade aromatic compounds. Prior to contamination most shallow aquifers
are aerobic, but anaerobic conditions rapidly develop once organic con-
taminants are introduced. Fe(III) is generally an abundant electron acceptor
for anaerobic degradation and early studies demonstrated a removal of
aromatic hydrocarbon contaminants from petroleum-contaminated ground-
water associated with geochemical signatures for Fe(III) reduction (Lovley
et al., 1989). Subsequent analysis demonstrated an abundance of Geobacter
species in the Fe(III) reduction zone (Anderson et al., 1998; Holmes et al.,
2004c, 2007; Lovley et al., 1989; Nevin et al., 2005; Rooney-Varga et al.,
1999), accounting for 41% of the active microbial community in the ground-
water (Holmes et al., 2007). In a similar manner, 25% of the microbial
community comprised Geobacter species in a landfill leachate-contaminated
aquifer (Röling et al., 2001). Quantifying specific genes or proteins known to
be involved in the degradation of aromatic compounds has further demon-
strated the importance of Geobacter species in naturally removing aromatic
contaminants (Hosoda et al., 2005; Kane et al., 2002; Kuntze et al., 2008, 2011;
Winderl et al., 2007, 2008; Yun et al., 2011b).
The finding that Geobacter species could reduce chelated Fe(III) faster
than Fe(III) oxides (Lovley and Phillips, 1988a; Lovley and Woodward,
1996) and that electron shuttles promoted Geobacter reduction of Fe(III)
oxide (Lovley et al., 1996a) led to studies evaluating whether the addition
of Fe(III) chelators could stimulate the degradation of aromatic
hydrocarbons (Lovley et al., 1994, 1996b). In the presence of these
stimulants, even benzene could be degraded as rapidly with Fe(III) as
the electron acceptor as it could with the introduction of oxygen.
The most practical method for enhancing electron-acceptor availability
to Geobacter species involved in the degradation of organic contaminants
in contaminated groundwater or aquatic sediments may be the concept
of “subsurface snorkels” (Lovley, 2011c). Studies with G. metallireducens,
as well as natural communities, demonstrated that providing an electrode
as an electron acceptor may be a good strategy for stimulating the degrada-
tion of aromatic hydrocarbon contaminants (Zhang et al., 2010). Graphite
electrodes may be the best option as they are inexpensive and durable and
have the added advantage of adsorbing aromatic contaminants on their
surface. This colocalizes the contaminant, the electron acceptor, and
the Geobacter species at the electrode surface. Initial studies focused on
the use of relatively complex systems in which the potential of the
electrode was electronically poised (Zhang et al., 2010). However, it may

be sufficient to insert conductive rods into contaminated sediments with
a portion of the conductive rod extending into aerobic soil, water, or the
atmosphere. With such subsurface snorkels, the portion of the rod in the
anaerobic soil can function as an electron acceptor to promote the growth
of Geobacter capable of degrading the contaminants while the portion of
the rod in the aerobic environment functions as a cathode (Lovley, 2011c).
11.1.2. Uranium and Related Metals and Metalloids
The ability of Geobacter to reduce soluble ions of metals to less soluble forms
shows promise as a bioremediation tool. Metals may be removed from water
in this manner in reactors, or stimulating the activity of Geobacter species for
in situ immobilization is an option. In some instances, Geobacter species
might naturally attenuate the movement of metals via reduction.
Uranium has been the contaminant metal of greatest focus because the
rapid kinetics of bacterial U(VI) reduction and low solubility of U(IV)
make this process an attractive option for removing uranium from
groundwaters below drinking water standards (Williams et al., 2011, and
references therein). The rather nonspecific nature in which Geobacter spe-
cies reduce U(VI) (see above) and the fact that even in uranium-con-
taminated environments U(VI) is likely to be a minor electron acceptor
(Finneran et al., 2002a) make it difficult to definitely determine if
Geobacter species are the agents for U(VI) reduction in studies in which
dissimilatory metal reduction has been stimulated to promote uranium bio-
remediation. However, the consistent pattern of effective U(VI) removal
being associated with increased growth and activity of Geobacter species
at least at some sites (Williams et al., 2011, and references therein) suggests
that Geobacter species play a role.
Stimulating the activity of Geobacter species may also remove a variety
of other toxic metals that Geobacter species have the potential to reduce
in pure culture, but the reduction of these contaminants may be indirect
in subsurface environments, because as noted above in Section 5, these
electron acceptors can also be reduced by Fe(II) that Geobacter species
generate during Fe(III) oxide reduction.
Although the commonly considered approach to stimulating the activity
of Geobacter species for bioremediation of uranium and related con-
taminants is to add organic electron donors, a more effective approach
might be to provide Geobacter species electrons with electrodes (Gregory
and Lovley, 2005). Long-term stimulation of anaerobic respiration has
several potential negative impacts (Williams et al., 2011). These include

(1) release of trace metals and arsenic that were associated with Fe(III)
oxides into the groundwater (Burkhardt et al., 2010), (2) deterioration of
the groundwater quality from accumulations of dissolved Fe(II) or sulfide,
and (3) aquifer plugging due to biomass or mineral accumulations
(Williams et al., 2011). Further, reductive immobilization of uranium in this
manner leaves the uranium contamination in the subsurface.
Therefore, a better alternative may be to feed Geobacter species
electrons with electrodes (Gregory and Lovley, 2005). Maintenance of
the electron addition to the subsurface with electrodes is much simpler
than complex pumping strategies for the controlled introduction of organic
electron donors and the electrode strategy is sustainable, easily powered
with solar panels. Further, this strategy specifically provides electrons for
the reduction of the soluble contaminant of interest and the U(IV) pro-
duced precipitates on electrodes. It would be a simple matter to periodi-
cally remove the electrodes, extract the U(IV) under aerobic conditions
in bicarbonate (Phillips et al., 1995), and return the electrodes to the
subsurface. This approach would alleviate all the negative side effects of
adding the organic electron donors listed above as well as remove the
uranium from the subsurface.
11.1.3. Chlorinated Contaminants
In a similar manner, providing electrons to Geobacter species in the subsur-

face with electrodes may be an effective strategy for stimulating reductive
dechlorination (Strycharz et al., 2008, 2010). Although no Geobacter spe-
cies are known to completely dechlorinate chlorinated solvents, electrodes
have the potential to specifically localize the electron donor and the
dechlorinating organisms. Therefore, cathodes colonized with Geobacter
species and positioned near source zones of chlorinated solvents could con-
vert the solvents to the much more soluble products, susceptible to aerobic
degradation, that could be degraded downgradient at the anode which
produces oxygen (Lovley and Nevin, 2011). As noted above, Geobacter
species have frequently been detected in subsurface environments con-
taminated with chlorinated solvents and in dechlorinating enrichments.
The formation of reactive Fe(II) minerals by Geobacter species during
Fe(III) oxide reduction may accelerate the removal of carbon tetrachloride
(McCormick et al., 2002). In a similar manner, humic substances may
provide an electron shuttle to promote carbon tetrachloride reduction
(Cervantes et al., 2004).
11.2. Producing Methane from Organic Wastes and

Hydrocarbon Deposits
Conversion of organic wastes and biomass to methane has been a long-

standing bioenergy strategy whose use could be expanded if the process
could be accelerated and made more consistently stable. Geobacter species
may have a role in this process development because the ability of Geo-
bacter species to function as syntrophs may permit them to significantly
contribute to rapid conversion of organic matter to methane.
Molecular analyses have demonstrated that Geobacter and closely related
species can account for over 20% of the microbial community in the
methanogenic aggregates that form in anaerobic digesters treating brewery
wastes (Morita et al., 2011; Werner et al., 2011). Detailed analysis of
aggregates from one of these digesters demonstrated that the aggregates
had a conductivity similar to that of Geobacter co-culture aggregates (Morita
et al., 2011). The temperature dependence of the conductance suggested an
organic metallic-like conductivity, similar to that observed (Malvankar et al.,
2011b) in pilin preparations of G. sulfurreducens and G. sulfurreducens bio-
films. Several lines of evidence suggest that methanogenic microorganisms
might be able to directly accept electrons and that direct electron transfer,
rather than interspecies hydrogen or formate transfer, was the primary mech-
anism for electron exchange with the aggregates (Morita et al., 2011). The
understanding that Geobacter species, and possibly other microorganisms,
may be directly transferring electrons to methanogens, via direct interspecies
electron transfer, may lead to new reactor designs to better promote this
interaction and accelerate the process (Lovley, 2011a,c).
It has also been proposed that the capacity of Geobacter species to func-
tion in methanogenic syntrophic interactions can be used to enhance the
recovery of hydrocarbons from coal and hydrocarbon deposits (Jones et al.,
2010; Siegert et al., 2010). In fact, in an enrichment culture converting coal
to methane, the most important microorganisms appeared to be Geobacter
and Methanosaeta species (Jones et al., 2010), which is similar to what was
found in wastewater methanogenic aggregates (Morita et al., 2011). This
suggests that principles for Geobacter contributions to methanogenic waste-
water treatment may apply to hydrocarbon recovery from the subsurface.
11.3. Microbial Fuel Cells, Electrosynthesis, and

Bioelectronics
There is significant interest in the development of large-scale microbial

fuel cell systems for wastewater treatment. Given the consistent
enrichment of Geobacteraceae on anodes of effectively operating microbial

fuel cells, pre-enrichment of anodes with Geobacter species may be an
important step in scale-up (Cusick et al., 2011).
There may be significant potential for increasing the current output of
microbial fuel cells via strain selection/design (Izallalen et al., 2008; Yi
et al., 2009). The anode of a microbial fuel cell is not a natural electron
acceptor, and thus it is unlikely that there has been significant selective
pressure on Geobacter species to optimize current production under the
conditions found in microbial fuel cells (Lovley, 2006a). For example,
increasing pilin expression of G. sulfurreducens, via strain selection or
genetic engineering, increased biofilm conductivity and current production
(Malvankar et al., 2011b). As more is learned about the mechanisms for
electron transfer to electrodes in Geobacter species, it may be possible to
further enhance power output.
Even without strain improvement there may be some short-term practical
applications for microbial fuel cells, such as powering electrical devices in
remote locations, such as at the bottom of the ocean (Tender et al., 2008).
The fact that Geobacter species are often the primary microorganisms col-
onizing electrodes harvesting current from a diversity of environments
suggests that they are likely to play an important role in any applications
of microbial fuel cells in which current is harvested in open environments
in which there will be competition for anode colonization. Geobacter-based
sensors may also be practical (Davila et al., 2010). Novel system designs
make it feasible to consider producing current with Geobacter species, even
in completely aerobic environments (Nevin et al., 2011b). Electrodes
deployed in subsurface environments are naturally colonized by Geobacter
species (Williams et al., 2010) and may function as sensors of subsurface
microbial activity (Tront et al., 2008; Williams et al., 2010).
Microbial electrosynthesis is a process in which electrons are provided to
microorganisms colonizing an electrode to support the reduction of carbon
dioxide to organic compounds that are excreted from the cells (Lovley,
2011b; Lovley and Nevin, 2011; Nevin et al., 2010, 2011a). When powered
with solar technology, microbial electrosynthesis is an artificial form of
photosynthesis in which sunlight drives the conversion of carbon dioxide
and water to organic compounds and oxygen. Proof-of-concept studies
have demonstrated acetate production with acetogenic microorganisms as
the catalysts (Nevin et al., 2010, 2011a).
Genome annotation led to the surprising discovery of enzymes for car-
bon dioxide fixation in some Geobacteraceae (Aklujkar et al., 2010). Within
the G. metallireducens genome, a pair of genes is predicted to encode an
ATP-dependent citrate lyase, which would allow the reverse TCA cycle
to produce acetyl-CoA. Further, genes for all of the identified enzymes of

the dicarboxylate/4-hydroxybutyrate cycle of carbon dioxide fixation
are predicted in the G. metallireducens genome. G. metallireducens is
also capable of electrosynthesis, and investigations with genetically
modified strains of other Geobacter species are ongoing because of the
ability of Geobacter species to interact so effectively with electrodes.
G. sulfurreducens can also use electrons derived from an electrode to
reduce protons to hydrogen (Geelhoed and Stams, 2011), potentially
providing a renewable catalyst that is much less expensive than the metal
catalysts typically employed for hydrogen production.
One of the most exciting practical applications for Geobacter species
could be bioelectronics. Electronically functional biomaterials are very
attractive because they can be synthesized from relatively inexpensive
feedstocks and do not contain toxic components (Hauser and Zhang,
2010). Further, conductive materials comprising living bacteria are self-
renewing because bacteria can self-repair and replicate. Initial studies have
already demonstrated the possibility of tuning the electronic properties of
Geobacter biofilms via simple genetic engineering and more sophisticated
modifications are feasible. Further elucidation of the mechanisms for elec-
tron transport along pili and ability of cytochromes to function as capacitors
could aid in the biomimetic design of new materials. Therefore, it is
expected that microbially based electronically functional materials will have
significant potential for next-generation biotechnological applications.
12. CONCLUSIONS
Studies to date have demonstrated the importance of Geobacter species to

the anaerobic degradation of organic matter in sedimentary environments
and its importance in iron, manganese, and trace-metal biogeochemistry.
Geobacter species can naturally attenuate the migration of organic and
metal contaminants, and strategies for artificially stimulating contaminant
removal by Geobacter species are being developed.
The novel electrical properties of Geobacter species, and their pili and
cytochromes, coupled with their ability to form direct electrical connections
with man-made electronics, are amazing and provide new paradigms for the
function of microbial communities and the development of next-generation
bioelectronics. It has been suggested that “if it were not for the bacterium
GS-15, we would not have radio and television today” because one of the
first discovered properties of G. metallireducens was its ability to make
magnetite and it was the study of magnetite lodestone that contributed to the
early understanding of electricity (Verschuur, 1993). Therefore, it may be
fitting that one of the most recently discovered properties of Geobacter
species, metallic-like conductivity along pili, has the possibility to make a
more direct contribution to further the development of electronics.
Our understanding of the ecology, physiology, biochemistry, and
bioelectronics of Geobacter is very rudimentary. Rapid advancements in
omic technologies greatly facilitated a substantial increase in the under-
standing of Geobacter over the past decade. Continued functional genomic
analyses of many aspects of Geobacter metabolism and genetic regulation
will be essential for continued progress. Further, contributions from other
fields, such as physics, materials science, and engineering, will be important
not only to increase basic understanding of Geobacter species but also for
the development of many promising, novel applications.
ACKNOWLEDGMENTS
Research on Geobacter species in our laboratory is currently funded by (1)

the Office of Science (BER) U.S. Department of Energy through Cooper-
ative Agreement No. DE-FC02-02ER63446, Award No. DE-SC0004114,
Award No. DE-SC0004080, Award No. DE-SC0004814, Award No. DE-
SC0004485, and Award No. DE-SC0006790; (2) the Advanced Research
Projects Agency-Energy (ARPA-E), U.S. Department of Energy, under
Award No. DE-AR0000087 and Award No. DE-AR0000159; and (3) the
Office of Naval Research Grant No. N00014-09-1-0190, Grant No.
N00014-10-1-0084, and Grant No. N00014-10-C-0184.
We thank Anna Palmissano and Dan Drell of the Department of
Energy and Linda Chrissey of the Office of Naval Research for
their invaluable insights, helpful discussions, and long-term support of
Geobacter research.
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Network Approaches to the Functional
Analysis of Microbial Proteins
J.S. Hallinan1, K. James1 and A. Wipat1,2
1
School of Computing Science, Newcastle University, Newcastle, United Kingdom
2
Institute of Cell and Molecular Biosciences, Newcastle University, Newcastle, United Kingdom
ABSTRACT
Large amounts of detailed biological data have been generated over the
past few decades. Much of these data is freely available in over 1000 online
databases; an enticing, but frustrating resource for microbiologists
interested in a systems-level view of the structure and function of microbial
cells. The frustration engendered by the need to trawl manually through
hundreds of databases in order to accumulate information about a gene,
protein, pathway, or organism of interest can be alleviated by the use of
computational data integration to generated network views of the system
of interest. Biological networks can be constructed from a single type of
data, such as protein–protein binding information, or from data generated
by multiple experimental approaches. In an integrated network, nodes
usually represent genes or gene products, while edges represent some form
of interaction between the nodes. Edges between nodes may be weighted
to represent the probability that the edge exists in vivo. Networks may also
be enriched with ontological annotations, facilitating both visual browsing
and computational analysis via web service interfaces. In this review, we
describe the construction, analysis of both single-data source and
integrated networks, and their application to the inference of protein
function in microbes.

DOI: 10.1016/B978-0-12-387661-4.00005-7
102 J.S. HALLINAN ET AL.
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2. Network Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2.1. Metabolic and Regulatory Networks . . . . . . . . . . . . . . . . . . . . . . . . . 104
2.2. Protein–Protein Interaction Networks . . . . . . . . . . . . . . . . . . . . . . . . 108
3. Functional Interaction Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.1. Resources for Network Construction and Integration . . . . . . . . . . . 119
4. Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
5. Using Networks for Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 120
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
1. INTRODUCTION
In the 1970s, a rule of thumb in microbiology was “one gene, one PhD”
(Murray, 2004). Determining the sequence of a gene and the structure,
function, and interactions of the protein or proteins it produced took years
of dedicated work by a skilled researcher. In the twenty-first century,
high-throughput (HTP) technologies produce gigabytes of sequence
data every year; corresponding advances in computational power and
algorithm development mean that the prediction of the function of most
of these proteins is done computationally, at least in the first instance.
The existence of more than 1000 freely available databases of biological
information on the web (Galperin and Cochrane, 2011) facilitates the
inference of protein function by the integration of information from a
range of sources.
Biological interaction data are often represented as networks, in which
nodes represent genes or gene products and edges represent one or more
types of interaction between pairs of nodes. Networks are easy to browse
visually, and this representation facilitates computational analysis using
techniques derived from graph theory. One approach, of particular value
for protein functional analysis, is the production and analysis of integrated
functional networks which combine multiple biological networks derived
from different experimental approaches, to give an overview of putative
functional relationships between proteins or genes.
Manually locating and downloading the relevant information for net-
work integration can take many hours for an individual researcher.
Automated data integration, whereby data is located, downloaded and
combined automatically by a computer, has become increasingly important
to systems biology over the past decade.
FUNCTIONAL ANALYSIS OF MICROBIAL PROTEINS 103
2. NETWORK BIOLOGY
Biologists have traditionally thought of gene or protein interactions in the

context of pathways: essentially linear sets of reactions, each with a com-
mon biological function or process. Although the existence of cross talk
between pathways has long been recognized, and, indeed, appears to play
a crucial role in cellular function and adaptation (Hazelbauer et al., 1989),
the pathway concept underlies much research in molecular biology.
However, a glance at a visual representation of, for example, a pathway
in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database
reveals a complex network of interactions, both within the pathway and
between different pathways (Fig. 1).
As experimental and computational technology improves, such network
diagrams can be constructed for increasingly large numbers of genes, up to
and including entire genomes. Genome-wide networks span a spectrum
Figure 1 The methane metabolism pathway in E. coli K-12 MG1655 as

represented in the KEGG database.
Table 1 Examples of genome-scale networks with different interaction types and

from different data sources.
Single data source Multiple data sources
Single Protein–protein interaction Protein–protein interaction

interaction network derived from Y2H data network derived from
type (Ito et al., 2001) orthology, gene fusion, etc.
(Marcotte et al., 1999)
Multiple Network downloaded from an Probabilistic functional
interaction integrative online database, for integrated network (PFIN)
types example, BioGRID (Hallinan (James et al., 2009;
et al., 2009) Lee et al., 2004)
from those derived from a single source, using a single type of data, to
those incorporating data generated by multiple technologies and
representing several different types of interaction (Table 1).
HTP technologies were first developed in the late 1980s, when the
development of yeast-two-hybrid screens (Fields and Song, 1989) and
DNA microarrays (Schena et al., 1995) introduced researchers to the con-
cept of genome-wide analysis of biological data. Network analysis was
instrumental in the exploration of these early datasets.
Much of the network analysis performed to date has involved networks
representing a single type of interaction, such as metabolic, protein-
protein, or genetic regulatory networks. Of these, large-scale metabolic
networks were the first to be developed.
2.1. Metabolic and Regulatory Networks
The reconstruction of metabolic networks is a topic of considerable interest

to the biotechnology industry. Microbes have been used for the industrial
production of enzymes and other small biomolecules since 1917 (Weizmann
and Rosenfeld, 1937). The existence of accurate dynamic models of micro-
bial metabolism opens the way to the optimization, by genetic engineering,
of metabolic networks for a particular purpose (Liu et al., 2010).
Techniques such as flux balance analysis (Varma and Palsson, 1994;
Kauffman et al., 2003), in conjunction with detailed domain knowledge,
allow researchers to explore ways to optimize the production, degradation
or other manipulation of molecules, prior to expensive laboratory
experiments. In order to genetically engineer metabolism in this way, the

regulation of gene expression must be altered at the transcriptional level.
Transcription factor-binding networks, gene regulatory networks, and the
coexpression networks derived from microarray data represent different,
complementary views of metabolism.
Prior to the advent of HTP technologies, metabolic networks were
constructed using data gleaned from the literature. Some of the earliest
metabolic reconstruction were for Clostridium acetobutylicum (Papoutsakis,
1984), Bacillus subtilis (Papoutsakis and Meyer, 1985a,b), and Escherichia
coli (Papoutsakis and Meyer, 1985a,b; Majewski and Domach, 1990; Varma
et al., 1993a,b). Today whole-genome scale metabolic networks can be
reconstructed using genomic, proteomic, and phenotypic data generated by
a wide range of technologies. For an excellent review of the techniques of
metabolic reconstruction see Feist et al. (2009).
Genetic regulatory networks have been the subject of considerable
interest, and have generated reams of computational models, starting with
the Random Boolean Networks of Kauffman in the 1970s (Kauffman,
1974). These networks are extremely abstract collections of nodes and
edges, in which nodes represent genes and edges genetic regulatory
interactions. Genes are either “on” or “off,” and interactions may be
inhibitory or excitatory. The network wiring is random, and genes have a
randomly assigned lookup table with which to update their state at each
time step. The update rules depend upon the states of a node's interactors
and the nature of the interactions. Surprisingly, these networks can exhibit
remarkably ordered behavior despite their random organization. Gene
regulatory networks, and related computational models, have been inten-
sively studied (Thieffry and Thomas, 1998; Chen et al., 1999; Garg et al.,
2009), and can provide unique insights into the behavior large, complex
systems of interactions. However, they are but are of limited application
to real biological systems.
With the advent of more powerful computers, large online databases,
and sophisticated search algorithms it has become possible to screen whole
genomes for transcription factor-binding sites. Consequently, genetic regu-
latory networks for real microbes can be built, based upon real rather than
theoretical data (Kim et al., 2009).
Microarrays were perhaps the first truly HTP technology. For the first
time, researchers could view a snapshot of the expression levels of all of
the genes in an entire genome at a given point in time (Spellman et al.,
1998). It was immediately apparent that time series microarray data might
be used to infer regulatory relationships between genes and proteins, and
considerable research has been done in this area in the past decade, based
upon the large amounts of freely available data online: the NCBI GEO1
database alone stores HTP gene expression data for over 500 organisms
in a standardized format (Edgar et al., 2002).
The assumption underlying much of this work is that genes which are
expressed in a similar manner are probably coregulated, or at least are part
of the same metabolic pathway. Potentially, an observed increase in the
levels of one RNA species, reliably followed by an increase in the levels
of a second RNA, might indicate a regulatory relationship between the
genes and proteins involved. However, microarray data is noisy, and detec-
tion of these relationships depends upon the availability of sufficiently
fine-grained time series data (Altman and Raychaudhuri, 2001).
Microarrays produce large amounts of coexpression data. A single HTP
experiment can generate gigabytes of data which must be stored, pre-
processed, and analyzed with algorithms sophisticated enough to handle
the inherently noisy nature of the data. In order to reduce computational
load when inferring network structures, expression patterns of different
genes are often clustered, and groups of genes treated as one. This simpli-
fication is widely used in all aspects of microarray analysis but has been
questioned (Allocco et al., 2004; Yeung et al., 2004). It is becoming increas-
ingly apparent that nontranscriptional factors such as posttranslational
modifications, and the formation of competing protein complexes must
be considered in the interpretation of such data. There is also growing evi-
dence that the relationship between RNA levels and protein levels, often
assumed to be linear in these studies, is considerably more complex (Qian
et al., 2001).
Another problem with the inference of transcriptional networks from
microarray data is the tacit assumption that a network topology which pro-
duces the correct output must be the topology of the target network, an
assumption which is almost certainly invalid. For example, Morishita et al.
(2003) investigated this issue. For a 5-node network, these workers found
207 different network structures which produced a given target output. Larger
networks have even more potential, biologically plausible, topologies.
These simplifications render doubtful the biological plausibility of regu-
latory networks derived from microarray data in isolation. However, gene
expression data can be layered over networks created using other data, for
visualization and to interpret these data in the context of metabolic and
regulatory pathways (Raghunathan et al., 2009). It is possible that the
advent of next-generation RNA sequencing approaches such as RNA-Seq,
1
http://www.ncbi.nlm.nih.gov/geo/
which use deep sequencing technologies to provide a far more precise

measurement of levels of transcripts and their isoforms than has previously
been possible (Wang et al., 2009), may overcome some of these issues.
Whole-genome metabolic and regulatory networks have been devel-
oped for a range of microbes, including Salmonella typhimurium
(Raghunathan et al., 2009), Escherichia coli (Feist et al., 2007), Clostridium
acetobutylicum (Lee et al., 2008), and the microbial eukaryote Saccharomyces
cerevisiae (Förster et al., 2003). Analysis of these networks has been used for a
number of applications, including prediction of gene function (Veeramani and
Bader, 2010), identification of virulence genes in Salmonella typhimurium
(Raghunathan et al., 2009), prioritization of candidate genes to complete
metabolic pathways (Kharchenko et al., 2004), prediction of phenotypes
(Famili et al., 2003), identification of bottleneck reactions within the cell
(Herrgårrd et al., 2006), and to aid in the design of metabolic engineering
schemas (Park et al., 2007).
The generation of metabolic and regulatory networks for multiple
species permits cross-species comparisons, which can provide valuable
insights into evolutionary processes (Pinter et al., 2005). For example,
Kreimer et al. (2008) compared the modular organization of more than
300 bacterial metabolic networks, and concluded that modularity is
affected by multiple factors including network size and environmental
factors. By reconstructing ancestral metabolic networks they concluded
that network modularity has increased over evolutionary time, probably
due to increasing niche specialization. In a separate metabolic comparison
study, Jeong et al. (2000) compared 43 metabolic networks covering all
three domains of life. Despite wide variation in the constituents of the
networks, the organization was fundamentally the same across all the
species, suggesting a “common blueprint” for metabolism.
Metabolic and regulatory networks do not have to be static snapshots.
Network models may be constructed in such way that they are simulatable,
usually using systems of ordinary differential equations (Wilkinson, 2009).
Such models allow hypotheses to be tested quickly and cheaply in silico
before the genetic circuits involved are implemented in vivo. For example,
in silico gene knockout experiments can be used to identify the phenotypic
effect of the removal of a single gene, or a combination of genes, much
more rapidly than would otherwise be possible. The major obstacle to
the use of such models is acquiring appropriate parameters, such as rate
constants, binding rates, and decay rates. An iterative approach is often
needed, with the model being used to generate hypotheses which are
tested in vivo, and the results of laboratory experimentation fed back to
improve the model (e.g., Raghunathan et al., 2009).
2.2. Protein–Protein Interaction Networks
Perhaps the most intensively studied networks are those representing phys-
ical protein–protein interactions (PPIs). The detection of physical binding
between proteins is the basis of many biological network analyses
(Eisenberg et al., 2000; Xia et al., 2004). PPIs can be either binary or can
represent protein complex interactions (Franzosa et al., 2009). Binary
interactions occur when pairs of proteins have one-to-one physical contact
(De Las Rivas and Fontanillo, 2010). In protein complex interactions, sev-
eral proteins are associated as members of the same complex. There may
or may not be a direct physical interaction between any pair of proteins
within the complex. The physical interactions that occur in a complex
may be indirect, mediated by other members of the complex, and thus
may not occur in a binary fashion.
Several experimental technologies, differing in their methodology and
interpretation, have been developed to detect binary and protein complex
interactions (Shoemaker and Panchenko, 2007; Lalonde et al., 2008;
De Las Rivas and Fontanillo, 2010). Initially these methods were designed
as small-scale experiments (Phizicky and Fields, 1995). Recently, however,
HTP techniques have been developed for the detection of PPIs on a
genome-wide scale (Berggard et al., 2007), allowing more sophisticated
analysis of cellular biology (Blackstock and Weir, 1999; Pandey and Mann,
2000). Two of these PPI detection methods in particular have been used to
produce genome-wide interaction networks in a number of species: yeast-
two hybrid (Y2H) for binary interactions and tandem affinity purification
(TAP) for complex detection.
Y2H screens can be low- or high-throughput (Walhout and Vidal, 2001).
Low-throughput Y2H involves examining selected pairs of proteins for the
detection of specific interactions. On a larger scale, a one-against-all
approach can be used to screen a specific protein or group of proteins
against the entire proteome (Drees et al., 2001). Finally, at the most HTP
level, Y2H can be applied in an all-against-all manner (Ito et al., 2000).
Fromont-Racine and coworkers carried out the first large-scale Y2H study
in 1997 (Fromont-Racine et al., 1997). Since then large-scale Y2H studies
have been carried out in a range of species (Table 2). In yeast, two
large-scale Y2H datasets are of have been the subject of systematic
meta-analysis (Mrowka et al., 2001; Deng et al., 2003a,b). Ito et al. (2001)
reported a comprehensive two-hybrid analysis which identified over 4500
two-hybrid interactions among approximately 3000 proteins using three
different reporter genes and multicopy plasmid constructs. Of these
Table 2 High-throughput yeast-two-hybrid studies.
Species Interactions Reference
Saccharomyces cerevisiae 167 Fromont-Racine et al. (1997)

Saccharomyces cerevisiae 875 Uetz et al. (2000)
Saccharomyces cerevisiae 848 core (4549_total) Ito et al. (2001)
Saccharomyces cerevisiae 1778 Yu et al. (2008)
Caenorhabditis elegans 4422 Li et al. (2004)
Drosophila melanogaster 20130 Giot et al. (2003)
Drosophila melanogaster 2185 Formstecher et al. (2005)
Campylobacter jejuni 11687 Parrish et al. (2007)
Helicobacter pylori 1280 Rain et al. (2001)
Plasmodium falciparum 2846 LaCount et al. (2005)
Homo sapiens 755 Colland et al. (2004)
Homo sapiens 2855 Rual et al. (2005)
Homo sapiens 2527 Stelzl et al. (2005)
Herpes virus 296 Uetz et al. (2006)
interactions they selected a core high-confidence dataset of approximately

800 interactions. In an earlier study by Uetz et al. (2000), two separate
Y2H screens were carried out using a single reporter and low-copy plasmid
construct (Uetz et al., 2000). Unexpectedly, the data of the Uetz and Ito
datasets did not overlap to a great extent (Ito et al., 2001). Two similar
large-scale Y2H studies of human proteins (Rual et al., 2005; Stelzl et al.,
2005) showed a similar lack of overlap with only six interactions in
common (Stelzl and Wanker, 2006).
Several theories have been postulated to account for the lack of overlap
between Y2H datasets (Ito et al., 2001; Futschik et al., 2007). Mutations in some
of the open reading frames may have affected the strength of protein binding in
one of the data sets, or the proteins may have misfolded due to the plasmid
construct. Differences in experimental design, such as the chosen reporter
gene or copy number of vectors, could impact the detection of interactions.
Additionally, while these studies are large-scale they are nonsaturating, in that
no single study has assessed the entire complement of potential interactions for
a species. Finally, it has been suggested that false positives may be present in
the datasets due to stochastic activation of the reporter genes. Conversely, it
has also been suggested that the low overlap is not due to false positives but
to poor sensitivity producing false negatives (Lemmens et al., 2010). It is highly
likely that the data contains both false positive and false negative interactions.
However, the lack of overlap has yet to be fully understood and, consequently,
systematic comparison of the datasets with other data types is essential for the
identification of true positive data.
Since the development of the Y2H technique, many variations have
been produced and its principles have been used to develop further PPI
detection techniques (Lemmens et al., 2010). Protein-fragment comple-
mentation assay (PCA) is an in vivo technique which uses a bait and prey
fused to two complementary reporter protein fragments, such as parts of
an enzyme or fluorescent protein, which will only assemble when in close
proximity (Michnik, 2003; Remy and Michnik, 2003; Tarassov et al.,
2008). PCA has the advantage of being carried out in the protein's natural
cellular environment, thus reducing false positives caused by interactions
between proteins that would not naturally meet in the cell. Fluorescence
resonance energy transfer and bioluminescence resonance energy transfer
are real-time Y2H variants in which the bait and prey are fused to two dif-
ferent fluorescent or bioluminescent molecules with distinct emission
factors. Interaction between the bait and the prey causes an energy transfer
that changes the signal from the cell (Xu et al., 1999; Damelin and Silver,
2000; Kaganman, 2007). Mammalian protein-–protein interaction trap is
an in vivo mammalian variation of Y2H that uses receptors, for instance,
the cytokine receptor, fused to the bait and prey (Eykerman et al., 2005;
Lievens et al., 2009).
Protein chips are also used for the detection of specific protein binding
in vitro (Zhu and Snyder, 2003). In this technique, large numbers of
proteins are immobilized by covalent bonding to a solid surface such as a
glass slide, and probed with a labeled substrate (Zhu et al., 2000). Protein
chips are produced by high-accuracy spotting robots, which can immobilize
a large number of proteins in a small space. The substrate probes can be
any type of biological molecule, including other proteins, antigens, small
molecules, drugs, and nucleic acids (Ge, 2000). Reporters such as fluores-
cent proteins are used to detect interactions. Whole-proteome chips are
now available, allowing genome-wide identification of specific binding
partners (Zhu et al., 2000, 2001).
TAP-MS and related methods can be used to identify potential protein
complexes. However, due to the nature of protein complex binding, analysis
of the data is difficult and the results can be interpreted in different ways
(Lu et al., 2010). There are two major algorithms used to identify binary
PPIs from TAP-MS data (Bader and Hohue, 2002; von Mering et al.,
2002). In the first, termed the spoke model, PPIs are inferred between the
bait protein and each of the identified preys. In the second, termed the matrix
model, pairwise PPIs are inferred between all proteins in the complex,
including the bait.
The two models represent different trade-offs between completeness

and accuracy (Bader and Hohue, 2002). The spoke model has reduced
false positives but increased false negatives, while the matrix model has
increased false positives (Hakes et al., 2007). Combined models have also
been developed for the interpretation of TAP-MS data. Some methods
vary the model chosen depending on the complex size (Hakes et al.,
2007). Others calculate probabilities for each individual interaction
(D'haeseleer and Church, 2004; Lee et al., 2007a,b). For instance,
probabilities can be calculated to downweight promiscuous proteins: those
proteins which have a larger number of in vitro interactions than are statis-
tically likely to occur in vivo (Lee et al., 2007a,b). This downweighting can
also be applied to other data types, such as Y2H datasets, to reduce false
positive results. Currently no model gives a clear picture of the true
physical interactions of the interactome, and analysis of the data in combi-
nation with other data types is necessary to accurately identify the protein
complex interactions of the interactome (Gilchrist et al., 2004). However,
all of the interactions of both the spoke and matrix models can be consid-
ered as functional linkages.
Affinity purification techniques have been used to detect complexes in a
number of species (Table 3). In yeast, three large-scale TAP-MS datasets
(Gavin et al., 2002; Ho et al., 2002; Krogan et al., 2006) have been widely
studied (Bader and Hogue, 2003; Dezso et al., 2003; Drewes and
Bouwmeester, 2003; Gagneur et al., 2006; Hart et al., 2007). Ho et al.
(2002) used a set of 725 baits to capture potential complexes. They detected
approximately 3500 interactions. Gavin et al. (2002) used a significantly
larger number of baits (1739); however, the final number of potential
complexed interactions was also approximately 3500. A later study using
approximately 4500 bait proteins used two distinct MS-based methods to
Table 3 High-throughput tandem affinity purification studies.
Species Interactions Reference
Saccharomyces cerevisiae 3400 Gavin et al. (2002)

Saccharomyces cerevisiae 3666 Ho et al. (2002)
Saccharomyces cerevisiae 7592 Gavin et al. (2006)
Saccharomyces cerevisiae 7079 Krogan et al. (2006)
Escherichia coli 5254 Butland et al. (2005)
Escherichia coli 11511 Arifuzzaman et al. (2006)
Homo sapiens 2068 Ewing et al. (2007)
Homo sapiens 2555 Hutchins et al. (2010)
increase accuracy, and integrated the results as probabilities using machine

learning, producing a final high-confidence set of 7123 interactions
(Krogan et al., 2006). However, the three TAP-MS datasets have little
overlap with one another, with known complexes and with the large-scale
Y2H datasets (von Mering et al., 2002; Goll and Uetz, 2006; Hart et al.,
2006; Krogan et al., 2006; Zhang, 2009). Therefore, in order to increase
coverage and reduce noise a separate combined dataset has been produced
by a probabilistic reanalysis of the available data (Collins et al., 2007).
In Y2H data some proteins appear to be naturally “sticky” and can
interact with a wide range of other proteins, many of which they will never
actually encounter in the cell. When assayed in vitro, such proteins
produce a large number of false positives; Sprinzak et al. (2003) found
that only around 50% of the interactions in the HTP Y2H datasets they
examined were true positives. False negatives may also be generated, for
example, when proteins such as membrane-spanning proteins do not fold
correctly under the experimental conditions, and therefore do not bind to
their usual partners.
In the pioneering work of Ito et al. (2000) the potential for combining a
set of two-protein interactions into a network was recognized. These
workers generated small integrated networks related to the spindle pole
body and vesicular transport, amongst others. Subsequently, the PPI net-
work of S. cerevisiae became a subject of intensive research and was
extended by numerous workers (Schwikowski et al., 2000; Ito et al.,
2001). PPI networks were subsequently generated for a wide range of
microorganisms (Rain et al., 2001).
HTP data can be extremely noisy, with high rates of both false positives
and false negatives (Sprinzak et al., 2003). The most reliable (although not
completely error-free) data is gleaned from the literature by experienced
curators. There are several databases of PPIs which include microbial data;
perhaps the most extensive is the microbial protein interaction database
from the J. Craig Venter Institute (Goll et al., 2008).
3. FUNCTIONAL INTERACTION NETWORKS
HTP datasets provide the raw material for large-scale in silico

investigations which are otherwise impossible, such as the genome-wide
identification of protein complexes (Gavin et al., 2002). Many HTP
datasets are generated to address a specific biological question, and much
of the data produced may not be published. Databases of partially analyzed
HTP data can be an excellent resource for reanalysis and meta-analysis.

However, such datasets can be difficult to work with. They tend to be very
large, and noisy. Both false positives and false negatives occur, and the
extent of noise and error in the datasets may be difficult to establish.
The existence of genome-scale, or near genome-scale datasets raises the
tantalizing prospect of using multiple, overlapping data sources to provide
a view over the entire set of interactions, whether physical, genetic, regu-
latory, synthetic lethal or other, which occur inside the cell of a given
organism. Such networks can provide insights which cannot be gained from
networks constructed from a single type of data; the fact that a single edge
represents several different types of interaction may have a significance
only apparent in an integrated network.
In general, nodes in an interactome graph correspond to genes or gene
products and edges represent a summary of all of the evidence for interac-
tion between them. More complex graph models can include nodes
representing other entities, such as pathways, ligands, annotations, and
publication references (Kohler et al., 2006). The simplest networks include
a connection between two nodes if there is evidence of a functional link
between them from at least one data source (Liu et al., 2008). These
networks can be used for a number of applications: for example, to detect
protein complexes (Bader and Hogue, 2003; Asthana et al., 2004; Brun
et al., 2004) to predict protein functions (Karaoz et al., 2004; Chua et al.,
2007) or to infer novel interactions that were not detected experimentally
(Gilchrist et al., 2004; Clauset et al., 2008). Edges in a network may be
weighted to represent the strength of the evidence for the edge (e.g., Lee
et al., 2004).
HTP datasets are widely distributed, existing in thousands of databases
with different file formats, degrees of curation, and accessibility. Searching
through all of the existing data for a gene of interest is a daunting task,
made even more challenging if large-scale analysis involving multiple
genes is required. Consequently, the advent of HTP technologies has been
accompanied by a surge of interest in automated data integration
algorithms and tools.
The conceptually simplest way to integrate gene or protein networks
from a range of sources is:
1. Identify data sources of interest. There are over 1300 publically available
online databases, but not all contain data for every organism. Many
databases will contain data that is not relevant to a given biological
question.
2. Download the latest release of each data set. Most databases permit bulk
download of data, although some require registration to do so. Different
databases use different file formats (flat files, XML, etc.)
3. Ensure that each dataset uses the same identifier for genes and proteins.
Each database may use a different form of identifier as its key. For
example, in B. subtilis the gene sinI has the EMBL identifier
EBBACG00000003862, the locus tag BSU24600, and the NCBI Gene
ID 938543.
4. Screen data for obvious errors: such as typographical errors and the
presence of data from another species.
5. Convert each dataset into a common format that can be read by your
analysis tool of choice. For example, XML files would have to be
converted into a tab-delimited list of interactions for input into Excel.
6. Merge the different datasets into a single file. Individual datasets can be
very large, so the combined file may exceed the memory limit of the
software used to manipulate it, leading to the risk of data corruption.
7. Import the merged dataset into an analysis tool. Errors can also be
introduced at this stage. Microsoft Excel, for example, may import
numbers as dates by default.
This process is clearly time-consuming and error-prone but has the
advantage of incorporating large amounts of data into a single repository,
where it can be browsed manually or analyzed computationally. There are
several web sites which contain data which has been imported and
integrated from a range of sources, reducing the workload involved in
steps 1–3. Such databases usually allow the user to select a subset of the
data and view it as a network in a browser window. For example, BioPixie2
contains data of several different types, including gene expression, interaction
data, HTP, and single experiments integrated using a Bayesian framework
(Myers et al., 2005). It generates subnetworks relating to query proteins using
a novel Bayesian algorithm. Similarly, STRING3 (Search Tool for the
Retrieval of Interacting Genes/Proteins) (Szklarczyk et al., 2011) contains
PPI data for over 1100 organisms and provides an interactive network viewer.
Although it only contains PPI data, users can project their own data onto a
STRING network.
Online integrated databases are valuable for exploring and visualizing
interactions between small numbers of genes, but they have a number of
drawbacks. Continued maintenance and development of the databases is
2
http://pixie.princeton.edu/pixie/
3
http://string-db.org/
dependent upon the funding acquired by their developers, and cannot be

guaranteed. Useful databases may also be acquired by private companies,
and access to the data forbidden or charged for. Some are only free for
academic use. Most of these databases concentrate on model organisms,
which are primarily eukaryotes, so data on prokaryotes is often sparse.
Given these limitations, there has been considerable interest over the past
few years in the development of automated data integration tools.
Another problem is that of computational access to the data. Running
an analytical tool such as a clustering algorithm over a genome-scale net-
work can provide new insights into the existence and organization of struc-
tural and functional modules within an interactome, leading to the
generation of hypotheses about pathways and interactions. Few of the
online databases provide computational access to the data in the form of
Web services. A Web service provides a well-defined set of commands in
a language such as Java, allowing a computer program resident on one
machine to execute commands, such as retrieving data, which are carried
out on a different computer somewhere on the Internet (Curbera et al.,
2002). Web services can be chained into workflows, in which the output
of one web service is automatically fed into another (Oinn et al., 2004).
Web services allow a complex, multistep analysis to be carried out auto-
matically and repeated as required. This type of automation of analysis is
known as the e-Science approach (Craddock et al., 2008).
One of the earliest attempts to create genome-wide functional networks
was carried out for S. cerevisiae by Marcotte et al. (1999). These workers
used a range of data types, including the function of homologous proteins,
correlated phylogenetic profiles, and patterns of domain fusion, to infer
relationships between proteins using the naïve approach. Network con-
struction was not automated, and edges were not weighted but were
designated “highest confidence,” “high confidence,” or neither, depending
upon the authors’ belief in the validity of the data source.
As well as being labor intensive, the naïve approach has the drawback of
lacking any incorporation of information about the properties of the nodes
and the types of interactions represented by edges between genes or
proteins. A single edge may be supported by several types of data, and
many tools will allow the inspection of annotations on nodes and edges.
However, these annotations are devoid of computationally available mean-
ing. The computer can access the facts that an edge exists between RAP1
and RIF2 in the S. cerevisiae network depicted in Fig. 2; that this edge has
been identified in a Y2H experiment; that the interaction data has been
deposited in the DIP database; and that it is described in the literature.
However, it requires human intelligence to recognize that this combination
Protein RIF2 (RAP1-interacting factor 2).
RIF2
DNA-binding protein RAP1 (SBF- Cocitation, 2-hybrid, DIP

E) (repressor/activator site-binding
protein) (TUF).
RAP1
Cocitation, DIP Casein kinase II subunit

beta' (CK II beta'). Casein kinase II su'bunit alpha' (EC
2.7.11.1) (CK II).
DNA repair protein RAD16 (EC 3.6.1.-) CKA2

(ATP-dependent helicase RAD16).
CKB2
CKA1
RAD16
DIP Casein kinase II subunit alpha

ABF1 (EC 2.7.11.1) (CK II alpha
subunit).
Transcription factor BAF1 (ARS-binding

factor 1) (Protein ABF1) (bidirectionally
acting factor) (SFB-B) (DNA replication
enhancer- binding protein OBF1).
Figure 2 Part of a Saccharomyces cerevisiae integrated network, indicating the

annotations on nodes and edges.
of evidence means that the interaction was generated in a HTP manner,

and probably verified in a low-throughput experiment before being publi-
shed. The edge may be highly weighted, indicating that there is a high
probability that it really exists, but reasoning over the evidence types pro-
vides more information than is inherent in the network annotations. In
contrast, the interaction between ABF1 and RAD16 is only present in
DIP, indicating that it might have been identified as part of a HTP screen
and may be less reliable.
These deficiencies can be overcome to some extent by the use of an
ontology. An ontology is an explicit specification of a conceptualization:
the objects, concepts, and other entities that are assumed to exist in some
area of interest, and the relationships that hold among them (Gruber,
1993). A set of objects, and the relationships between them, are
represented by a controlled vocabulary, which can be manipulated by a
computational reasoner to deduce information implicit, but not necessarily
explicit, in the domain of interest. The development of biomedical
ontologies has become an active research area. There is a structured hier-

archy of biomedical ontologies, centered around the Open Biological and
Biomedical Ontologies project4 (Smith et al., 2007). The incorporation of
ontological annotations into an integrated network facilitates automated
reasoning over the network. There are currently a number of research
groups working on automated integration, knowledge induction, and
model annotation for biological systems.
One such tool is Ondex5 (Kohler et al., 2006). Ondex is a tool for
integrating data from a range of databases into a single network enriched
with semantic metadata based upon an underlying ontology. Ondex
includes parsers for reading data from many of the most popular databases
and converting them into Ondex format. Nodes, interactions, and
attributes have types, which are organized in a hierarchical fashion. For
example, the node type Protein is a subtype of Molecule, which is itself a
subtype of Thing. Every Protein is therefore also a Molecule and a Thing.
Similarly, the relationship type catalyzes is a subtype of actively participates
in. The statement that p::Protein catalyzes r::Reaction implies that that
p actively participates in r. Adding this information means that the
computer stores not only data but also its meaning, providing a “semantic
representation” of the data. The amount of semantic annotation in Ondex
is sufficient to support subsequent semi-automatic and systematic computa-
tional analysis of the resulting knowledge graph. However, in contrast to
many semantic data integration exercises based on ontologies, the
ontologies underlying Ondex are not so detailed that the computational
requirements necessary for reasoning preclude the use of reasoning on
large networks.
To date, Ondex networks have been developed for model eukaryotes
such as Arabidopsis thaliana and Homo sapiens, with application in sys-
tems studies ranging from characterizing plant stress response genes to
predicting repurposing opportunities for therapeutic agents (Cockell
et al., 2010; Hassani-Pak et al., 2010). More recently, Ondex networks for
enhancing systems approaches to the study of microorganisms have
become available. Networks for studying telomere function in for
S. cerevisiae have been described by Weile and coworkers (2011) and
knowledge graphs for supporting systems biology studies of B. subtilis have
also been described (Misirli et al., 2011). Further, microbial Ondex
networks are planned for release as the system development progresses.
4
http://www.obofoundry.org/
5
http://www.ondex.org/
Adding annotation to a network can be nearly as time-consuming as

manually integrating the network itself. To this end various strategies have
been developed to draw information from remote data sources into a sin-
gle dataset which can then be used to annotate an integrated network or
network representation of a dynamic model. The Taverna scientific
workflow system6 is being used by the Ondex project both to add
annotations to existing Ondex graphs and to eventually extend or create
new networks. In addition, Web based tools such as Saint7 (Lister et al.,
2010) have been designed to mark up models such as networks with data
drawn from a range of sources (Lister et al., 2009). The user is presented
with possible annotations to the data and may select which annotations
are the most appropriate to transfer to an existing model.
Another extension to the simple integrated network is the incorporation
of edge weights. As discussed above, some interactions in a network are
more strongly supported by data than others. Including a numerical
representing of this strength of belief in the edges means that networks
can be thresholded for visual inspection, displaying only high-confidence
edges and reducing the complexity of the graph. Weighted edges can also
be used by computational algorithms, giving more credence to clusters or
paths between nodes containing many high-confidence edges. Such
networks are generally referred to as probabilistic functional integrated
networks (PFINs).
Edge weights are usually computed by comparison with a high-confidence,
manually curated Gold Standard dataset. Data from the Gene Ontology
Consortium8 (The Gene Ontology Consortium, 2000) or the KEGG9
(Kanehisa et al., 2010) are frequently used for this purpose. Each dataset is
scored against the Gold Standard to obtain a measure of its genome coverage
and the extent of true and false positives, and then the individual datasets
are integrated. The simplest form of integration is simply to sum the weights
of each dataset for each edge, but some workers use a system of scaling
the weights, to account for possible correlations between the datasets
(Lee et al., 2004).
6
http://www.taverna.org.uk/
7
http://saint-annotate.sourceforge.net/
8
http://www.geneontology.org/
9
http://www.genome.jp/kegg/
3.1. Resources for Network Construction and Integration
There are a large number of databases containing information about dif-

ferent types of interactions between genes and gene products. A useful list,
organized by type of interaction, is online at Pathguide.10 Of the 24
organisms covered, 10 are microbes, making this resource a valuable first
port of call for microbiologists. In addition the journal Nucleic Acids
Research produces an annual Database Issue and online Database Collec-
tion11 currently with links to over 1300 databases.
As mentioned above, different databases have different organizing
schemas, and import and export data using different file formats. The practi-
cal difficulties caused by this diversity have been recognized by the commu-
nity, and the Proteomics Standards Initiative (PSI) was formed to establish
community standards for data representation in proteomics. Their PSI
MI12 format is a data exchange format for PPIs. Many of the major databases
accept and output data in this format. Another important community stan-
dard is the minimum information required for reporting a molecular interac-
tion experiment (Orchard et al., 2007). These are reporting guidelines by
which researchers are encouraged to abide, in order to “produce
publications of increased clarity and usefulness to the scientific community”
and to “support the rapid, systematic capture of molecular interaction data in
public databases, thereby improving access to valuable interaction data.”
4. FUNCTIONAL ANALYSIS
A major application of network analysis is the inference of function for

unknown genes. The concept of “function” is not clear-cut for proteins.
Proteins may have many, overlapping functions at different levels of orga-
nization in the cell. We adopt the all-encompassing definition suggested by
Rost et al. (2003): “function is everything that happens to or through a
protein.”
The best-studied microbe, the baker's yeast Saccharomyces cerevisiae,
has 6607 ORFs, of which the function of 1707 (25.8%) was listed as
“uncharacterized” or “dubious” as of February 2011.13 For most
10
http://www.pathguide.org/
11
http://www.oxfordjournals.org/nar/database/a/
12
http://psidev.sourceforge.net/mi/xml/doc/user/#further-info
13
http://www.yeastgenome.org/cache/genomeSnapshot.html
microorganisms the situation is far worse; in Bacillus subtilis 168 only

35.6% of CDS have had their function experimentally demonstrated in
that strain.14 For experimentally uncharacterized genes functionality is usu-
ally inferred from homology with genes in other species, using the BLAST
tool (Altschul et al., 1990). However, every new genome sequenced yields
a significant number of novel genes; in a typical metagenomics experiment
it has been estimated that up to 90% of the DNA sequenced may remain
unannotated because of lack of homology to existing sequence (Huson
et al., 2009). Functional analysis via homology can therefore be of limited
value.
Numerous attempts have been made to determine function from
sequence ab initio. Methods used include identification of sequence motifs
associated with posttranslational modifications such as phosphorylation
(Blom et al., 1999) and prediction of functional classes, rather than
functions, from sequence (Jensen et al., 2002).
Another widely used approach is the computational prediction of PPIs, on
the assumption that proteins which physically bind perform the same functions
as part of a complex. Prediction of protein–protein binding is an active area of
research, and numerous algorithms have been developed to predict such
interactions either from sequence alone or using additional data such as gene
neighborhood information, phylogenetic profiles, or gene fusion occurrence.
Recent reviews include Plewczy nski and Ginalski (2009) and Skrabanek
et al. (2008). However, computational prediction of protein–protein binding
is still far from perfect. The most recent available report from the CASP (Crit-
ical Assessment of Protein Structure) competition describes a range of met-
hods with varying results, and only small improvements over the
performance at the previous competition (Moult et al., 2009).
5. USING NETWORKS FOR FUNCTIONAL ANALYSIS
The effectiveness of functional inference based on data of a single type is

limited, since protein function can be reflected in many types of
interactions. One experimental approach may pick up indications which
may be missed by another approach. Integrated functional networks,
which bring together data from a wide range of sources, contain a wealth
of data that may be used for inferring protein function (Marcotte et al.,
1999; Lee et al., 2004, 2007a,b; Myers et al., 2005; Costello et al., 2009).
14
http://www.cns.fr/agc/microscope/genomic/overview.php?
Network-based functional prediction is often referred to as guilt-by-

association, since annotations are transferred between pairs of genes which
are connected in the network (Aravind, 2000; Oliver, 2000; Wolfe et al.,
2005). There are many functional prediction algorithms, differing in com-
plexity and accuracy (Sharan et al., 2007). The simplest guilt-by-association
algorithm involves simply transferring to a node all of the annotations car-
ried by nodes to which it is linked. However, HTP data is noisy, as
discussed above, and this process may transfer a significant proportion of
false positive annotations, particularly for highly connected “hub” proteins.
Several workers have extended this simple approach in an attempt to
deal with the noise in the dataset. One such approach is the Majority Rule
(Schwikowski et al., 2000). In this approach the annotation that is most
highly represented in a gene's neighborhood (the set of nodes to which it
is directly connected) is transferred to the gene. A cutoff may also be
applied to the number of annotations on the neighboring genes, so that
only genes with a number of annotations greater than the cutoff are eligi-
ble for annotation transfer.
Another extension to the basic algorithm is to incorporate edge weight
information. Edge weights may be calculated to reflect the probability that
a pair of genes is related, usually using machine learning algorithms (Deng
et al., 2003a,b, 2004a,b; Jansen et al., 2003; Letovsky and Kasif, 2003;
Troyanskaya et al., 2003; Chen and Xu, 2005; Date and Stoeckert, 2006;
Nariai et al., 2007; Kao and Huang, 2010). Annotations may be transferred
between genes if the weight on the edge between them exceeds a given
cutoff (Bork et al., 1998; Aravind, 2000; Oliver, 2000). Alternatively, the
sum of the edge weights can be calculated, so that both the frequency of
an annotation among the gene's neighbors and the confidence with which
those neighbors are linked to the gene are taken into account (McDermott
et al., 2005). Finally, annotations may be transferred only from the neigh-
bor with the highest edge weight, an approach known as the Maximum
Weight rule. This approach has been shown to improve accuracy over
other guilt-by-association algorithms (Linghu et al., 2008).
The guilt-by-association algorithms discussed so far use information
from a gene's immediate neighbors—those to which it is directly linked.
These algorithms are therefore of limited value in areas of the network
containing a high proportion of poorly annotated genes. Several functional
prediction methods which take into account a wider neighborhood have
been developed. Proteins which share annotation partners have a high
probability of having a common function. Consequently, annotations can
be transferred between nodes which are connected by a path consisting
of two edges (Chua et al., 2006, 2007). A combination of level-1 and
level-2 annotation transfer has been shown to produce improved perfor-

mance over level-1 alone (Chua et al., 2007). Alternatively, the chi-squared
statistic can be used to extend the majority rule to a specified radius
around the gene of interest (Hishigaki et al., 2001).
Annotations may also be transferred across the entire network, using
methods which maximize the edge weights of functionally associated
proteins (Karaoz et al., 2003; Vasquez et al., 2003; Massjouni et al., 2006).
Global functional prediction can also be combined with local methods
using machine learning in order to optimize performance (Chen and Xu,
2004). Functional flow is a widely used graph theoretic algorithm that
simulates the flow of annotations globally through the network, from
annotated to unannotated genes, based on edge weights (Nabieva et al.,
2005). This algorithm is particularly useful in poorly annotated areas of a
network, since the propagation of annotations is not impeded by the pres-
ence of unannotated nodes in a network.
Another, widely used approach to functional inference is clustering of
the network. Clusters are sets of nodes which are more tightly linked to
each other than they are to the rest of the network. In an integrated func-
tional network, clusters represent functional modules. Nodes of unknown
function which cluster with a set of nodes sharing an annotation are likely
to have the same biological function. Literally hundreds of clustering
algorithms have been described (for a comprehensive overview, see
Hartigan, 1975), most of which can be modified to operate upon networks
if a node distance metric can be specified, and many algorithms have been
developed specifically for network analysis.
6. CONCLUSIONS
PFINs may be used for a number of applications, including detection of

protein complexes (Bader and Hogue, 2003; Asthana et al., 2004; Brun
et al., 2004), prediction of protein functions (Karaoz et al., 2004; Nabieva
et al., 2005; Hu et al., 2010, 2009), identification of evolutionary
relationships (Kelley et al., 2003), and inference of novel interactions that
were not detected experimentally (Gilchrist et al., 2004; Clauset et al.,
2008).
Probabilistic integrated functional networks are of particular value to
the functional analysis of microbial proteins because they bring together
large amounts of data which would otherwise be distributed amongst
online databases and papers into a compact format which is easily
visualized and computationally analyzed. Such large-scale data integration

permits the automated assignment of putative function to proteins which
would otherwise be largely unannotated, facilitating the development of
testable hypotheses about the functions and interactions of proteins within
the microbial cell.
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The Diversity of Microbial Responses to
Nitric Oxide and Agents of Nitrosative Stress:
Close Cousins but Not Identical Twins
Lesley A.H. Bowman1, Samantha McLean1, Robert K. Poole1
and Jon M. Fukuto2
1
Department of Molecular Biology and Biotechnology, The University of Sheffield,
Sheffield, United Kingdom
2
Department of Chemistry, Sonoma State University, Rohnert Park, California, USA
ABSTRACT
Nitric oxide and related nitrogen species (reactive nitrogen species) now
occupy a central position in contemporary medicine, physiology,
biochemistry, and microbiology. In particular, NO plays important
antimicrobial defenses in innate immunity but microbes have evolved
intricate NO-sensing and defense mechanisms that are the subjects of a
vast literature. Unfortunately, the burgeoning NO literature has not always
been accompanied by an understanding of the intricacies and complexities
of this radical and other reactive nitrogen species so that there exists
confusion and vagueness about which one or more species exert the
reported biological effects. The biological chemistry of NO and derived/
related molecules is complex, due to multiple species that can be generated
from NO in biological milieu and numerous possible reaction targets.
Moreover, the fate and disposition of NO is always a function of its
biological environment, which can vary significantly even within a single
cell. In this review, we consider newer aspects of the literature but, most
importantly, consider the underlying chemistry and draw attention to the
distinctiveness of NO and its chemical cousins, nitrosonium (NOþ),

DOI: 10.1016/B978-0-12-387661-4.00006-9
136 LESLEY A.H. BOWMAN ET AL.
nitroxyl (NO, HNO), peroxynitrite (ONOO), nitrite (NO2), and

nitrogen dioxide (NO2). All these species are reported to be generated in
biological systems from initial formation of NO (from nitrite, NO
synthases, or other sources) or its provision in biological experiments
(typically from NO gas, S-nitrosothiols, or NO donor compounds). The
major targets of NO and nitrosative damage (metal centers, thiols, and
others) are reviewed and emphasis is given to newer “-omic” methods of
unraveling the complex repercussions of NO and nitrogen oxide assaults.
Microbial defense mechanisms, many of which are critical for
pathogenicity, include the activities of hemoglobins that enzymically
detoxify NO (to nitrate) and NO reductases and repair mechanisms (e.g.,
those that reverse S-nitrosothiol formation). Microbial resistance to these
stresses is generally inducible and many diverse transcriptional regulators
are involved—some that are secondary sensors (such as Fnr) and those
that are “dedicated” (such as NorR, NsrR, NssR) in that their
physiological function appears to be detecting primarily NO and then
regulating expression of genes that encode enzymes with NO as a
substrate. Although generally harmful, evidence is accumulating that NO
may have beneficial effects, as in the case of the squid-Vibrio light-organ
symbiosis, where NO serves as a signal, antioxidant, and specificity
determinant. Progress in this area will require a thorough understanding
not only of the biology but also of the underlying chemical principles.
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3. Origins of Reactive Nitrosative Species in Biology . . . . . . . . . . . . . . . . . 140
3.1. Nitrite Reduction and Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.2. Nitrate-Derived Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3.3. NO Synthases and the Nitrosative Burst . . . . . . . . . . . . . . . . . . . . . 142
3.4. Non-NOS Sources of NO in Microbes . . . . . . . . . . . . . . . . . . . . . . . 145
3.5. The Combined Reactive Species Response . . . . . . . . . . . . . . . . . . 147
4. The Biological Chemistry of NO and Related Species . . . . . . . . . . . . . . 148
4.1. NO, Its Redox Chemistry, and NO2 . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.2. The Reaction of NO with Superoxide Anion . . . . . . . . . . . . . . . . . . . 151
4.3. Reaction with Metal Centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.4. Products of NO Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.5. The Reactions of HNO with Biological Targets . . . . . . . . . . . . . . . . 154
5. Laboratory Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
5.1. The Use of Nitrogen Oxide Donors . . . . . . . . . . . . . . . . . . . . . . . . . . 156
5.2. NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
5.3. S-Nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
5.4. Other Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
5.5. HNO Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 137
5.6. Use of ONOO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

5.7. NO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.8. NO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.9. Other Nitrogen Oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
6. Bacterial Responses to RNS: Effectors and Regulators . . . . . . . . . . . . . 166
6.1. Targets of RNS in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . 166
6.2. Microbial Defenses: The Microbe Strikes Back . . . . . . . . . . . . . . . . 167
6.3. Microbial Globins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
6.4. NO and RNS Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
6.5. Other Proteins Implicated in NO Tolerance . . . . . . . . . . . . . . . . . . . 176
6.6. Beneficial Effects of NO in Microbial Symbioses . . . . . . . . . . . . . . . 177
6.7. Microbial Responses to ONOO Stress . . . . . . . . . . . . . . . . . . . . . . 178
7. Microbial Sensing of NO and Gene Regulation . . . . . . . . . . . . . . . . . . . . 181
7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
7.2. Fnr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
7.3. NorR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
7.4. NsrR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
7.5. Others (DOS, FixL, GCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
8. Global and Systems Approaches to Understanding Responses to NO
and RNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
8.1. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
8.2. Outcomes from Global Transcriptomic Approaches . . . . . . . . . . . . 188
8.3. Responses of Other Microbes to RNS . . . . . . . . . . . . . . . . . . . . . . . 193
8.4. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
ABBREVIATIONS
CPTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-
oxyl-3-oxide
EPR electron paramagnetic resonance
FNR ferredoxin-NADPþ reductase
Fnr fumarate and nitrate reduction regulator, encoded by
fnr gene
GSH glutathione
GSNO S-nitrosoglutathione
GSSG glutathione disulfide (oxidized glutathione)
GTN glyceryltrinitrate
Hcy homocysteine
iCAT isotope-coded affinity tag
iTRAQ isobaric tags for relative and absolute quantitation
NHE normal hydrogen electrode
NOC-5 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-
triazene
NOC-7 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-
methyl-1-triazene
NOR-3 ()-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-
hexeamide
DEA/NO diethylamine NONOate
DETA diethylenetriamine NONOate
NONOate
norV, norW genes involved in nitric oxide reduction and its
regulation (norR)
NOS NO synthase
PTIO 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide
RNS reactive nitrogen species
SNAP S-nitroso-N-acetyl-D,L-penicillamine
SNO S-nitrosothiol
SNOCAP S-nitrosothiol capture
SNP sodium nitroprusside
SOD superoxide dismutase
1. OVERVIEW
Nitric oxide (NO) is a small and freely diffusible species once known pri-
marily as a toxic component of air pollution. In physiology and biochemis-
try, it was well known as a poison and ligand for heme proteins. The
discovery of the enzymic generation of NO in mammalian systems and
its cell signaling functions represents a watershed moment in the evolution
of our understanding of biological signal transduction. The importance of
NO as a molecule of real biological significance cannot, however, have
escaped the attention of any microbiologist, although the realization is rel-
atively recent. To illustrate this point, consider that a multi-authored,
edited book Microbial Gas Metabolism published in 1985 (Poole and
Dow, 1985) contained only three index entries for ‘nitric oxide’, namely,
‘denitrification’, ‘mass spectrum cracking pattern’ and ‘reaction with cyto-
chrome d.’ The general topics addressed by these specific terms illustrate
well the dominant interests of microbiologists in NO a quarter of a century
ago—NO as a possible (but far from proven) intermediate in the microbe-
catalyzed conversion of nitrate to dinitrogen, the biochemical analysis and
detection of the gas, and the use of NO as an experimental tool in the

study of heme proteins.
In the intervening years, a large literature has grown up around NO and
related species, fueled by the recognition that NO plays important antimi-
crobial defenses in innate immunity and that, in turn, and not unexpect-
edly, microbes have intricate NO-sensing and defense mechanisms.
Unfortunately, the burgeoning publication of information on NO has not
always been accompanied by an understanding of the intricacies and com-
plexities of NO and other “agents of nitrosative stress” so that there exist
some confusion and vagueness about which one or more species exert the
reported, interesting biological effects. There is, for example, a tendency
for authors to write “NO” when it is actually meant “in a generic sense.”
It should not be necessary to write “NO radical” to eliminate the possibil-
ity that one is actually meaning NOþ or some other congener. There is
only one NO.
Butler and Nicholson (2003) suggest that “there are not many small
molecules about which a whole book could be written,” although in biol-
ogy, we doubt the veracity of this. Consider the gases oxygen, carbon diox-
ide, methane, and nitrogen. Nevertheless, we agree that NO is certainly
one such species: several books could be, and have been, written on this
one gas. As a result, we are forced to be brief and focused when revisiting
some familiar aspects of NO in microbiology and apologize for any over-
sights or omissions. Our objective in this review is not only to review newer
aspects of this vast literature but, most importantly, to consider the under-
lying chemistry and draw attention to the distinctive chemistry of NO and
its chemical cousins, NOþ, NO, HNO, ONOO, NO2, and NO2.
2. HISTORICAL PERSPECTIVE
The modern era of NO research may be considered to begin in the 1980s

when NO was identified as the endothelium-derived relaxing factor
(EDRF). This remarkable story and its culmination in a Nobel Prize and
the designation of NO as “molecule of the year” by Science in 1992 are
covered well elsewhere, especially in accounts by the laureates (Furchgott,
1999; Ignarro, 1999, 2005; Murad, 1999). NO was also shown to participate
in the regulation of the nervous and immune systems, and it was soon dis-
covered that NO also plays a vital role in the resistance of mammalian hosts
to microbial infections. Activated macrophages were shown to form nitrite
and nitrate from arginine (Iyengar et al., 1987; Marletta et al., 1988)
via the formation of NO (Stuehr et al., 1989) and to have a powerful cyto-
static effect in vitro on the fungal pathogen Cryptococcus neoformans
(Granger et al., 1986). Activated macrophages also destroyed the intracellu-
lar parasite Leishmania major in vitro by an L-arginine-dependent mecha-
nism (Green et al., 1990) and mice infected with L. major developed
exacerbated disease when the lesions were injected with the NOS inhibitor
L-NMMA, providing the first compelling evidence for the attenuation by
NO of an infectious microorganism in vivo (Liew et al., 1990).
A direct role for NO against intracellular bacteria was soon established,
initially with Mycobacterium bovis (Flesch and Kaufmann, 1991). Shortly
after, we showed that NO dramatically upregulated expression of the
Escherichia coli flavohemoglobin (Poole et al., 1996), and an enzymic func-
tion in NO detoxification was demonstrated by Gardner et al. (1998). A glo-
bin mutant was NO sensitive unambiguously demonstrating a physiological
role (Membrillo-Hernández et al., 1999). In murine macrophages, NO was
shown to have a role in bacterial clearance (Shiloh et al., 1999; Vazquez-
Torres et al., 2000). Also, flavohemoglobin-catalyzed NO detoxification by
Salmonella enterica serovar Typhimurium protected the bacterium from
NO-mediated killing in human macrophages (Stevanin et al., 2002).
Flavohemoglobin (Hmp) was shown to catalyze the reaction of NO with oxy-
gen to give innocuous nitrate via a dioxygenase (Gardner et al., 1998, 2000,
2006) or denitrosylase (Hausladen et al., 1998a, 2001) mechanism, and
further gene reporter experiments showed that hmp gene transcription is
activated on exposure of bacteria to NO or nitrosating agents (Poole et al.,
1996; Poole and Hughes, 2000; Gilberthorpe et al., 2007). Mutants were used
to demonstrate unequivocally the key role of flavohemoglobin in defense
against NO not only in vitro (Membrillo-Hernández et al., 1999) but also
in vivo (Stevanin et al., 2002). Other globins intensively studied (Wu et al.,
2003) now include the two globins in each of Mycobacterium tuberculosis
(Couture et al., 1999; Pathania et al., 2002) and Campylobacter jejuni
(Lu et al., 2007a,b). These are covered further in Section 8.3.
3. ORIGINS OF REACTIVE NITROSATIVE SPECIES

IN BIOLOGY
3.1. Nitrite Reduction and Denitrification
The major source of NO in man is via the action of NOS (see Section 3.3),
but other sources should be briefly considered. Nitrite is protonated under
acidic conditions (as in the stomach) and the resulting nitrous acid will
yield NO and other nitrogen oxides; the beneficial effects of acidified
nitrite in killing ingested pathogens, gastric mucosal integrity and other
effects are discussed elsewhere (Lundberg et al., 2004). Acidified nitrite
is sometimes, but perhaps not ideally, used as a source of NO in bacterial
experiments in vitro (as covered elsewhere in this review).
The bacterial reduction of nitrite is a key reaction in anaerobic bacterial
metabolism and the subject of an immense literature (Potter et al., 2001;
Lundberg et al., 2004). There are three classes of nitrite reduction. In
the first (denitrification), reduction of nitrite to NO is catalyzed by either
copper-containing NirK or cytochrome cd1 nitrite reductase, NirS. The
periplasmic enzymes involved have been extensively characterized and
are outside the scope of this contribution (for an authoritative review,
see Potter et al., 2001). Second, when bacteria utilize nitrite as a terminal
electron acceptor, NADH- and siroheme-dependent reduction is the pri-
mary pathway, encoded in E. coli by the nirBDCcydG operon. Third,
nitrite can be reduced to ammonia by a widely distributed cytochrome c
nitrite reductase Nrf (Potter et al., 2001), which can also, however, produce
low levels of NO when nitrite is in excess under anoxic conditions (Corker
and Poole, 2003).
3.2. Nitrate-Derived Stress
Nitrate is often regarded mainly as a water pollutant and its presence in the
diet of man is seen as an unfortunate consequence of the use of nitrogen
fertilizers in agriculture. Increasingly stringent regulations to limit nitrate
intake suggest that nitrate has wholly undesirable effects including forma-
tion of N-nitrosamines, infantile methemoglobinemia, carcinogenesis, and
possibly teratogenesis (McKnight et al., 1999). However, nitrosamine
formation is via nitrite (nitrosation chemistry) not nitrate; nitrite is the cul-
prit. An alternative view is that the products of nitrate metabolism
have beneficial effects, especially in host defense. The argument is made
elsewhere (Lundberg et al., 2004) that nitrate-reducing commensal bacteria
play a symbiotic role in mammalian nitrate reduction to nitrite, NO, and
other products. Nitrate reduction to nitrite and thence to NO are the topics
of a vast literature, beyond our scope (for a review, see Lundberg, 2008).
It is clear, though, that large amounts of NO and other reactive nitrogen
species (RNS) are generated in vivo from salivary nitrite in the acidic stom-
ach (Benjamin et al., 1994) and may contribute to killing of ingested
pathogens (Lundberg et al., 2004). Depending on nitrite concentration in
the saliva (McKnight et al., 1999), the NO concentration in the stomach

headspace gas may be around 20 ppm (McKnight et al., 1997).
3.3. NO Synthases and the Nitrosative Burst
3.3.1. The NOS Family
NO synthases (NOSs) are highly regulated multidomain metalloenzymes

that catalyze the conversion of L-arginine (L-Arg) to L-citrulline and NO
with the consumption of NADPH and O2. They were first identified in
mammals, and three forms are identified: endothelial NOS (eNOS or
NOSIII), neuronal NOS (nNOS or NOSI), and inducible NOS (iNOS or
NOSII) (reviewed in Alderton et al., 2001). The nitrosative burst, triggered
by pathogenic agonists and inflammatory mediators (Lowenstein et al.,
1993), succeeds the oxidative burst and is mediated by NO production fol-
lowing activation of iNOS. The first two isoforms are constitutively
expressed and are calcium dependent, whereas iNOS is stimulated to pro-
duce NO at markedly higher levels than the constitutive isoforms following
infection (Lowenstein and Padalko, 2004). iNOS is most important in a
microbial context and is known to occur in a wide variety of stimulatable
cells such as macrophages, neutrophils, vascular smooth cells, and glial
cells in the CNS (Bogdan, 2001). On microbial infection, the NO produced
has diverse, apparently conflicting functions; on the one hand, NO exerts
antimicrobial and anti-inflammatory host defense effects and, on the other,
proinflammatory and cytotoxic activities (Fang, 2004). The host defense
function is exemplified by the effects of NO in microbial infections,
whereas NO-mediated inflammation and pathogenesis are known in cer-
tain diseases including arthritis, encephalitis, ulcerative colitis, and viral
infections.
These enzymes are homodimeric with each monomer containing an N-
terminal oxygenase domain with binding sites for heme, L-arginine, and
tetrahydrobiopterin (H4B) and a C-terminal reductase domain, which has
binding sites for FMN, FAD, and NADPH (Stuehr et al., 2009). The reduc-
tase domain transfers electrons sourced from NADPH via flavin carriers to
heme in the oxygenase domain. This drives the oxidation of L-arginine in
the presence of O2 to NO, citrulline, and NADPþ (Daff, 2010). Electron
flow is strictly between the reductase domain of one monomer and the
oxygenase domain of the other (Siddhanta et al., 1996).
As iNOS is confined to the cytoplasm, NO must diffuse to the
phagosome to react with internalized microorganisms. Unlike O2
generation, the onset of NO production is late in the defensive process,

starting around 8 h postinfection as demonstrated in murine macrophages
challenged with S. enterica (Eriksson et al., 2003). NO makes a significant
contribution to the host’s innate immune response, exemplified by a recent
study revealing that macrophages derived from murine bone marrow gen-
erate an approximately 2-log reduction in C. jejuni viability relative to
iNOS mutants over a 24-h period (Iovine et al., 2008).
In a recent example of the intricacies of iNOS function, a newborn
mouse model (Mittal et al., 2010) of meningitis revealed that infection with
E. coli K1 resulted in iNOS expression in the brain. iNOS/ mice, how-
ever, were resistant to infection and showed normal brain morphology
and a reduced inflammatory response. An iNOS inhibitor (ami-
noguanidine) also prevented meningitis and brain damage, and further,
peritoneal macrophages and polymorphonuclear leukocytes from such
mutant mice showed enhanced killing of bacteria. These data suggest that
NO from iNOS is actually beneficial for E. coli K1 survival in the macro-
phage and suggests a therapy for neonatal meningitis.
3.3.2. Bacterial NOS
NOS enzymes are now also recognized in several bacteria (Table 1) and,
more controversially, plants (for a review, see Wilson et al., 2008). The pro-
karyotic NOS enzymes (Crane et al., 2010) are in many ways similar to
their mammalian counterparts, catalyzing the conversion of L-Arg to NO
via the intermediate No-hydroxy-L-arginine (NOHA), but the role(s) of
the NO so formed is far less clear.
In 1994, the first report of a bacterial NOS-like activity was published
(reviewed in Crane et al., 2010). However, the first definitive evidence
for NOS-like proteins came from genome mining just over 10 years ago,
revealing bacterial ORFs with high sequence similarity to mammalian
NOS. Most recent data suggest that bacterial and mammalian NOS
enzymes have similar reactivities with almost identical catalytic active sites.
This has greatly facilitated research into the core features of all NOS
proteins because many bacterial NOS are readily expressed in E. coli
and provide protein for crystallographic, various spectroscopic, and kinetic
studies.
The main differences between NOS of mammals and bacteria reside in
cofactor specificity and the nature of the reductase partners for these
enzymes (Crane et al., 2010). Not all bacterial NOS contain the pterin
cofactor (tetrahydrobiopterin, H4B) associated with mammalian NOS.
Table 1 Bacterial NO synthasesa.
Biological function Reductase Comments Referenceb
Nocardia species First report of bacterial Chen and Rosazza

NOS, but no obvious NOS (1994, 1995)
gene homologue
Lactobacillus No obvious NOS gene Morita et al. (1997)
fermentum homologue
Salmonella No obvious NOS gene Choi et al. (2000)
enterica homologue
Typhimurium
Staphylococcus NO or NOS protect cells from NOS homologue Hong et al. (2003);
aureus H2O2 Gusarov and Nudler
(2005)
Deinococcus 1. Reaction with tryptophan Active with surrogate Adak et al. (2002b);
radiodurans tRNA synthetase II to form mammalian reductase Buddha et al. (2004);
4-nitro-Trp-tRNATrp, involved Patel et al. (2009)
in protein or secondary
metabolite synthesis?
2. Response to UV radiation
exposure, with NO
upregulating transcription of
growth factor?
Bacillus subtilis, 1. NO or NOS protects cells Dedicated reductase not First crystal structure of a Gusarov and Nudler
Bacillus anthracis from H2O2 required (?) bacterial NOS (2005); Shatalin et al.
2. Protection against (2008); Gusarov et al.
antibiotics (see text) (2009)
Streptomyces NOS encoded on No obvious reductase Kers et al. (2004)
species, S. pathogenicity island associated partner proteins
turgidiscabies with potato scab disease. NOS encoded by NOS
involved in thaxtomin (toxin) pathogenicity island
production (see text)
Sorangium Covalently bound Agapie et al. (2009)
cellulosum unique reductase
a
NOSs have also been reported in eukaryotic microbes, notably the protozoa Entamoeba histolytica and Toxoplasma gondii, but neither genome contains
an NOS homologue (Crane et al., 2010).
b
For further details, see Crane et al. (2010).
In the latter enzymes, H4B delivers electrons to the active site for oxygen
activation in two steps. In the first, oxygen bound to heme is activated by
a single electron transfer, and in the second (final) stage of the reaction,
NO is liberated. The mechanism of action of the mammalian enzymes is
beyond our scope but well covered in reviews (Daff, 2010). It is worth not-
ing, however, that a common feature of bacterial NOS is that the NO dis-
sociation rate is 15- to 25-fold slower than in the mammalian enzyme
(Adak et al., 2002a; Wang et al., 2004). This favors oxidation of NO to
more reactive species raising the intriguing possibility that synthesis of
these alternative species (HNO, NO) is the true biological function, as
discussed below. Nonetheless, NO does appear to be the major product
in at least three cases (Johnson et al., 2008; Shatalin et al., 2008; Patel
et al., 2009).
A case of special interest in the context of the present review is that of
Streptomyces turgidiscabies, where the NOS is encoded on a pathogenicity
island associated with potato scab disease (Crane et al., 2010). NOS has
been shown by mutation studies to be involved in production of thaxtomin
(a plant toxin), and it is thought that NOS might be involved in a biosyn-
thetic nitration reaction (Johnson et al., 2008). Indeed, a feeding study with
15
N-Arg showed that the thaxtomin nitro group nitrogen does originate
from the terminal guanidinium nitrogen of Arg, strongly implicating an
NOS activity. Importantly, however, NO will not itself react directly to
nitrate substrates such as the tryptophanyl moiety of thaxtomin, whereas
oxidation products of NO (NOþ, NO2þ, ONOO, NO2; see Section 4)
are known to nitrate aromatic groups (Hughes, 2008).
What are the potential targets of endogenously generated NO and
related reactive species in bacteria? The examples in Table 2 include tran-
scription factors, biosynthetic enzymes, metalloproteins, and kinases. How-
ever, the true biological roles of NOS-derived NO in microbes remain
elusive. The fact that NOS enzymes are restricted to certain species and
genera suggests a complexity that we do not yet understand.
3.4. Non-NOS Sources of NO in Microbes
Classically, NO has been regarded as a mammalian signaling molecule or a

component of the host’s innate immune response to infection. However, a
number of papers have demonstrated that bacteria also have the capacity
to synthesize NO. This ability was first demonstrated by Hollocher and
others: bacteria grown anaerobically with nitrate produced NO from nitrite
and the NO was detected by nitrosation of 2,3-diaminophthalene.
Table 2 Bacterial targets of NO and nitrosative stress.
Target Basis Examples Selected examples
Heme cofactors Iron nitrosylation Hemes of cytochromes, globins Fu et al. (2009); Pixton et al.
(2009)
Diverse S-nitrosation Binding of NO moiety 5S-nitrosylated proteins in Qu et al. (2011)
events to Cys residue to give Helicobacter pylori
S-nitrosothiol
10S-nitrosylated proteins in E. coli Brandes et al. (2007)
29S-nitrosylated proteins in Rhee et al. (2005)
M. tuberculosis
Unidentified S-nitrosation events in Wang et al. (2011)
Moraxella catarrhalis
Free and Zn-bound Cys thiols in Bourret et al. (2011)
Borrelia burgdorferi
Transcription factors Heme binding DosS/DosT Kumar et al. (2007)
and sensor kinases
Shewanella oneidensis H-NOX- Price et al. (2007)
histidine kinase pair
SNO formation E. coli OxyR Hausladen et al. (1996)
Fe–S cluster reaction E. coli SoxR, Fnr Ding and Demple (2000);
with NO Cruz-Ramos et al. (2002);
Landry et al. (2010); Smith
et al. (2010), for an
overview, see Tonzetich
et al. (2010)
Actin Actin nitrosation GSNO nitrosates key proteins Flamant et al. (2011)
involved in S. flexneri invasion,
perhaps actin and GTPase
Outer membrane SNO formation Bacillus subtilis Morris et al. (1984)
proteins
The responsible enzymes have been identified as nitrite reductase

(Corker and Poole, 2003) or nitrate reductase (Ji and Hollocher, 1989;
Gilberthorpe and Poole, 2008). There are parallels with the recently
described ability of mammalian neuroglobin to generate NO from nitrite,
which is suggested to be a primordial hypoxia- and redox-regulated source
of NO for physiological functions (Tiso et al., 2011). The generation of NO
by Salmonella has been demonstrated not only in vitro but also inside
infected cancer cells and in implanted tumors in live mice using mic-
rosensors. In strains mutated in hmp and norV, the bacteria generated
more NO and killed cancer cells more effectively in a nitrate-dependent
manner (Barak et al., 2010).
3.5. The Combined Reactive Species Response
ROS and RNS act in concert, exemplified by murine macrophages doubly

immunodeficient in iNOS and NAPDH oxidase (Phox) that were
completely unable to quash S. enterica growth (Vazquez-Torres et al.,
2000). In other words, the roles of iNOS and Phox are nonredundant
(Nathan and Shiloh, 2000). An earlier study also clearly demonstrated syn-
ergistic killing of E. coli by the combination of NO and H2O2 (Pacelli
et al., 1995). Additionally, NO and O2 can rapidly react to form the highly
toxic species peroxynitrite (ONOO-) (Reaction 5), which will be discussed in
Section 4.2. On account of diffusion constraints, particularly the negative
charge on O2, ONOO generation tends to colocalize with NADPH oxi-
dase. Peroxynitrite is often stated to be the principal nitrating agent for tyro-
sine residues in proteins, yet the yield in vitro of nitrated tyrosine at pH 7.4
is less when O2 and NO are cogenerated than when a bolus of ONOO is
given. Goldstein et al. (2000) demonstrated that maximal nitrosation of tyro-
sine occurred when NO and O2 fluxes were equal. Although ONOO is
formed in vivo from these two radicals, and the temporal concurrence of
these precursors, as is found at inflammatory sites, favors ONOO forma-
tion in vivo, ONOO itself is not likely to be the nitrating species. It is gen-
erally thought that decomposition of the conjugate acid, peroxynitrous acid
(ONOOH), gives the two one-electron oxidants nitrogen dioxide (NO2)
and hydroxyl radical (HO), either of which is capable of oxidizing tyrosine
to the tyrosyl radical. Subsequent addition of NO2 to the tyrosyl radical then
gives nitrotyrosine (e.g., Gunaydin and Houk, 2009). Thus, this mechanism
of tyrosine nitration is highly dependent on the presence of NO2, and since
NO2 can be formed from peroxynitrite-independent pathways, tyrosine
nitration is not necessarily a marker for ONOO formation.
4. THE BIOLOGICAL CHEMISTRY OF NO AND

RELATED SPECIES
4.1. NO, Its Redox Chemistry, and NO2
The biological chemistry of NO and derived/related molecules is poten-

tially complex due to a multitude of species that can be generated from
NO in a biological milieu and the multiple possible reaction targets
associated with these derived species (for an overview, see Lehnert and
Scheidt, 2010). Moreover, the fate and disposition of NO is always a func-
tion of its biochemical environment, which can vary significantly even
within a single cell. The redox relationship between NO and related/
derived nitrogen oxides is given in Fig. 1 and all of the species shown have
been reported to be generated in biological systems from initial formation
of NO. Herein will be described the chemical properties of NO and
derived agents important to their biological functions and effects.
The biological utility of NO is based on its unique chemistry. First and
foremost, NO has an unpaired electron and is, therefore, considered to
be a free radical (for the sake of simplicity, we have adopted the definition
of Halliwell and Gutteridge (2007)) for the term “free radical” which is any
species that can exist independently (i.e., free) and contains one or more
unpaired electron. The presence of an unpaired electron is immediately
evident from the Lewis dot depiction for NO (Fig. 2). However, the
unpaired electron is not associated solely with the nitrogen atom of NO
(as indicated by the Lewis structure), but rather is delocalized throughout
+e− +e− +e− +e− +2e−

NO3− + 2H+ NO+ NO HNO H2NO NH2OH NH3(+ H2O)
−e− −e− −e− −e− −2e−
−e− +e−
−H2O H2O
H2O
−
+e
NO2 NO2− + 2H+
−e−
Figure 1 Redox relationship between biologically relevant nitrogen oxides.

Note: protons omitted from several processes for the sake of simplification.
N + O N O
Figure 2 Lewis dot depiction of NO.

the molecule in a p* antibonding orbital as indicated by the molecular

orbital diagram (Fig. 3).
As discussed below, the free radical nature of NO as well as the orbital
location on the unpaired electron are important factors in the biological
chemistry of NO. Although the term free radical is often associated with
extreme reactivity (e.g., strong oxidant), this is not the case with NO.
The lack of oxidizing capability of NO is evidenced by its relatively low
reduction potential (E ¼ 0.55 V vs. NHE for the NO,Hþ/HNO couple;
Bartberger et al., 2002; Shafirovich and Lymar, 2002). For comparison,
the hydroxyl radical (HO, a prototypical strong one-electron oxidant)
has a reduction potential of 2.3 V versus NHE for the HO,Hþ/H2O couple
at pH 7 (Sawyer, 1991). Consistent with the idea that NO is a poor
one-electron oxidant, NO is also poor at H-atom abstraction since the
bond formed from this reaction (the HNO bond) is weak, approximately
47 kcal/mol (Dixon, 1996). Again, for comparison, when HO abstracts a
hydrogen atom, the strength of the bond made (HOH) is very strong,
119 kcal/mol. Thus, NO is generally difficult to reduce and, therefore, poor
at initiating radical chemistry via oxidation pathways. However, NO will
σ∗
π∗
2p
2p
σ∗
2s
2s
σ
N O
Figure 3 Molecular orbital diagram for NO. Schematic depiction of molecular

orbitals shown.
rapidly react with existing radicals. As such, NO has been found to be a

potent antioxidant capable of quenching otherwise deleterious/oxidizing
radical chemistry (Rubbo et al., 1995). NO also reacts with dioxygen
(which has two unpaired electrons and so, according to the definition
above, is considered a free radical or, in this case, a diradical). The reaction
of NO with O2 results in the generation of nitrogen dioxide (NO2)
(Reaction 1).
2NO þ O2 ! 2NO2 ð1Þ

Like NO, NO2 is a free radical species. However, unlike NO, NO2 is
a fairly strong oxidant, as indicated by a reduction potential of 1.04
(vs. NHE) for the NO2/NO2 couple (Stanbury, 1989). Therefore, reaction
of NO with O2 takes two relatively weak oxidants (the reduction potential
for the O2/O2 couple is 0.33 V (Sawyer, 1991)) and forms a reasonable
one-electron oxidant. NO2 is known to oxidize a variety of biologically rel-
evant functional groups such as cysteine thiols, tyrosines, and polyunsatu-
rated fatty acids, just to name a few. In the absence of NO2-reactive
species (i.e., reductants), the fate of NO2 generated via the reaction of
NO with O2 is to react with another NO (since they are both radicals) to
give dinitrogen trioxide (N2O3) (Reaction 2).
NO2 þ NO (
+ N2 O3 ð2Þ
N2O3 (which is not a radical) is electrophilic and in aqueous systems will
react with H2O to give two equivalents of nitrite (NO2) (Reaction 3).
If other nucleophiles are present (e.g., thiols, amines), in a reaction analo-
gous to the reaction of H2O, these nucleophiles can be nitrosated by N2O3
(Reaction 4).
N2 O3 þ H2 O (
+ 2NO2 þ 2Hþ ð3Þ
N2 O3 þ Nuc ! Nuc NO þ NO2 ð4Þ
Thus, the combination of Reactions 1, 2, and 4 can result in the

NO-mediated nitrosation of biological nucleophiles. For example,
S-nitrosothiols (SNOs) can be generated from thiols via this chemistry.
S-nitrosation and S-nitrosylation are terms often used interchangeably in
the biological literature, yet it must be stressed that they have distinct
meanings. S-nitrosation is a mechanism whereby NOþ-like species mediate
an attack upon thiol side groups, whereas S-nitrosylation is a mechanistically
ambiguous term for any process that results in the generation of an SNO.
Thus, the term nitrosyl does not describe a mechanism but instead refers to
NO bound to a metal, for example. It needs to be noted that the biological
accessibility of this nitrosation chemistry is likely to be rare or, at the very
least, very restricted due to the reaction kinetics. As shown in Reaction 1,
two equivalents of NO are required for the O2-dependent generation of
NO2. Thus, the kinetics of this reaction has a second-order dependence on
NO (Ford et al., 1993). This second-order dependence indicates that this pro-
cess is significant only at high concentrations of NO. Since both NO and O2
favorably partition into membranes where concentrations of both are likely
to be significantly higher than in the aqueous compartments of cells, it has
been proposed that nitrosation chemistry of the type described above may
have special relevance in lipophilic/membrane environments (Liu et al.,
1998). As an example of the effect that the second-order dependence on
NO can have, for purely aqueous, aerobic (assuming 200 mM O2) solutions
of NO, a 10 mM solution will degrade to one half its original concentration
in approximately 1 minute, whereas a 10 nM solution of NO will degrade
to half its concentration in over 70 h (note that the term “half-life” was not
used since this term is only applicable to first-order processes).
4.2. The Reaction of NO with Superoxide Anion
One of the most highly studied reactions of NO is with the reduced

dioxygen species superoxide (O2), generating, initially, peroxynitrite
(ONOO) (Reaction 5).
NO þ O2 ! ONOO ð5Þ
There are numerous excellent reviews on the chemistry, biology, and
(patho)physiology of this reaction and ONOO (see, e.g., Pryor and
Squadrito, 1995; Beckman and Koppenol, 1996; Ferrer-Sueta and Radi,
2009). This reaction readily occurs via a near diffusion-controlled process.
The pKa of peroxynitrous acid (ONOOH) is 6.8, indicating that, at pH 7,
the anion is the predominant species. In pure aqueous solution at physio-
logical pH, ONOO will eventually decompose to nitrate (NO3). This
rearrangement occurs presumably via ONOOH and involves the genera-
tion of oxidizing intermediates (e.g., Gunaydin and Houk, 2008). Indeed,
ONOO/ONOOH is capable of performing oxidation chemistry on, for
example, thiols (i.e., cysteine), phenols (i.e., tyrosine), and other biologi-
cally relevant reducing species. A primary fate of ONOO in many
biological systems is reaction with carbon dioxide (CO2) giving, initially,
nitrosoperoxycarbonate (ONOOCOO) (Reaction 6). Rearrangement of
this species generates nitrocarbonate (Reaction 7), which then hydrolyzes

to give NO3 and carbonate (CO32).
ONOO þ CO2 ! ONOO CðOÞO ð6Þ
ONOO CðOÞO ! O2 NO CO2 ð7Þ

As with ONOOH decomposition, the rearrangement of nitrosoperoxy-
carbonate to nitrocarbonate involves reactive intermediates that are also
capable of oxidizing a number of biological molecules (e.g., Gunaydin
and Houk, 2009).
4.3. Reaction with Metal Centers
In a reaction that is analogous to its reaction with O2, NO also reacts with
dioxygen-bound metal species such as oxyhemoglobin or oxymyoglobin to
form NO3. In both oxymyoglobin and oxyhemoglobin, the bound
dioxygen has significant O2 character due to extensive donation of
electrons from the metal to the bound O2. Thus, reaction of NO with
the heme-bound O2 is analogous to the reaction of NO with “free”
O2 (Reactions 8 and 9), although recent studies indicate no intermediacy of
ONOO on the millisecond time scale in this chemistry (Yukl et al., 2009).

MbFeII þ O2 ! MbFeII O2 $ MbFeIII O2 ð8Þ

MbFeII O2 $ MbFeIII O2 þ NO ! MbFeIII þ NO3 ð9Þ
Along with its reaction with dioxygen and dioxygen-derived species, NO
also binds metals. Most notable in biological systems is the reaction of NO
with hemeproteins (e.g., Ford, 2010), although NO can bind other nonheme
metalloproteins as well. Unlike O2 and CO, which will only bind to ferrous
(Fe2þ) hemes, NO is capable of binding both ferric (Fe3þ) and ferrous
hemeproteins (provided there is an open or exchangeable coordination site).
The reaction with ferrous hemes results in a species that favors a 5-coordinate,
square pyramidal geometry. Indeed, a major site of action of NO in mamma-
lian systems is the hemeprotein soluble guanylate cyclase (sGC) which binds
NO via its ferrous heme leading to the formation of a 5-coordinate ferrous
nitrosyl that is presumably responsible for enzyme activation (although the
process may be more complicated) (Poulos, 2006). Significantly, when O2
and CO bind most ferrous hemes, a 6-coordinate geometry is preferred
making NO a unique ligand among these small molecule diatomic signaling
species (Traylor and Sharma, 1992). Binding of NO to a ferric heme leads to

the generation of a nitrosyl complex whereby the NO ligand becomes
electrophilic (possesses nitrosonium (NOþ) character). Attack of the bound
NO by a nucleophile (such as water, thiols, etc.) then leads to reduction of
the ferric iron to ferrous and generation of a nitrosated species (Reaction 10).
III
Fe NO $ FeII NOþ þ Nuc H ! FeII þ Nuc NO þ Hþ ð10Þ
This process is referred to as reductive nitrosation whereby nitrosation of
water (generating NO2) and thiols (generating nitrosothiols) can readily
occur. Significantly, nitrosation via these metal catalyzed processes is not sec-
ond order in NO and therefore is not as kinetically restricted as the autoxida-
tion process described earlier. However, since a reduced metal is a product
(i.e., FeII) and an oxidized metal (i.e., FeIII) is required for the chemistry,
there will be a requirement for reoxidation of the metal if the process is to
be catalytic.
As mentioned above, NO2 can be generated via the autoxidation of
NO (Reactions 1–3) or reductive nitrosation (Reaction 10, where the
nucleophile is water). Significantly, Reactions 3 and 2 are reversible,
indicating that NO can be formed from high concentrations of NO2 under
acidic conditions. NO2 can also be reduced directly to make NO under
appropriate conditions. The one-electron reduction of NO2 to NO is
highly proton dependent and can be very favorable since the reduction
potential for the NO2,Hþ/NO couple is 0.98 V, versus NHE.
4.4. Products of NO Reduction
Thus far, the discussion of nitrogen oxide chemistry has concentrated on

species that are oxidized relative to NO (i.e., NO2, N2O3, NO2, ONOO,
etc.). Indeed, in mammalian systems, oxidation appears to be the major fate
of NO. However, reduction of NO is well established in prokaryotes. One-
electron reduction of NO generates nitroxyl (NO/HNO). As mentioned
previously, the reduction potential of the NO,Hþ/HNO couple is only
0.55 V (vs. NHE at pH 7), indicating that direct, proton-assisted one-
electron reduction of free NO is relatively difficult. The chemistry of NO/
HNO has been the topic of numerous recent investigations and is reviewed
elsewhere (e.g., (Fukuto et al., 2005; Miranda, 2005)). The pKa of HNO has
been determined to be 11.4 (Shafirovich and Lymar, 2002), indicating that
HNO is the predominant species at pH 7. A unique aspect of the acid–base
chemistry of HNO is that the two equilibrium partners do not have the same
electronic ground state. That is, HNO is a ground state singlet, while NO is
a ground state triplet (3NO) (akin to O2) (Reaction 11).
HNO (
+ 3 NO þ Hþ ð11Þ

Thus, the protonation of NO and the deprotonation of HNO are slow
compared to other acid–base processes due to the requirement for a spin-
flip. It is generally thought that, in biological systems where HNO or NO
are formed, no chemistry associated with the conjugate will be observed
since other reactions are faster than protonation–deprotonation.
Both the one- and two-electron standard reduction potentials for the
HNO,Hþ/H2NO and HNO,2Hþ/NH2OH couples are reported to be
approximately 0.7 and 0.9 V versus NHE, respectively (the two-electron
process, the HNO,2Hþ/NH2OH couple, at pH 7, has a reduction potential
of approximately 0.3 V vs. NHE) (Shafirovich and Lymar, 2002; Dutton
et al., 2005). These values predict that HNO could be easily reduced in
biological systems. Nitroxyl itself, however, can be very reducing. The
reported reduction potential for the NO/3NO couple is 0.81 V versus
NHE, indicating that 3NO can be a very potent reducing agent (Bartberger
et al., 2002). As mentioned earlier, the N H bond dissociation energy for
HNO is also only approximately 47 kcal/mol. This low bond strength
predicts that HNO would be a very good hydrogen atom donor.
4.5. The Reactions of HNO with Biological Targets
One of the most prevalent biological targets for HNO appears to be thiols
and thiolproteins. Both experimental and theoretical work indicates that
HNO is highly thiolphilic (Doyle et al., 1988; Bartberger et al., 2001).
Attack of a nucleophilic thiol at the electrophilic nitrogen atom of HNO
results in the formation of a fleeting N-hydroxysulfenamide (Fig. 4).
The N-hydroxysulfenamide intermediate has two possible fates. In the
presence of excess or vicinal thiols, the N-hydroxysulfenamide reacts to
form a disulfide and hydroxylamine. A competing rearrangement can also
occur resulting in the formation of a sulfinamide. Significantly, in biological
systems, disulfides are generally considered to be readily reduced back to
the corresponding thiols, whereas sulfinamides are likely to be resistant
to reduction. Thus, reaction of HNO with thiols can result in either
reversible or irreversible modifications.
Another likely class of biological targets for HNO is metals. Akin to
other small molecule metal ligands (e.g., O2, NO, CO, H2S, CN), HNO
NH2
Protein S Sulfinamide
O
H + H Rearrangement
H O N OH
N Protein S
Protein S
RSH
N-Hydroxysulfenamide
Protein S SR
+
Disulfide
NH2OH
Figure 4 Reaction of HNO with thiols.
is capable of coordinating a variety of metals and/or metalloproteins.

Heme-containing proteins are currently the most highly studied among
possible metalloprotein targets (Farmer and Sulc, 2005). HNO is capable
of binding both ferrous and ferric heme proteins. For example, reaction
of HNO with ferrous myoglobin results in the formation of a stable
MbFeII–HNO complex (Sulc et al., 2004) (Reaction 12). Reaction of
HNO with ferric myoglobin results in the formation of a stable ferrous-
nitrosyl MbFeII–NO complex (Doyle et al., 1988) (Reaction 13).
MbFeII þ HNO ! MbFeII NðHÞO ð12Þ
MbFeIII þ HNO ! MbFeII NO þ Hþ ð13Þ

Although most previous biological studies involving nitroxyl presumably
involve the protonated HNO species, it should be noted that formation of
the anion, 3NO, will not result in immediate HNO generation due to the
spin restriction to protonation (remember that the pKa of HNO is 11.4).
Therefore, if 3NO is generated in a biological system, the chemistry of this
species will be prevalent. As discussed briefly above, 3NO is a strong reduc-
tant and should reduce/coordinate metals. Also, 3NO will react with O2, in a
process that is isoelectronic with Reaction 5, giving ONOO (Reaction 14).
3
NO þ O2 ! ONOO ð14Þ
The above discussion of the nitrogen oxides and the associated chemical
descriptions is not meant to be comprehensive but considered to serve as a
starting point for understanding the diversity and complexity of biological
nitrogen oxide chemistry. For more complete descriptions of these and
other aspects of nitrogen oxide chemistry, readers are encouraged to find
one of the available reviews (e.g., Wink and Mitchell, 1998; McCleverty,
2004; Hughes, 2008; Thomas et al., 2008).
5. LABORATORY METHODS
Working with many of the nitrogen oxides described above can be accom-
plished using either authentic compounds, or in many cases, it is more
convenient to use donor species. Herein, we discuss briefly aspects of
working with the nitrogen oxides that appear to be of the most current
interest—NO, NO2, N2O3, NO2, HNO, and ONOO. For several nitro-
gen oxide species discussed below, the use of donor compounds is prevalent
and even necessary. There are several important factors that need to be
considered when using donors, however. Thus, prior to a discussion of the
individual donors, a general comment on the use of donors is warranted.
5.1. The Use of Nitrogen Oxide Donors
With the use of any donor species, there are at least four important con-
siderations that need to be accounted for when interpreting the experimen-
tal results (e.g., Fukuto et al., 2008): (1) It must be determined that the
biological actions of the donor are due to the species released (i.e., NO)
and not due to the donor itself; (2) it is important to understand that the
biological activity can be due to donor coproducts (i.e., other species gen-
erated alongside the species of interest); (3) there is the possibility that
impurities in the donor may be the active species; and (4) for donors that
require biological activation, there is the possibility that the activation pro-
cess itself (i.e., oxidation or reduction) can be at least partially responsible
for the activity. One way to control most of the above-mentioned
possibilities is to use structurally distinct donors from different compound
classes (and with different mechanisms of release) and to incorporate con-
trol experiments using fully decomposed donors. Since donors from vary-
ing classes are structurally distinct, their syntheses and mechanisms of
release are also distinct. Thus, it is highly likely that the only thing they will
have in common is the release of the species of interest. Therefore, if simi-

lar biological activity is observed with the varied donors, it is likely that the
donated species (i.e., NO) is responsible. Controlling for possible activity
associated with impurities and/or coproducts can be accomplished by sim-
ply decomposing the donor and testing the decomposed donor solution.
Since NO is a fleeting species in aqueous aerobic solution (decomposes
to NO2), allowing time for both donor decomposition and NO degrada-
tion to NO2 is necessary. For example, Dukelow et al. (2002) showed that
exhausted DETA NONOate had the same growth inhibitory effect toward
Pseudomonas aeruginosa in vitro as DETA NONOate, showing that NO
was not responsible for the antibacterial effects observed (Dukelow et al.,
2002). In contrast, investigators examining the ability of S. enterica to
mount an acid tolerance response in the presence of RNS revealed that
spermine NONOate sensitizes cells to acid stress, a feat dependent upon
NO release, as the parent compound spermine did not elicit a reduction
in cell viability (Bourret et al., 2008).
5.2. NO
Although NO can be purchased in the gaseous form in a pressurized tank

(Aga and Hughes, 2008), this is often not convenient to use in many
biological studies since gas-handling systems need to be in place and deter-
mining the concentrations of NO in solutions made by passing NO gas
through aqueous mixtures is not trivial. One solution to providing the gas
involves the use of a gas-permeable membrane (Silastic) immersed in the cul-
ture and through which gas mixtures containing NO are continuously passed.
Dose rates of NO delivery are directly proportional to the length of the
immersed tubing (Tamir et al., 1993). This method has been used to study
induction of the E. coli SoxRS regulon and was found to be more effective
than bolus additions of NO (Nunoshiba et al., 1995), while also mimicking
better the continuous fluxes of the gas that occur in macrophages. In one
recent development, Skinn et al. (2011) fabricated a small (65 ml) stirred
reactor that incorporates a flat porous membrane for NO delivery (sitting
below a stirrer) and a loop of gas-permeable tubing for O2 delivery. In trials
using a 10% NO mixture and a buffer that was initially air-equilibrated, con-
stant rates of NO2 (i.e., the end product of NO oxidation accumulation)
were observed (53 mM/h). Such a system has great potential in microbial
physiology experiments but have not yet been reported. Other means for
exposing microbial cultures to NO include incubating Petri dishes in a con-
stantly replenished atmosphere containing 10% air, 960 ppm NO, and the
balance as nitrogen. The additional presence of the redox cycling agent

phenazine methosulfate appeared necessary for consistent NO-dependent
killing but the basis for this observation seems obscure (Gardner et al.,
1998). In other papers, a three-way valve was used to deliver mixtures of
O2, N2, and NO, and gas mixtures were passed through a trap containing
NaOH pellets to remove NO2 and higher oxides of nitrogen formed prior
to entering reaction vessels (Gardner et al., 1997). A laboratory method for
generating NO by the self-decomposition through disproportionation of
nitrous acid is described in useful detail by Aga and Hughes (2008).
However, most experimentalists examining the biology of NO utilize
NO donors. There are many NO donors available and their use and chem-
istry has been reviewed previously (Wang et al., 2002; Miller and Megson,
2007; Aga and Hughes, 2008). Currently, one of the most utilized class of
NO donors are the diazeniumdiolates (also referred to as “NONOates”)
(e.g., Keefer, 2003) (Reaction 15).
R2 N½NONO þ Hþ ! R2 NH þ 2NO ð15Þ
Their utility is based on the wide variety of donors available with vary-
ing NO release rates and the fact that NO release is spontaneous at physi-
ological pH (i.e., does not require bioactivation). The varied NO release
rates for the diazeniumdiolates have been attributed to a competition
between several protonation sites on the molecule, of which only one leads
to NO generation (Dutton et al., 2004a). The half-lives of these compounds
range from 1 min to several hours. All of these factors make this class of
NO donor highly preferable for biological studies. The only coproduct
for the diazeniumdiolates is a secondary amine, which can be controlled
by testing either the amine itself or the decomposed donor.
In some studies mixtures or cocktails of NO donors with complementary
properties may be used. For example, we have used a mixture of NOC-5
and NOC-7 in studies of E. coli to maintain a sustained output of NO in studies
of hmp gene transcription (Cruz-Ramos et al., 2002). NO release from NOC-5
(half-life of 25 min at 37 C) was combined with NO release from NOC-7
(half-life of 5 min at 37 C) to provide NO release over a period of 1 h or more.
In practice, NO loss through biological or nonbiological routes limited the
presence of NO in cultures to about 30 min (Cruz-Ramos et al., 2002).
Sodium nitroprusside (SNP, Na2Fe(CN)5NO) is a clinically relevant and
commonly used donor of NO. In spite of its clinical utility, the mechanism
of NO release from SNP in biological systems is not completely established
and likely to be complex (Wang et al., 2002; Miller and Megson, 2007).
However, it is known that SNP will not spontaneously release NO in a
biological system. Release of NO from SNP requires either light or reduc-

tive metabolism. These factors make it difficult to accurately predict the
levels of NO generated from SNP and/or compare experiments where
SNP is used since light exposure and the degree of reductive metabolism
can differ significantly between experimental systems. Moreover, SNP
contains five cyanide ligands which can also be released and which may
also have biological activity. In in vivo systems, the release of cyanide from
biologically active levels of SNP is typically not a problem (at least in the
short term) since NO is such a potent vasorelaxant that the levels of
released cyanide are tolerated. However, considering all of these factors
(possible photochemical release, the requirement for reductive metabo-
lism, the release of cyanide) as well as others (i.e., the addition of iron),
the use of SNP as an NO donor in a research setting is not optimum.
5.3. S-Nitrosothiols
Another often-used class of NO donor is S-nitrosothiols (SNOs). Although

SNO species are clearly biologically relevant (e.g., Hess et al., 2005), they
can do much more than simply release NO. The release of NO from
SNO compounds is not spontaneous and requires light or reducing metals
(i.e., Cu1þ) (Singh et al., 1996) (Reactions 16 and 17).
RS NOðþlightÞ ! RS þ NO ð16Þ
RS NO þ Cu1þ ! RS þ NO þ Cu2þ ð17Þ

Moreover, SNO compounds can react with other thiols to give, instead,
HNO (Reaction 18) or transfer the equivalent of nitrosonium ion (NOþ) to
another thiol (Reaction 19) (Wong et al., 1998):
RS NO þ R0 SH ! RSSR0 þ HNO ð18Þ
RS NO þ R0 SH ! RSH þ R0 S NO ð19Þ
The possibility of all of this chemistry occurring in a biological system
makes mechanistic interpretation of experiments difficult. Thus, in studies
where only the administration of NO is desired, SNO species are not ideal.
Controls are clearly needed but the design of these experiments is not
always straightforward. For example, GSNO is widely used as a convenient
S-nitrosothiol and it might be imagined that glutathione would be a useful
control molecule. However, GSH is not an ideal control molecule since it
may not be the product of GSNO metabolism (see below). Nevertheless,
where GSH has been used (as well as GSSG) it has been shown to be
relatively ineffective at eliciting upregulation of GSNO-inducible genes in
C. jejuni (Monk et al., 2008), yet GSH (5 mM) is toxic to M. tuberculosis
(Venketaraman et al., 2005).
The evidence to date suggests that, when GSNO is added to bacterial
cultures, intracellular outcomes are preceded by enzymic transformations
of GSNO. The mechanisms of communication between extracellular and
intracellular pools of SNOs are poorly understood but transport
mechanisms involving cell-surface protein disulfide isomerases (Zai et al.,
1999; Ramachandran et al., 2001), g-glutamyl transpeptidase (De Groote
et al., 1995; Hogg et al., 1997), or anion exchangers (Pawloski et al., 2001)
have been proposed. GSNO is proposed to transfer NOþ to outer
membrane thiols in Bacillus (Morris and Hansen, 1981) but other studies
suggest that active transport of the compound is required for toxicity.
In S. enterica, GSNO (0.5 mM) is bacteriostatic but GSNO is not itself
transported into the cell. Highly GSNO-resistant mutants were isolated
from a MudJ transposon library and the insertions shown to be in dppA
and dppD (De Groote et al., 1995). These genes are part of an operon
encoding dipeptide permease, an ABC-family transporter responsible for
L-dipeptide import. It is therefore suggested that a periplasmic
transpeptidase encoded by the ggt gene removes the g-glutamyl moiety.
Indeed, ggt mutants of E. coli (De Groote et al., 1995) and M. tuberculosis
(Dayaram et al., 2006) are also GSNO resistant. In E. coli, the residual
dipeptide, S-nitroso-L-cysteinylglycine, is then transported inward using
the Dpp-encoded dipeptide permease. Figure 5 summarizes the proposed
mechanisms. A similar mechanism appears to operate in E. coli since reg-
ulatory perturbations inducible by GSNO are dependent on the presence
of the Dpp system (Jarboe et al., 2008). In other words, the toxic agent
in the cytoplasm is not GSNO but the nitrosated dipeptide.
In M. bovis, the oligopeptide permease operon (oppBCDA) is
implicated in GSNO transport (Green et al., 2000). Mutation of the oppD
gene encoding the ATPase component of this binding protein-dependent
transport system elicits resistance to 4 mM GSH in the external medium,
a concentration that inhibits the wild-type strain. Importantly, similar
results were found with 0.5 mM GSNO, which is bactericidal for the
wild-type strain but not the oppD mutant. The resistance of the mutant
to GSH is due to diminished import of the thiol, as shown by transport
studies using [3H]GSH. In view of the finding that, in S. enterica, it is trans-
port of the dipeptide that carries the NOþ group (De Groote et al., 1995),
Green et al. (2000) tested whether the opp system in M. bovis transports
A. Salmonella enterica B. Mycobacterium bovis
Glu-CysNO-Gly
(GSNO)
Outer membrane
GGT Glu-CysNO-Gly
SBP Periplasmic space
CysNO-Gly
Glu
Dpp Opp Inner membrane
CysNO-Gly GSNO
Transnitrosation
reactions
Figure 5 Transport mechanisms for SNOs in bacteria leading to intracellular

transnitrosation reactions. (A) In S. enterica, GSNO passively enters the periplasm,
where a transpeptidase (GGT) removes the g-glutamyl moiety (De Groote et al.,
1995). The residual dipeptide, S-nitroso-L-cysteinylglycine, is then transported
inward using the Dpp-encoded dipeptide permease. (B) In M. bovis, the
oligopeptide permease (Opp) affects GSNO transport (Green et al., 2000). A peri-
plasmic substrate-binding protein (SBP) is a product of the same operon.
dipeptides, but none of the tested components of GSH (L-Cys-Gly, L-Cys,

Gly) showed toxicity toward this bacterium.
Note that in mammalian systems, the cellular impact of GSNO or
S-nitroso-N-acetyl-D,L-penicillamine (SNAP) is influenced by the availability
of cysteine or cystine. Indeed, the degradation of GSNO is absolutely depen-
dent on extracellular cystine, which is reduced to cysteine, which in turn
reacts with GSNO to form S-nitrosocysteine (CysNO) in a transnitrosation
reaction. CysNO is then imported by the amino acid transport system
L-AT (Zhang and Hogg, 2004).
5.4. Other Donors
There are several clinically used NO donors that have also been utilized as
NO donors in biological experiments. Organic nitrate esters such as
glyceryltrinitrate (GTN) and the iron–nitrosyl compound sodium
nitroprusside (SNP) are used as sources of NO in a clinical setting or in
microbiology (Joannou et al., 1998; Murray et al., 1998; Lloyd et al.,
2003). However, both require reductive bioactivation, and like RSNO
species, both are capable of other chemistries (see Section 5.2).
5.5. HNO Donors
Recent findings of possible pharmacological applications for HNO have

stimulated the use and development of HNO donors in biological systems
(Miranda, 2005; Paolocci et al., 2007; Fukuto et al., 2008). The most conve-
nient and prevalent HNO donor is Angeli’s salt (Na2N2O3) (Reaction 20).
N2 O3 2 þ Hþ ! HNO þ NO2 ð20Þ

Similar to the diazeniumdiolates, the release of HNO from Angeli’s salt
occurs via specific protonation on one of several possible protonation sites
(Dutton et al., 2004b). The half-lives of Angeli’s salt at 25 and 37 C are
approximately 17 and 2.5 min, respectively. As shown in Reaction 20, the stoi-
chiometric coproduct in Angeli’s salt decomposition is NO2. Since NO2 is
readily available, controlling its release is straightforward and easy using
authentic NO2 (or a better alternative, albeit more expensive, is to examine
the effects of decomposed Angeli’s salt). Thus, Angeli’s salt is a convenient
and well-defined HNO donor for use in biological systems. It should be noted,
however, that at acidic pH (< 4), Angeli’s salt becomes an NO donor.
Another previously utilized HNO donor is Piloty’s acid. Piloty’s acid is a
representative of the N-hydroxylsulfonamide class of donor. The mecha-
nism of decomposition of Piloty’s acid requires the deprotonation of a
weakly acidic proton (Reaction 21), and therefore only generates HNO
at a significant rate under basic conditions.
R SðOÞ2 NHOH ! R SðOÞ2 NHO ! R SðOÞO þ HNO ð21Þ

Thus, in typical biological experiments (e.g., pH 5–7.5), Piloty’s acid is
very slow at HNO release. Also, at neutral pH where Piloty’s acid decom-
position is slow, autoxidation can occur in aerobic solutions, leading to NO
release (Reaction 22) (Zamora et al., 1995).
R SðOÞ2 NHOH ! R SðOÞ2 NHO ! R SðOÞOH þ NO ð22Þ

The slow release under typical biological conditions and the possible
autoxidation makes Piloty’s acid (and derived species) less than optimum
donors of HNO for many biological preparations.
Recent reports indicate the utility of other HNO donors. Acyloxy
nitroso compounds reported by Sha et al. (2006) require an ester hydrolysis
step prior to HNO release but offer a wide array of structural diversity and
diazeniumdiolates made from primary amines appear to offer structural
diversity and spontaneous HNO release in vivo (Miranda et al., 2005).
Significantly, the HNO-donating prodrug cyanamide (H2NCN) has been
used clinically in antialcoholism therapy. Cyanamide can be oxidized to
N-hydroxy cyanamide, which spontaneously decomposes to HNO and
HCN (Reaction 23).
H2 N CN ! HONH CN ! HNO þ HCN ð23Þ

Oxidation of cyanamide can be carried out by catalase/H2O2 (Nagasawa
et al., 1990). Thus, HNO release from cyanamide requires oxidative
bioactivation and simultaneous cyanide release, both of which can repre-
sent complications in biological experiments.
Relatively few studies can be cited to illustrate the identification of HNO
as a species directly responsible for biological effects. However, a clear
example is provided by work on the thiol-containing, metal-responsive yeast
(Saccharomyces cerevisiae) transcription factor Ace1. Ace1 is activated by
binding copper via multiple Cys thiols, resulting in the transcription of genes
encoding proteins involved in copper sequestration (Shinyashiki et al., 2005).
Activation of Ace1 by copper addition to cultures in the presence of various
nitrogen oxides has been studied in depth. Both diethylamine NONOate
(DEA/NO) and Angeli’s salt inhibited Ace1, but the inhibition by NO was
oxygen dependent while the effect of the HNO donor was not (Cook
et al., 2003). The results are interpreted as thiol modification by NO via
the generation of nitrosating species, whereas HNO is able to react directly
with protein thiols. The work also provides an example of the use of
decomposed donors as controls (see Section 5.1): decomposed Angeli’s salt
was without effect and the product of HNO dimerization, nitrous oxide
(N2O), was also inactive, further indicating that HNO is the active species.
5.6. Use of ONOO
Peroxynitrite has been studied extensively using both the authentic com-
pound and via the use of donors. The most commonly utilized ONOO
donors are the sydnonimines (Fig. 6). SIN-1 (R ¼ morpholino, Fig. 6) is a
R R
N N
–
– N NH N NH
O O
Sydnonimines, R = morpholino (SIN-1)
Figure 6 General structure of sydnonimines.
prototypical sydnonimine and its decomposition has been studied exten-

sively (Bohn and Schonafinger, 1989). In aqueous, aerobic solution SIN-1
will ring open followed by one-electron oxidation by O2, generating O2.
The oxidized, ring-opened species then spontaneously releases NO. Thus,
SIN-1 generates NO and O2 in a one-to-one stoichiometry. Since the
reaction of NO and O2 is near diffusion controlled, aerobic SIN-1 decom-
position results in formation of ONOO.
Many in vitro studies have employed these generators to examine the
reactivity and toxicity of ONOO, but findings have often been contradic-
tory. For example, superoxide dismutase (SOD) has been shown to pro-
vide considerable protection toward SIN-1-mediated killing in E. coli,
which is due to O2 scavenging, preventing ONOO formation (Brunelli
et al., 1995). Conversely, it has been demonstrated that SOD actually pro-
motes SIN-1 toxicity. In one study, SOD potentiated the cytotoxicity of
SIN-1 toward the human hepatoma liver cell line (HepG2) by elevating
H2O2 production through the catalyzed dismutation of O2 (Gergel et al.,
1995). However, in another study, SOD enhanced SIN-1 killing of the
parasite L. major by scavenging O2 and increasing the half-life of the true
noxious species, NO (Assreuy et al., 1994). Consequently, it is apparent
that when employing ONOO generators, appropriate control
experiments must be conducted in order to establish whether observations
are attributable to ONOO per se or its reactants.
5.7. NO2
Nitrogen dioxide is available as a compressed gas. Since NO2 is an oxidant

and toxic, use of authentic gas can be problematic if proper gas handling
equipment is not available. Moreover, solutions of NO2 are not stable since
it will dimerize to N2O4, which hydrolyzes to give NO2 and NO3
(Reactions 24 and 25).
2NO2 ! N2 O4 ð24Þ
N2 O4 þ H2 O ! NO2 þ NO3 þ 2Hþ ð25Þ

Thus, experiments aimed at examining specifically NO2 in a biological
system are not always straightforward. In instances where the observed
biological activity is thought to be a result of NO2 generation via autoxida-
tion of NO, a convenient reagent is available for the facile conversion of
NO to NO2. Nitronyl nitroxides (e.g., PTIO) or the water soluble analog
carboxy PTIO (cPTIO), which are widely used research tools for scaveng-
ing NO (Akaike et al., 1993), are capable of converting NO directly to NO2
in an O2-independent manner (thus avoiding the high-order kinetics of
autoxidation) (Akaike and Maeda, 1996) (Reaction 26).
PTIO þ NO ! NO2 þ PTI ð26Þ

Generation of NO2 from NO allows the possibility that N2O3 is the bio-
logically active species since NO and NO2 react quickly to give N2O3. Dis-
tinguishing between the actions of NO2 and N2O3 can be addressed by
examining the effect of PTIO concentration on the biological activity
(e.g., Shinyashiki et al., 2004). At low PTIO/NO ratios, the NO2 formed
will have the opportunity to react with remaining NO to generate N2O3.
However, at high PTIO/NO ratios, most of the NO will be converted to
NO2, precluding formation of N2O3.
Nitronyl nitroxides are also used to detect NO since the reaction of the
EPR-active PTIO with NO generates another distinct EPR-active species
(PTI) (Akaike and Maeda, 1996). Thus monitoring the conversion of
PTIO to PTI via EPR can serve as a quantitative indication of the NO
levels. When using the nitronyl nitroxides as research tools (either for
detection/quantitation of NO or as a reagent to convert NO to NO2), it is
important to realize that the ultimate products generated from the reaction
of NO with PTIO depend significantly on the relative concentrations of the
reactants (Goldstein et al., 2003). For example, the NO2 formed from the
reaction of NO and PTIO can also react with PTIO to give NO2 and
the corresponding oxoammonium cation (PTIOþ). PTIOþ can further
react with NO (in aqueous solution) to give PTIO and NO2. Thus, quan-
titation of NO via PTIO is valid only when NO is in low concentration and
alternative scavengers for NO2 are important considerations when PTIO is
used to convert NO to NO2. Regardless, with a reasonable understanding
of this chemistry, the nitronyl nitroxides are important tools for examining
many aspects of nitrogen oxide chemistry and biology.
5.8. NO2
Nitrite is commercially available as a stable salt. Generally, it is easily han-

dled and amenable to direct use in biological experiments. It is important
to note, however, that high concentrations of NO2 (especially under acidic
conditions) can generate N2O3 (the anhydride of nitrous acid, HONO) which
is also a source of NO and NO2. Despite this, many studies use, as a matter of
convenience, acidified nitrite as an agent of nitrosative stress (see, e.g.,
Mendez et al., 1999; Kim et al., 2003; Mukhopadhyay et al., 2004;
Iovine et al., 2008). However, these conditions are far from ideal in
experiments aimed at determining the mode of action of NO and RNS. In
one report, killing of Mycobacterium ulcerans, which causes ulcerative skin
disease, was reported within 20 min but the concentration of nitrite was very
high (40 mM) (Phillips et al., 2004). More recently, it has been reported that
nrfA and ytfE mutants were sensitive to “NO donors” but the reagents used
were actually GSNO and acidified nitrite (10 mM) (Harrington et al., 2009).
5.9. Other Nitrogen Oxides
Other nitrogen oxides of possible interest include NO3 and NH2OH, both
of which are commercially available and fairly easy to work with in
aqueous solutions. An overview of the relationship between the nitrogen
compounds discussed in Sections 4 and 5 is presented as Fig. 7.
6. BACTERIAL RESPONSES TO RNS: EFFECTORS

AND REGULATORS
6.1. Targets of RNS in Microorganisms
It is frequently stated that NO is a highly reactive gas and must interact

with numerous and diverse biological targets. In fact, as we outline in Sec-
tion 4.1, NO is not especially reactive but it is true that its targets are not as
restricted as those of another “gasotransmitter,” carbon monoxide (CO)
(Davidge et al., 2009a). The direct cellular effects of NO are incompletely
understood because of the complexity of NO chemistry introduced in
Section 4. Nevertheless, various biomolecules are targeted by NO and
the resulting RNS. Some examples from this vast literature are listed in
Table 2 and a detailed analysis is presented in Stamler et al. (2001).
2e− Powerful 1e−

oxidant oxidant
RS-NO + H+
e−, 2H+ e−, H+
−e− O2− H2O2 HO . + H2O
RSH
e− e−, H+
L-Arginine NO O2
H2O
−e−
H2O
N2O3 ONOO− Small %
2nd order
NO−2 + H+ in [NO]
Nitrosation
−e− H+
chemistry
("NO+"-donor)
NO2 ONOOH
−e−, H2O Small %
NO−3 + 2H+
1e− oxidant Major
reaction
Figure 7 The biological and integrative chemistry and fate of NO- and O2-
derived species. Of particular importance is the generation of the one-electron
oxidants NO2 and HO and nitrosating species N2O3. This figure is not meant to
be comprehensive, but rather merely serves to illustrate the integrated nature of
NO and O2 chemistry in biological systems. Not shown here is the possible (and
likely important) role of biological redox metals in this system.
6.2. Microbial Defenses: The Microbe Strikes Back
Many mechanisms are known to confer NO resistance in bacteria, such as

flavohemoglobin-catalyzed NO detoxification in enteric bacteria and
numerous other species as well as other globins. Confusing the literature,
however, is that many experimental studies to unravel the details of NO
resistance are performed with proxies for NO, often SNOs and especially
GSNO. It is important to recognize that most of the enzymic detoxification
systems studied, as far we can judge from current experiments, are first and
foremost concerned with NO detoxification, not with SNO detoxification.
The best understood of these, flavohemoglobin, is an NO-detoxifying
enzyme and has no significant activity against SNOs. Although the earliest
papers on flavohemoglobin regulation (Poole et al., 1996) and enzymic
function (Gardner et al., 1998) were conducted with “real” NO, many later
studies have substituted these agents. Hausladen et al. (1998a) provide a

clear distinction between NO metabolism and SNO metabolism. It was
known that SNO decomposition by E. coli generates nitrite and nitrate
and that NO could arise from the former via chemistry described above.
A quest for NO decomposition showed that although the NO-metabolizing
activity of E. coli was stimulated by treatment of the culture with CysNO,
the SNO-lyase activity was not.
The major diverse mechanisms identified were reviewed previously
(Poole, 2005b) and are updated in Table 3. This is a rapidly developing
field: regulation of gene expression by NO and RNS and the functional
identification of resistance mechanisms are being studied in numerous bac-
teria, as well as fungi and protozoa. Here we focus on a small number of
systems that have emerged as paradigms for future study and bring to
readers’ attention selected new papers that illustrate novel or particularly
interesting examples.
6.3. Microbial Globins
An exciting development in biological NO biochemistry over the past

15 years has been the realization that a major mechanism for NO resis-
tance and detoxification in bacteria and eukaryotic microbes is hemoglo-
bin-mediated NO chemistry. Microbial globins have consequently been
extensively studied and reviewed (Poole and Hughes, 2000; Frey et al.,
2002; Wittenberg et al., 2002; Frey and Kallio, 2003; Wu et al., 2003; Poole,
2005b; Vinogradov et al., 2006; Lu et al., 2008). Globin function is typically
defined by reactivity toward small ligands, such as O2, CO, and NO, which
bind to the heme distal site. Reactivity with, and biological activity in rela-
tion to, each of these ligands has been reported for various microbial
globins. Here we focus only on NO.
6.3.1. Flavohemoglobins
Three classes of bacterial globin are recognized, namely, the flavohemoglobins,

the single-domain “myoglobin-like” globins, and the truncated globins.
Members of the best understood class, the flavohemoglobins, are distin-
guished by the presence of an N-terminal globin domain (a three-on-
three a-helical fold similar to myoglobin) with an additional C-terminal
domain with binding sites for flavin adenine dinucleotide (FAD) and
nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Widely
Table 3 Examples of proteins implicated in tolerance to NO and nitrosative stress via consumption of NO or S-nitrosothiols.
Function/reactions
Class Protein Organism(s) catalyzed Comments Referencea
1. Globins Myoglobin, Higher animals Transient Nitrite reduction; Brunori (2001);

hemoglobin formation of physiological role in Flogel et al. (2001);
peroxynitrite- NO tolerance? NO Hendgen-Cotta et al.
globin; quantitative scavenging? OONO (2008); Ascenzi et al.
formation of nitrate scavenging? (2009)
without nitration of
globin
Truncated globin Mycobacterium Conversion of NO NO-inducible NO Pawaria et al. (2007);
(HbN) species to nitrate? uptake Martr et al. (2008);
Lama et al. (2009)
Vitreoscilla globin Vitreoscilla sp. NO consumption Mechanism unknown; Kallio et al. (2007);
(Vgb) globin confers growth Frey et al. (2011)
tolerance to SNP
Single-domain Campylobacter Confers enhanced Mechanism presumed Monk et al. (2008);
globin (Cgb) jejuni, C. coli resistance to NO to be “NO Shepherd et al.
and nitrosating dioxygenase” (2010a, 2011); Smith
agents converting NO to et al. (2011)
nitrate
Flavohemoglobin E. coli, Enzymic hmp expression Stevanin et al.
(Hmp) Salmonella, detoxification of upregulated by NO (2007); Laver et al.
B. subtilis, NO by conversion and nitrosating agents; (2010); Svensson
Erwinia, many to nitrate mutants are NO et al. (2010); Wang
others sensitive et al. (2010b)
(continued)
Table 3 (continued)
Function/reactions
Class Protein Organism(s) catalyzed Comments Referencea
2. Flavorubredoxin E. coli, NO reduction and Upregulated in Mills et al. (2005,

Reductases (NorVW) Salmonella detoxification response to NO and 2008); Pullan et al.
RNS (2007)
Cytochrome c E. coli, C. jejuni NO reduction and Nrf mutant strains Pittman et al. (2007);
nitrite reductase detoxification show higher NO Mills et al. (2008);
(NrfA) sensitivity van Wonderen et al.
(2008); Einsle (2011)
SNO-lyase; GSH- E. coli, yeast, GSNO or SNO Controls cellular levels Foster et al. (2009b);
dependent mammalian reductase of S-nitrosothiols and Tavares et al. (2009)
formaldehyde cells S-nitrosylated proteins
dehydrogenase
Cytochrome and Mitochondria NO reduction at Activity may be Butler et al. (2002);
quinol oxidases of higher CuB restricted to oxidases Borisov et al. (2009)
organisms, in heme-CuB family;
bacteria physiological
significance unclear
3. Others Cytochrome c0 Rhodobacter NO reductase, CycP mutants are Stevanin et al.
(CycP) capsulatus forming N2O hypersensitive to (2005); Heurlier
nitrosothiols and NO et al. (2008)
a
Illustrative only; the most recent papers are cited in most cases.
distributed in bacteria and lower eukaryotes, flavohemoglobins confer

protection from NO and nitrosative stresses by direct consumption of
NO (Poole and Hughes, 2000). Indeed, flavohemoglobins appear to have
no direct role in the metabolism of oxygen or other gaseous ligands
and such proteins have not been reported in higher animals.
Flavohemoglobins oxidize NAD(P)H and transfer an electron to the
N-terminal heme domain via a noncovalently bound FAD in the reduc-
tase (or FNR, ferredoxin-NADP reductase-like) domain. Reduced heme
catalyzes the reaction between NO and O2 generating nitrate; that is,
Hmp acts as an NO-detoxifying enzyme. There remains controversy over
the reaction mechanism at the heme: either NO (denitrosylase mecha-
nism) (Hausladen et al., 1998b, 2001) or O2 (dioxygenase mechanism)
(Gardner et al., 1998, 2000, 2006) has been claimed to bind first to the
heme.
Flavohemoglobins are critical for pathogenicity in some species; for
example, E. coli and Salmonella mutants lacking Hmp are compromised
for survival in mouse and human macrophages (Stevanin et al., 2002,
2007). In the plant pathogen Erwinia chrysanthemi, HmpX not only
protects against nitrosative stress but also attenuates host hypersensitive
reaction during infection by intercepting NO produced by the plant for
the execution of the hypersensitive cell death program (Favey et al.,
1995; Boccara et al., 2005). In accordance with the role of Hmp in limiting
NO-related toxicity, expression of the protein occurs only when NO is
present in the cell environment. Indeed, hmp gene expression is tightly
regulated at the transcriptional level by NO-responsive transcription
factors, notably NsrR and Fnr (Spiro, 2007). NO regulation of gene expres-
sion is discussed more fully below (Section 7). Tight control of Hmp
synthesis and function appears critical since constitutive Hmp expression
and function in E. coli in the absence of NO generates oxidative stress
by virtue of oxygen reduction by the heme to superoxide anion (Poole
et al., 1997; Wu et al., 2004). Similarly, constitutive expression of Hmp in
Salmonella renders cells hypersensitive to paraquat and H2O2
(Gilberthorpe et al., 2007), as well as ONOO (McLean et al., 2010b);
remarkably, the toxicity of ONOO is in part alleviated by NO, presum-
ably because NO diverts Hmp function to nitrate formation, rather than
generation of oxidative stress, which exacerbates the stress caused by
ONOO. The flavohemoglobins from diverse bacteria have protective
functions against RNS, including Ralstonia eutropha, Bacillus subtilis,
P. aeruginosa, Deinococcus radiodurans, S. enterica, and Klebsiella
pneumoniae (Frey et al., 2002). Note, however, that in this study the agent
of RNS used was SNP (1 mM), an unfortunate choice.
6.3.2. Single-Domain 3/3 Globins
The second class of bacterial globins comprises the single-domain globins.

These also exhibit a three-on-three a-helical fold similar to myoglobin
but no separate C-terminal reductase domain. This class is typified by the
globin of Vitreoscilla (named Vgb, VtHb, or Vhb), an obligate aerobic
bacterium that grows in low oxygen environments (Webster, 1987). This glo-
bin was the first bacterial hemoglobin to be crystallized, and the 3D structure
(of the ferric homodimer) conforms to the classical globin fold (Bolognesi
et al., 1999). This protein has been implicated in redox chemistry and NO
detoxification in vivo, but the mechanism by which the protein is rereduced
after a catalytic cycle is obscure (see Section 6.3.3). The disordered CD region
(i.e., the vicinity of the C and D helices) in the crystal structure of Vgb is a
potential site of interaction with the putative FAD/NADH reductase partner.
Considerable interest has been directed at Vgb because of its possible role in
facilitating oxygen transport and metabolism and the consequent biotechno-
logical implications (Frey et al., 2011). Interestingly, a chimeric protein com-
prising the Vitreoscilla hemoglobin and a flavoreductase domain from a
flavohemoglobin (Fhp) relieves nitrosative stress in E. coli (Frey et al., 2002).
A more comprehensive molecular genetic view of bacterial non-
flavohemoglobins is offered by the microaerophilic, foodborne, pathogenic
bacterium C. jejuni, which is exposed to NO and other nitrosating species
during host infection (Iovine et al., 2008; Tarantino et al., 2009). This
single-domain globin, Cgb, is dramatically upregulated by the transcription
factor NssR in response to nitrosative stress (Elvers et al., 2005; Monk
et al., 2008; Smith et al., 2011). Cgb has been shown to detoxify NO and
possess a peroxidase-like heme-binding cleft. In marked contrast to
Vitreoscilla Vgb, there is no evidence to date that Cgb functions in oxygen
delivery. Cgb can provide an electronic “push” from the proximal ligand
and an electronic “pull” from the distal binding pocket, creating a favor-
able environment for the isomerization of a putative ONOO intermediate
in the NO dioxygenase reaction (Shepherd et al., 2010a). We have recently
demonstrated that the mechanism of NO detoxification is unlikely to pro-
ceed via the formation of an oxyferryl (Fe(IV)¼¼O) species but that NO
interacts with the Fe(III) and Fe(II) species (Shepherd et al., 2011).
6.3.3. Truncated Globins
The third class of globins comprises the truncated proteins, which are the
most recently discovered and appear widely distributed in bacteria,
microbial eukaryotes, and plants (Wittenberg et al., 2002; Milani et al.,

2003; Ascenzi et al., 2007). Instead of the classical 3-over-3 a-helical sand-
wich motif adopted by single-domain globins and the heme domains of
flavohemoglobins, trHbs adopt a 2-over-2 a-helical structure and are typ-
ically 20 residues shorter than 3-over-3 globins. Sequence analysis of
more than 200 trHbs indicates that they can be divided into three groups:
I, II, and III (sometimes referred to as N, O, and P, respectively)
(Wittenberg et al., 2002). Most studies on trHbs have focused on trHb
groups I and II, although the type-III trHb from C. jejuni, Ctb, has
recently been structurally and kinetically characterized (Wainwright
et al., 2005, 2006; Nardini et al., 2006, Lu et al., 2007b, 2008; Bolli et al.,
2008). The function of this globin (named Ctb but also trHbP) remains
enigmatic: although its expression is elevated on exposure to NO and
RNS, via the action of the NO-responsive regulator NssR (Elvers et al.,
2005; Monk et al., 2008), mutation of the ctb gene does not give an
NO-sensitive phenotype (Wainwright et al., 2005). Recently we have
suggested (Smith et al., 2011) that binding of NO or oxygen (Ctb having
an especially high affinity for the latter) may serve to modulate the intra-
cellular availability of NO for NssR activation. Further work is required
to define a clear function; indeed, the same is true for many bacterial
globins. Work conducted on M. bovis revealed that trHbn stoichiometri-
cally oxidizes NO to NO3 and protects aerobic respiration from NO
inhibition (Ouellett et al., 2002).
It is speculated that single-domain and truncated globins associate with
host reductases as a source of electrons for NO or O2 reduction. A recent
study conducted on a mouse neuroglobin (Ngb) that plays a role in
neuroprotection showed that NADH:flavorubredoxin oxidoreductase
from E. coli could reduce the globin in the presence of NADH as the
electron donor (Giuffre et al., 2008). Despite early reports (Jakob et al.,
1992) of a reductase purified from Vitreoscilla that reduced the
Vitreoscilla hemoglobin (Vgb, Vhb) in vitro, this aspect of the mode of
action of NO-detoxifying globins remains obscure. It appears that many
globins could, in principle, serve an NO scavenging function if provided
with a mechanism for heme iron re-reduction such as, in vitro, ferre-
doxin-NADP reductase (E. coli) (Smagghe et al., 2008). The reduction
mechanism may, however, involve not a specific, cognate reductase but
the general reducing environment of the bacterial cytoplasm governed,
for example, by the GSH pool. A similar idea has been proposed for the
reduction of bacterial NOS enzymes that lack a reductase domain
(Gusarov et al., 2008).
6.4. NO and RNS Reductases
6.4.1. NO Reductases
In E. coli, NorR (see Section 7.3) activates the transcription of the norVW
genes encoding a flavorubredoxin and an associated flavoprotein, respec-
tively, which together have NADH-dependent NO reductase activity
(Gardner et al., 2003). Confusingly, these proteins have also been referred
to as FlRd and FlRd-red (flavorubredoxin reductase) (Gomes et al., 2002),
names that do not relate to any E. coli gene. E. coli NorV is perhaps the
most extensively characterized reductase that detoxifies NO under anaero-
bic and microaerobic conditions (Gardner and Gardner, 2002; Gardner
et al., 2002; Gomes et al., 2002). NorV is an oxygen-sensitive flavor-
ubredoxin with an NO reactive di-iron center that reduces NO to (N2O)
as well as oxygen to water (Gomes et al., 2002). Thus, anaerobically a norV
mutant exhibits impaired growth in the presence of NO and is sensitive
also to SNP (Hutchings et al., 2002). E. coli also possesses a dedicated
NAD(P)H flavorubredoxin oxidoreductase, NorW, whose function is
believed to be rereduction of the NorV protein.
Expression of E. coli NorV and NorW is regulated at the transcriptional
level, via the regulator NorR, by a variety of NO-related species including
GSNO (Mukhopadhyay et al., 2004; Flatley et al., 2005), NO (from a saturated
solution of the gas) (Justino et al., 2005), and NO released from NOC-5 and
NOC-7 (Pullan et al., 2007). A NorR protein was first discovered in R.
eutropha (for a review, see Spiro, 2007). It has been suggested that NorR is a
heme-based sensor (Gardner, 2005), but instead NoR is activated by the for-
mation of a mono-nitrosyl iron center located in the GAF domain (i.e., a
domain related to cyclic GMP-regulated cyclic nucleotide phosphodiesterases,
adenylyl cyclase and FhlA) of the protein (D’Autreaux et al., 2005). It has
been suggested that NorR responds exclusively to NO (Spiro, 2006), since
treatment of the ferrous NorR protein with NO leads to activation in vitro
(D’Autreaux et al., 2005). However, its activity can also be inferred in vivo
by upregulation of norVW during growth with GSNO (e.g., Flatley et al.,
2005; Pullan et al., 2007). NorR is a critical sensor of NO-related stress not only
in E. coli but also in R. eutropha and P. aeruginosa (Spiro, 2007).
Periplasmic cytochrome c nitrite reductase, nrfA, is expressed by many
enteric pathogens including E. coli and S. enterica under microaerobic or
anaerobic conditions where electron acceptors are limited. The protein cata-
lyzes a six-electron reduction of NO2 to ammonium (NH4þ) with NO as a
proposed intermediate (Simon, 2002). Indeed, we have shown that NrfA is
responsible for much of the NO evolution that occurs in vitro when anaero-
bically grown cells are treated with high concentrations of nitrite (Corker
and Poole, 2003) (see below). However, the role of NrfA in NO biochemistry
is controversial. It has also been known for some time that NrfA can drive a
five-electron reduction of exogenous NO and a physiological relevance of
this function has been proposed in NO consumption (Poock et al., 2002).
Wild-type cells cultured anaerobically consumed 300 nmol of NO per mg
of protein per min, whereas NO reduction was negligible in nrfA mutants.
Additionally, growth of nrfA mutants was completely attenuated following
the addition of 150 mM NO, whereas wild-type growth was completely
unperturbed (Poock et al., 2002). A recent comprehensive study of the NO
detoxification machinery of Salmonella has revealed roles for all three
proteins so far invoked in anaerobic NO tolerance (Mills et al., 2008). A suite
of double and triple mutants with deletions in hmp, norV, and nrfA was used
to conclude that the NO reductase NorV and the nitrite reductase NrfA are
required additively for NO tolerance under anaerobic fermentative
conditions as well as under anaerobic respiratory (glycerol with fumarate
and nitrate) conditions. NO solutions (40 mM final concentration) were used
(Mills et al., 2008). A minor role for Hmp was also demonstrated, consistent
with the low activity of this protein in anoxic NO consumption (Kim et al.,
1999). It is proposed that the periplasmic location of NrfA enables NO
reduction prior to cell entry while any NO that does enter the cell is
metabolized by cytoplasmic NorV (Mills et al., 2008). A constitutively
expressed NrfA has also been described in C. jejui, which is proposed to pro-
vide a basal level of NO detoxification (Pittman et al., 2007).
6.4.2. Detoxification and Metabolism of SNOs
“NO biology” involves not only NO but also a family of NO-related

molecules, including S-nitrosothiols (SNOs). SNO formation is considered
to be a form of post-translational protein modification of importance in
numerous cell signaling events (Hess et al., 2005; Foster et al., 2009a).
In one early case, an enzyme capable of metabolizing GSNO ( a “SNO-
lyase”) as well as protein SNOs was identified as the glutathione-
dependent formaldehyde dehydrogenase of mammals, yeast, and E. coli
(Liu et al., 2001). Subsequently, several other alcohol dehydrogenase
proteins (AdhC) were identified as exhibiting GSNO reductase activity
in N. meningitidis, H. influenza, and S. pneumoniae (Kidd et al., 2007;
Potter et al., 2007; Stroeher et al., 2007). Such enzymes may have important
functions in alleviating stress imposed by nitrosative species. In yeast, the
combined effects of flavohemoglobin and GSNO reductase (consuming

NO and GSNO, respectively) modulate SNO levels. Nitrosative stress
may be mediated principally by the S-nitrosylation of a particular subset
of protein targets (Foster et al., 2009b). The levels of low molecular weight
SNOs may be diminished not only by SNO-lyases but also by the activities
of NO-consuming enzymes. Thus, the meningococcal NO reductase NorB
increases the rate of SNO (GSNO) degradation in vitro and SNO forma-
tion in murine macrophages is reduced by infection with NorB-expressing
meningococci or with Hmp-expressing Salmonella or E. coli (Laver et al.,
2010). It appears that it is the removal of NO by these enzymes and the
prevention of new SNO formation that is critical. These mechanisms may
contribute to bacterial pathogenesis since S-nitrosylation is critically
involved in, for example, apoptosis, signaling cascades involving neuronal
NOS (Jaffrey et al., 2001), and regulation of gene expression.
The reduction of GSNO has also been demonstrated by the
nitroreductase, NtrA, from Staphylococcus aureus (Tavares et al., 2009),
which binds GSNO with a higher affinity than does the glutathione-
dependent formaldehyde dehydrogenase of E. coli. Additionally, the
thioredoxin system has a demonstrated role in GSNO reduction, as shown
by the accelerated breakdown of this nitrosating species in vitro by
thioredoxin/thioredoxin reductase isolated from both E. coli (Nikitovic
and Holmgren, 1996) and M. tuberculosis (Attarian et al., 2009). Further
to this, Helicobacter pylori strains deficient in either of its two thioredoxin
proteins have been shown to be more sensitive to GSNO and SNP than the
isogenic wild-type strain, suggesting that the thioredoxin system may be
involved in SNO depletion (Comtois et al., 2003).
6.5. Other Proteins Implicated in NO Tolerance
Mutational and transcriptomic approaches have revealed additional

proteins with known or inferred roles in protection from NO or RNS.
Rather than speculate here on the possible roles of the numerous genes
identified as upregulated by these stresses, we focus on a subset that have
received more careful consideration.
A transcriptomic analysis of anaerobically grown E. coli treated with
NO (50 mM, from a solution of the gas) revealed upregulation not only
of hmp, the norVW operon (Sections 6.3.1 and 6.4.1), and genes of inter-
mediary metabolism, but also of ytfE (see below) and the gene for a
LysR-type regulator, yidZ (Justino et al., 2005). Mutation of either gene
rendered cells hypersusceptible to NO.
The ytfE gene has subsequently been shown to be regulated by NO or

other RNS in several other studies and is one of a very small group of
genes that is consistently and massively upregulated on bacterial exposure
to NO and nitrosative stress. It is widely accepted that the YtfE protein
plays an important role in Fe–S cluster metabolism/repair (Justino et al.,
2007) and it has been proposed that the protein should be renamed RIC
(Repair of Iron Centers) (Overton et al., 2008). A recent reanalysis of
strains carrying a “clean” ytfE mutation confirms a role in iron–sulfur clus-
ter repair and also points to a role for YtfE in H2O2 resistance (Vine et al.,
2010). The E. coli protein is a dimer with two Fe atoms per monomer.
Spectroscopic analysis of the purified protein reveals a nonheme dinuclear
iron center having m-peroxy and m-carboxylate bridging ligands and six His
residues coordinating the irons (Todorovic et al., 2008). Homologues of
YtfE occur in numerous bacteria (Overton et al., 2008). In Haemophilus
influenzae, for example, ytfE mutants are sensitive to agents of nitrosative
stress, although very high concentrations of acidified sodium nitrite
(10 mM) and GSNO (5 mM) were used and described as “NO donors”
by Harrington et al. (2009). A ytfE mutant was also sensitive to macro-
phage assaults, an effect that was abrogated by an inhibitor of NOS. Inter-
estingly, in this organism, activation of ytfE is dependent on positive
control by Fnr (see Section 7.2), despite the fact that Fnr is itself an
iron–sulfur cluster protein, whereas in E. coli, ytfE expression is negatively
regulated by Fnr although the effect may be indirect (Justino et al., 2006).
Several transcriptomic studies suggest a role in NO responses of the hcp
and hcr genes, encoding the hybrid cluster protein (an iron–sulfur protein)
and its cognate reductase. These proteins may form a hydroxylamine oxi-
doreductase that converts NH2OH to ammonia (Wolfe et al., 2002; Cabello
et al., 2004) but a role in resisting peroxide stress has also been proposed
(Almeida et al., 2006).
6.6. Beneficial Effects of NO in Microbial Symbioses
NO is not only a toxic radical but may have beneficial effects in certain
microbial lifestyles, particularly biofilm formation and symbioses (for a
review, see Wang and Ruby, 2011). The first report of a bacterial NO
response in symbiosis was that of Meilhoc and others who showed that
Sinorhizobium meliloti upregulates > 100 genes in response to NO, including
hmp, and that an hmp mutant showed decreased nitrogen fixation in planta.
It is proposed that this detoxification system overcomes the inhibitory effects
of NO on nitrogen fixation during symbiosis (Meilhoc et al., 2010).
In one well-studied example, the squid-Vibrio light-organ symbiosis, NO

serves as a signal, antioxidant and specificity determinant (Wang and
Ruby, 2011). In the early stages of the symbiosis, NOS is active and this
enzyme and NO are detectable in vesicles of the mucus secretion where
Vibrio fischeri cells aggregate. Experiments with NO scavengers demon-
strate that this NO influences specificity of the interaction since NO
removal permits nonsymbiotic Vibrio species to form hyperaggregates.
As V. fischeri penetrates the deep crypts of the light organ, the bacterium
experiences higher levels of NO and colonization irreversibly reduces the
NO and NOS levels there. The host therefore uses NO to sense and
respond to the correct symbiont.
The genetic and biochemical evidence suggests that V. fischeri possesses
an NO sensor, H-NOX (heme NO/oxygen binding; Wang et al., 2010a),
which governs the response of at least 20 genes to the NO in the squid.
Ten of these genes are Fur-regulated, presumably reflecting the iron-
limited environment of the host and the fact that the host supplies iron
in the form of hemin (Wang et al., 2010a). To manage the NO levels to
which V. fischeri is exposed, it possesses several NO detoxification systems
including those encoded by the hmp, norVW, and nrf genes (Wang et al.,
2010b). The bacterium also contains an NO-insensitive terminal oxidase,
AOX (Dunn et al., 2010; Spiro, 2010), with an unidentified redox center(s)
and mode of action. This fascinating symbiosis will surely reveal new aspects
of NO as a signaling molecule in modulating symbioses.
6.7. Microbial Responses to ONOO Stress
It is frequently supposed that superoxide anion from the oxidative burst

(Phox or NOX2, the NADH-dependent phagocytic oxidase) and NO from
iNOS combine to generate ONOO, which exerts greater toxicity than
either radical alone. However, the roles of Phox and iNOS are both tempo-
rally (Vazquez-Torres et al., 2000) and genetically (Craig and Slauch, 2009)
separable during Salmonella infection, so it is questionable whether
ONOO is a major antimicrobial species in this scenario. Peroxynitrite
may also arise from the activity of a single enzyme: exposure of murine
macrophages to Bacillus anthracis endospores upregulates NOS2. This
isozyme generates not only NO but also superoxide, and the anticipated
product, ONOO, is detectable by the dihydrorhodamine assay
(peroxynitrite-mediated oxidation of dihydrorhodamine). In this case,
ONOO does not appear to have microbicidal activity (Weaver et al.,
2007). In other examples, ONOO demonstrates toxicity toward a broad
range of pathogens, including E. coli (Zhu et al., 1992; Brunelli et al., 1995),
H. pylori (Tecder-Unal et al., 2008), and Trypanosoma cruzi (Alvarez
et al., 2004). However, evidence is increasing that pathogens possess sys-
tems that directly detoxify ONOO, allowing them to evade this species
and thrive. In Salmonella typhi, an rpoS mutant is more susceptible to
ONOO than a wild-type strain but the molecular basis of this is at present
obscure (Alam et al., 2006). Detailed examples will be discussed below.
Peroxiredoxins are typically associated with the reduction of H2O2 and
organic hydroperoxides and are widespread throughout prokaryotic and
eukaryotic systems (Poole, 2005a). The peroxiredoxin alkylhydroperoxide
reductase subunit C (AhpC) isolated from S. enterica enabled catalytic
breakdown of ONOO to NO2 with a second-order rate constant of
1.51 106 M 1 s 1. This proceeds via oxidation of a cysteine residue toward
the N-terminus of the protein, and catalytic turnover is achieved by reduction
of AhpC by the flavoprotein AhpF. Catalysis was shown to be efficient
in protecting plasmid DNA from single-strand breaks (Bryk et al., 2000).
Peroxiredoxins with peroxynitritase activity, enzymes with the ability to
break down ONOO, have also been identified in other microbes, including
S. cerevisiae. This eukaryote possesses two peroxiredoxins, thioredoxin
peroxidase I and II (Tsa1 and Tsa2), which share 86% identity at the amino
acid level. These proteins catalyze the breakdown of ONOO with reported
second-order rate constants in the region of 105 M 1 s 1 but lack a dedicated
reductase partner. Enzymatic activities are thus recycled by the thioredoxin/
thioredoxin reductase system (Ogusucu et al., 2007).
Catalase-peroxidases offer an alternative to peroxiredoxins in the cata-
lytic turnover of ONOO. Classically, these heme-containing enzymes
are characterized by their bifunctional capacity to break down H2O2 and
organic peroxides (Claiborne and Fridovich, 1979). However, the cata-
lase-peroxidase KatG from M. tuberculosis (Wengenack et al., 1999) and
S. enterica (McLean et al., 2010a) have also demonstrated peroxynitritase
activities with reported second-order rate constants of 1.4 105 M 1 s 1
and 4.2 104 M 1 s 1, respectively. Following incubation with ONOOH,
Wengenack et al. (1999) demonstrate an initial reduction in the Soret band
of KatG followed by recovery upon exhaustion of ONOOH. This implic-
ates the involvement of heme in the catalysis of ONOO breakdown.
Table 4 lists the second-order rate constants of proteins isolated from path-
ogenic organisms that exhibit peroxynitritase activity. In light of the
peroxynitritase activities of certain peroxiredoxins and catalase-
peroxidases, it would appear that these enzymes have the capacity to
detoxify a repertoire of reactive species, making them potentially powerful
virulence factors.
Table 4 Second-order rate constants of proteins with peroxynitritase activities.
10 5 Second-order
Protein Organism/Source
Temperature ( C)/pH rate constant (M 1 s 1) Reference
KatG E. coli 25/7.40 0.33 L. Bowman and S. McLean,

unpublished finding
KatG S. enterica 25/7.40 0.42 McLean et al. (2010a)
KatG M. tuberculosis 37/7.40 1.40 Wengenack et al. (1999)
AhpC H. pylori RT/6.75 12.10 Bryk et al. (2000)
AhpC M. tuberculosis RT/6.75 13.30 Bryk et al. (2000)
AhpC S. enterica RT/6.75 15.10 Bryk et al. (2000)
Thioredoxin S. cerevisiae 25/7.40 5.10 Ogusucu et al. (2007)
peroxidase II
Thioredoxin S. cerevisiae 25/7.40 7.40 Ogusucu et al. (2007)
peroxidase I
Tryparedoxin T. cruzi 37/7.40 7.20 Trujillo et al. (2004)
peroxidase
Tryparedoxin T. brucei 37/7.40 9.00 Trujillo et al. (2004)
peroxidase
RT—room temperature.
7. MICROBIAL SENSING OF NO AND GENE REGULATION
7.1. Introduction
Since the discovery that expression of the flavohemoglobin Hmp of E. coli

is upregulated by NO and that this protein constitutes an effective NO
detoxification enzyme, there has been an explosion of interest in the molec-
ular mechanisms underpinning the regulation of this gene and the many
others now implicated in NO and RNS detoxification. Excellent reviews
have recently appeared (Spiro, 2006, 2007), and the reader is referred to
those articles for details. In brief, NO activates gene expression via SoxR
(Ding and Demple, 2000), Fnr (Cruz-Ramos et al., 2002), OxyR (Kim et al.,
2002), NorR (D’Autreaux et al., 2005), Fur (D’Autreaux et al., 2004), and
NsrR (Bodenmiller and Spiro, 2006). Spiro usefully distinguishes between
those that are secondary sensors and those that are “dedicated” in that their
physiological function appears to be detecting primarily NO and then
regulating expression of genes that encode enzymes with NO as a substrate.
The archetype is NsrR, which appears in enterobacteria to be the major
regulator for NO-detoxifying proteins like flavohemoglobin. In addition to
sensing NO in solution, these regulators may also sense the small levels of
NO that may emanate from SNOs. Here we present only a few comments
on some of the major regulators, emphasizing the sensing of NO. NO sensors
are also known in higher organisms such as the mammalian circadian protein
CLOCK (Lukat-Rodgers et al., 2010).
7.2. Fnr
Fnr is an example of the large Fnr-CRP family of transcriptional regulators

widely distributed in bacteria (Green et al., 2001). The family also includes
the CO sensor CooA and the first clear example of an NO sensor, namely,
the NnrR protein of Rhodobacter sphaeroides (Tosques et al., 1996). There
is strong evidence that it is NO that is sensed by NnrR and other members
of the family (Spiro, 2007). The first clue to the involvement of Fnr in hmp
regulation was the finding that a lysogenic hmp–lacZ reporter construct
was more highly expressed in an fnr mutant than in the parent strain
(Poole et al., 1996), indicating that Fnr represses hmp transcription and
consistent with the presence of an Fnr binding site close to the 10
sequence of the hmp promoter, to which Fnr binds (Cruz-Ramos et al.,
2002). NO modifies the [4Fe–4S] cluster of Fnr in vitro to form
dinitrosyl-iron-dithiol (DNIC) complexes (for a review, see Tonzetich

et al., 2010), relieving repression of hmp. The Fnr-like protein of Azotobac-
ter vinelandii, CydR, is also NO sensitive (Wu et al., 2000).
A further member of this family is the NssR protein of C. jejuni that
controls a small regulon; some of the genes therein, notably that encoding
the hemoglobin Cgb, are known to be directly involved in NO detoxifica-
tion. Although NssR has been reported so far to respond only to GSNO,
it is known that Cgb gene regulation is activated by NO. However, we have
been unable to date to demonstrate any effect of NO or GSNO on DNA
binding by the purified NssR protein (Smith et al., 2011).
7.3. NorR
NorR is a s54-dependent enhancer binding protein. In R. eutropha, it elicits

NO-triggered activation of a two-gene operon (norAB) encoding the respi-
ratory nitrate reductase. The homologue in E. coli activates the divergently
transcribed norVW operon in response to NO, SNP, GSNO, or acidified
nitrite (see Section 6.4.1). These genes encode a flavorubredoxin and its
cognate reductase that reduce NO to N2O. NorR is activated by formation
of mono-nitrosyl complex at a mono-nuclear iron center situated in the
GAF domain (D’Autreaux et al., 2005; Tucker et al., 2005). Evidence that
NorR senses NO comes from experiments in vitro that demonstrate activa-
tion on treating the Fe(II) form with NO. In P. aeruginosa, NorR activates
the divergent hmp-like gene fhp (Arai et al., 2005).
7.4. NsrR
NsrR is widely distributed in Gram-negative bacteria, but in the

gammaproteobacteria, its function is assumed by NorR, which controls
expression of the hmp gene in P. aeruginosa and Vibrio cholerae (for a review,
see Tucker et al., 2010). NsrR was first identified genetically in E. coli in 2005
and is now considered a major regulator of the hmp, ygbA, nrfA, and ytfE
genes and others in E. coli (Mukhopadhyay et al., 2004; Rodionov et al.,
2005; Bodenmiller and Spiro, 2006). In E. coli and Salmonella, NsrR represses
its regulated genes so that, in an nsrR mutant, exceptionally high levels of
Hmp, for example, are made (Gilberthorpe et al., 2007), exceeding those
observed in a wild-type strain in the presence of 1 mM GSNO. Recent studies
on three NsrR proteins (from Streptomyces coelicolor, B. subtilis, and Neisseria
gonorrhoea), expressed in and purified from E. coli, show that NsrR is an
iron–sulfur protein, although the cluster is not the same in each case (for a
review, see Tucker et al., 2010). The present picture is that NsrR proteins con-
tain an O2-stable [2Fe–2S] cluster that is sensitive to NO. The discrepancies in
the literature raise the concern that, on expression in E. coli, inappropriate
clusters, relative to the native bacterium, are incorporated. Alternatively,
clusters in vivo may become degraded during aerobic purification. Whatever
the answer, NsrR senses NO via an iron–sulfur cluster which is nitrosylated,
leading to derepression of target genes.
7.5. Others (DOS, FixL, GCS)
M. tuberculosis survives hypoxia and NO by entering a dormant state of non-

replicating persistence. The dormancy regulon that allows this transition is
activated by the response regulator component DevR of the two-component
regulatory mechanism called DevSRT (also known as DosSRT) (Dasgupta
et al., 2000; Sardiwal et al., 2005). DevS and DosT each possess heme cofactors
to sense O2, being inactive when heme(II) binds the ligand. Anoxia, but also
the presence of NO or CO, leads to activation. The stability of the Fe(II)–O2
complex is enhanced by interdomain interactions, making DevS an efficient
gas sensor. The proteins autophosphorylate and then transfer a phosphate
group to the gene regulator DevR. Recent studies (Yukl et al., 2011) show that
the heme–O2 form of DevS reacts efficiently with NO to produce nitrate in a
reaction reminiscent of the dioxygenase activity of Hmp and some other
globins. The resultant Fe(III) form of DevS is inactive but has a high affinity
for NO. The data suggest that, on exposure to NO, the inactive oxy–heme
complex is rapidly converted to Fe(II)–NO, triggering the onset of the dor-
mant phase and promoting mycobacterial survival.
8. GLOBAL AND SYSTEMS APPROACHES TO

UNDERSTANDING RESPONSES TO NO AND RNS
Due to the complex nature of the interactions between microorganisms

and various RNS, many studies now utilize a variety of “omic” techniques
available to more fully understand the targets of and responses to these
stresses. Global/systems approaches have the advantage of being able to
highlight predicted as well as unexpected and novel responses of the
microbe. These approaches are able to show us the interactions, robust-
ness, and modularity of the complex microbial systems in place for the
sensing and detoxification of RNS.
Both transcriptomic and proteomic techniques have been used to inves-

tigate nitrosative stress in microorganisms. Computational modeling is also
increasingly utilized to interpret the vast amount of data generated by such
approaches. However, care must be taken in the design of experiments so
that interpretation is able to accurately reflect the events occurring in vivo.
8.1. Methodology
8.1.1. Culture Conditions
The majority of experiments on growing microbial cultures are conducted

under batch culture conditions. Here, an initial volume of media and cul-
ture is added to a closed system and growth is monitored over time with
no further addition or expulsion of media. In such a system, nutrients will
become limited and metabolite levels will increase over time. In response,
the cell population physiology will adapt to the changing conditions. These
changes are in addition to adaptations made in response to the addition of
a stressor such as NO.
However, for detailed transcriptomic and proteomic analysis, it is highly
preferable to keep all conditions constant so that gene/protein alterations
can be attributed solely to addition of the stress and not to other factors
such as changes in growth rate or differences in nutrient levels. In continu-
ous chemostat culture, fresh medium is pumped into the culture vessel and
surplus culture medium removed to maintain a constant volume. In this
system, growth becomes limited by 1 or more nutrients in the medium.
After this “steady state” is reached, growth rate is controlled by the addi-
tion of fresh medium and hence fresh delivery of the limiting nutrient. In
this way, growth rate and therefore population density can be controlled.
This approach can be utilized with complex media; however, the use of a
defined medium allows identification and tight control of a single limiting
nutrient, frequently the carbon or nitrogen source. Defined media can also
ensure the bioavailability of all micronutrients, which could otherwise be
sequestered by components of a complex medium such as Luria broth
(Hughes and Poole, 1991). Other forms of continuous culture (e.g., tur-
bidostat, pH auxostat) have been developed (Pirt, 1985); however, work
detailed in this chapter, unless otherwise stated, will focus upon data
accumulated using batch or continuous chemostat culture, which has been
utilized for investigation of a variety of nitrosative stresses.
An important criticism of continuous culture is the accumulation of
loss-of-function rpoS mutations, which can overtake the culture (Notley-
McRobb et al., 2002). However, further analysis found that this phenome-
non does not occur in anaerobic cultures (King and Ferenci, 2005), nor
does it occur, for example, in the E. coli MG1655 strain (King et al.,
2004) used in many microarray studies though it is clear that strict monitor-
ing of cell cultures is required. This, and other evolutions, may affect
the usefulness of chemostat cultures in providing a physiologically stable
population for examination compared to batch culture. However, it is
almost certain that undesirable and uncontrolled changes in growth rate
will affect the outcome and interpretation of batch growths when growth
inhibitory compounds are added. This is highlighted in the literature where
batch culture experiments are more variable than those conducted under
continuous culture conditions (Piper et al., 2002).
8.1.2. Transcriptomics
Microarray analysis is a valuable tool for investigating microbial targets of

and responses to nitrosative stress. This method of measuring global
changes in gene expression in response to RNS has been utilized in a num-
ber of studies with a variety of bacteria, including M. tuberculosis (Ohno
et al., 2003), B. subtilis (Moore et al., 2004; Rogstam et al., 2007),
P. aeruginosa (Firoved et al., 2004), C. jejuni (Elvers et al., 2005),
C. neoformans (Chow et al., 2007), S. aureus (Richardson et al., 2006;
Schlag et al., 2007), and Neisseria meningitidis (Heurlier et al., 2008). How-
ever, it is perhaps unsurprising that the responses of E. coli to nitrosative
stress have been most widely studied (Mukhopadhyay et al., 2004; Flatley
et al., 2005; Justino et al., 2005; Pullan et al., 2007; Bower et al., 2009;
McLean et al., 2010c). It is in this literature that the stark differences
between data sets utilizing differing culture conditions can be readily
identified with very few transcriptional units being identified in common.
In addition to culture conditions, it is also important to distinguish between
the different agents of RNS-mediated stress used as they cannot be used
interchangeably (e.g., NOþ, NO, NO), given that they have unique reac-
tion chemistries (Hughes, 1999; Aga and Hughes, 2008) (Section 4.5) and
elicit quite different responses (Flatley et al., 2005; Pullan et al., 2007).
8.1.3. Modeling
Due to the ever-increasing amounts of data derived from “omic” studies

and the desire to obtain information on the regulatory networks that cause
changes in gene transcription, new methods are required to assess and
accurately interpret data. Computational modeling allows a quantitative

estimation of the regulatory relationship between transcription factors
and genes (Sanguinetti et al., 2006).
Probabilistic state space modeling has been utilized in a number of
recent microbial studies concerned with monitoring E. coli responses to
various environmental changes (Partridge et al., 2007; Davidge et al.,
2009b; Shepherd et al., 2010b). McLean et al. (2010c) used this modeling
technique to highlight changes in the activity of four transcription factors
of E. coli due to ONOO exposure (OxyR, ArgR, CysB, and PhoB) and
were also able to confirm a lack of activity of some transcription factors
that the microarray data suggested may have been altered (e.g., FNR
and IHF). A comparison of this microarray data was also made with data
obtained from exposure to H2O2 using mathematical modeling, which
allowed the similarities and differences between the two stresses to be
highlighted at transcription factor level. However, as with any comparison,
experimental design and continuity are of paramount importance in order
to ensure the models generated are meaningful.
8.1.4. Proteomics
The measurement of changing protein levels has classically been measured

using techniques such as 2D gel electrophoresis or liquid chromatography
followed by mass spectrometry. For the former, protein samples are
separated by isoelectric point (pI) and/or molecular weight and detected
by staining. Protein levels are quantified according to staining intensity
and subsequent identification performed by isolation of protein bands,
sample digestion, and mass spectrometry. This technique has been widely
used for decades and is the basis for several proteomic analyses of
nitrosative stress in microbial cultures (Monk et al., 2008; Qu et al., 2009).
Early techniques focused upon the identification and characterization of
proteins; however, more recently, the focus has extended to the quantita-
tive and comparative measurement of global changes of protein transcrip-
tion via the use of chromatography, mass spectrometry, and bioinformatics.
One technique used for investigation of the response of Desulfovibrio
vulgaris to nitrate stress was via a shotgun proteomic method utilizing iso-
baric tags for relative and absolute quantitation (iTRAQ) (Redding et al.,
2006). This method, commercialized by Applied Biosystems in 2004,
enables the simultaneous identification and quantification of peptides
using mass spectrometry and allows the parallel proteome analysis of up
to four samples via the labeling of primary amines with amine-specific
isobaric reagents (Thompson et al., 2003; Ross et al., 2004). Once tagged,
the labeled peptides from different samples (e.g., pre- and poststress) are
mixed, separated using 2D liquid chromatography and analyzed using mass
spectrometry and tandem mass spectrometry. The iTRAQ-tagged peptides
release reporter ions, the quantities of which are recorded and the peak
area used to quantitate the relative abundance of their originating proteins
(Ross et al., 2004).
Approaches have also been developed to specifically identify
S-nitrosated targets across a given organism’s proteome following chal-
lenge with RNS. The biotin switch method developed by Jaffrey et al.
(2001) was pioneering in singling out SNO-bound proteins. It operates by
blocking free cysteine thiols via S-methylthiolation followed by reduction
of S-nitrosothiols with ascorbate. Thiols are subsequently labeled with a
sulfhydryl-specific biotinylating agent. Labeled proteins are isolated by a
streptavidin pull-down assay and separated by SDS-PAGE. Bands of inter-
est are cut from the gel and subjected to MALDI-TOF to uncover modified
proteins. Jaffrey et al. (2001) used this technique to identify endogenously
S-nitrosated proteins in a rat model by comparing data derived from a
wild-type mouse and an nNOS mutant. Findings revealed that targets
included metabolic, structural, and signaling proteins (Jaffrey et al., 2001).
Modifications to the Biotin Switch method have lead to the development
of several novel techniques that couple labeling of NO-bound thiols to direct
peptide capture methods that automate the detection of altered proteins.
A prime example is the relatively novel S-nitrosothiol capture (SNOCAP)
technique (Paige et al., 2008) that links attributes of the Biotin Switch
method with the isotope-coded affinity tag (iCAT) approach. iCAT was
developed to differentially label two biological samples with discrete isotope
labels that were subsequently subjected to liquid chromatography tandem
mass spectrometry (LC-MS/MS) to identify and quantify differences in pro-
tein expression levels. The tag comprises a thiol reactive group, a heavy or
light isotope linker and a biotin affinity tag. Labels were designed to react
with free sulfhydryl groups that are ubiquitous in proteins, enabling wide
coverage of the proteome (Gygi et al., 1999). SNOCAP, on the other hand,
was developed to solely label thiol groups derived from the reduction of
S-nitrosothiols (Paige et al., 2008). In brief, free thiols are blocked and
S-nitrosothiols are reduced with ascorbate to form thiols. Heavy and light
isotopically labeled thiol biotinylating agents are used to tag newly formed
thiols from cell populations subjected to two different biological conditions.
These samples are mixed and tryptically digested, and tagged proteins are
purified using neutravidin. The sample mixture is subsequently subjected
to LC-MS/MS, which allows for the identification of modified proteins
(without the need for gel electrophoretic separation) and enables the relative
quantification of SNO sites between two different samples. Liquid chroma-
tography is employed to reduce the complexity of the sample by separating
peptides before MS analysis. The mass spectrophotometer is subsequently
set up to measure the relative signal intensities of identical peptide pairs that
only differ in mass by a fixed value dictated by the mass difference of the
heavy and light isotope labels. Operating in MS/MS mode, the amino acid
sequences of individual fragmented peptides contained in the digest mixture
can be determined. Databases are then queried to identify proteins from
which the sequenced peptides originated. Paige et al. (2008) successfully
employed SNOCAP to uncover glutathione-reducible and nonreducible
proteins in cultured cells. This method was the first to enable the relative
quantification of S-nitrosation on a proteome-wide scale between two
different samples.
8.2. Outcomes from Global Transcriptomic Approaches
8.2.1. Responses of E. coli
8.2.1.1. Responses of E. coli to NO and RNS

In E. coil, a number of nitrosative stresses have been analyzed using
transcriptomics. In particular, the response to NO, GSNO, and ONOO
has been examined in the Poole laboratory utilizing the same continuous
culture and defined minimal media conditions, which allows for a direct
comparison of the effects of the three reactive nitrogen species.
Transcriptome profiling experiments were used to investigate the tran-
scriptional basis of the response to the presence of GSNO in both aerobic
and anaerobic cultures of E. coli MG1655 (Flatley et al., 2005). Aerobically,
17 genes were upregulated, most notably those involved in the detoxification
of NO and methionine biosynthesis. Among the NO detoxification genes
upregulated were hmp and norV, which encode an NO-consuming
flavohemoglobin and flavorubredoxin, respectively (Sections 6.3 and 6.4).
Additionally, the transcription of six genes involved in methionine biosyn-
thesis or regulation was significantly elevated. Mutants of metN, metI,
and metR exhibited growth sensitivity to GSNO, and exogenously
provided methionine was found to rescue this phenotype. This supports the
hypothesis that GSNO nitrosates homocysteine, withdrawing it from the
methionine biosynthesis pathway. Anaerobically, 10 genes were significantly
upregulated in response to GSNO, of which, norV, hcp, metB, metR, and
metF were also upregulated aerobically, suggesting that the response to

GSNO is broadly similar under both conditions. These data revealed new
genes important for GSNO tolerance and demonstrated that methionine
biosynthesis is a casualty of nitrosative stress, an observation not seen in
previous studies utilizing batch culture conditions and complex media
(Mukhopadhyay et al., 2004).
Further transcriptomic work aimed to compare and contrast the responses
of E. coli to the nitrosating agent GSNO with that of NO. Previous studies
investigated the effects of NO under both aerobic and anaerobic conditions,
but these observations were made only in batch culture (Mukhopadhyay
et al., 2004; Justino et al., 2005) and thus could not be legitimately compared
to data obtained in chemically defined medium under continuous culture
conditions. The resulting study (Pullan et al., 2007) made use of the same
defined minimal medium and identical continuous culture conditions as the
GSNO work. Addition of NO in this study utilized the addition of the NO-
releasing compounds, NOC-5 and NOC-7, which release NO with half-lives
of 5 and 25 min, respectively, at pH 7.0 and 37 C. The data revealed some
similarities and several marked differences in the transcriptional responses
to these distinct nitrosative stresses. NO causes an upregulation of nitrosative
stress response genes such as hmp and norV, as in the anaerobic study.
Responses appeared to be regulated by global regulators including Fnr, IscR,
Fur, SoxR, NsrR, and NorR. Anaerobically, evidence for the NO inactivation
of Fnr was seen, with upregulation of Fnr-repressed genes and downregulation
of Fnr-activated genes being observed. Notably, expression of none of the met
genes was altered, suggesting that homocysteine nitrosation does not occur
and so, in contrast to GSNO, methionine biosynthesis is not a target of NO
stress under anoxic conditions.
Jarboe et al. (2008) extended these observations by applying network
component analysis to transcriptomic data sets and showed that GSNO
targets homocysteine (Hcy) and cysteine with disruption of the methionine
biosynthesis pathway. Reaction of GSNO with Hcy and Cys resulted in
altered regulatory activity of MetJ, MetR, and CysB, activation of the
stringent response, and growth inhibition. Supplementation with methio-
nine abrogated the GSNO effects (Pullan et al., 2007; Jarboe et al., 2008)
but was without effect on NO sensitivity (Pullan et al., 2007). Hcy was ear-
lier reported to be an effective endogenous antagonist of GSNO-mediated
cytotoxicity (Degroote et al., 1996). Further distinction between the effects
of NO and GSNO is provided by the observation that the upregulation of
Hmp via the transcriptional regulator NsrR may be demonstrated to arise
from the submicromolar levels of NO released from GSNO, but GSNO
internalization is not required for this (Jarboe et al., 2008).
E. coli K-12 samples were also investigated for their anaerobic response
to NO gas in minimal salt medium in batch cultures (Justino et al., 2005).
The authors found that norVW and hmp were significantly upregulated,
highlighting the importance of Hmp for the detoxification of NO in anaer-
obic conditions as shown by Kim et al. (1999). In addition, Fur- and FNR-
mediated repression was relieved and genes responsible for iron–sulfur
cluster assembly/repair were upregulated (ytfE and the isc and suf
operons), in broad agreement with the later study by Pullan et al. (2007).
E. coli MG1655 was also the strain used for a study of aerobic transcrip-
tional responses to GSNO and acidified sodium nitrite (Mukhopadhyay
et al., 2004) in rich medium and batch culture conditions. Again, upregulation
of the nitrosative stress detoxification genes norVW and hmpA was seen in
response to both acidified nitrite and GSNO. This study suggested that,
under these conditions, the sensing of NO was also mediated by modification
of the transcription factor, Fur implicating iron limitation as a consequence
of nitrosative stress. However, involvement of the Fur regulator was not seen
in later work by Flatley et al. (2005); this could be due to the choice of media
used in each case as the bioavailability of iron in the Luria broth used by
Mukhopadhyay and coworkers was likely low, due to a lack of chelators in
the medium (Hughes and Poole, 1991). There was also no evidence for
upregulation of the methionine biosynthesis pathway in the data derived
from rich medium, reflecting the potential discrepancies between data sets
collected under differing experimental conditions.
Uropathogenic Escherichia coli (UPEC), responsible for many urinary
tract infections in humans, encounter multiple stresses during their transit
through the body including RNS. Recently, data have been presented to
show that UPECs preconditioned with acidified sodium nitrite were better
able to colonize the bladders of mice than nonconditioned bacteria (Bower
et al., 2009). Microarray analysis of the UPEC response to acidified sodium
nitrite suggested that upregulation of NsrR-regulated genes, multiple genes
involved in the transport and metabolism of polyamines, and other stress
responsive factors may be responsible for the competitive advantage. This
data suggest that the route of infection and hence the stresses encountered
by the bacterium (e.g., RNS) have a major impact upon host colonization
and bacterial survival.
8.2.1.2. Responses of E. coli to ONOO

Transcriptomic analysis of the E. coli response to ONOO (McLean et al.,
2010c) was also undertaken using defined minimal media and continuous
culture conditions in order to assess the effects of this highly reactive species
and to allow direct comparison to the GSNO and NO data sets. This study
utilized a bolus addition of commercial ONOO to the vessel and feed line.
The resulting microarray data revealed that, in contrast to GSNO and NO, a
number of oxidative stress response genes were upregulated, most notably
katG and ahpCF. KatG has been proposed to act as a peroxynitritase in
M. tuberculosis (Wengenack et al., 1999) as have KatG and AhpCF in
S. enterica (Bryk et al., 2000) (McLean et al., 2010a). Interestingly, the
expression of none of the recognized nitrosative stress response genes was
altered after ONOO exposure, indicating that this reactive species does
not act as a classical nitrosative stress, as do NO and GSNO (Flatley et al.,
2005; Pullan et al., 2007). However, the lack of response by some of the clas-
sical nitrosative stress response genes is not surprising as proteins such as
Hmp and NorVW specifically detoxify NO, levels of which would not be
expected to increase during ONOO exposure. Further, an increase in
Hmp expression during ONOO stress has deleterious effects on S. enterica
(McLean et al., 2010b), causing hypersensitivity to the stress. This is probably
due to the Hmp-catalyzed production of superoxide in the absence of NO
(Orii et al., 1992; Membrillo-Hernandez et al., 1996; Wu et al., 2004). The reg-
ulation of other nitrosative stress response genes including those responsible
for nitrite detoxification (nrfA and hcp) was unaltered by ONOO exposure,
suggesting that, while undoubtedly levels of nitrite will increase upon expo-
sure to ONOO, levels were either insufficient to upregulate these genes
or detoxification of ONOO is more significant than removal of the compar-
atively inert nitrite. Some genes that were upregulated are of unknown func-
tion and could play a role in response to the apparent S-nitrosylation of thiols
or in response to the nitration of tyrosine residues in proteins.
Other targets of ONOO suggested by interpretation of the microarray
data included cysteine (cys) and arginine (arg) biosynthesis as well as
genes involved in iron–sulfur cluster assembly/repair (suf and isc genes),
the high-affinity phosphate transport system (pst), and the (gsi) glutathione
import system. The transcription levels of several genes encoding
membrane and transport proteins were also altered both positively and
negatively, which suggests that ONOO reacts with proteins in the
membrane as well as causing lipid oxidation and nitration (Radi et al.,
1991; Szabo et al., 2007) during its passage into the cell.
8.2.1.3. An emerging picture of the NO and RNS responses in E. coli

There are only a small subset of genes altered in response to more than
one of the above stresses and none whose transcript levels are altered by
all three (Fig. 8.). Upregulated in response to GSNO and NO are genes
Figure 8 The global responses of E. coli to discreet nitrosative stresses. A

simplified comparative overview of E. coli transcript levels altered in response to
GSNO, NO, and ONOO are represented in the Venn diagram as well as areas
of common response between stresses.
involved in NO resistance and those that elicit nitrosative stress resistance

(NorR and NsrR regulons, respectively). Unique to the NO and ONOO
responses are those genes responsible for iron–sulfur cluster assembly
and repair, indicating a common target of these two species. The only gene
significantly altered by both GSNO and ONOO codes for a poorly
characterized protein, YeaJ. This protein is suggested to be a putative
diguanylate cyclase due to the presence of a characteristic GGDEF motif
and is responsible for synthesis of the second messenger, cyclic di-GMP.
Many proteins containing the GGDEF domain also contain other domains
that can receive signals. YeaJ contains an upstream motif suggested to be
an S-nitrosation site (TDCD) (Stamler et al., 1997), which could provide
a signal for some of the cellular responses to RNS.
8.3. Responses of Other Microbes to RNS
8.3.1. C. jejuni
C. jejuni, a predominant causative agent of bacterial gastrointestinal dis-

ease worldwide (Friedman et al., 2000), was found to significantly
upregulate eight genes in response to GSNO stress (Elvers et al., 2005)
including cgb, which codes for a single-domain globin known to protect
the bacteria from nitrosative stress; ctb (Cj0465c), a truncated globin; and
six genes of unknown function. Further work confirmed the rapid response
of cgb upregulation as a protective measure against GSNO (Monk et al.,
2008). In this study, other genes showing enhanced transcription levels
were the truncated globin ctb and a variety of heat-shock response, iron
transport, and oxidative stress response genes including trxA, trxB, ahpC,
and two putative oxidoreductases. Although the function of the hemoglo-
bin Cgb is well established in NO detoxification (see Section 6.3.2), the
function of the truncated globin Ctb is less clear. When ctb is mutated,
no major compensatory transcriptomic adaptations are evident (Smith
et al., 2011). One hypothesis is that, by binding NO or O2 avidly, Ctb
dampens the response to NO under hypoxic conditions, perhaps because
Cgb function (NO detoxification) is O2 dependent (Smith et al., 2011).
Further work is needed to understand the role of this truncated globin.
8.3.2. B. subtilis
The Gram-positive soil bacterium B. subtilis contains an NOS (Adak

et al., 2002a) and also coexists with denitrifying bacteria, so it is likely
to have developed mechanisms of detoxification of both exogenously
and endogenously formed RNS. Microarray analysis of B. subtilis expo-
sure to NO-saturated solutions under aerobic conditions revealed that
the most strongly induced genes were hmp and members of the sB, Fur,
and to a lesser extent, PerR regulons (Moore et al., 2004). Anaerobically,
the same genes were upregulated, however; the strongest responses were
those of hmp and members of the Fur and PerR regulons with sB genes
being only slightly upregulated. B. subtilis cultures exposed to SNP
showed induction of hmp as well as the sB, ResDE (Ye et al., 2000)
and Rex (Larsson et al., 2005) regulons (Rogstam et al., 2007). The
ResDE and Rex regulons are upregulated by changes in redox status or
lowered oxygen availability.
8.3.3. M. tuberculosis
The response of M. tuberculosis to RNS has also been investigated (Ohno

et al., 2003). Microarray analysis using two separate NO donors, NOR-3
and Spermine NONOate, identified the upregulation of 36 genes including
those playing roles in small molecule metabolism (including a nitrate
reductase, narX, and ferredoxin, fdxA), macromolecule metabolism (sigE),
and cell processes including a putative nitrite extrusion protein narK2 and
genes coding for probable transmembrane proteins. Genes downregulated
in the same study included those encoding for putative transcriptional
regulators and cell envelope/energy metabolism proteins, suggesting a
metabolic downshift in response to the NO donors.
The transcriptomic response of M. tuberculosis to macrophage attack has
found evidence of a response to nitrosative stress (Schnappinger et al., 2003;
Cappelli et al., 2006), including upregulation of RNS detoxification genes
including alkyl hydroperoxidase (ahpC), which has peroxynitritase activity
(Bryk et al., 2000), and nitrate reductase (narX), as well as other genes
previously hypothesized to alter their transcriptional activity in response
to nitrosative stresses.
8.3.4. P. aeruginosa
P. aeruginosa upregulates a number of genes in response to GSNO stress

(Firoved et al., 2004). Of these, many are directly responsible for the detox-
ification of oxides of nitrogen. The flavohemoglobin, fhp, is most highly
upregulated; other genes coding for Nor, MoaB1, and NarK1 are also
upregulated.
8.3.5. S. aureus
When exposed to SNAP, S. aureus genes involved in iron homeostasis,

hypoxic/fermentative metabolism, the flavohemoglobin hmp and the
2-component system srrAB were upregulated. SrrAB has been shown to
regulate the expression of many NO-induced metabolic genes (Throup
et al., 2001). When exposed to nitrite, biofilm formation is inhibited in
S. aureus and a clear response to both oxidative and nitrosative stress
can be seen via increases in genes involved in DNA repair, detoxification
of ROS and RNS, and iron homeostasis (Schlag et al., 2007).
8.3.6. N. meningitidis
Activity of the NO-sensitive repressor NsrR from N. meningitidis has been

assessed by microarrays in response to Spermine NONOate (Heurlier
et al., 2008). Target genes under the control of NsrR included norB (NO
reductase), dnrN (repair of nitrosative damage to iron–sulfur clusters),
aniA (nitrite reductase), nirV (nitrite reductase assembly protein), and
mobA (possible molybdenum metabolism) and were all upregulated in
response to the NO donor. Evidence also suggests that the anaerobic-
response regulator Fnr is sensitive to NO, but not to the extent of NsrR.
8.3.7. Yersinia pestis
Microarray analysis of the causative agent of the bubonic plague, Y. pestis,

isolated from the buboes of rats showed a definite response to nitrosative
stress (Sebbane et al., 2006). Most striking was the 10- to 20-fold increase in
expression of hmp. Upregulation of metLRBF, nrdHIEF, ytfE, hcp, hcr,
and tehB was also observed. The latter four genes and hmp are repressed
in E. coli by the transcription factor NsrR (Bodenmiller and Spiro, 2006),
the homologue of which (YPO0379) was downregulated 1.7- to 4-fold in
the bubo. Upregulation of the methionine biosynthesis pathway may also
indicate a response to S-nitrosylation in buboes as identified in the transcrip-
tional analysis to GSNO in E. coli (Flatley et al., 2005).
8.4. Proteomics
One study utilizing C. jejuni sought to identify both transcriptomic and

proteomic alterations in response to GSNO (Monk et al., 2008). Using
2D gel proteomic analysis (Holmes et al., 2005), the authors found that
levels of the two globins, Ctb and Cgb, were enhanced at both the trans-
criptomic and proteomic level as was the heat-shock protein DnaK. Some
proteins were upregulated that did not appear in the transcriptomic data
(e.g., Cj0383c and Cj0509c) and vice versa. This could be a result of the dif-
fering timescales used for each approach; for microarray analysis, cells
were incubated for 10 min, whereas proteomic analysis utilized samples
that had been incubated three times longer. These discrepancies could
reflect responses on differing timescales, a lack of translation or rapid pro-
tein degradation.
A proteomic analysis of the response of H. pylori to the NOþ releasing

molecule SNP using 2D proteomics revealed 38 proteins with altered expres-
sion levels (Qu et al., 2009). Among these were proteins involved in
processing, antioxidation (including TrxR), general stress response, and
virulence.
The study of nitrosative stresses in microbial species using modern
proteomic techniques is still in its infancy. However, quantitative
proteomic analysis has been undertaken to investigate nitrate stress in
the anaerobic sulfate-reducing bacterium D. vulgaris Hildenborough using
iTRAQ (Redding et al., 2006). Changes in the protein profile of this organ-
ism were analyzed upon addition of sodium nitrate using iTRAQ labeling
and tandem liquid chromatography separation coupled with mass spec-
trometry detection. The authors found that the use of 103 mM sodium
nitrate produced only a mild effect upon the proteome, with proteins
involved in central metabolism and the sulfate reduction pathway being
unperturbed. Unsurprisingly, proteins involved in nitrate stress detoxifica-
tion were increased as well as those for transport of proline, glycine-betaine,
and glutamate, suggesting that nitrate stress also induced salt stress. In
addition, levels were increased for several oxidative stress response, ABS
transport system, and iron–sulfur cluster containing proteins.
As discussed previously (Section 8.1.4), not only have proteomic met-
hods been applied to monitor changes in protein expression levels across
the proteome in response to stress, they have also been used to unravel
the S-nitrosoproteome of organisms following challenge with RNS.
Recently, a novel fluorescence-based approach has been developed and
tested on E. coli cell lysates incubated with GSNO to identify S-nitrosated
proteins across the proteome. Twenty modified proteins were uncovered,
with functions in protein synthesis and folding, global regulation, quorum
sensing, signal transduction, and bacterial attachment (Wiktorowicz et al.,
2011). Investigators examining the S-nitrosoproteome of M. tuberculosis,
produced in response to cellular challenge with NaNO2, uncovered 29
modified proteins, a large proportion of which were found to be involved
in intermediary and lipid metabolism. Proteins involved in the defense
against oxidative and nitrosative stresses were also nitrosated (Rhee
et al., 2005). In 2007, Brandes and coworkers applied a method to identify
all reversibly modified thiols mediated by the NO donor, DEA/NO,
including S-nitrosothiols, disulfide bonds, and sulfenic acids. Investigators
uncovered 10 altered proteins, six of which were encoded by essential
genes (Brandes et al., 2007). More recently, researchers have uncovered
five S-nitrosated proteins in H. pylori following incubation of cell lysates
with GSNO, namely, GroEL, a chaperone and heat-shock protein; UreA,
urease alpha subunit; TsaA, alkylhydroperoxide reductase; and two coding

sequences of unknown function (Qu et al., 2011).
Other techniques, such as isotope-coded affinity tags (iCATs) and
S-nitrosothiol capture (SNOCAP), are currently being developed for use
in studying the interaction between RNS and microbes but so far there
appears to be no published work in this field.
9. CONCLUSIONS
The past 15 years have seen a remarkable transformation of our apprecia-

tion of the assaults on microbes by NO and RNS and also the elaborate
and effective defense measures mounted. Certain recurrent themes are
evident. Microbes (in most cases, the information relates to bacteria) are
able to resist NO in their environments by a relatively small number of
detoxification mechanisms, the best understood being globins that catalyze
NO conversion to nitrate and reductases that produce nitroxyl anion, and
ultimately, nitrous oxide. The species sensed that induces the expression
of these enzymes is probably the same, that is, NO, but the possibility
exists that activation of the defense response may be achieved by protein
nitrosation, for example, rather than sensing of NO by a metal center. A
major weakness in our understanding is how bacteria sense and detoxify
other RNS, such as those commonly used in experimental studies, notably,
S-nitrosothiols (especially GSNO), SNP, and acidified nitrite. In the case of
GSNO, SNO reductases are known to denitrosylate affected proteins, but
there do not appear to be any detoxification mechanisms recognized so
far that detoxify the products of SNP and acidified nitrite, but only the
resultant NO. Several approaches may assist in tackling this problem. First,
investigators should exercise great care when designing experiments to
ensure that the nitrosative stress applied is well characterized. SNP, for
example, is an agent that, although easy to obtain, will have quite unpre-
dictable effects in terms of the extent and rates of NO release and may
even release other biologically active species (cyanide, in this case).
In the case of peroxynitrite, it appears that this species should probably
not be regarded as an agent of NO-related stress, but as a species capable
of nitration, nitrosylation, and oxidative stress. Second, sound principles of
microbial physiology should be applied in experimental design, particularly
so that culture conditions are reproducible and consistent with the use of
the RNS species employed. Finally, much may be learned by adopting a
systems or modeling approach to unraveling the complexities of the
microbial response. Since the outcomes of NO or RNS exposure are so

pervasive, affecting directly and indirectly a host of cellular processes, such
approaches have great potential but are in their infancy.
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Author Index
Abergel, C., 30 Albertson, L.K.

Abraham, P., 11 Alderton, W.K., 142
Abusoud, H.M., 142 Alfreider, A., 6–12, 57–58
Achenbach, L.A., 6–12 Allaker, R.P., 166
Adachi, M., 19 Alley, E.W., 142
Adai, A.T., 104, 111, 113, 118, 120 Allocco, D.J., 106
Adak, S., 143–145, 193 Alm, E.J., 182–183
Adams, L., 18, 44 Almeida, C.C., 177
Adams, L.A., 11, 31, 47, 52–53 Alonso, V., 38
Adams, L.K., 11, 57 Alquicira-Hernandez, S.
Adams, S.L., 111–112 Altenberend, F., 185, 194
Addis, M., 115 Altman, R.B., 106, 121
Adinolfi, S., 146 Altschul, S.F., 119–120
Adkins, J.N., 30 Alvarez, M.N., 178–179
Adkins, R.M., 17, 31, 49 Ambs, S., 156
Adriaens, P., 60 Amos, B.K., 6–12, 14, 20
Aebersold, R., 187–188 Anand, A., 169
Aelterman, P., 12 Anastacio, A.S., 6–12
Afkar, E., 30, 34 Anders, K., 105–106
Aga, R.G., 157–158, 185 Andersen, G.L., 6–12
Agapie, T., 144 Anderson, R.T., 6–12, 18, 20, 42, 57–58
Agarwal, A., 122 Andrade, A., 49
Ahn, C.Y., 14 Andras, C., 12
Ahrendt, A.J., 17 Andrew, C.R., 146
Akaike, T., 165, 178–179 Andrews, J.R., 120
Akerley, B.J., 166, 177 Angenent, L.T., 11
Akimenko, V., 23 Angove, H., 141
Akiyama, M., 11 Anjum, M.F., 140, 167–168, 181–182
Akkermans, A.D., 6–12 Annan, R.S., 194
Aklujkar, M., 13, 15–17, 24, 48, 50, Anneser, B., 6–12
62–63 Ansari, S., 20–21
Akob, D.M., 6–12 Antommattei, F.M., 17
Akuta, T., 178–179 Anton, J., 11
Al-Mamun, A. Anzi, C., 171
Alam, M.S., 178–179 Apel, W.A.
Albert, R., 107 Apkarian, R.P., 11
222 AUTHOR INDEX
Aragones, C.E., 11 Baillie, L., 178–179

Arai, H., 182 Bailly, X., 168
Arakawa, T., 32 Bain, T., 25
Araki, N. Baldwin, B.R., 6–12
Aravind, L., 121 Balkwill, D.L., 11
Arese, M., 169 Banci, L., 30
Argen, R., 104–105 Banerjee, D., 6–12
Arkin, A.P., 182–183 Banfield, J.F., 6–12
Armstrong, J., 119 Baptista, J.M., 177
Arnoux, P., 30 Barabasi, A.L., 107, 111–112
Arredondo-Peter, R., 168 Barak, Y., 145–147
Arslan, H., 178–179 Barbe, J., 47–48
Arya, S., 169 Barbe, J.F., 17, 31, 49
Asakawa, S., 11 Bard, J., 116–117
Ascenzi, P., 169, 172–173 Barkay, T., 20–21
Ashburner, M., 116–117 Barlett, M., 7, 25
Asso, M., 30 Barnes, S., 149–150
Assreuy, J., 164 Baron, D.B., 19
Asthana, S., 113, 122 Barragán, M.J., 49
Attarian, R., 176 Barrett, C.L., 7, 13, 29
Aubert, P., 146 Barrett, J., 169, 174, 185, 188–189,
Auch, A.F., 119–120 190–191, 195
Augusto, O., 179, 180 Barrick, J.E., 51
Aulak, K.S., 143–145, 193 Bartberger, M.D., 149–150, 153–154
Av-Gay, Y., 176 Barynin, V., 169, 172
Avetissova, E., 143–145 Basseguy, R., 19
Azarov, I., 145–147 Basu, P., 17
Aziz, N., 169, 185, 195 Basu, S., 145–147
Azizian, M.F., 6–12 Battaglia, V., 109, 112
Batton, A.R., 178
Babnigg, G., 17 Bauer, A., 111–113
Babu, M., 122 Bauer, I., 11
Bach, H., 176 Baumann, R.G., 12
Bacher, A., 25–26 Baumann, S., 25–26
Bader, G.D., 107, 110, 111–112, 113, Baumbach, J., 113, 117
120, 122 Baxter, S., 108
Bader, J.S., 107 Beadle, I., 11
Baedecker, M.J., 23 Beanan, M.J., 30
Baek, K.H., 14 Beason, S.M., 120
Baek, S.M., 6–12 Becher, S., 169
Bailey, V.L. Beck, A., 27
AUTHOR INDEX 223
Beckman, J.S., 151–152, 164, 178–179, Boesch, B.W., 105

191 Boffi, A., 172
Bedard, D.L., 11 Bogdan, C., 142
Bedzyk, L.A., 166, 174, 182–183, 185, Bogle, M.A., 11
188–189, 190, 193 Bohn, H., 163–164
Beers, A.R., 11 Boles, A.R., 60
Behrends, A., 11 Bolivar, F.
Behrens, S., 6–12 Boll, M., 6–12, 25–27, 49, 57–58
Beller, H.R., 57–58 Bolli, A., 172–173
Benanti, E.L., 47 Bollinger, C.J.T., 146, 168, 171, 172
Benjamin, N., 140–142, 166 Bolognesi, M., 168, 172–173
Benlloch, S., 11 Bolton, H., 20
Bennett, K., 111–112 Bonavides, C.
Bennie, C., 176 Bond, D.R., 12, 13, 17, 18–19, 24–25, 36,
Bento, F.M., 11 37, 39, 52–53
Bergel, A., 19 Boon, N., 12
Berggard, T., 108 Boothman, C., 11, 57
Berná, A., 37, 39 Bordel, S., 104–105
Bernhardt, P.V., 169, 172 Borenstein, E., 107
Berthomieu, C., 181 Borisov, V.B., 169
Bertini, I., 30 Bork, P., 110, 111–112, 114, 121
Bertone, P., 110 Borole, A.P., 12
Bethke, C.M., 11 Bosch, J., 18
Beveridge, T.J., 30 Bosche, M., 111–113
Beyer, W.F., 181 Bostick, B., 6–12
Bhupathiraju, V.K., 6–12 Botstein, D., 105–106, 121
Bibb, M., 46–47 Böttcher, A., 25–26
Bidon-Chanal, A., 169 Botton, S., 6–12, 57–58
Bilwes, A.M., 144 Botuyan, M.V., 181
Birkenstock, T.A., 185, 194 Boucher, L.
Bischoff, S., 6–12 Boukhalfa, H., 20
Blackstock, W.P., 108 Bourreille, A., 146
Blakeney-Hayward, J.D., 14 Bourret, T.J., 146, 156–157
Blaser, M.J., 142–143, 166, 172 Boutilier, K., 111–112
Blázquez, B., 49 Bouwer, E.J., 60
Block, K.F., 51 Bouwmeester, T., 111–112
Blom, N., 120 Bower, J.M., 185, 190
Blothe, M., 6–12 Bowman, L.A.H., 171, 179, 180, 185,
Blunt-Harris, E.L., 21, 22, 34 186, 190–191
Boccara, M., 171 Boyartchuk, V., 166, 177
Bodenmiller, D.M., 181, 182–183, 195 Boylan, J.A., 146
224 AUTHOR INDEX
Boylen, C.W., 11 Bukhalid, R.A., 144

Braeken, K., 50–51 Bumgarner, R., 121
Brainard, J.R., 20 Bunker, D.J., 41–42
Brandes, N., 146, 196–197 Burgard, A., 62
Brashear, W.T., 140, 168–171 Burkhardt, E.-M., 6–12, 59–60
Braster, M., 6–12 Burlat, B., 169
Brause, J.E., 140 Burnham, M.K.R., 194
Brazeau, E., 108–109 Busalmen, J.P., 37, 39
Breaker, R.R., 51 Busby, S.J., 47–48
Breitkreutz, B.J. Bush, K.M., 191
Brembeck, F.H., 108–109 Bussey, H., 110
Brettske, I., 11 Butala, M., 47–48
Briée, C. Butcher, P.D., 194
Brito, A.G., 11 Butchins, L.J.C., 41–42
Britt, R.D., 144 Butland, G., 122
Bro, C., 184–185 Butler, A., 139
Broadbelt, L.J., 107 Butler, C.S., 12, 169
Brocchieri, L., 45 Butler, J.E., 7, 15–17, 23–24, 27–28, 30,
Brockman, F.J., 11 31, 32, 33–34, 36, 44, 49–50
Brodie, E.L., 6–12 Butt, J.N., 169
Broene, R.D., 154–155 Butte, A.J., 106, 121
Brofft, J.E., 6–12 Buxton, R.S., 146
Brown, P.O., 17, 104, 105–106 Byung, H.K., 12
Bruand, C., 177
Bruix, M., 31 Cabello, P., 177
Brun, C., 113, 122 Cabezas, A., 12
Brunak, S., 120 Caccavo, F. Jr., 6–12, 20
Brunelli, L., 164, 178–179 Cagney, G., 108–109, 111–112
Brunori, M., 169, 173, 174 Cahyani, V.R., 11
Bruschi, M., 30 Cai, T., 158–159
Bruun, A.-M., 6–12 Call, D.F., 12
Bryan, B., 61–62 Callister, S.J., 6–12, 52–54
Bryant, A.P., 194 Cam, Y., 177
Bryk, R., 179, 180, 190–191, 194 Camacho, P., 11
Buchel, G., 6–12 Camargo, F.A.O., 11
Buchholz-Cleven, B.E., 7 Cameron, K.D., 144
Buchner, A., 11 Cammack, R., 162
Budde, H., 180 Campbell, G., 160–161
Buddha, M.R., 144 Campoy, S., 47–48
Bug, W., 116–117 Can, F., 178–179
Bui, O., 17, 55–56 Cantor, C.R., 113, 122
AUTHOR INDEX 225
Cao, G.L., 178–179 Cheesman, M.R., 169

Cappelli, G., 194 Chemama, Y., 109, 112
Cardenas, E., 6–12 Chen, C.Y., 146, 186, 196–197
Carley, J., 6–12 Chen, G.C., 108–109
Carlson, H.K., 178 Chen, H., 143–145
Carmona, M., 49 Chen, J., 108
Carr, S.A., 194 Chen, K.
Carroll, S.L., 6–12 Chen, M., 193
Carver, D., 115 Chen, T., 105, 108–109, 121
Casamayor, A., 110 Chen, X.P., 11
Cashel, M., 50–51 Chen, Y.J., 121, 144
Castillo, F., 177 Cheng, S.A., 12
Catarino, T., 48 Chesnes, J., 41–42
Catteuw, D., 110 Chevenet, F., 113, 122
Cavistin, J.P., 108–109 Chiba, T., 104, 108–110, 112
Cederbaum, A.I., 164 Chiffer, M., 34
Ceol, A., 119 Childers, S.E., 29, 32, 33–34, 44
Cervantes, F.J., 6–12, 60 Chin, K.J., 6–12, 31, 53
Chae, K.J., 12 Chiriac, C., 114, 120
Chakraborty, R., 23 Chivers, P.T., 47
Champine, J.E., 13, 23, 30 Cho, B.K., 17
Champion, H.C., 153–154, 162, 163 Cho, D.H., 14
Chan, J., 185, 194 Choi, D.W., 144
Chandler, D.P., 6–12 Choi, M.J., 12
Chandran, S., 122 Choi, W.S., 144
Chang, H.W. Choo, Y.F., 12
Chang, I.S., 12, 30 Chou, K.J.Y., 160, 189
Chang, S., 110 Chow, E.D., 185
Chang, Y.J., 6–12 Christopoulos, C., 122
Chao, L.Y., 146 Chu, D., 185, 194
Chapelle, F.H., 6, 15, 57, 58 Chu, J., 23
Chapman, S.K., 30–31 Chua, H.N., 113, 121–122
Charnock, J.M., 11, 41–42, 57 Chung, S., 121
Charoensawan, V. Church, G.M., 105, 107, 111
Chatr-aryamontri, A., 119 Churchill, P.F., 11, 24
Chatterjee, D., 11 Ciaccio, C., 169, 172–173
Chaudhuri, S.K., 17, 32 Cichocka, D., 61
Chaurasia, G., 109–110 Cifuentes, A., 11
Chaussonnerie, S., 11 Ciufo, S., 29
Chavan, M.A., 7, 11, 13, 14, 17, 18 Claiborne, A., 179
Chazelle, B., 122 Clark, D.L., 20
226 AUTHOR INDEX
Clauset, A., 113, 122 Couture, M., 140

Clauwaert, P., 12 Covalla, S.F., 14, 17, 18–19, 29, 37
Coates, J.D., 6–12, 16, 21, 22, 24, 57 Covert, M.W.
Cochrane, G.R., 102 Cowan, R., 11
Cole, J., 141, 177 Cox, A.G., 186
Cole, J.A., 140–142, 172–173, 174–175, Cox, M.M., 47–48
177, 193 Crack, J.C., 146, 158, 181–182
Cole, J.R., 14 Craddock, T., 115
Coletta, M., 169, 172–173 Craig, M., 178–179
Colizzi, V., 194 Crane, B.R., 143–145
Collado-Vides, J. Crea, R.
Collart, F.L., 24 Crespo, A., 169
Collins, S.R., 111–112 Criddle, C.S., 11
Cologna, S.M., 153–154, 162, 163 Cronin, C.N., 27
Colton, C.A., 156 Cross, A.S., 178–179
Comtois, S.L., 176 Crow, J.P., 164, 178–179
Conlon, E.M., 17 Cruciat, C.M., 111–113
Connell, N.D., 159–161 Cruz-Ramos, H., 146, 158,
Conrad, R., 11 181–182
Contag, C.H., 145–147 Cuesters, W., 116–117
Cook, G.M., 186 Cui, X.Y., 162
Cook, J.A., 147 Cuifo, S., 13
Cook, N.M., 163 Cummings, D.E., 6–12
Coopamah, M.D., 140 Cummings, T.A., 11
Cooper, C.E., 142 Cunha, F.Q., 164
Coppi, M.V., 13, 17, 18, 23–25, 31, 32, Curbera, F., 115
33, 47 Cusick, R.D., 12, 61–62
Corbel, C., 38 Czapski, G., 147
Corbin, G.A., 11 Czjzek, M., 30
Cord-Ruwisch, R., 21–22, 34
Cordes, E.E., 12 D’ausilio, C.A., 34
Corker, H., 141, 145–147, 169, 171, D’Autreaux, B., 182
174–175, 190–191 D’haeseleer, P., 111
Cornell, M., 110, 111–112 D’imperio, S.
Corral, A.M., 11 D’mello, R.A.M., 171
Correia, C., 181 D’Autreaux, B., 174, 181, 182
Corum, M.D., 61 Daefler, S., 106–107
Costantino, G., 157–158 Daff, S., 142, 143–145
Costello, E.K., 11 Dalkilie, M.M., 120
Costello, J.C., 120 Dalton, D.D., 6–12
Costello, K., 11 Damelin, M., 110
AUTHOR INDEX 227
Dandekar, T., 121 Delerce, S., 11

Daniels, R., 50–51 DeLisi, C., 121
Daniels, S., 186–187 Delledonne, M., 171
Daprato, R.C., 14 DeMaster, E.G., 159, 163
Dar, S.A., 42 Dementieva, I.S., 34
Daran-Lapujade, P., 184–185 Demirbilek, M., 178–179
Darchen, A., 38 Demoncheaux, E.A.G., 140, 171
Das, T.K., 183 Demple, B., 146, 157–158, 181
Dasgupta, N., 183 Den Camp, H.J.M.O., 6–12
Date, S.V., 104, 111, 113, 118, 120, 121 Deng, M., 108–109, 121
Datta, N., 111–112 Deng, Y., 11
Daugherty, S., 55–56 Dennis, P.C., 14
David, L., 111–112 Derakshani, M., 11
Davidge, K.S., 166, 186 Deretic, V., 185, 194
Davidson, G., 31 DeReuse, H., 109, 112
Davidson, V.L., 146 Derojaswalker, T., 157–158
Davila, D., 62 Desantis, T.Z., 6–12
Davis, J.A., 11 Desvignes, V., 11
Davis, J.P. Detter, J.C.
Davis, M., 7 Dewilde, S., 168
Davis, R.W., 104 Dey, S., 186–187
Day, C.S., 163 Dezso, Z., 111–112
Dayaram, Y.K., 159–160 Dheilly, A., 38
Dayvault, R., 6–12 Di Cara, A., 105
De Groote, M.A., 160–161 di Masi, A., 169
de Hoon, M.J.L. Dias, A.V., 47
De Las Rivas, J., 108 Díaz, E., 49
De Lorenzo, V., 47 Diaz-Lazcoz, Y., 121
De Marinis, E., 169 Díaz-Mejía, J.J., 122
De Micheli, G., 105 Dibartolo, G., 17
de Rojas Walker, T., 157–158 Dibden, D.P., 186
De Schamphelaire, L., 12 Didonato, L.N., 17, 31, 50–51
de Vries, S., 152 Didonato, R.J. Jr., 15–17, 34, 47–48, 62
De Wever, H., 7, 14 Dikshit, K.L., 140, 169
DeAngelo, J., 146, 181 Dilly, G.F., 12
deBeer, D., 145–147 Dinauer, M., 140
Deboy, R., 55–56 Ding, C., 113, 122
Decking, U.K.M., 169 Ding, H.G., 146, 181
Deen, W.M., 157–158, 159 Ding, R., 12
Degraff, W., 147 Ding, Y.H., 13, 15–17, 30
Degroote, M.A., 189 Dionisi, A.M., 172
228 AUTHOR INDEX
Dixon, R.N., 149–150, 174, 181, 182–183 Dupont, R.R., 11

Djordjevic, S., 183 Durack, D.T., 139–140
Dobarro, J., 62 Dutton, A.S., 153–154, 158, 162, 163
Dodson, R.J., 30 Dzikovski, B., 143–145
Doerks, T., 114
Dohnalkova, A., 30 Eads, B.D., 120
Dolganov, G., 194 Edgar, R., 105–106
Dolinski, K., 114, 120 Edwards, E.A., 14
Dolinsky, K., 121 Edwards, J.S., 104–105
Dolled-Filhart, M., 106 Edwards, M.R., 162
Dollhopf, S.L., 11 Efron, B., 194
Domach, M.M., 105 Egawa, T., 140, 168, 169, 172–173
Domrachev, M., 105–106 Egger, L.A., 48
Dong, J., 11 Ehrhardt, D.W., 108
Donnelly, A., 11 Ehrt, S., 194
Donohue, T.J., 178 Einsle, O., 169
Donzelli, S., 156 Eisen, J.A., 30
Dörner, K., 25–26 Eisen, M.B., 105–106
Dou, Y., 140, 168–171 Eisenberg, D., 104, 115, 120
Dougall, H., 141–142 Eisenhaber, F., 121
Dow, C.S., 138–139 Eisenreich, W., 25–26
Doyle, M.P., 154–155 Ekberg, S.A., 20
Drake, H.L., 6–12 Elifantz, H., 6–12, 23, 53–54
Drees, B.L., 108–109 Ellis, D.J., 21
Drewes, G., 111–112 Elshahed, M.S.
Dricot, A., 108–109 Elvers, K.T., 169, 172–173,
Drummond, R.S., 141–142 174–175, 193
Duan, X.J. Emili, A., 121, 122
Duan, X.W., 146 Emrich, S.
Dubchak, I.L., 182–183 Engelen, F.A.A., 162
Duffy, E.B., 11 England, R., 50–51
Dufour, Y.S., 178 Englert, A., 12
Duftler, M., 115 Entight, A.J., 120
Duhamel, M., 14 Epperlein, M., 164
Duke, N.E., 31, 34, 48 Erdjument-Bromage, H., 146, 175–176,
Dukelow, A.M., 156–157 187, 196–197
Dumas, C., 19, 24–25, 40 Erickson, J., 31, 34
Duncan, C.W., 141–142 Eriksson, S., 142–143
Dunman, P.M., 185 Erxleben, A., 26–27
Dunn, A.K., 178 Escolar, L., 47
Duong-Dac, T., 6–12 Esquivel, J.P., 62
AUTHOR INDEX 229
Esteve-Núñez, A., 17, 30, 36–37, 39, Finster, K., 6–12

42, 53 Firestone, M.K., 6–12
Estrin, D.A., 169 Firoved, A.M., 185, 194
Eykerman, S., 110 Flamant, M., 146
Ezaki, T., 178–179 Flammini, A., 122
Flatley, J., 174, 185, 188–189, 190–191,
Fahland, T.R., 17 195
Falkow, S., 166 Fleming, E.J., 57
Famili, I., 17, 107 Flesch, I.E.A., 140
Fang, F.C., 140, 142, 146, 147, 160–161, Fletcher, K.E., 11
166, 177, 178–179, 185, 189 Flickinger, M.C., 19
Fang, M., 186, 196 Flogel, U., 169
Fang, Y., 17, 54 Flohe, L., 180
Farmer, P.J., 149–150, 154–155 Flores-Santana, W., 156
Farres, J., 146, 168, 171, 172 Flynn, D., 62
Favey, S., 171 Flynn, T.M., 11
Fechter, P., 51 Fogarty, H.E., 22
Feelisch, M., 162 Fong, S.S., 107
Feist, A.M., 105, 107, 120 Fontanillo, C., 108
Feisthauer, S., 61 Ford, E., 149–150, 154
Feliu, J.M., 37, 39 Ford, P.C., 150–151, 152–153
Fendorf, S., 6–12 Förster, J., 107
Ferenci, T., 184–185 Forster, W., 11
Fernandes, A.P., 34 Forte, E., 169, 174
Fernandez De Henestrosa, A.R., Fortin, D., 11
47–48 Fortner, J.D., 62
Fernando, L., 11 Foster, M.W., 169, 175–176
Ferrer-Sueta, G., 151–152 Fraga, J.L., 6–12, 21, 22
Ferris, C.D., 175–176, 187 Franceschini, A., 114
Ferris, J., 115 Francis, C.A., 11
Fertig, S.J., 12 Francisco, M.M., 17
Festy, D., 38 Frank, B., 34
Fettig, M.R., 14 Franke, A., 146
Fetzer, I., 11 Franklin, R.R., 39
Fidelis, K., 120 Franks, A.E., 11, 12, 13–14, 17, 22, 29,
Field, E.K. 32–33, 37, 38, 40, 58–59, 60, 62
Field, J.A., 6–12, 60 Franzosa, E., 108
Fields, M.W., 11 Fredrickson, J.K., 11, 29
Fields, S., 104, 108, 110, 111–112, 121 Freeman, B.A., 149–150, 191
Findlay, R.H., 14 Freguia, S., 12
Finneran, K.T., 11, 20, 24, 55–56 Frenzel, P., 11
230 AUTHOR INDEX
Frey, A.D., 146, 168, 169, 171, 172 Gao, C.J., 186, 196
Fricke, K., 12, 39 Gao, Y., 12
Fridovich, I., 179 Garavelli, J.S., 177
Friedman, C.R., 193 García, J.L., 49
Friedman, N., 147 Garcia, M.L., 11
Friedrich, M.W., 6–12 Garcia-Sotelo, J.S.
Friedrich, T., 25–26 Gardner, A.M., 140, 157–158,
Frischer, M.E., 11 167–171, 174
Frizzell, S., 145–147 Gardner, P.R., 140, 157–158,
Frommer, W.B., 108 167–171, 174
Fromont-Racine, M., 108–109 Garg, A., 105
Fu, R., 146 Garg, S., 17
Fuchs, B.M., 11 Garifulin, O., 166, 177
Fuchs, G., 25–26, 27 Garton, E.M., 146
Fukuhara, H., 11 Gaspard, S., 31
Fukui, M., 11 Gault, A.G., 11, 57
Fukumori, Y., 30 Gavin, A.C., 111–113
Fukuto, J.M., 149–150, 153–154, Gaw, C.V., 6–12, 21
156–157, 158, 159, 162, 163, 165 Gazzola, G., 39
Fuller, J.H., 27 Ge, H., 110
Fullstone, G.J., 146 Geary, L., 145–147
Fulthorpe, R.R., 14 Geelhoed, J.S., 24–25, 62–63
Fulton, J.R., 21 Geesey, G.G., 31
Furchgott, R.F., 139–140 Gehlhausen, J.R., 120
Furumichi, M., 118 Gelb, M.H., 187–188
Futamata, H., 11 Gelfand, M.S., 182–183
Futcher, B., 105–106 Gelpi, J.L., 169
Futschik, M.E., 109–110 Gentry, T.J., 6–12
Gerber, S.A., 187–188
Gagneur, J., 111–112 Gergel, D., 164
Galdenzi, S. Gerlach, R.
Gallien, S., 25–27 Gerstein, M., 106–107, 108,
Galmiche, J.P., 146 110, 121
Galouchko, A., 23 Gesellchen, V., 110
Galperin, M.Y., 48, 102 Gessner, C.R., 174
Galushko, A.S., 21, 46 Getzoff, E.D., 144
Gama-Castro, S., 44 Geyer, R., 6–12, 14
Gambarelli, S., 181 Gherardini, F.C., 146
Gambhir, V., 169 Ghosh, D.K., 142
Gammeltoft, S., 120 Gibbons, F.D., 113, 122
Gannon, S.M., 24–25, 58–59, 60 Gibson, D.M., 143–145
AUTHOR INDEX 231
Gidley, M.D., 169, 174, 176, 185, 189, Goschel, K., 6–12
190–191 Goth, K.A., 143
Gihring, T.M., 6–12 Goto, S., 118
Gilberthorpe, N.J., 140, 145–147, 171, Gotz, F., 185, 194
182–183 Gough, J., 168
Gilberthorpec, N., 169 Gow, A.J., 140, 167–171
Gilchrist, M.A., 111, 113, 122 Grandi, P., 111–113
Gill, F., 57 Granger, D.L., 139–140, 160–161
Gilmour, C.C., 57 Grassi, M., 194
Giloteaux, L., 7, 11 Gray, S.M., 62
Ginalski, K., 120 Greco, T., 156–157
Ginder-Vogel, M., 6–12 Green, J., 146, 158, 169, 172–173, 174,
Giometti, C.S., 17, 30 181–182, 185, 186, 188–189, 190–191,
Giovannoni, S.J., 13 193, 195
Girguis, P.R., 12 Green, P.G., 57
Gischkat, S., 11 Green, R.M., 160–161
Gish, W., 119–120 Green, S.J., 6–12, 139–140
Giuffre, A., 169, 173, 174 Greenbaum, D., 108, 121
Gladwin, M.T., 169 Greenblatt, J.F., 111–112, 121
Glasauer, S., 41–42 Greenman, J., 18–19
Glaven, R.H., 13, 17, 24–25, 29, 33–34 Greenwood, C., 169
Glockner, F.O., 11 Greenwood, M., 115
Glot, L., 108–109 Gregory, K.B., 24–25, 59–60
Glover, K., 115 Griebler, C., 6–12
Goeddel, D.V. Griffin, P., 179, 180, 190–191, 194
Goedecke, A., 169 Groen, J.
Goehler, H., 108–109 Gross, S.S., 139–140
Goeltom, M.T. Groveman, E., 62
Golden, M.H., 141–142 Gruber, T.R., 116–117
Goldstein, S., 147, 165 Gruhler, A., 111–112
Goll, J., 111–112 Grundger, F., 61
Golova, J., 6–12 Grundl, T.J., 11
Gomes, C.M., 174 Grzesiok, A., 162
Goncalves, V.L., 177 Gu, B.H., 6–12, 57
Gonzalez-Gomez, I., 143 Guallar, V., 169
Goodwin, S., 13, 23 Guenoche, A., 113, 122
Goon, D.J., 163 Guermazi, S., 11
Gophna, U., 107 Guertin, M., 140, 168, 172–173
Gorby, Y.A., 6, 12, 13, 20, 30, 34 Guessan, A.L., 11
Gordon-Raagas, H.B., 185, 190 Guessan, L.A., 53–54
Gore, J., 51 Guest, J.R., 181–182
232 AUTHOR INDEX
Gugliuzza, T., 17 Hansel, C.M., 11

Guigliarelli, B., 30 Hansen, E.J., 146, 177, 178
Gunaydin, H., 147, 151–152 Hansen, J.N., 146, 160
Gunn, C., 178–179 Hao, T., 108–109
Gunnlaugsson, H.P., 6–12 Haque, M.M., 142
Gunsalus, R.P., 21–22 Hardle, J.M., 166
Guo, W., 108–109 Hargrove, M.S., 173
Guo, X., 111–112, 122 Harhangi, H.R., 6–12
Gusarov, I., 143–145 Harms, H., 27
Gustafson, A.M., 121 Harnisch, F., 12, 39
Gutiérrez-Garrán, C., 39 Harrington, J.C., 166, 177
Gutteridge, J.M., 148–149 Harris, C.C., 156
Guy, J.K., 154–155 Harris, J.C., 162
Gygi, S.P., 187–188 Harrison, J.M., 11
Hart, G.T., 111–112
Ha Nguyen, T.T. Hart, J., 18–19
Ha, P.T., 12 Hartigan, J.A., 122
Haaijer, S.C.M., 6–12 Harvey, E., 146
Hacheri, E.L., 11 Harwood, C.R., 115
Haderlein, S.B., 18 Haser, R., 30
Hadjiev, D., 38 Hashimoto, K., 12
Haenig, C., 108–109 Hatcher, P.G., 21
Hagedoorn, P.L., 25–26 Hattan, S., 186–187
Hagen, W.R., 25–26 Hattori, M., 11, 104, 108–110, 112
Haggman, H., 169, 172 Hatzimanikatis, V., 107
Hakes, L., 111 Hauser, C.A.E., 63
Halden, R.U. Hausinger, R.P., 47
Hallberg, K.B., 11 Hausladen, A., 140, 146, 167–171,
Haller, H., 6–12 175–176, 181
Hallinan, J., 115 Haveman, S.A., 15–17, 40–41, 53
Halliwell, B., 148–149 Hayashi, M., 182
Halloy, S.R.P., 11 Hayes, L.A., 21
Halm, H., 6–12 Hazelbauer, G.L., 103
Hama, K., 11 Hazen, T.C., 6–12, 23, 186–187, 196
Hamamura, N., 57–58 He, H.L., 105
Hamilton, C.Y., 12 He, Q., 17
Hamon, C., 186–187 He, Z.L., 11, 17
Han, C.S., 13 Hedderich, R., 23
Han, J.W., 144 Hedrick, D.B., 11
Hancock, J.T., 143 Heidelberg, J.F., 30
Hänninen, P.J., 6–12 Heider, J., 27
AUTHOR INDEX 233
Heidrich, J., 146 Hirose, T.

Heilbut, A., 111–112 Hirozane-Kishikawa, T., 108–109
Heintz, D., 25–27 Hishigaki, H., 121–122
Heinze, J., 25–26 Hixson, K.K., 30
Heipieper, H.J., 14 Ho, C., 145–147
Heister, K., 18 Ho, Y., 111–112
Heitman, J., 175–176 Hobbs, A.J., 162
Heitmann, K., 27 Hodges-Myerson, A.L., 13, 31
Heller, A., 39 Höfert, C., 111–113
Helmann, J.D., 185, 193 Hofmann, T., 18
Helmick, R.A., 174 Hogan, D.A., 14
Helms, C.A., 11 Hogg, N., 159, 160, 161
Hendgen-Cotta, U.B., 169 Hogg, P.J., 160
Henry, C.S., 107 Hogue, C.W., 107, 110, 111–112, 113, 122
Hensley, S.A., 62 Holliger, C., 31
Heo, J., 177 Hollocher, T.C., 145–147
Herbert, R.A., 11 Holloway, D.T., 121
Herbert-Guillou, D., 38 Holmes, D.E., 6–12, 13, 14, 15–17,
Herman, D.J., 6–12 18–19, 23, 25–26, 32–34, 35, 36,
Herrgårrd, M.J., 105, 107 37–38, 47–48, 52–54, 57–58
Herrmann, C. Holmes, K., 195
Herrmann, S., 61 Holmgren, A., 176
Héry, M., 11, 57 Holstege, F.C., 111–112
Herzel, H., 109–110 Hong, I.S., 144
Hesham, A.E., 12 Hong, S.Y., 144
Hess, D.T., 159, 160, 169, 175–176 Hoogewijs, D., 168
Hettich, R.L., 6–12 Hori, T., 6–12
Heurlier, K., 169, 185, 195 Horner, O., 181
Heyneker, H.L. Hosfield, D., 144
Hibbs, J.B. Jr., 139–140 Hosoda, A., 57–58
Hibbs, M.A., 114, 120 Houk, K.N., 149–150, 151–152,
Hibbs, S., 178–179 153–154, 158, 162
Higney, P. Housewright, M.E., 24
Hildebrandt, P., 39, 177 Houtepen, A.J., 40
Hill, S., 181–182 Hozalski, R.M., 19
Hinds, J., 172–173, 193 Hu, P., 122
Hinlein, E.S., 57–58 Hu, X., 110
Hinnebusch, B.J., 195 Hua, Y.J., 143–145
Hinton, J.C.D., 142–143, 169, 174–175 Huang, H., 146
Hiraishi, A., 11 Huang, J.Y., 121
Hirakawa, M., 118 Huang, Y.N., 186–187
234 AUTHOR INDEX
Huber, R. Ishihama, A., 43–44, 184–185

Hugenholtz, P. Ishii, M., 182
Hughes, J.B., 14, 62 Ishii, S., 11, 12
Hughes, M.N., 140, 145, 146, 156, Ishijima, Y., 11
157–158, 162, 167–171, 174–175, Islam, F.S., 6–12, 57
181–182, 184, 185, 188–189, 190–191, Ismail, W., 40–41, 55–56
195 Isoyama, N., 31
Hunt, D.M., 146 Istok, J.D., 6–12
Huot, H., 55–56 Itakura, K.
Huson, D.H., 119–120 Ito, T., 104, 108–110, 112
Hussain, P., 156 Ivanova, A.E., 6–12
Hutchings, M.I., 174, 182–183 Iyengar, R., 139–140
Huynen, M., 121 Izallalen, M., 32–33, 37, 41, 55–56, 62
Hvitved, A.N., 140, 168–171 Izquierdo-Lopez, A.V., 24
Hwang, C.C., 11
Hyduke, D.R., 160, 189 Jackson, M.I., 156–157, 162, 163
Hyun, J., 159 Jacobs, W.R., 185, 194
Jacq, B., 113, 122
Icopini, G.A., 20 Jaffe, P., 42
Ideker, T., 122 Jaffrey, S.R., 175–176, 187–188
Ieropoulos, I.A., 18–19 Jahn, M.K., 14, 18
Igarashi, Y., 11, 182 Jain, A., 39
Ignarro, L.J., 139–140 Jakob, U., 146, 196–197
Ignatchenko, A., 111–112 Jakob, W., 173
Ikeda, A., 11 James, K.
Illiopoulos, I. James, P., 108
Imade, H., 106 Janczuk, A.J., 158–159
Imfeld, G., 11 Janga, S.C., 122
Immoos, C.E., 154–155 Jansen, R., 108, 121
In, S.C., 12 Jara, M., 47–48
Inoue, K., 13–14, 29, 32–33, 36, 40, Jarboe, L.R., 160, 189
41–42 Jardine, P.M., 6–12
Inouye, M., 48 Jayaraman, T., 145–147
Ioannidis, N., 190–191 Jeandey, C., 181
Ioanoviciu, A., 183 Jehmlich, N., 26–27
Iovine, N.M., 142–143, 166, 172 Jennings, M.P., 175–176
Irigoin, F., 178–179 Jensen, L.J., 114, 120
Isbell, T.S., 163 Jensen, M.P., 179, 180, 190–191
Ischiropoulos, H., 140, 147, 156–157, Jenter, H.L., 6–12, 16
178–179, 191 Jeong, H., 107
Isenberg, J.S., 156 Jetten, M.S.M., 6–12
AUTHOR INDEX 235
Ji, X.B., 145–147 Kai, F., 12

Jia, H., 18–19, 37 Kaiser, D., 45
Jia, J.H., 146, 186, 196–197 Kaiya, S., 11
Jiang, D., 175–176 Kallio, P.T., 146, 168, 169, 171, 172
Jiang, H., 122 Kaludercic, N., 156–157
Jiang, J., 11, 21 Kalyanaraman, B., 149–150, 159, 160
Jiang, X.M., 160 Kane, S.R., 57–58
Jim, K., 122 Kanehisa, M., 118
Jimenez-Jacinto, V. Kang, T.J., 178–179
Jiyoung, L., 12 Kao, K.C., 121
Joannou, C.L., 162 Kappler, A., 11, 21
Jobb, G., 11 Kapur, V., 183
Johannes, J., 25–26 Karabay, G., 178–179
Johnsen, C.V., 14, 55–56 Karachi, T.A., 156–157
Johnson, E.G., 143–145 Karaoz, U., 113, 122
Johnson, G., 31 Karlin, S., 45
Johnson, J.P., 11, 14, 17, 18–19, 29, 60 Karp, K., 6–12
Johnston, J.M., 23 Karp, P.D., 107
Johnstone, R., 186–187 Karp, R.M., 122
Jokipii-Lukkari, S., 169, 172 Karr, E.A., 178
Jones, E.J., 61 Kasai, Y., 57–58
Jones, M.A., 169, 174–175 Kashefi, K., 20–21
Jones, R.A., 169, 174, 185, 189, 190–191 Kasif, S., 113, 121, 122
Jones-Carson, J., 140, 147, 178–179 Kato, S., 12
Jrgensen, K.S., 6–12 Kato-Marcus, A., 12
Joseph, C., 32 Katori, T., 153–154, 162, 163
Joseph, J., 159, 160 Kauffman, K.J., 104–105
Joseph, M.G., 156–157 Kauffman, P., 62
Joshi, M.S., 150–151 Kauffman, S.A., 105
Joyce, A.R., 107 Kaufmann, F., 18, 33
Joyner, D.C., 6–12, 186–187, 196 Kaufmann, S.H.E., 140
Juárez, J.F., 49 Kearsey, M.J., 110
Juarez, K., 17, 49 Keasling, J.D., 23, 186–187, 196
Judson, R.S., 108–109 Keefer, L.K., 158
Juhasz, P., 186–187 Keller, J., 12
Jung, S.H., 12 Keller, M., 55–56
Justino, M.C., 174, 176, 177, 185, 189, 190 Kelley, B.P., 122
Jyothisri, K., 183 Kelly, A., 140
Kelly, D.J., 169, 174–175, 176
Kaden, J., 21, 22 Kelly, J.J., 40
Kaganman, I., 110 Kelm, M., 169
236 AUTHOR INDEX
Kemmeren, P., 111–112 Kinsall, B., 11

Kendall, S.L., 183 Kirk, M., 149–150
Keng, T., 146, 181 Kiss, H.E., 13
Kerin, E.J., 57 Kitajima, S., 11
Kerkhof, L.J., 6–12, 42 Kitanidis, P.K., 6–12
Kerrien, S., 119 Kjelgaard, P., 185, 193
Kers, J.A., 144 Klapper, L., 21
Ketley, J.M., 195 Klare, J.P., 169
Keulers, M., 162 Kleid, D.G.
Khainovski, N., 186–187 Kleinsteuber, S., 27
Khalaf, R., 115 Klemic, J.F., 110
Kharchenko, P., 107 Klemic, K.G., 110
Khare, T., 17, 30 Klimes, A., 7, 13–14, 29
Kidd, S.P., 175–176 Kluger, Y., 121
Kiely, P.D., 12, 61–62 Knickerbocker, C., 6–12
Kiéné, L., 38 Knight, E.M.
Kienle, S., 186–187 Knight, J.R., 108–109
Kikuchi, M., 11 Knight, R., 11
Kim, B.C., 13–14, 17, 29, 31, 32–33, Knights, D., 11
41, 49 Knittel, K., 11
Kim, B.H., 12, 14, 30 Knowles, R.G., 142
Kim, C.C., 166 Knudsen, S., 184–185
Kim, H., 39 Kobayashi, A.
Kim, H.D., 105 Koeppen, S., 108–109
Kim, H.S., 14 Koffman, B.
Kim, I.S., 12, 113 Koh, S.C., 14
Kim, J., 27 Kohane, I.S., 106, 121
Kim, J.N., 51 Kohler, J., 113, 117
Kim, J.R., 12 Kohno, M., 165
Kim, K.S., 30 Kohno, S., 185, 194
Kim, S.M. Kojima, H., 11
Kim, S.O., 140, 159, 167–168, 174–176, Kolaczyk, E.D., 121
181–182, 190 Kolesar, S.E., 21
Kim, T.Y., 107 Kolk, A.H.J., 166
Kim, Y.K., 144 Kollah, B., 11
Kimura, M., 11 Komatsu, M., 19
King, O.D., 113, 122 Konishi, H., 11, 31
King, S.B., 163 Konorev, E., 160
King, T., 184–185 Konstantinov, A., 169
Kinkel, T., 146, 177, 178 Koppenol, W.H., 151–152
Kinniburgh, D.G., 57 Korenevsky, A.A., 31
AUTHOR INDEX 237
Kori, A., 184–185 La Duc, M.T., 11

Koskenkorva, T., 146 Labarre, M., 172–173
Kosson, D.S., 11 Labesse, G., 171
Kostka, J.E., 6–12 Labib, M.E., 11
Kothari, S., 55–56 Labigne, A., 109, 112
Kovacik, J.W.P., 11 Laempe, D., 25–26
Kozminski, K.G., 108–109 Lai, T., 11
Krader, P., 14 Lalonde, S., 108
Krajmalnik-Brown, R., 12, 38 Lalowski, M., 108–109
Krasnoff, S.B., 144 Lam, P., 6–12
Kraszewski, A. Lama, A., 169
Krause, R., 110, 111–113 Lamas, S., 166
Kreimer, A., 107 Lamb, B.T., 112
Kretzschmar, A.K., 26–27 Lamb, C., 171
Krishna, M.C., 147 Lancaster, J.R. Jr., 146, 150–151
Krogan, N.J., 111–112, 121 Land, M.L., 13, 17
Kroneck, P.M.H., 173 Landa, E.R., 6, 60
Kruger, M., 61 Landry, A.P., 146
Krumholz, L.R., 11 Landry, C.R., 110
Krummenacker, M., 107 Lange, U., 40
Krushkal, J., 17, 31, 49, 50–51 Langley, S., 11
Kryshtafovych, A., 120 Langlois, R., 6–12
Kube, M., 27 Lapidus, A., 17
Kuhara, S., 108–109, 112 Laroche, J., 6–12
Kuhn, K., 186–187 LaRossa, R.A., 166, 174, 182–183, 185,
Kuhn, M., 114 188–189, 190
Kuijper, S., 166 Larrahondo, M.J., 11, 14, 17, 18,
Kukhtin, A., 6–12 33–34, 47
Kukkadapu, R.K., 11, 24 Larsen, J.T., 6–12, 53–54
Kumar, A., 146 Larsson, J.T., 185, 193
Kummerfeld, S.K. Lash, A.E., 105–106
Kunapuli, U., 7, 14 Latour, J.M., 181
Kung, J.W., 25–27 Lau, M.W., 108–109
Kunimaru, T., 11 Laver, J.R., 169, 175–176
Kuntze, K., 6–12, 25–26, 49, 57–58 Lavik, G., 6–12
Kuriyama, H., 162 Law, N., 20–21
Kuroi, A., 182 Lawrence, J.R., 6–12
Kurosky, A., 196–197 Lawrence, K.A., 146
Kusel, K., 6–12 Lawrence, N.D., 185–186
Kuypers, M.M.M., 6–12 Le Brun, N.E., 146, 182–183
Kyrpides, N.C. Leach, E.R., 174–175
238 AUTHOR INDEX
Leach, L.J., 110 Li, N., 108–109

Leaf, C.D., 139–140 Li, P., 115
Leak, D., 12 Li, T.S., 11, 140, 168–171
Leal, F.A., 163 Li, W.J., 186, 196
Leang, C., 13, 17, 18, 22, 29, 31, Li, Y., 108–109, 177
32–33, 53 Li, Z., 111, 120
Lear, G., 11 Liang, L.Y., 57
Lee, B.D. Liao, J.C., 160, 189
Lee, E.R., 51 Liesack, W., 11
Lee, H.S., 12, 38 Lievens, S., 109–110
Lee, H.W., 144 Liew, F.Y., 139–140, 164
Lee, H.Y., 144 Lilly, W.W., 23
Lee, I., 104, 111–112, 113, 118, 120 Lim, C.H., 159
Lee, J., 12, 107, 120 Lin, B., 6–12, 57–58
Lee, K.H., 107 Lin, J., 106
Lee, L., 169, 174–175 Lin, W.C., 17, 47–48
Lee, M.E., 140, 169, 171, 175–176, Lin, Y., 140
182–183 Lind, J., 147
Lee, S.Y., 107, 120 Lindberg, C., 17
Lefebvre, O. Lindley, P.F., 177
Legall, J., 42 Linghu, B., 108, 121
Legler, T.C., 57–58 Link, K.H., 51
Legrain, P., 108–109 Linossier, I., 38
Lehnert, N., 148 Linse, S., 108
Leichert, L.I., 146, 196–197 Lintinen, P.T.J., 6–12
Leifert, C., 141–142 Lioliou, E., 51
Leigh, M.B., 6–12 Lipman, D.J., 119–120
Lemaitre, N., 195 Lippard, S.J., 143–145, 181–182
Lemmens, I., 109–110 Lipton, M.S., 11, 30, 52–53
Lenzen, G., 109, 112 Lipton, S.A., 191–192
Letovsky, S., 113, 121, 122 Liseo, B., 194
Lettinga, G., 6–12, 60 Liss, S.N., 14
Leung, K.M., 12 Lister, A.L., 118
Levar, C.E., 13 Liu, A.M., 15–17, 18, 24–25, 32, 34,
Leveinen, J., 6–12 47–48, 146
Levi, R., 139–140 Liu, F.G., 146
Lewis, R.S., 157–158 Liu, G.L., 12, 61–62
Li, D., 12 Liu, J.L., 12, 119
Li, J., 12, 111–112 Liu, L.M., 104–105, 169, 175–176
Li, L., 193 Liu, M.Y., 42
Li, M.C., 12 Liu, O.W., 185
AUTHOR INDEX 239
Liu, R., 12 Ludwig, W., 11

Liu, W., 149–150, 154 Lueders, T., 6–12, 14
Liu, X., 150–151 Lukat-Rodgers, G.S., 181
Liu, Y., 12, 39, 113, 194 Lundberg, J.O., 140–142
Liu, Z.F., 186, 196 Lunn, A.D., 169, 175–176
Livens, F.R., 11, 20–21, 41–42 Lunsford, R.D., 194
Livstone, M.S. Luo, J., 6–12
Lloyd, D., 162, 174–175, 190 Luo, Y., 12
Lloyd, J.R., 11, 13, 20–21, 30–31, 34, Luo, Z.W., 110
41–42, 57 Luque, F.J., 169
Lockshon, D., 108–109 Luque, J., 169
Löffler, C., 25–27 Luzzi, I., 172
Loffler, F.E., 11, 14, 20, 60 Ly, H.K., 39
Logan, B.E., 12, 19, 61–62 Lymar, S.V., 149–150, 153–154
Lojou, E., 30 Lyon, E.H.
Londer, Y.Y., 30–31, 34, 48
Lonergan, D.J., 6–12, 15, 16, 23 Macalady, J.L.
Long, P.E., 6–12, 17, 52–54 Macaskie, L.E., 20
Long, W.C., 34 Macbeth, T.W., 11, 14
Lonsdale, J.T., 194 Mack, E.E., 57
Lopez, B.E., 162, 163, 165 MacMicking, J.D., 140
Lopez-Fuentes, A. Macrae, J.D., 11
López-García, P. Madhani, H.D., 185
Loque, D., 108 Madsen, E.L.
Lord, P., 118 Madupu, R., 30
Loria, R., 143–145 Maeda, H., 165
Loscalzo, J., 160 Magnuson, T.S., 31
Louro, R.O., 30–31 Mahadevan, R., 17, 23, 36–37, 54,
Lovell, S.C., 111 55–56, 62
Lovley, D.R., 6–12, 13–14, 15–17, 16, Mahapatro, S.N., 154–155
18–19, 20–22, 23, 24–25, 29, 31, Majewski, R.A., 105
32–34, 36–37, 39, 40–42, 43, 44, Makita, Y.
47–48, 49, 50–51, 52–54, 55–59, 60, Malanoski, A.P., 39
61, 62 Maliskaya, Y., 110
Lowe, M., 6–12 Malvankar, N., 37
Lowenstein, C.J., 142 Malvankar, N.S., 11, 22, 29–30, 38–39,
Lowy, D.A., 12, 37, 62 40, 43, 61
Lu, C.Y., 110, 140, 168, 169, 172–173 Mancardi, D., 153–154, 162, 163
Lu, S.P., 11 Mandhana, N., 174
Lucchini, S., 142–143 Mangan, J.A., 194
Ludden, P.W., 177 Manickam, N., 6–12
240 AUTHOR INDEX
Manley, K., 53–54 Matsumoto, A., 159, 175–176

Mann, B.E., 166, 186 Matthies, D., 11
Mann, M., 108 May, I., 41–42
Mansfield, T.A., 108–109 Mayilraj, S., 6–12
March, A.W., 6–12 Mcarthur, J.V., 6–12
Marchese, J.N., 186–187 Mccarthy, K.D., 19
Marcotte, E.M., 104, 111–112, 113, 115, McClelland, M., 156–157
118, 120 McCleverty, J.A., 156
Marcus, A.K., 12, 38 McCormack, D.G., 156–157
Margalit, H., 112 Mccormick, M.L., 60
Mariani, F., 194 McDermott, J., 121
Mariani, S. McDevitt, D., 194
Marino, M., 140 McDonald, J.F., 144
Maritan, A., 122 McEwan, A.G., 175–176
Marletta, M.A., 139–140, 144, 146, 178 McFall-Ngai, M.J., 178
Maroney, M.J., 31, 32 Mcguinness, L.R., 11
Maroulis, S., 162 Mcinerney, M.J., 6–12, 21–22, 57–58
Marsden, G., 172–173, 193 Mcintire, W.S., 27
Marshall, H.E., 159, 175–176 McKenzie, A., 108–109
Marshall, I.P.G., 6–12 McKenzie, H., 141–142
Marsili, E., 19, 37, 39 Mckinley, J.P., 6–12
Martens-Habbena, W., 146, 177, 178 Mcknight, D.M., 21
Marteyn, B., 146 McKnight, G.M., 141–142
Marti, M., 169 Mclean, J.E., 11
Martin, D., 113, 122 Mclean, J.S., 30
Martin, H.G., 23 McLean, S., 171, 179, 180, 185, 186,
Martin, L.A., 140, 157–158, 167–171 190–191
Martinez, C.L.T., 162 Mclellan, M., 11
Martins, G., 11 McLendon, W.J., 153–154, 162, 163
Martr, M.A., 169 McLeod, C.W., 186
Marutzky, S., 6–12 McNicholl-Kennedy, J., 195
Marvin, D., 115 McQuade, L.E., 143–145, 146, 181–182
Marzioch, M., 111–113 Meckenstock, R.U., 6–12, 14, 18,
Marzorati, M., 12 21, 22
Mas, J., 38, 62 Megson, I.L., 158–159
Mason, R.P., 57 Mehanna, M., 61–62
Mason, S.P. Mehta, S., 108–109, 121, 156–157
Massjouni, N., 121–122 Mehta, T., 32, 33–34
Mastroeni, P., 140, 147, 178–179 Meier, H., 11
Mathur, E.J. Meijerink, B.B., 6–12
Matin, A., 145–147 Meilhoc, E., 177
AUTHOR INDEX 241
Melhado, J., 62 Miller, M.R., 158–159

Melhuish, C., 18–19 Miller, W., 119–120
Melo, A.M.P., 169, 176, 177 Millo, D., 39
Meltzer, M.S., 139–140 Millott, S., 139–140
Membrillo-Hernández, J., 140, 167–168, Mills, C.E., 171
181–182, 190–191 Mills, H.J., 6–12
Mendez, L.S.S., 166 Mills, P.C., 169, 174–175
Mer, G., 181 Minguez, P., 114
Merchant, K., 181 Mintzlaff, S., 108–109
Merenyi, G., 147 Miranda, K., 162
Merino, E., 17 Miranda, K.M., 149–150, 153–154,
Merrill, M.D., 19, 61–62 162, 163
Merx, M.W., 169 Mirsky, V.M., 40
Mesbah, N.M., 11 Misik, V., 164
Messenger, S.L., 169, 175–176 Mitchell, A., 162
Messier, V., 110 Mitchell, J.B., 147, 156
Mester, T., 24, 29, 30, 31, 32–34, 43 Mitra, S., 119–120
Meszaros, E., 11 Mittal, R., 143
Méthé, B.A., 6–12, 13, 17, 18, 23, 30, 31, Mittelberger, T., 25–26
32, 34, 43–44, 45–46, 48, 49, 50–51, Miya, A., 19
52–54 Miyakoda, H., 11
Metzler, D.R., 6–12 Miyamoto, Y., 165
Meurette, G., 146 Miyazaki, K., 165
Meyer, C., 105 Mizuno, T., 48
Meyer, K. Mo, K., 14
Meyer-Klaucke, W., 177 Moenne-Loccoz, P., 152, 183
Michalsen, M.M., 11 Moens, L., 168
Michaud-Soret, I., 181 Mohammed, A.K., 186–187
Michiels, J., 50–51 Mohan, V.P., 185, 194
Michnik, S.W., 110 Mohanram, K., 105
Michon, A.M., 111–113 Mohanty, S.R., 11
Middha, S., 120 Moir, J.W.B., 169, 174–176, 185, 195
Mikoulinskaia, O., 23 Mokhiber, R., 6–12
Milani, M., 172–173 Molina, M.M., 110
Milano, V., 11 Monack, D., 166
Miletto, M., 52–53 Monahan, I.M., 194
Millar, A., 111–112 Moncada, S., 139–140, 164
Millar, K., 6–12 Mondal, D., 57
Miller, A.R. Monk, C.E., 159–160, 169, 172–173,
Miller, L.D., 55–56 182, 186, 193, 195
Miller, M.J., 150–151 Montecchi-Palazzi, L., 119
242 AUTHOR INDEX
Moore, C.M., 113, 122, 185, 193 Muñoz, F.J., 38

Moore, L., 111–112 Muñoz-Berbel, X., 38
Moore, S., 119 Murad, F., 139–140
Moreau, M., 143–145 Murali, T.M., 113, 121–122
Moreira, D. Murase, J., 11
Morel, F.M.M., 57 Murgida, D.H., 177
Moreno- Vivian, C., 177 Murillo, F.M., 17
Morgado, L., 30–31, 32–33, 34 Murphy, W.J., 142
Moris, M., 50–51 Murray, A.W., 102
Morishita, R., 106 Murray, D.B., 162
Morita, H., 144 Musat, F., 6–12
Morita, M., 11, 22, 61 Musat, N., 6–12
Mormile, M.R., 11 Muta, S., 108–109, 112
Morris, S.L., 146, 160 Mutus, B., 160
Moschetti, T., 173 Myers, C.L., 114, 120
Mosher, J.J., 55–56 Myers, E.W., 119–120
Moskow, J.J., 108–109
Motterlini, R., 166, 186 N’guessan, A.L., 11, 47, 53–54
Moult, J., 120 N’guessan, L.A., 6–12, 17, 42
Mouser, P.J., 6–12, 47–48, 53–54, 55–56 Nabieva, E., 122
Moutakki, H., 57–58 Nacy, C.A., 139–140
Movahedzadeh, F., 183 Naganuma, T., 11
Mowat, C.G., 30–31 Nagarajan, H., 7
Moy, R.H., 51 Nagasawa, H.T., 159, 163
Moyles, D., 30 Nagata, Y., 144
Mozzarelli, A., 172 Nagy, W., 115
Mrazek, J., 45 Naik, R.R., 17
Mukai, M., 140, 168, 172–173 Najar, F.Z.
Mukhi, N., 115 Nakai, K., 121–122
Mukhopadhyay, A., 186–187, 196 Nakamura, R., 12
Mukhopadhyay, P., 166, 174, 182–183, Nakano, M.M., 183, 185, 193
185, 188–189, 190 Narayan, V., 108–109
Mulholland, F., 159–160, 169, 172–173, Nardini, M., 172–173
186, 193, 195 Nariai, N., 121
Muller, A., 11 Nathan, C., 140, 147, 179, 180,
Muller, J., 114 190–191, 194
Müller, M., 25–26 Nathan, C.F., 139–140, 146, 196–197
Mulrooney, S.B., 47 Navani, N.K., 140
Mulvey, M.A., 185, 190 Nealson, K.H., 11
Munhoz, D.C., 179, 180 Neill, S.J., 143
Muniz- Rascado, L. Neimann, J., 193
AUTHOR INDEX 243
Nelson, D.C., 57 Notley-McRobb, L., 184–185

Nelson, K.E., 30 Nowlin, D.M., 103
Nelson, W., 30 Nudelman, R., 181
Neph, S., 51 Nudler, E., 143–145
Nerenberg, R., 12 Nunez, C., 17, 33, 36–37, 44,
Nerz, C., 185, 194 47–48
Nesbo, C.L., 24 Núñez, C., 17
Netto, L.E., 179, 180 Nunoshiba, T., 157–158
Neu, M.P., 20
Neumann, T., 186–187 O’Brien, S., 185
Neunlist, M.R., 146 O’Donnell, C.A., 164
Nevin, K.P., 6–12, 13–14, 17, 18–19, O’Driscoll, F., 141–142
20–21, 23, 24–25, 29, 31, 32–34, 35, O’Neil, R.A., 11, 12, 14, 17, 18, 33–34,
36, 37, 47, 49, 50–51, 52–54, 57–59, 47, 52–54
60, 62 O’Neill, K.R., 6–12
Newby, D.T., 6–12 O’Shae, E.K., 105
Newcomer, D., 11 Oddou, J.L., 181
Newman, M.E., 113, 122 Oesterheld, M., 119
Newton, T.W., 20 Ofran, Y., 119
Ng, H.Y. Ogles, D.M., 6–12
Nicholson, R., 139 Ogusucu, R., 179, 180
Nicholson, S., 140 Oh, H.M., 14
Nicoll, J.S., 11, 12, 37, 39 Oh, H.Y., 144
Nicora, C.D., 6–12, 52–53 Ohno, H., 185, 194
Nielsen, J., 184–185 Oinn, T., 115
Nielson, J., 104–105, 107 Okamoto, M., 106
Nierzwicki-Bauer, S.A., 11 Oliver, S.G., 110, 111–112, 121
Niggemyer, A.M., 6–12 Olmo-Mira, M.F., 177
Nijenhuis, I., 11 Olson, J.S., 140, 168–171
Nikitovic, D., 176 Oltvai, Z.N., 107, 111–112
Nikolausz, M., 11 Olvera, L., 49
Nishizawa, M., 108–109, 112 Ondrias, K., 164
Nixon, J. Ono, I., 106
Nobre, L.S., 169, 176 Ono, N., 106
Nogueira, R., 11 Ono, T., 121–122
Noll, M., 11 Orchard, S., 119
Nord, G.L., 6 Orem, W.H., 61
NØrnberg, P., 6–12 Orii, Y., 171, 174–175, 190–191
Noronha-Dutra, A., 164 Orloff, A.L., 18–19, 23
North, N.N., 11 Ornatowski, W., 185, 194
Norton, J.M., 11 Orshonsky, L., 34
244 AUTHOR INDEX
Orshonsky, V., 31, 34 Park, Y.S., 17

Ortiz de Montellano, P.R., 183 Parker, D.S., 61–62
Ortiz-Bernad, I., 6–12, 20 Parker, K., 186–187
Oshima, K., 11 Parkinson, C., 139–140
Ostendorf, D.W., 57–58 Parry, R.J., 144
Otsuka, S., 11 Parsons, J.R., 6–12
Ou, J., 62 Parthasarathy, S., 149–150
Ouellett, H., 172–173 Partridge, J.D., 186
Ouellett, Y., 140, 172–173 Pasquali, P., 172
Oughtred, R. Patel, B.A., 143–145
Ouzounis, C.A. Patel, R.P., 146, 163
Overton, T.W., 177 Pathak, A., 6–12
Owen, A.B., 121 Pathania, R., 140
Ozawa, R., 104, 108–110, 112 Patil, S.A., 39
Paton, J.C., 175–176
Pacelli, R., 147 Patwardhan, R., 120
Pacheco, I., 42 Paul, A., 11
Padalko, E., 142 Paulsen, I.T., 30
Paige, J.S., 187–188 Pawaria, S., 169
Paley, S. Pawloski, J.R., 160
Palmer, P.D., 20 Peacock, A.D., 6–12, 42
Palmer, R.M.J., 139–140 Pearson, B.M., 159–160, 169, 172–173,
Palsson, B.O., 7, 13, 17, 104–105, 186, 193, 195
106–107, 120 Pederick, R.L., 57
Palumbo, A.V., 6–12, 55–56 Peduzzi, R., 6–12
Pan, C.J., 165 Peduzzi, S., 6–12
Panchenko, A.R., 108 Pellegrini, M., 104, 115, 120
Pancost, R.D., 57 Pelletier, E., 11
Panda, K., 144 Peluffo, G., 178–179
Pandey, A., 108 Penn, C.W., 172–173, 193
Pant, K., 143–145 Peplies, J., 11
Panzera, A., 39 Peralta-Gil, M.
Paolocci, N., 153–154, 156–157, 162 Percent, S.F., 11
Papoutsakis, E.T., 105 Peregrín-Alvarez, J.M., 111–112
Parameswaran, P., 12, 38 Pereira, I.A., 42
Park, C., 103 Perez- Polo, J.R., 196–197
Park, H., 48 Perez-Martin, J., 47
Park, I., 13 Perez-Rueda, E., 44
Park, J.H., 107 Perfect, J.R., 139–140
Park, M., 6–12 Perpetua, L.A., 14, 17, 24–25, 33–34,
Park, S.F., 169, 172–173, 174–175, 193 47–48
AUTHOR INDEX 245
Persson, K., 169 Podar, M., 55–56

Pervitsky, D., 154–155 Pogoutse, O., 122
Pesce, A., 172–173 Pokkuluri, P.R., 30–31, 34, 48
Pessanha, M., 30–31, 34, 48 Pol, A., 6–12
Petel, F., 109, 112 Poljakovic, M., 169
Peters, F., 25–27 Pollina, R.B., 39
Petersen, C.A., 146 Polya, D.A., 11, 57
Petillot, Y., 30 Pommerenke, B., 11
Petrecca, R., 62 Poock, S.R., 174–175
Petrie, L., 11 Poole, L.B., 179
Petrucci, P., 172 Poole, R.K., 138–139, 140, 141,
Petzke, L.M., 11 145–147, 158, 159–160, 166, 167–171,
Peyton, B.M. 169, 172–173, 174–176, 179, 180,
Pfiffner, S., 6–12 181–183, 184, 185, 186, 188–189,
Phanse, S., 122 190–191, 193, 195
Pharkya, P., 17 Porcella, S.F., 195
Phelps, T.J., 55–56 Porron-Sotelo, L.
Philippi, S., 113, 117 Porwollik, S., 156–157
Phillips, E.J.P., 6–12, 13, 15, 16, 18, 19, Postier, B.L., 15–17, 34, 47–48, 62
21, 23, 41–42, 60 Poté, J., 6–12
Phillips, R., 166 Potrykus, K., 50–51
Phizicky, E.M., 108 Potter, A.J., 175–176
Phung, N.T., 12 Potter, L., 141
Piacenza, L., 178–179 Potter, S., 140
Pillai, S., 186–187 Poulos, T.L., 152–153
Pilobello, K., 12 Prakash, O., 6–12
Pin, C., 195 Prakash, P., 104–105
Pineyro, M.D., 180 Prasadarao, N.V., 143
Pino, C., 177 Price, M.S., 146
Pinter, R.Y., 107 Prince, E.K., 20
Piper, M.D.W., 184–185 Prince, R., 160–161
Pirt, S.J., 184 Privalle, C.T., 146
Pistoia, C., 172 Pronk, J.T., 184–185
Pittman, M.S., 169, 174–175 Pruesse, E., 11
Pixton, D.A., 146 Pryor, W.A., 151–152
Plaia, T.W., 14 Pu, S., 111–112
PlewczyNski, D., 120 Puljic, M., 17, 31, 49
Plotze, M., 62 Pullan, S.T., 169, 174, 185, 188–189,
Plugge, C.M., 21–22 190–191, 195
Pochart, P., 108–109 Punna, T., 111–112
Pocock, M.R., 115, 118 Purkayastha, S., 186–187
246 AUTHOR INDEX
Pursnani, S., 142–143, 166, 172 Regan, J.M., 12, 18–19

Puzrin, A.M., 62 Regenberg, B., 184–185
Regev, A., 105
Qian, J., 106 Reguera, G., 17, 29, 31, 34, 36, 37, 38,
Qian, X., 31, 32–34, 35, 41–42 39, 49
Qiu, Y., 7, 13, 17, 29, 44 Reguly, T.
Qu, W., 146, 186, 196–197 Reiche, M., 11
Qu, Y., 17, 31, 49 Reilly, S.D., 20
Quast, C., 11 Reimers, C.E., 12
Que, L., 175–176 Reinhardt, R., 27
Quintana, L., 20 Remor, M., 111–113
Qureshi-Emili, A., 108–109 Ren, Z., 18–19
Renshaw, J.C., 20–21, 41–42
Rabaey, K., 12 Resch, C.T., 6–12
Rabus, R., 27 Reski, R., 26–27
Rader, G., 12 Rettori, D., 179, 180
Radi, R., 178–179, 180, 189, 191 Reverdy, C., 109, 112
Radinovic, S., 110 Rhee, K.Y., 146, 196–197
Raghunathan, A., 106–107 Rhee, S.Y., 108
Rain, J.C., 108–109, 112 Rhen, M., 142–143
Raines, K.W., 178–179 Ribeiro, D.C., 11
Rajagopala, S.V., 112 Rice, D.W., 169, 172
Rajamohan, G., 169 Richard, C., 172–173
Ramachandran, N., 160 Richardson, A.R., 185
Ramani, A.K., 111–112 Richardson, D.J., 141, 169, 174–175
Ramos-Hernandez, N., 11 Richardson, P.M., 6–12
Rassaf, T., 169 Richnow, H.H., 6–12, 57–58, 61
Rattray, M., 185–186 Richter, D.C., 119–120
Raval, P., 142 Richter, H., 18–19, 37, 39, 40, 55–56
Rawlings, C., 113, 117 Richter, L., 11
Raychaudhuri, S., 106 Rick, J.M., 111–113
Razavi, H.M., 156–157 Ridnour, L.A., 153–154, 156, 162, 163
Rea, H., 196–197 Riggs, A.D.
Read, R.C., 140, 169, 171, 174, 175–176, Riggs, A.F., 140, 168–171
182–183, 185, 186, 189, 190–191 Riley, M.
Rebeil, R., 195 Rinck, A., 146, 196–197
Redding, A.M., 186–187, 196 Ringelberg, D., 6–12
Redfern, B., 163 Rison, S.C.G., 183
Reed, J.L., 105, 106–107 Risso, C., 7, 12, 13, 23, 53–54, 55–56
Reed, M.A., 110 Rist, B., 187–188
Reed, S.C., 11 Ritalahti, K.M., 11
AUTHOR INDEX 247
Rittmann, B.E., 12, 38 Rotello, V.M., 19, 29

Rivera, C.G., 121–122 Roth, A., 51, 114
Riviere, D., 11 Roth, F.P., 113, 122
Robello, C., 180 Rothermich, M., 36–37
Roberts, R.E., 186 Rousseau, D.L., 140
Robertson, D.L., 111 Rowland, H.A.L., 11, 57
Robson, D., 114, 120 Rowley, G., 169, 174–175
Rocks, S.S., 57 Rual, J.F., 108–109
Roden, E.E., 11, 18, 21, 24, 57 Rubbo, H., 149–150
Rodgers, K.R., 181 Ruby, E.G., 146, 177, 178
Rodionov, D.A., 182–183 Rudd, M.A., 160
Rodriguez, C.E., 163 Rudolph, C.
Rodriguez-Valera, F., 11 Rudy, G., 6–12
Roe, B.A. Ruegg, A., 113, 117
Roesch, W., 11 Rugor, A., 25–26
Roest, K., 6–12 Ruppin, E., 107
Rogers, N.J., 162, 171 Rusin, P.A., 20
Rogstam, A., 185, 193 Rusnak, F., 179, 180, 190–191
Rokhlenko, O., 107 Russell, S.W., 142
Roldan, M.D., 177 Russo, A., 165
Röling, W.F.M., 6–12, 57–58 Rust, J.M.
Rollefson, J.B., 13, 19, 36 Ryckelynck, N., 12
Rolli-Derkinderen, M., 146
Romao, C.V., 177 Saavedra, J.E., 153–154, 162, 163
Romby, P., 51 Sabate, N., 62
Romilly, C., 51 Sachdeva, S., 183
Rooney-Varga, J.N., 6–12, 57–58 Saier, M.
Root, D.E., 122 Saini, H.K., 120
Root, P., 160 Saini, H.S., 6–12
Rosadini, C.V., 166, 177 Sakaki, Y., 104, 108–110, 112
Rosazza, J.P.N., 144 Sakata, R., 144
Rosen, G.M., 178–179 Sakuma, I., 139–140
Rosenberg, M., 194 Salama, J.J., 119
Rosenfeld, B., 104–105 Salamov, A.A.
Rosenzweig, R., 6–12 Salgardo, H.
Ross, P.L., 186–187 Salgueiro, C.A., 30–31, 32–33, 34, 48
Rosse, C., 116–117 Salminen, J.M., 6–12
Rosso, K.M., 30 Salter, L.A., 111, 113, 122
Rost, B., 119, 120 Salwinsky, L., 119
Rotaru, A.E., 11 Salzman, A.L., 140, 157–158, 167–171
Rotaru, C., 11 Samudrala, R., 121
248 AUTHOR INDEX
Samuni, A., 147, 165 Schink, B., 15–17, 21–22, 34, 46

Sanchez-Rosario, Y., 11 Schlag, S., 185, 194
Sandler, S.J., 13 Schleinitz, K.M., 27, 28
Sanford, R.A., 11, 20, 60 Schloss, P.D.
Sanguinetti, G., 185–186, 190–191 Schmeling, S., 27
Sansonetti, P.J., 146 Schmidt, G., 186–187
Santos-Zavaleta, A., 44 Schmidt, S.K., 11
Saraiva, I.H., 30–31 Schmidt, T.M., 16
Saraiva, L.M., 169, 174, 176, 177, 185, Schmitz, J., 169
189, 190 Schnappinger, D., 194
Sardiwal, S., 183 Schoenherr, A., 108–109
Sarnighausen, E., 26–27 Schon, T., 169
Sarti, P., 169 Schonafinger, K., 163–164
Sasamoto, K., 165 Schoolnik, G.K., 194
Sato, H., 11 Schrader, J., 169
Sato, K., 165 Schreiber, F., 145–147
Sattath, S., 112 Schröder, U., 12, 39
Sauer, R.T., 47 Schubert, C.J., 6–12
Save, S., 169 Schultz, J., 111–113
Savidge, T.C., 146, 196–197 Schulz, A., 44
Sawers, G., 181–182 Schulz, D., 11
Sawyer, D.T., 149–150 Schumann, W., 44
Sayyar, B., 17 Schuster, S.C., 119–120
Scala, D.J., 11 Schwarz, J., 186–187
Scalfone, N.B., 11 Schwikowski, B., 112, 121
Scandurra, F.M., 169 Scott, C., 146, 158, 181–182
Schachter, V., 109, 112 Scott, D.T., 21
Schadt, C.W., 55–56 Scribner, A.W., 160
Schaefer, J.K., 57 Sebbane, F., 195
Schaefer, S., 6–12 Sedelnikova, S.E., 169, 172
Schaeffer, C., 26–27 Seeliger, S., 34
Schafer, J., 186–187 Segura, D., 17, 23, 46
Scheibe, T.D., 17, 54, 55 Sekiguchi, Y., 12
Scheid, D., 11 Selembo, P.A., 12, 19
Scheidt, W.R., 148 Selig, L., 109, 112
Scheinost, A.C., 6–12 Semprini, L., 6–12
Schena, M., 104 Senger, M., 115
Schenkman, L.R., 108–109 Senko, J.M., 11, 17
Schiffer, M., 30–31, 34, 48 Senoo, K., 11
Schilling, C.H., 17, 55–56, 62 Seo, D.W., 144
Schindler, K. Seringhaus, M., 108
AUTHOR INDEX 249
Setchell, K.D.R., 140, 168–171 Shoemaker, B.A., 108

Seth, A., 160–161 Shoeman, D.W., 159
Sghir, A., 11 Short, J.M.
Sha, X., 163 Shrivastava, S., 55–56
Shafirovich, V., 149–150, 153–154 Siddhanta, U., 142
Shah, M.B., 11 Sieber, J.R., 21–22
Shalon, D., 104 Siegel, D.I., 23
Shames, I., 110 Siegert, M., 61
Shamir, R., 121 Sierro, N.
Shanker, R., 6–12 Sietmann, R., 12
Shao, C., 146, 196–197 Silver, P.A., 110
Shapiro, C., 145–147 Simon, J., 174–175
Shapleigh, J.P., 181–182 Simon, S., 109, 112
Sharan, R., 121, 122 Simonovic, M., 114
Sharma, M.L., 31, 32, 33–34, 36–37, Simplaceanu, V., 145–147
143–145 Singh, K.K., 183
Sharma, V.S., 152–153 Singh, M., 122
Shatalin, K., 143–145 Singh, R.J., 11, 159, 160
Shatalina, Y., 143–145 Singha, B., 6–12
Shay, T., 105 Sitte, J., 6–12
Sheik, C.S. Sittler, T., 122
Shelobolina, E.S., 7, 14, 15–17, 18, 31, Sivaramakrishnan, S., 183
33, 41–42 Sivtsov, A.M., 18
Shepard, W., 30 Skapski, A., 177
Shepherd, M., 169, 172–173, 182, 186, 193 Skinn, B.T., 159
Sherlock, G., 105–106 Skovgaard, M., 120
Shi, J., 181–182 Skrabanek, L., 120
Shi, L., 29 Skusa, A., 113, 117
Shi, X. Slauch, J.M., 178–179
Shiau, S.C., 112 Sleep, B.E., 14
Shiloh, M.U., 140, 147 Smagghe, B.J., 173
Shimizu, S., 11 Smedley, P.L., 57
Shimkets, L.J., 6–12 Smets, B.F., 11
Shimomura, T., 19 Smith, B., 116–117
Shimoyama, T., 12 Smith, C.
Shin, S., 106–107 Smith, D.L., 11, 110
Shinoda, Y., 57–58 Smith, H.K., 159–160, 169, 172–173,
Shinyashiki, M., 163, 165 182, 186, 193, 195
Shiono, T., 11 Smith, J.A., 7, 12
Shirota, F.N., 159, 163 Smith, L., 141–142
Shiva, S., 145–147, 169 Smith, L.J., 146
250 AUTHOR INDEX
Smith, L.M., 141–142 Stafford, S.L., 175–176, 196–197

Smith, R.D., 6–12, 30 Stahl, D.A., 146, 177, 178
Smith, S., 11 Stamler, J.S., 140, 146, 159, 160, 166,
Smithgal, A.N., 6–12 167–171, 169, 175–176, 181, 191–192
Smolders, A.J.P., 6–12 Stams, A.J., 24–25
Snel, B., 110, 111–112 Stams, A.J.M., 21–22
Snider, R.M., 39 Stanbury, D.M., 150–151
Snitkin, E.S., 121 Stancevic, B., 187–188
Snoeyenbos-West, O.L., 6–12, 62 Stanley, A., 30
Snowman, A.M., 142 Stapleton, M.R., 146
Snyder, M., 106–107, 110, 121 Stark, C.
Snyder, S.H., 142, 175–176, 187 Stark, M., 114
Solana-Lira, H. Starodubtseva, M., 144
Sole, V.A., 20 Stauffer, G., 189
Soman, K., 196–197 Stcuki, J.W., 6–12
Sompornpisut, P., 30 Stecher, H.A., 12
Song, B., 11 Steefel, C.I., 11
Song, O., 104 Stehr, M., 180
Sontineni, S., 17 Stein, L.Y., 11
Sorensen, D.L., 11, 14 Steinhoff, H.J., 169
Sorenson, K.S. Jr., 11 Steinmetz, L.M., 111–112
Sosnik, J., 42 Stelzl, U., 108–109
Sowell, P., 11 Stephen, C.S., 36
Spain, A.M., 11 Stephen, J.R., 6–12
Sparks, J.P., 143–145 Steppi, S., 11
Specht, M., 113, 117 Stern, M.K., 179, 180, 190–191
Spellman, P.T., 105–106 Stevanin, T.A., 169, 174, 185, 189,
Spencer, F., 111–112 190–191
Spiro, S., 169, 171, 174–175, 178, Stevanin, T.M., 140, 169, 171, 175–176,
181–183, 195 182–183
Spitznagel, J.K. Jr., 11 Stevens, B.M., 11
Spormann, A.M., 6–12 Stewart, V., 140, 167–168,
Spring, S., 6–12 181–182
Springael, D., 61 Steyn, A.J.C., 146
Sprinzak, E., 112 Stine, O.C., 11
Squadrito, G.L., 151–152 Stockwell, B.R., 122
Squier, T.C., 29 Stoeckert, C.J., 121
Srikanth, S., 19, 39 Stoesser, R., 11
Srinivasan, M., 108–109 Stoker, N.G., 183
Staats, M., 6–12, 57–58 Stoll, S., 144
Stabb, E.V., 178 Stolz, J.F., 6, 7, 17
AUTHOR INDEX 251
Storz, G., 51, 166, 174, 182–183, 185, Szabo, C., 157–158, 191
188–189, 190 Szklarczyk, D., 114
Straub, K.L., 7, 34, 35
Strid, S., 169 Tae, B., 12
Strietelmeier, B.A., 20 Tainer, J.A., 144
Stroedicke, M., 108–109 Tait, C.D., 20
Stroeher,U.H., 175–176 Takagi, T., 121–122
Strous, M., 6–12 Takahashi, N., 11
Strunk, O., 11 Takahata, Y., 57–58
Strycharz, S.M., 24–25, 39, 41, 42, 60 Takai, K., 11
Stubner, S., 11 Taki, K., 11
Stuehr, D.J., 139–140, 142, Talaue, M.T., 159–160
143–145, 193 Tamir, S., 157–158
Stults, J.R., 6–12 Tan, H., 42
Stumpflen, V., 119 Tanabe, M., 118
Sturdevant, D.E., 195 Tanaka, H., 144
Sublette, K.L., 6–12 Tang, X., 158–159
Sucher, N.J., 191–192 Tang, Y.J., 23
Sudarsan, N., 51 Tanigami, A., 121–122
Sudhamsu, J., 143–145 Tannenbaum, S.R., 157–158
Sukharnikov, L.O. Tao, T., 144
Sulc, F., 154–155 Tao, W., 193
Sullivan, S.A., 31, 50–51 Tarantino, M., 172
Summers, Z.A., 15–17, 46 Tarassov, K., 110
Summers, Z.M., 11, 13, 22, 32, 40–41, Tarricone, C., 172
46, 53, 62 Tashiro, K., 108–109, 112
Sun, F., 108–109, 121 Taubert, J., 113, 117
Sun, G.X., 11 Taubert, M., 26–27
Sun, J., 17, 19, 55–56, 62 Tauxe, R.V., 193
Sun, Y.D., 146, 186, 196–197 Tavares, A.F.N., 169, 176
Sundin, B., 108–109 Tavernier, J., 109–110
Sung, H.C., 12 Taylor, P., 111–112
Sung, W.K., 113, 121–122 Tecder-Unal, M., 178–179
Sung, Y., 7, 11, 14, 20 Teh, E.H., 12
Suseno, S., 144 Teichmann, S.A.
Sutton, S.R., 11 Teixeira, M., 42, 174, 176, 177, 185,
Suzuki, M.T., 57 189, 190
Suzuki, T., 140, 168–171 Tejero, J., 142
Svensson, L., 169 Tempst, P., 146, 175–176, 187, 196–197
Switzer, C.H., 149–150, 154, 156 Tender, L., 39
Syutsubo, K. Tender, L.M., 12, 13–14, 37, 62
252 AUTHOR INDEX
Tender, L.R., 12 Tran, H.T., 17, 29, 45, 50

Terada, A., 11 Tran, L.M., 160, 189
Testerman, T., 189 Traylor, T.G., 152–153
Thauer, R.K., 23 Tremblay, P.L., 13, 33, 36, 41, 46–47, 51
Theesfeld, C.L., 114, 120 Trent, J.T., 173
Thieffry, D., 105 Tribollet, B., 38
Thiele, I., 105 Tringe, S.G.
Thomas, B.C., 11 Triplett, E.W., 11
Thomas, D.D., 150–151, 156 Tront, J.M., 62
Thomas, L., 153–154, 162, 163 Troyanskaya, O.G., 114, 120, 121
Thomas, R., 105 Trujillo, M., 180
Thomas, S.H., 20 Tsai, P., 178–179
Thompson, A., 142–143, 186–187 Tsokos, M., 147
Thompson, M.J., 104, 115, 120 Tsushima, I.
Thomson, A.J., 146, 158, 181–182 Tu, Z., 121
Thomson, M.J., 169, 185, 195 Tucker, N.P., 174, 181, 182–183
Thorne, S.H., 145–147 Tufan, H., 178–179
Throup, J.P., 194 Tuominen, M.T., 29, 43
Tiede, D.M., 34 Turano, P., 30
Tiedje, J.M., 14 Turecek, F., 187–188
Tien, M., 36 Turner, S.M., 172–173, 193
Tikuisis, A.P., 111–112 Tyagi, J.S., 183
Timm, J., 108–109 Tyce, R.C., 62
Timmins, G.S., 185, 194
Tiso, M., 145–147 Ueda, S., 165
Tocchetti, C.G., 153–154, 162, 163 Ueki, T., 13–14, 17, 28, 34, 40–41,
Todorovic, S., 177 42–43, 44, 46, 49, 50, 52–53
Tokunaga, T.K., 6–12 Ueno, Y., 12
Toledo, J.C., 146 Uetz, P., 108–109, 111–112, 121
Tollaksen, S.L., 17, 30 Ujihara, T., 11
Tombor, B., 107 Ulitsky, I., 121
Tompa, M., 51 Ullmann, A.K., 26–27
Tong, A., 108–109 Underhill, B., 23
Tonolla, M., 6–12 Urrutia, M.M., 11, 24
Tonzetich, Z.J., 146, 181–182 Urvil, P., 196–197
Toone, E.J., 191–192
Tor, J.M., 20–21 Vallone, B., 173
Torres, C.I., 12, 38 Van Auken, K.
Torres, C.L., 153–154, 162, 163 Van Breukelen, B.M., 6–12
Tosques, I.E., 181–182 Van der Heyden, J., 110
Tramontano, A., 120 Van Dien, S.J., 23
AUTHOR INDEX 253
Van Dongen, B.E., 57 Vielotte, N., 162

Van Dorsselaer, A., 25–27 Vigués, N., 38
van Eldik, R., 146 Villanueva, L., 15–17, 53
Van Harmelen, M., 6–12 Vine, C.E., 177
Van Nostrand, J.D., 11 Vinogradov, S.N., 168
Van Praagh, C.V., 20 Virtaneva, K., 195
Van Stempvoort, D.R., 6–12, 57–58 Visca, P., 172–173
Van Verseveld, H.W., 6–12 Visconti, P., 42
van Wonderen, J.H., 169 Vishnivetskaya, T., 12
Vandekerckhove, J., 110 Vitkup, D., 107
Vanderleyden, J., 50–51 Vodyanitskii, Y.N., 18
Vanderroost, N., 110 Vogel, J., 110
Vanengelen, M.R. Vogt, C., 6–12, 27, 57–58
Vanfleteren, J.R., 168 Voldman, A., 142–143, 166, 172
Vanmaekelbergh, D., 40 Volpe, E., 194
Vanpraagh, C.G., 14 Von Bergen, M., 25–27
Vargas, M., 29 von Mering, C., 110, 111–112, 114
Varma, A., 104–105 von Wachenfeldt, C., 185, 193
Varshney, G.C., 169 Voordeckers, J.W., 41
Vasquez, A., 122 Voskuil, M.I., 194
Vaughan, D.J., 11, 57 Vouille, V., 171
Vazquez, F., 31 Voytek, M.A., 61
Vazquez, J.R., 25–26 Vrionis, H.A., 6–12, 14, 17, 20, 47,
Vazquez-Torres, A., 140, 147, 156–157, 52–53
178–179 Vu-Thi-Thu, L., 60
Vecoli, C., 156 Vugman, N.V., 162
Veeramani, B., 107
Venkatesan, K., 108–109 Wach, M.J., 144
Venkateswaran, A., 55–56 Wadley, R.B., 162
Venketaraman, V., 159–160 Wagner, A., 111, 113, 122
Verberkmoes, N.C., 6–12 Wagner, R.C., 12
Verhee, A., 110 Wainwright, L.M., 140, 168, 172–173,
Verrier, P., 113, 117 193
Verschuur, G.L., 63–64 Walhout, A.J., 108–109
Versichele, M., 12 Walsh, R.C., 146
Verstraete, W., 12 Wan, J.M., 6–12
Verwegen, K., 6–12 Wang, G., 110
Vescio, P.A., 11 Wang, J.X., 51
Vespigniani, A., 122 Wang, P.G., 158–159
Vicente, J.B., 174, 176, 185, 189, 190 Wang, S.C., 47
Vidal, M., 108–109, 110 Wang, T., 185, 193
254 AUTHOR INDEX
Wang, W., 146, 177, 178 Wells, J.M., 195

Wang, X. Wen, Z., 158–159
Wang, X.D., 145–147 Wengenack, N.L., 179, 180, 190–191
Wang, X.J., 11 Werner, J.J., 11, 61
Wang, Y., 11 Westerhoff, H.V., 6–12
Wang, Y.H., 172–173 Westram, R., 11
Wang, Y.L., 146, 177, 178 Whaling, P., 12
Wang, Z.Q., 11, 106–107, 143–145 White, D.C., 6–12, 13
Wanger, G., 6–12 White, H.K., 12
Wanker, E.E., 108–109 Wiatrowski, H.A., 20–21
Wanner, G. Wible, A., 114, 120
Wansbrough-Jones, M., 166 Widdel, F., 11, 27
Ward, J.A., 7, 11 Widman, P.K., 6, 41–42
Ward, J.E., 15–17, 32, 33–34, 47, 52–53 Widom, J., 143–145
Ward, N., 30 Wietzorrek, A., 46–47
Ward, P.M., 20–21 Wiktorowicz, J.E., 196–197
Ward, T.E., 18–19 Wildi, W., 6–12
Wasserman, G., 142–143, 166, 172 Wilkes, H., 11
Watanabe, K., 12, 57–58 Wilkins, M.J., 6–12, 50, 52–54
Waters, L.S., 51 Wilkinson, D.J., 107
Watson, D.B., 6–12 Wilks, M., 166
Weaver, J., 178–179 Williams, H.N., 11
Weber, H., 162 Williams, K.H., 6–12, 42, 52–54, 59–60,
Weber, K.A., 11, 24 62
Webster, D.A., 172, 173 Williamson, B., 186–187
Webster, J., 17, 31, 32 Wilneff, J., 178
Weerawarana, S., 115 Wilson, D.
Wegener, H.C., 193 Wilson, I.D., 143
Wei, C.C., 143–145 Wilson, J.L., 166, 169, 172
Weicker, S., 156–157 Wilton, R., 48
Weinberg, Z., 51 Wincott, P.L., 57
Weinrauch, Y., 142–143, 166, 172 Winderl, C., 6–12, 57–58
Weir, M.P., 108 Winfield, J., 19
Weis, R.M., 17, 50 Wink, D.A., 147, 149–151, 153–154,
Weiss, V. 156, 162
Weissenbach, J., 11 Wipat, A., 115, 118
Weissman, J.S., 111–112 Wischgoll, S., 26–27, 49–50
Weitzberg, E., 140–142 Wishnok, J.S., 139–140, 157–158
Weizmann, C., 104–105 Wittenberg, B.A., 140, 168, 172–173
Weldon, J.M., 11, 57 Wittenberg, J.B., 140, 168, 172–173
Wellenreuther, G., 177 Woebken, D., 11
AUTHOR INDEX 255
Wojcik, J., 109, 112, 113, 122 Yabuki, S., 12

Wolf, M., 21 Yadhukumar., 11
Wolfe, C.J., 121 Yakuwa, H., 19
Wolfe, M.T., 177 Yamamoto, K., 108–109, 112
Wolting, C., 111–112 Yamamoto, M., 11
Wong, L., 113, 121–122 Yamazawa, A., 12
Wong, P.S., 122, 159 Yan, B., 17, 31, 44
Wong, S.M.S., 166, 177 Yang, J., 11
Wood, S.J., 34, 48 Yang, L., 11, 111–112
Wood, S.R., 185, 194 Yang, M., 12
Woodard, T.L., 14, 17, 18–19, 32–33, 37, Yang, S., 110
47, 52–54, 57–58, 60, 62 Yang, T.H., 17, 55
Woodward, J.C., 21, 41–42, 58 Yang, W., 122
Woodward, J.J., 144 Yang, X., 31, 34
Woollard, P., 119 Yang, Z.K., 11, 55–56
Worm, U., 108–109 Yanina, S., 30
Woudstra, M., 30 Yansura, D.G.
Wrzeszczynskia, K.O., 119 Yao, Z., 51
Wu, C.Q., 142 Yarasheski, K., 11
Wu, D., 30 Yates, J.R., 17
Wu, G.H., 140, 146, 158, 168, 169, 171, Ye, R.W., 185, 193
172, 181–182, 190–191 Yeates, T.O., 104, 115, 120
Wu, H., 112 Yee, C.H., 186
Wu, L.Y., 11 Yeger-Lotem, E., 107
Wu, M., 30 Yeh, S.R., 140, 168, 169, 172–173
Wu, Q., 20 Yi, H., 13–14, 18–19, 39, 62
Wu, S., 12 Yidd, S.P., 175–176
Wu, W.M., 6–12 Yin, L.F., 143–145
Wu, X., 158–159 Yoochatchaval, W.
Yoon, J.W., 144
Xavier, A.V., 42 Yoon, P.S., 139–140
Xenarios, I. Yoshida, H.
Xia, Y., 108, 121 Yoshida, M., 104, 108–110, 112, 165
Xian, M., 158–159 Yoshida, N., 11
Xing, D.F., 12 Yoshikawa, H., 144
Xu, A., 15–17 Yoshitake, J., 178–179
Xu, D., 121 Young, L.Y., 11
Xu, G.Q., 187–188 Young, N.D., 7, 13, 15–17
Xu, H., 11, 31 Young, T., 193
Xu, M.Y., 11 Youssef, N.H.
Xu, Y.S., 160–161, 189 Yu, H., 106, 108, 111–112, 121, 186, 196
256 AUTHOR INDEX
Yuan, Y., 121 Zhang, P., 62

Yukl, E.T., 152, 183 Zhang, Q., 146, 196–197
Yun, H., 107, 120 Zhang, R.D., 12
Yun, J., 34, 52–54, 57–58 Zhang, S., 11, 63
Yuretich, R., 18 Zhang, T., 7, 25, 58–59, 63
Zhang, X., 27
Zaa, C.L.Y., 11 Zhang, Y.H., 11, 12, 161
Zachara, J.M., 29 Zhao, H., 108, 113
Zagyanskiy, Y., 34 Zhao, J., 54
Zai, A., 160 Zhao, R., 156–157
Zaki, M.H., 178–179 Zhao, X.C., 111–112
Zamarro, M.T., 49 Zheng, M., 166, 174, 182–183, 185,
Zamble, D.B., 47 188–189, 190
Zamora, R., 162 Zheng, W., 57
Zanoni, M., 39 Zheng, Y., 113, 122
Zappacosta, F., 194 Zheng, Z., 11
Zeier, J., 171 Zhong, G., 111–112
Zeiger, S., 11 Zhou, J.Z., 11, 17
Zeng, J.P., 186, 196 Zhou, Y.B., 146, 186, 196–197
Zeng, M., 169, 175–176 Zhu, G.F., 185, 194
Zengler, K., 7, 13, 17, 29 Zhu, H., 110, 140, 168–171
Zenkner, M., 108–109 Zhu, L., 178–179
Zgur-Bertok, D., 47–48 Zhu, W., 17, 30
Zhang, A., 111–112 Zhu, Y.G., 11
Zhang, C.P., 12 Zhuang, K., 55–56
Zhang, H.S., 12 Ziv-Ukelson, M., 107
Zhang, J.L., 142 Zopfi, J., 6–12
Zhang, K., 108–109, 121 Zuber, U., 44
Zhang, M., 18–19, 105–106
Subject Index
Note: The page numbers taken from figures and tables are given in italics.
A E
Affinity purification techniques, Elemental sulfur, 21
111–112 Extracellular electron transfer,
Angeli’s salt, 162–163 Geobacter
cytochromes
B capacitor role, 42–43
G. sulfurreducens, 34
Bacterial NOS, 120–122
MacA, 33
Bioremediation, Geobacter
OmcB, 31, 32
aromatic hydrocarbons, 57–59
OmcE, 32
chlorinated contaminants, 60
OmcZ, 32–33
uranium and metals, 59–60
OmpB and OmpC, 33–34
Bottom-Up Genome-Scale (BUGS)
PgcA, 33
modeling, 54–56
PpcA, 30–31
electrodes, 37–40
C extracellular electron acceptor, 41–42
Chemotaxis, 50 Fe(III) Oxide, 34–37
Co(III)-EDTA, 20 microbial nanowires, 29–30
Cytochromes syntrophy, 40–41
capacitor role, 42–43
G. sulfurreducens, 34 F
MacA, 33
Flavohemoglobins, 168–171
OmcB, 31, 32
OmcE, 32
OmcZ, 32–33 G
OmpB and OmpC, 33–34 Geobacter species
PgcA, 33 aromatic compound usage, 14
PpcA, 30–31 biogeochemical impacts, 56–57
bioremediation
D aromatic hydrocarbons, 57–59
chlorinated contaminants, 60
Deltaproteobacteria, 16f
uranium and metals, 59–60
258 SUBJECT INDEX
Geobacter species (Continued) microbial nanowires, 29–30

distribution and abundance syntrophy, 40–41
bioremediation, 6–12 Geobacter sulfurreducens
culture isolates, 6–12, 7t DL-1 strain, 13
Fe(III) reduction, 6 KN400 strain, 13–14
low maintenance energy G. metallireducens, 13
requirement, 6 methane production, 61
molecular analysis, 6–12 microbial electrosynthesis, 62–63
16S rRNA gene sequences, 11f microbial fuel cell systems, 61–63
electron acceptors phylogeny and genomic resources
anaerobic growth, 17 Deltaproteobacteria, 15, 16f
Co(III)-EDTA, 20 Pelobacter species, 15–17
electrodes, 18–19 subsurface clade, 17
elemental sulfur, 21 pure culture isolates, 13, 14
Fe(III), 18 regulatory networks
humic substances, 21 chemotaxis, 50
metal ions, 19–20 nucleotide-based second
Mn(IV) oxides, 19 messenger, 50–51
soluble U(IV), 20 Sigma factors, 43–45
Tc(VII) reduction, 20 transcription factors, 45–48
V(V), 20 two-component system, 48–50
electron donors subsurface clade I, 14–15
acetate, 23–24 subsurface clade II, 15
aromatic compounds, 25–28 syntrophy, 21–22
G. bemidjiensis, 24
hydrogenase, 24 H
long-chain fatty acid metabolism, 24
HNO donors
environmental systems biology
Ace1, 163
BUGS modeling, 54–56
acyloxy nitroso compounds, 163
transcriptomics and proteomics,
Angeli’s salt, 162
52–54
cyanamide, 163
extracellular electron transfer
Piloty’s acid, 162
cytochromes and multicopper
Humic substances, 21
proteins, 30–34
Hydrogenase, 24
cytochromes, capacitor role, 42–43
electrodes, 37–40
extracellular electron acceptor, I
41–42 Isobaric tags for relative and absolute
Fe(III) Oxide, 34–37 quantitation (iTRAQ), 186–187
SUBJECT INDEX 259
L affinity purification techniques,

Long-chain fatty acid metabolism, 24 111–112, 111t
HTP data, 112
physical interactions, 108
M protein chips, 110
Methane production, 61 protein-fragment complementation
Microarrays, 105–106 assay, 110
Microbial electrosynthesis, 62 spoke and matrix model, 111
Microbial fuel cell systems, 61–63 TAP-MS data, 110
Microbial nanowires, 29–30 yeast-two hybrid, 108–109, 109t
Microbial proteins Mn(IV) oxides, 19
functional analysis, 119–122
functional interaction networks, 113
computational access, 115 N
DIP databas, 115–116 Nitric oxide (NO). See also Reactive
edge weights, 118 nitrogen species
genome-scale datasets, 113 antimicrobial defenses, 139
geome-wide functional networks, biological chemistry, 148–156
115 biological utility, 148–149
HTP data, 112–113 characteristics, 138–139
network construction and E. coli response
integration, 119 E. coli K-12, 190
Ondex5 tool, 117, 118 E. coli MG1655, 190
Open Biological and Biomedical GSNO-mediated cytotoxicity, 189
Ontologies project4, 116–117 GSNO tolerance, 188–189
sources, 113–114 nitrosative stresses, 191–192
STRING3, 114 Uropathogenic Escherichia coli,
metabolic and regulatory networks 190
cross-species comparison, 107 free radical nature, 149–150
DNA microarrays, 104 historical perspective, 139–140
genome-scale networks, 104t HNO
microarrays, 105–106 ferric myoglobin, 154–155
RNA sequencing approach, nitroxyl reaction, 155
106–107 thiols, 154, 155f
in silico gene knockouts, 107 Lewis dot depiction, 116f
single type protein interaction, 104 metal centers, 152–153
transcriptional networks, 106 microbial cultures, 184–185
yeast-two-hybrid screen, 104 microbial sensing
methane metabolism, E. coli K-12 Fnr, 181–182
MG1655, 103 NorR, 182
protein–protein interaction networks NsrR, 182–183
260 SUBJECT INDEX
Nitric oxide (NO). See also Reactive nitrite, 166

nitrogen species (Continued) nitrogen dioxide
microbial symbioses, 177–178 dimerization and hydrolysis,
molecular orbitals, 149f 164–165
nitrosation, 150–151 nitronyl nitroxides, 165
nitrosative species (see Nitrosative NO
species) donors, 158
NO2, 150 laboratory method, 157–158
N2O3, 150 mixtures/cocktails, 158
probabilistic state space modeling, sodium nitroprusside, 158–159
185–186 organic nitrate esters, 162
proteomics peroxynitrite, 163–164
biotin switch method, 187 S-nitrosothiols, 159–161
2D gel electrophoresis, 186 Nitrosative species
isobaric tags for relative and nitrate-derived stress, 141–142
absolute quantitation, 186–187 nitrite reduction and denitrification,
S-nitrosothiol capture, 187–188 140–141
redox chemistry, 148–151 NO synthases and nitrosative burst
reductases bacterial NOS, 143–145, 144t
E. coli NorV and NorW, 174 combined reactive species
periplasmic cytochrome c nitrite response, 147
reductase, 174–175 non-NOS sources, NO, 145–147
reduction, 153–154 NOS Family, 142–143
SNO, detoxification and metabolism, Nucleotide-based second messenger,
175–176 50–51
superoxide anion
NO3- and carbonate, 151–152 O
peroxynitrite, 151–152
Ondex5 tool, 117
tolerance, 176–177
Open Biological and Biomedical
transcriptomics, 185
Ontologies project4, 116–117
Nitrogen oxide
donors
DETA-NO, 156–157 P
experimental consideration, Pelobacter species, 15–17
156–157 Peroxynitrite, 163–164
HNO donors E. coli response, 190–191
Ace1, 163 microbial responses
acyloxy nitroso compounds, 163 catalase-peroxidases, 179
Angeli’s salt, 162 dihydrorhodamine assay, 178–179
cyanamide, 163 peroxiredoxins, 179
Piloty’s acid, 162 toxicity, 178–179
SUBJECT INDEX 261
second-order rate constants, 180t chemotaxis, 50

Piloty’s acid, 162 nucleotide-based second messenger,
Protein-fragment complementation 50–51
assay, 110 Sigma factors, 43–45
Protein–protein interaction (PPI) transcription factors, 45–48
networks two-component system, 48–50
affinity purification techniques, RNA sequencing approach, 106–107
111–112, 111t
HTP data, 112 S
physical interactions, 108 Sigma factors
protein chips, 110 FliA homolog, 45
protein-fragment complementation RpoD homolog, 44
assay, 110 RpoE homolog, 45
spoke and matrix model, 111 RpoH, 44
TAP-MS data, 110 RpoN, 45
yeast-two hybrid, 108–109, 109t RpoS homolog, 44
Proteomics, 52–54, 195–197 Single-domain 3/3 globins, 172
biotin switch method, 187 S-nitrosothiols (SNOs), 150–151
2D gel electrophoresis, 186 STRING3, 114
isobaric tags for relative and absolute Superoxide dismutase (SOD), 164
quantitation, 186–187
S-nitrosothiol capture, 187–188 T
Transcription factors
R BgeR, 48
Reactive nitrogen species (RNS) Fur, 47
B. subtilis, 193 GSU0514, 46
C. jejuni, 193 GSU1771, 46–47
microbial defenses, 167–168 HgtR, 46
microbial globins LexA homologs, 47–48
flavohemoglobins, 168–171 NikR family, 47
single-domain 3/3 globins, 172 Transcriptomics, 52–54
truncated globins, 172–173 Truncated globins, 172–173
M. tuberculosis, 194 Two-component system, 48–50
N. meningitidis, 195
P. aeruginosa, 194 U
S. aureus, 194 Uropathogenic Escherichia coli, 190
targets of, 166
Yersinia pestis, 195 Y
Regulatory networks, Geobacter
Yeast-two-hybrid screen, 104
species

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Academic Press is an imprint of Elsevier

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First edition 2011

Copyright # 2011 Elsevier Ltd. All rights reserved.

No part of this publication may be reproduced, stored in a retrieval system or

For information on all Academic Press publications

Printed and bound in the United Kingdom

MUKTAK AKLUJKAR, Department of Microbiology and Environmental Bio-

LESLEY A.H. BOWMAN, Department of Molecular Biology and

JESSICA E. BUTLER, Department of Microbiology and Environmental Bio-

KELLY A. FLANAGAN, Department of Microbiology and Environmental

ASHLEY E. FRANKS, Department of Microbiology and Environmental Bio-

JON M. FUKUTO, Department of Chemistry, Sonoma State University,

LUDOVIC GILOTEAUX, Department of Microbiology and Environmental

J.S. HALLINAN, School of Computing Science, Newcastle University,

DAWN E. HOLMES, Department of Microbiology and Environmental Bio-

K. JAMES, School of Computing Science, Newcastle University, Newcastle,

DEREK R. LOVLEY, Department of Microbiology and Environmental Bio-

NIKHIL S. MALVANKAR, Department of Microbiology and Environmental

SAMANTHA MCLEAN, Department of Molecular Biology and Biotechnology,

KELLY P. NEVIN, Department of Microbiology and Environmental Biotech-

ROBERTO ORELLANA, Department of Microbiology and Environmental

ROBERT K. POOLE, Department of Molecular Biology and Biotechnology,

CARLA RISSO, Department of Microbiology and Environmental Biotechnol-

AMELIA-ELENA ROTARU, Department of Microbiology and Environmental

PRAVIN M. SHRESTHA, Department of Microbiology and Environmental

TOSHIYUKI UEKI, Department of Microbiology and Environmental Biotech-

A. WIPAT, School of Computing Science, Newcastle University, Newcastle,

TIAN ZHANG, Department of Microbiology and Environmental Biotechnol-

Department of Microbiology and Environmental Biotechnology Center, University of

Geobacter species specialize in making electrical contacts with extracellular

ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 59 Copyright # 2011 by Elsevier Ltd.

appears to be an important process in anaerobic wastewater digesters.

7.3. Model for Extracellular Electron Transfer to Fe(III) Oxide . . . . . . . 34

Geobacter species represent a rare example of a genus of microorganisms

magnetite from microbial Fe(III) reduction, (4) anaerobic oxidation of an

Dominance in Current Metallic-like

1985 1990 1995 2000 2005 2010 2015

U(VI) Humics as Electrode-

Figure 1 Time line of important discoveries associated with Geobacter species.

Figure 2 Publications and citations each year with Geobacter as a topic

2. DISTRIBUTION AND ABUNDANCE OF GEOBACTER

The hallmark physiological capability of Geobacter species is their ability

Genome Electron donors Other Optimal

Genome Electron donors Other Optimal

Geobacter Contaminated Ac, Buty, Bzo, PCIO, Fe AQDS, 30 Coates et al.

Genome Electron donors Other Optimal

“Geobacter Acetate- Ac, Act, Bzo, PCIO, 25–30 Holmes

Figure 3 Neighbor-joining tree showing the phylogenetic relationship within

3. BRIEF DESCRIPTION OF GEOBACTER SPECIES

A significant number of pure culture isolates of Geobacter species are

important physiological differences. In addition to producing more current

G. plymouthensis. Some of these isolates were recovered from surface

4. PHYLOGENY AND GENOMIC RESOURCES

Geobacter species are in the family Geobacteraceae, which is within the

Geobacter species can use a diversity of electron acceptors to support anaer-

At the circumneutral pH in most environments in which Geobacter thrive, Fe

After Fe(III) reduction, electron transfer to electrodes is probably the

5.3. Other Extracellular Electron Acceptors

Fe(III)-reducing microorganisms reduced Au(III), the Geobacter species

5.4. Other Microorganisms—Syntrophy

In the absence of alternative electron acceptors, Geobacter species can

6.1. Acetate, Other Fatty Acids, Hydrogen, Electrodes,

The universal ability of all Geobacter species to oxidize acetate with Fe