Prescott's Microbiology 12th Edition

Joanne Willey
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 twelfth edition

Prescott’s Microbiology

Joanne M. Willey
HOFSTRA UNIVERSITY

Kathleen M. Sandman
Dorothy H. Wood
E D U C AT I O N & T R A I N I N G S Y S T E M S
I N T E R N AT I O N A L

wil88396_fm_i-xxiv.indd 1 06/12/21 3:01 PM

 PRESCOTT’S MICROBIOLOGY

Published by McGraw Hill LLC, 1325 Avenue of the Americas, New York, NY 10019. Copyright © 2023 by McGraw
Hill LLC. All rights reserved. Printed in the United States of America. No part of this publication may be reproduced or
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ISBN 978-1-265-12303-1
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 Brief Contents

About the Authors xiv 24 Fungi 518

25 Viruses 532

Part One Introduction to Microbiology

1 The Evolution of Microorganisms and
Microbiology 1
2 Microscopy 23
3 Bacterial Cell Structure 44
4 Archaeal Cell Structure 80
5 Eukaryotic Cell Structure 91
6 Viruses and Other Acellular Infectious Agents
Part Six Ecology and Symbiosis
26 Exploring Microbes in Ecosystems 556
27 Microbial Interactions 571
28 Biogeochemical Cycling and Global Climate
Change 584
29 Microorganisms in Marine and Freshwater
109 Ecosystems 599
30 Microorganisms in Terrestrial Ecosystems 617

Part Two Microbial Nutrition, Growth, and Control

7 Bacterial and Archaeal Growth 127 Part Seven Pathogenicity and Host Response
8 Control of Microorganisms in the Environment 162 31 Innate Host Resistance 636
9 Antimicrobial Chemotherapy 179 32 Adaptive Immunity 663
33 The Microbe-Human Ecosystem 696
34 Infection and Pathogenicity 714
Part Three Microbial Metabolism
10 Introduction to Metabolism 201
11 Catabolism: Energy Release and Conservation 219 Part Eight M
 icrobial Diseases, Detection, and
12 Anabolism: The Use of Energy in Biosynthesis 255 Their Control
35 Epidemiology and Public Health Microbiology 730
36 Clinical Microbiology and Immunology 750
Part Four Microbial Molecular Biology and Genetics 37 Human Diseases Caused by Viruses and Prions 769
13 Bacterial Genome Replication and Expression 277 38 Human Diseases Caused by Bacteria 801
14 Regulation of Cellular Processes 310 39 Human Diseases Caused by Fungi and Protists 845
15 Eukaryotic and Archaeal Genome Replication and
Expression 336
16 Mechanisms of Genetic Variation 353 Part Nine Applied Microbiology
17 Microbial DNA Technologies 377 40 Microbiology of Food 871
18 Microbial Genomics 397 41 Biotechnology and Industrial Microbiology 887
42 Applied Environmental Microbiology 898

Part Five The Diversity of the Microbial World Appendix 1  Review of the Chemistry of
A
19 Archaea 419 Biological Molecules A-1
20 Nonproteobacterial Gram-Negative Bacteria 433 Appendix 2 Common Metabolic Pathways A-9
21 Proteobacteria 455 Appendix 3 Microorganism Pronunciation Guide A-17
22 Gram-Positive Bacteria 479
Glossary G-1
23 Protists 498
Index I-1

iii

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Remote proctoring and browser-locking capabilities, hosted by
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vii
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 A Modern Approach to Microbiology

Evolution as a Framework
Introduced immediately in chapter 1 and used as an overarching Brief Contents
theme throughout, evolution helps unite microbiological con-
cepts and provides a framework upon which students can build
their knowledge.
About the Authors xiv 24 Fungi 518
25 Viruses 532

Part One Introduction to Microbiology

1 The Evolution of Microorganisms and Part Six Ecology and Symbiosis
Microbiology 1
26 Exploring Microbes in Ecosystems 556
2 Microscopy 23
27 Microbial Interactions 571
3 Bacterial Cell Structure 44
28 Biogeochemical Cycling and Global Climate

An Introduction to the Entire Microbial World

4 Archaeal Cell Structure 80 Change 584
5 Eukaryotic Cell Structure 91 29 Microorganisms in Marine and Freshwater
6 Viruses and Other Acellular Infectious Agents 109 Ecosystems 599

Covered in chapters 3–6, separate chapters on the structure and Part Two Microbial Nutrition, Growth, and Control
30 Microorganisms in Terrestrial Ecosystems 617

function of bacteria and archaea are followed by the discussion 7 Bacterial and Archaeal Growth 127
8 Control of Microorganisms in the Environment 162
Part Seven Pathogenicity and Host Response
31 Innate Host Resistance 636
of eukaryotic cells and viruses. 9 Antimicrobial Chemotherapy 179 32
33
Adaptive Immunity 663
The Microbe-Human Ecosystem 696
34 Infection and Pathogenicity 714
Part Three Microbial Metabolism
10 Introduction to Metabolism 201
11 Catabolism: Energy Release and Conservation 219 Part Eight Microbial Diseases, Detection, and
12 Anabolism: The Use of Energy in Biosynthesis 255 Their Control
35 Epidemiology and Public Health Microbiology 730

Broad Coverage of Microbial Ecology Part Four Microbial Molecular Biology and Genetics
13 Bacterial Genome Replication and Expression 277
36
37
Clinical Microbiology and Immunology 750
Human Diseases Caused by Viruses and Prions 769
38 Human Diseases Caused by Bacteria 801
The importance and multidisciplinary nature of microbial ecology 14 Regulation of Cellular Processes 310
15 Eukaryotic and Archaeal Genome Replication and
39 Human Diseases Caused by Fungi and Protists 845

are demonstrated by content that ranges from global climate Expression 336
16 Mechanisms of Genetic Variation 353 Part Nine Applied Microbiology

change to the human microbiome. 17 Microbial DNA Technologies 377

18 Microbial Genomics 397
40 Microbiology of Food 871
41 Biotechnology and Industrial Microbiology 887
42 Applied Environmental Microbiology 898
34.4 Damage to the Host 725
Part Five The Diversity of the Microbial World Appendix 1 A Review of the Chemistry of
19 Archaea 419 Biological Molecules A-1
20 Nonproteobacterial Gram-Negative Bacteria 433 Appendix 2 Common Metabolic Pathways A-9
the gene for the toxin that causes toxic shock syndrome. The move- Exterior
pH ~ 7.0 21 Proteobacteria 455 Appendix 3 Microorganism Pronunciation Guide A-17
ment of genes from one bacterial cell to another is discussed in 1
Neutral pH causes
chapter 16, while the detection of pathogenicity islands within a dissociation of B
A Binding 22 Gram-Positive Bacteria 479
Plasma site from binding site Glossary G-1
microbe’s genome is explained in chapter 18. Microbes use membrane
Dimeric B B 23 Protists 498
exotoxin Index I-1
mechanisms other than mutation to create genetic variability A
(section 16.4); Comparative genomics (section 18.7) B
iii
Clathrin released Clathrin-coated pit
Toxins Are Biological Poisons 2
A toxin (Latin toxicum, poison) is a substance that disrupts A
B
the normal metabolism of host cells with deleterious effects
on the host. Toxigenicity is the pathogen’s ability to produce
B

A Coated 4
toxins, and intoxications are diseases that result from a spe- pH ~ 7.0 B vesicle
B component is
cific toxin produced by the pathogen. Intoxication diseases do Uncoated A 3 recycled to cell
not require the presence of the actively growing pathogen— vesicle surface.
B pH ~ 5.0
just its toxin, as in the case of botulism. Bacteria produce two (endosome)
B
structurally different types of toxins—exotoxins (proteins)
A A
and endotoxin (lipopolysaccharide)—and some fungi produce Uncoupling vesicle (CURL)
potent mycotoxins. 5 separation of A from
A
B component
NAD+ Nicotinamide
Exotoxins
Molecular Microbiology and Immunology
EF-2 ADP-Ribosyl-EF-2
Exotoxins are soluble, heat-labile proteins (inactivated at 60°
to 80°C) usually released into host tissues as the bacterial path- Inhibition of protein synthesis: Cell death
ogen metabolizes. Often exotoxins travel from the site of infec-

The twelfth edition includes updates on genetics, biotechnology,

(a)
tion to other body tissues or target cells, where they exert their
effects (figure 34.7). Exotoxins are often encoded by genes
carried on plasmids or prophages within certain bacteria. They Cytoplasmic
are associated with specific diseases and often are named for
the disease they produce (e.g., the diphtheria toxin). Some are
Pore-
forming
exotoxin Pore
contents out
(low osmolarity) genomics and metagenomics, immunology, and the human mi-
crobiome. An up-to-date discussion of immunity, with enhanced
among the most lethal substances known—toxic in nanogram- Extracellular space protein
per-kilogram of body weight concentrations (e.g., the botuli-
num toxin).
Exotoxins exert their biological activity by specific mecha-
nisms and are grouped by either mechanism of action (e.g., a detail between innate and adaptive linkages, helps students grasp
the complexity and specificity of immune responses. The micro-
cytotoxin kills cells) or their protein structure. A common struc-
tural type is the AB toxin, which gets its name from the fact that Cytoplasm H 2O
it has two distinct toxin subunit types, an “A” (or active) compo- Exotoxin forms Swelling, host
nent and a “B” (or binding) component. The B portion of the
toxin binds to a host-cell receptor and triggers endocytosis. Thus
(b)
pore in membrane. cell lysis, death
(high osmolarity) biome and its impact on human homeostasis is introduced in
the B component determines the cell type infected. Once inter-
nalized, the A and B components dissociate from one another.
The A component, which functions as an enzyme, is now free to
Figure 34.7 Two Examples of Exotoxin Mechanisms. (a) 1. The B
subunit of the diphtheria AB cytotoxin binds to the cell receptor in the
chapter 33, The Microbe-Human Ecosystem.
catalyze a reaction that will cause host cell toxicity (figure 34.7a). clathrin-coated pit. 2. The intact toxin is endocytosed. 3. The pH change within
AB toxins act on cells by different mechanisms. Many A subu- the endosome causes the subunits to separate. An endosome in which this
nits have ADP ribosylation activity, which catalyzes the transfer separation occurs is sometimes called a compartment of uncoupling of
548 CHAPTER 25 | Viruses
of adenosine diphosphate and ribose moieties of host NAD+ to receptor and ligand (CURL). 4. The B subunit is then recycled. 5. The active
target host molecules (see figure 10.7). Certain exotoxins that are toxin (A) subunit blocks protein synthesis by adding an ADP-ribose group to
grouped by mechanism of action have the ability to disrupt mem- host elongation factor-2 (EF-2), which leads to cell death. (b) Here, a channel-
branes. Examples of this type of functional exotoxin are the forming (pore-forming) toxin such as α-hemolysin produced by S. aureus,
channel (pore)-forming toxins (figure 34.7b). They destabilize inserts itself into the host-cell membrane, forming a channel (or pore). Multiple
Endoplasmic
membrane integrity so that the host cell lyses. The general prop- membrane pores result in an osmolarity shift, as water enters the cell and
reticulum
erties of several exotoxins are presented in table 34.4. As cytoplasmic contents move out. The resulting effect of this toxin is cell lysis. Structural Exocytosis
2 proteins 4
M
E N 3
1 S

SARS-CoV-2 and the Impact of COVID-19 Double membrane Positive-strand Cytoplasmic

Students are introduced to the virology of SARS-CoV-2 and the vesicle RNA genome vesicles

pathobiology of COVID-19 in chapters 25 and 37, respectively. Figure 25.19 Replication and Viral Exit. (1) Viral replication occurs in cytoplasmic double membrane vesicles that shelter the genome and concentrate
ribonucleotide precursors. (2) Structural proteins are synthesized on the endoplasmic reticulum. (3) The nucleocapsid protein in complex with the positive-strand

Throughout the text, the relevance of concepts to the pandemic RNA genome is engulfed by membranes to form cytoplasmic vesicles (4) that exit the cell by exocytosis.

are noted as easy-to-find text boxes. Assembly of mature SARS-CoV-2 virions begins when the
positive-strand RNA genome joins with nucleocapsid (N) pro-
teins to form ribonucleoprotein complexes (figure 25.19). The
spike (S), envelope (E), and membrane (M) proteins are retained
in the endoplasmic reticulum (ER) membranes. The nucleocap-
sid transits from the ER, and the M protein coordinates envelope
viii assembly. E and S proteins are inserted in the membrane, and E
mediates membrane curvature around the nucleocapsid. A host
cell protease, furin, cleaves the S protein as it is inserted in the
membrane. This promotes more rapid entry into the next host
cell. Mature virions accumulate in cytoplasmic vesicles and exit
by exocytosis.
Tobacco Mosaic Virus
Most plant viruses are RNA viruses, and of these, positive-strand
RNA viruses are most common. Tobacco mosaic virus (TMV;
family Virgaviridae) is the best-studied positive-strand RNA plant
wil88396_fm_i-xxiv.indd 8
virus. TMV virions are filamentous with coat proteins arranged in
protomers form disks composed of two layers arranged in a
helical spiral. Association of coat protein with TMV RNA be-
gins at an initiation site close to the 3′ end of the genome. The
capsid grows by the addition of protomers, probably as disks, to
the end of the rod. As the rod lengthens, the RNA passes
through a channel in its center and forms a loop at the growing
end. In this way, the RNA easily fits as a spiral into the interior
of the helical capsid.
Multiplication of plant viruses depends on the virus’s abil-
ity to spread throughout the plant. Viruses move to new sites
through the plant vasculature; usually they travel in the phloem.
They can also spread locally in nonvascular tissue. Because
plant cells have tough walls, viruses move from cell to cell
through plasmodesmata. These are slender bridges of cytoplas-
mic material, including extensions of the ER. Plasmodesmata
extend through holes in cell walls to join adjacent cells. Viral
movement proteins are required for transfer through the plas-
modesmata. Movement proteins are associated with ER mem- 06/12/21 3:02 PM
 A Modern Approach to Microbiology

390 CHAPTER 17 | Microbial DNA Technologies

Cas9 nuclease
21st-Century Microbiology
gRNA

Prescott’s Microbiology leads the way with text devoted to

CRISPR genome engineering, global climate change, and micro-
DNA:RNA hybrid

Genomic DNA PAM

Cas9 nuclease cuts

both DNA strands.
bial fuel cells. For more, see chapters 17, 28, and 42.

The double-strand break is repaired The double-strand break is repaired by

by nonhomologous end joining. homologous recombination with donor DNA.

Figure 17.12 Genome Editing with Cas9 Nuclease. Hybridization between the guide RNA (gRNA) and the chromosome activates the nuclease activity of
Cas9. PAM, protospacer adjacent motif.
MICRO INQUIRY How could you assemble the donor DNA molecule for homologous recombination?
Chemistry) and the other by Feng Zhang, sought to adapt Cas9
for genome editing. In this process, genomic DNA can be di-
rectly modified and the procedures are general enough to be
used for any cell into which DNA can be introduced and ex-
pressed. Responses to viral infection (section 14.6)
Like restriction enzymes, Cas9 is an endonuclease that cuts
both strands of a target DNA. However, unlike restriction en-
zymes, which recognize four to eight base pairs through con-
tacts between the DNA and the enzyme active site, Cas9 is a
ribonucleoprotein consisting of a polypeptide and a guide RNA
(gRNA). Recognition of target DNA for cleavage occurs by hy-
bridization of about 20 bases between the gRNA and its com-
plementary DNA sequence in the genome (figure 17.12). A
second short series of bases, the protospacer adjacent motif
(PAM), is located next to the hybridizing region on the opposite
DNA strand.
In microbes, the CRISPR locus is the source of the gRNA
(see figure 14.26), and the Cas9 nuclease protects the cell from
viral attack. Sequences in the CRISPR locus derive primarily
from mobile genetic elements (bacteriophage and plasmids), so
the Cas9 nuclease in a microbial cell specifically targets in-
vading DNA for destruction. The extreme specificity con-
ferred by the gRNA is the key to genome editing because each
20-base target sequence almost certainly occurs only once in
any given genome. In contrast, a restriction enzyme that recog-
nizes a few nucleotides will cut the genome, on average, every
few thousand bases.
Donor DNA
Metagenomics and the Human Microbiome
Expanded coverage of metagenomics and its importance in
understanding the role of microbes in all environments and in
exploring symbionts of invertebrates is threaded throughout the
text. Chapter 33, The Microbe-Human Ecosystem, explores the
Cas9 enzymes can be engineered to carry gRNAs with spec-
ified nucleotide sequences, thereby programming the recogni- human microbiome and its role in health and disease.
tion sequence for the nuclease. The gRNA directs Cas9 to
hybridize with a defined site in a genome, making it the most
precise mechanism available for targeting and cutting DNA. In
eukaryotes, all of which lack a CRISPR/Cas system, the editing
process begins by introducing the two components of the mature
Cas9 endonuclease, the apoenzyme and the gRNA, to the host
cells. These molecules may be added directly, or they may be
added as cloned DNA regulated by an inducible promoter. In the
latter case, upon induction, the Cas9-gRNA complex assembles
Laboratory Safety
and performs its DNA cutting function.
Figure 17.12 illustrates how Cas9 recognizes and hydrolyzes Reflecting recommendations from the Centers for Disease Con-
a specific DNA sequence. A portion of the gRNA protrudes from
the enzyme, available for hybridization. Upon locating its
complement, the gRNA induces a conformational shift in the
trol and Prevention, along with the American Society for
nuclease (protein) portion of Cas9, which then hydrolyzes phos-
phodiester bonds in both DNA strands, leaving blunt ends. In the ­Microbiology, chapter 36 provides specific guidance for labora-
simplest case, a point mutation occurs as the cell attempts to re-
pair the damage. Some bacteria and archaea and all eukaryotes tory best practices to help instructors provide safe conditions
have a nonhomologous end joining (NHEJ) system to rejoin
the two chromosome pieces. If the repair re-creates the original
sequence, it is again susceptible to Cas9 cutting. As a result, im-
during the teaching of laboratory exercises.
perfect repairs with a deletion or insertion of a few base pairs is
the typical outcome. The consequence is usually a frameshift
mutation in the gene that results in an inactive protein. A limita-
tion to this method is that the outcome differs in each cell.

Special Interest Essays 1.2 Microbes Have Evolved and Diversified for Billions of Years 7

Organized into four themes—Microbial Diversity & Ecology,

Techniques & Applications, Historical Highlights, and Disease— 9.7 Antiprotozoan Drugs 195

MICROBIAL DIVERSITY & ECOLOGY

these focused and interesting essays provide additional insight
Plasmodium reproduction and are effective in eradicating asexual
stages of the protozoan’s life cycle that occur in red blood cells.
into relevant topics.
An essential part of the Plasmodium spp. life cycle is degradation
of hemoglobin. During this process, the microbe polymerizes a
toxic heme metabolite into a nontoxic form, called hemazoin.
Quinine drugs block this process and the toxic metabolite accu-
mulates. During the 2020 COVID-19 pandemic, chloroquine and
DISEASE
9.1 Chloroquine and COVID-19: A Cautionary Tale
In the early days of the COVID-19 pandemic, caused by the
coronavirus SARS-CoV-2, the search to repurpose existing
drugs as a treatment (or even cure) was intense. As the pan-
demic grew, the notion of waiting for new drugs to be devel-
oped and tested seemed untenable. During the SARS
epidemic of 2003, chloroquine and its derivative hydroxy-
chloroquine (box figure) were shown to block replication of
the causative coronavirus, SARS-CoV in vitro. Although the
antiviral mechanism of these drugs remains debated, one
leading theory was that they prevent progression of the viral
life cycle by increasing the pH of the endosome in which the
virus resides upon entering a host cell.
Unfortunately, the desire for a treatment collided with a
lack of understanding of how drug trials must proceed to pro-
tect the public from ineffective and unsafe drugs. By rushing
clinical trials of dubious quality into print, the scientific com-
munity bears some of the blame in the ensuing confusion.
Some of the events surrounding chloroquine and hydroxyclo-
roquine in the first half of 2020 include:
February 4: The journal Cell Research publishes a letter to
the editor by Chinese scientists, including a notable coronavi-
rus expert, suggesting that the antimalarial drug chloroquine
might be effective in combating COVID-19.
February 15: A group of French scientists publishes a simi-
lar editorial in the International Journal of Antimicrobial
Agents.
Mid- to late February: Several news outlets report promis-
ing results from early Chinese clinical trials using chloro-
quine and the more bioavailable hydroxychloroquine in
treating COVID-19 patients.
March 16: Entrepreneur Elon Musk tweets that chloroquine
might be effective in treating COVID-19.
March 20: U.S. President Donald Trump announces chloro-
quine is a “game changer.”
March 23: Patients who take chloroquine and hydroxy-
chloroquine for autoimmune diseases report shortages in
getting these medications, which many have been taking for
years.
March 24: An Arizona man dies and his wife becomes
gravely ill after ingesting chloroquine-containing fish tank
cleaner in an effort to prevent contracting COVID-19.
its derivative hydroxychloroquine garnered much publicity as a
potential treatment or prophylaxis (Disease 9.1).
Another drug, mefloquine, has been found to swell P. falci-
parum food vacuoles, where it may act by forming toxic com-
plexes that damage membranes and other plasmodial components.
Primaquine is active against a dormant form of the protists (hyp-
nozoites) found in the liver, so it prevents relapses of malaria
March 25: The World Health Organization announces a
large, international clinical trial to test the safety and effec-
tiveness of hydroxychloroquine in treating COVID-19.
March 28: The U.S. Food and Drug Administration (FDA)
issues emergency use authorization allowing widespread use
of the drug.
April 10: Reports from frontline health-care workers suggest
the drug is not effective and may be causing adverse events in
some patients.
April 13, April 22: Two clinical trials report that hydroxy-
chloroquine failed to demonstrate any potential benefit in
treating COVID-19 patients.
April 24: The FDA issues a warning against using hydroxy-
chloroquine if not hospitalized.
May 18: Donald Trump announces he is taking hydroxy-
chloroquine prophylactically.
May 26: The medical journal The Lancet publishes a large
clinical trial that concludes hydroxychloroquine is not effec-
tive in treating COVID-19 and increases the risk of death.
June 5: The Lancet retracts the paper published on May 26
due to concerns about data quality.
June 15: The U.S. FDA rescinds its emergency use authoriza-
tion for chloroquine and hydroxychloroquine.
June 20: The NIH closes a clinical trial due to lack of evi-
dence that the drugs are effective in treating COVID-19.
Since that time, chlroroquine and hydroxychloroquine
have largely returned to treating malaria and autoimmune
diseases. Let’s hope we don’t have to wait for the next
pandemic to find out if all the stakeholder agencies learned
anything from the rise and fall of these drugs.
CH3 OH
N
HN
CH3
Cl N
Chloroquine and Hydroxychloroquine. Hydroxychloroquine bears a
hydroxyl group, as shown in purple.
1.1 Hydrothermal Vents: Did Life Begin Under the Sea?
Whether or not early life was RNA-based, one thing is clear:
the origin of life needed energy to synthesize biomolecules.
So, perhaps the most fundamental evolutionary question is
“Where did biomolecules and the energy needed to build
them come from?” Three hypotheses have been suggested.
First, the panspermia theory speculates that meteorites
bombarded our planet, bringing with them other-worldly bio-
molecules. Second, the more familiar primordial soup theory
suggests that organic molecules were spontaneously assem-
bled by an input of energy, such as lightning strikes. The last
theory, which has gained evidence in recent years, hypothe-
sizes that both the energy and the molecules originated in
hydrothermal vents. Let’s explore the hydrothermal vent
theory.
Hydrothermal vents are geothermally active deep-sea
chasms thousands of meters below the surface of the ocean.
Their discovery in 1977 sparked tremendous excitement as im-
ages of entirely new ecosystems with mysterious organisms cap-
tured the attention of scientists and the public (see section 27.2).
These vents pump 400oC sulfide-rich water into cold ambient
water, causing the sulfide to instantly precipitate, so these
chimneylike structures are dubbed “black smokers.” In
2000, scientists made yet another deep-sea discovery with a
different kind of vent system. These are cooler (45–90oC)
and alkaline (pH 9–11). When these waters mix with the
surrounding seawater (pH about 8.0), calcium carbonate pre-
cipitates, forming white chimneys, as seen in the Lost City
vents (box figure).
This pH gradient is critical to the hypothesis that a vent
system, such as Lost City, could be the origin of biomolecules.
As you may have learned when studying mitochondria or
batteries, the separation of positive and negative charges cap-
tures potential energy (remember that energy can’t be cre-
ated). In Lost City vents, the thin walls of the chimneys serve
(tRNA) to construct proteins. Also rRNA itself catalyzes pep-
tide bond formation during protein synthesis. Thus RNA seems
to be well poised for its importance in the development of pro-
teins (figure 1.5). Because RNA and DNA are structurally simi-
lar, RNA could have given rise to double-stranded DNA. It is
suggested that once DNA evolved, it became the storage facility
for genetic information because it provides a more chemically
stable structure. Two other pieces of evidence support the RNA
world hypothesis: the fact that the energy currency of cells,
ATP, is a ribonucleotide and the discovery that RNA can regu-
late gene expression. ATP: the major energy currency of
cells (section 10.2); Riboswitches: effector-mRNA interactions
D.Kelley, University of Washington.
to separate these fluids with as much as a 3-unit pH differ-
ence. The question now being asked is “Was this potential
energy tapped to convert CO2 in seawater to simple carbon-
based molecules, such as amino acids, short hydrocarbons,
and others?”
If the answer is yes, a 2019 study shows that a mixture of
molecules called single-chain amphiphiles (SCAs), which are
simpler versions of more familiar phospholipids, can form
vesicles in hot, alkaline pH seawater that mimics that of Lost
City. Putting this together, we can hypothesize a series of
events that occurred 3.7–4.0 billion years ago. First, the pres-
ence of the pH gradient across geological barriers in the Lost
City drove the formation of random organic molecules, some
of which were SCAs. These SCAs accumulated and formed
vesicles that entrapped fluids preserving the pH gradient.
These vesicles had the energy to test the formation of differ-
ent molecules. Was one of them RNA?
regulate transcription (section 14.3); Translational riboswitches
(section 14.4)
However, the RNA world hypothesis is not without prob-
lems, and more recent experiments suggest the first nucleic ac-
ids may have been a mix of DNA and RNA molecules. Another
area of research also fraught with considerable debate is the
evolution of metabolism. The early Earth was a hot environment
that lacked oxygen. Thus cells that arose there must have been
able to use the available energy sources under these harsh con-
ix
ditions. Today there are heat-loving archaea capable of using
inorganic molecules such as FeS as a source of energy. Some
suggest that this interesting metabolic capability is a remnant of

wil88396_fm_i-xxiv.indd 9 06/12/21 3:02 PM

 Student-Friendly Organization

CHAPTER

38
Human Diseases
Caused by Bacteria

VCG Wilson/Corbis/Getty Images

The Plague Family Tree
A lmost everyone has heard of the Black Death—the outbreak of
plague in Europe from 1347 to 1351 when as much as 40% of
the population died. Caused by the Gram-negative bacterium Yersinia
pestis, it was called Black Death because victims bled under the skin
and experienced necrosis (cell death) of the extremities, which turned
skin purple-black.
For almost as long as the Black Death has been studied, it has been
believed that the outbreak in 1347 and other waves of plague that
followed over the next several hundred years were independent
introductions of Y. pestis from Asia. But new evidence suggests that this
may not be the case. Europe may have reciprocated and provided Asia
with a more virulent Y. pestis strain.
How do you study a disease outbreak that happened over
500 years ago? You pair a team of archaeologists, who can find,
document, and exhume graves, with a team of forensic anthropolo-
gists and bacteriologists, who can extract, sequence, and analyze
Y. pestis DNA from teeth and bones. Studies of plague victims in
Spain, England, Germany, and Russia now give a more complete,
albeit complex, story of Y. pestis evolution. It is broadly agreed that
the story starts about 1320 with a plague outbreak in Mongolia; from
there it spread to China in the 1330s. It came to Europe in 1347 on a
dozen ships that docked in Sicily, where accounts from the time
describe dead sailors covered in black boils. From Sicily, the disease
quickly migrated north, reaching Russia by 1351.
After the plague epidemic receded in the mid-1350s, it reemerged
every few generations for the next three centuries, as depicted in the
adjacent painting by Josse Lieferinxe dated 1497. If Europeans were the
victims of independent waves of Y. pestis from Asia, bacterial DNA from
Micro Focus—Each chapter begins with a real-life story
victims of each outbreak should demonstrate genetically different strains.
However, Y. pestis DNA from victims spanning the fourteenth to seven-
illustrating the relevance of the content covered in the up-
teenth centuries is highly similar, suggesting it was the same strain
that persisted right up until the last outbreak in France in 1722. Some coming text.
scientists believe that the Black Death Y. pestis strain became more
virulent and made its way back to China, where it caused outbreaks in
the nineteenth and twentieth centuries.
Why does anyone even care about a disease that happened so long
ago? COVID-19 has certainly shown us that diseases emerge and move.
We need to understand the past to respond in the future. In this chapter
we learn about bacteria that cause human disease. Although it is good
to remember that only a tiny fraction of bacteria are pathogens, it is wise
to become acquainted with those that adversely impact humans.
Readiness Check: Readiness Check—The introduction to each chapter in-
cludes a skills checklist that defines the prior knowledge stu-
Based on what you have learned previously, you should be able to:
✓ Describe basic bacterial cell biology (sections 3.2–3.10)
✓ Describe the mechanisms of action of the major classes of antibiotics
(section 9.4) dents need to understand the material that follows.
✓ Compare and contrast the general principles of innate and adaptive
immunity (chapter 31; sections 32.1–32.8)
✓ Explain how key pathogens cause infection (chapter 34)
✓ Differentiate between different types of vaccines (section 35.6)
✓ Explain (in general) methods by which pathogenic bacteria are identified
(chapter 36)

801
Comprehension Check—Questions within the narrative of
each chapter help students master section con­cepts before
moving on to other topics.
16.9 Evolution in Action: The Development of Antibiotic Resistance in Bacteria 373

excises incorrectly to generate a specialized transducing parti- RTF

cle, these bacterial genes are most often present (figure 16.24).
Cross-Referenced Notes—In-text references refer Bacteriophage lambda: a temperate bacteriophage
(section 25.2) Specialized Transduction

students to other parts of the book to review. Comprehension Check

1. Describe generalized transduction and how it occurs. What is an
abortive transductant?

Animation Icon—This ­symbol indicates that material 2. What is specialized transduction and how does it come about? IS1
3. How might one tell whether horizontal gene transfer was mediated IS1 Cm
by generalized or specialized transduction?
presented in the text is accompanied by an animation
Km
Sm, Su
4. Why doesn’t a cell lyse after successful transduction with a Amp
temperate phage?
within Instructor Resources in Connect. Create a file 5. How are conjugation, transformation, and transduction similar?
How are they different? Tn4

attachment assignment in Connect to have your students Tn3

view the animation, or post it to your Learning Manage- 16.9 Evolution in Action: The tnpA tnpR bla

Development of Antibiotic
ment System for students. Resistance in Bacteria
After reading this section, you should be able to:
a. Describe an R plasmid and its associated genetic elements
b. Distinguish integrative conjugative elements, transposons, and
Learning Outcomes—Every section in each chapter
conjugative plasmids
c. Describe how genetic elements mobilize portions of chromosomes
begins with a list of content-based activities students
Figure 16.25 An R Plasmid. Plasmid R1 is an R plasmid that contains
the replicative transposon Tn3. Tn3 contains the gene for β-lactamase (bla),
an enzyme that confers resistance to ampicillin (Amp). Note that Tn3 is
inserted into another transposable element, Tn4. Tn4 carries genes that
provide resistance to streptomycin (Sm) and sulfonamide (Su). The R1
plasmid also carries resistance genes for kanamycin (Km) and
chloramphenicol (Cm). The RTF region of R1 codes for proteins needed for
plasmid replication and transfer. Transposase and resolvase are encoded
by tnpA and tnpR, respectively.

should be able to perform after reading.

Within 3 years after the widespread use of penicillin began in the
1940s, penicillin-resistant bacteria were found in clinical speci-
mens. To understand the origin of drug resistance, it is important
to recall that in nature, antibiotic-producing microbes must also
protect themselves from the antibiotics they secrete. In other
words, microbes capable of synthesizing antibiotics must also
Micro Inquiry—Selected figures in every chapter
have mechanisms to resist their effects—otherwise they would
succumb to the effects of the antibiotic. In antibiotic-producing
MICRO INQUIRY As a replicative transposon, what would happen if
Tn3 hopped from this R1 plasmid into a different plasmid?
Frequently a bacterial pathogen is drug resistant because it
has a plasmid bearing one or more resistance genes; such plas-
mids are called R plasmids (resistance plasmids; figure 16.25).
Plasmid-borne resistance genes often code for enzymes that
destroy or modify drugs. Plasmid-associated genes have been
implicated in resistance to antibiotics in most classes. Once a cell

contain probing questions, adding another assess-

microorganisms, the genes that encode resistance proteins are
often referred to as immunity genes. Immunity genes are usually
coordinately regulated with genes that encode antibiotic biosyn-
ment opportunity for the student.
thetic enzymes. It is believed that many genes encoding antibi-
otic resistance in bacteria were captured from antibiotic-producing
bacteria and moved by HGT to nonproducers, giving rise to a
large pool of resistance-encoding genes outside the producing
microorganisms. It is therefore not surprising that in nonproduc-
possesses an R plasmid, the plasmid (or its genes) may be trans-
ferred to other cells quite rapidly through HGT. Because a single
plasmid may carry genes for resistance to several drugs, a patho-
gen population can become resistant to several antibiotics
simultaneously, even though a patient may be treated with only
one drug.
Antibiotic-resistance genes are also located on genetic ele-
ments other than plasmids. Many transposons contain genes for

x ing bacteria, genes for drug resistance may be present on bacte-

rial chromosomes, plasmids, transposons, and other MGEs.
Because they are often found on MGEs, they can easily move
between cells. In addition to acquiring resistance genes by HGT,
some nonproducers can become resistant due to spontaneous
chromosomal mutations. Usually such mutations result in a
change in the drug target; therefore the antibiotic cannot bind
and inhibit growth. There are several mechanisms of drug
resistance (section 9.8)
antibiotic resistance and can move rapidly between plasmids and
through a population (figure 16.25). Tn3, a transposon active in
many Gram-negative bacteria, is of particular note because it is a
replicative transposon. Thus it not only moves but also leaves a
copy of itself in its original location.
Antibiotic-resistance genes also reside in and move with
genetic elements termed integrative conjugative elements
(ICEs) and mobilizable genomic islands (MGIs). These ele-
ments share components and mechanisms with transposons and

wil88396_fm_i-xxiv.indd 10 06/12/21 3:02 PM

 Quorum Sensing by Vibrio spp.
Cell-to-cell communication among bacterial cells often occurs by
the exchange of chemicals often termed signals or signaling mole-
cules. The exchange of signaling molecules is essential to coordi-
nate gene expression in microbial populations. Cell communication
plays an essential role in the regulation of genes whose products are
needed for the establishment of virulence, symbiosis, biofilm pro-
duction, and morphological differentiation in a wide range of bac-
teria. Quorum-sensing mechanisms vary among microbes. Here
we focus on two of the best-studied systems.
The marine bioluminescent bacterium Vibrio fischeri uses
quorum sensing to regulate light production within the light
organ of its squid host; cells produce light only at a high den-
sity (see figure 7.27). Quorum sensing is also used to control
genes whose products are needed for maintenance of the sym-
simple feedback loop is created. Without AHL-activated LuxR,
the luxI gene is transcribed only at basal levels. When V. fischeri
cell density within the squid light organ is low, the small
amounts of AHL produced by the bacterial cells freely diffuse
out of each cell and accumulate in the environment. As cell
density increases, the concentration of AHL outside each cell
eventually exceeds that inside the cell, and the concentration
gradient is reversed (see figure 7.26). As the intracellular AHL
concentration increases, it binds and activates LuxR. LuxR then
increases transcription of luxI and the genes whose products are
needed for bioluminescence (luxCDABEG). Quorum sensing is
often called autoinduction, and the AHL signal is termed the
autoinducer (AI) to reflect the autoregulatory nature of this
system. Quorum Sensing
Another kind of quorum sensing depends on an elaborate

Student-Friendly Organization
biotic relationship between V. fischeri and its host. As a result, phosphorelay signal transduction system. It is found in both
the squid/V. fischeri symbiosis has become an important model Gram-negative and Gram-positive bacteria, and has been best
for understanding animal–bacterial associations. Our focus is studied in the bioluminescent bacterium Vibrio harveyi.
on the regulation of a single operon, that involved with biolu- Unlike V. fischeri, V. harveyi responds to three autoinducer
minescence. However, it should be kept in mind that quorum molecules. As shown in figure 14.24, AI-1, AI-2, and CAI-1 are
sensing regulates multiple genes and operons. Cell-cell secreted by cells, which then use separate proteins called
communication within microbial populations (section 7.6) LuxN, LuxPQ, and CqsS, respectively, to detect their presence.
Quorum sensing in V. fischeri and many other Gram- LuxN, LuxPQ, and CqsS are sensor kinases. At low cell den-
negative bacteria uses an N-acylhomoserine lactone (AHL) sity in the absence of any autoinducer, the three sensor kinases
signal (figure 14.23). Synthesis of this small molecule is cata- autophosphorylate and converge on a single phosphotrans-
lyzed by an enzyme called AHL synthase, the product of the ferase protein called LuxU. LuxU accepts phosphates from any
luxI gene. The luxI gene is subject to positive autoregulation; of the three sensor kinases and then phosphorylates the re-
that is, transcription of luxI increases as AHL accumulates in sponse regulator LuxO. Phosphorylated LuxO in turn activates
the cell. This is accomplished through the transcriptional acti- the transcription of genes encoding several small RNAs that
vator LuxR, which is active only when AHL binds to it. Thus a destabilize luxR mRNA. LuxR is a transcriptional activator of

Vivid Instructional Art—Three-dimensional

renditions and bright, attractive colors enhance Autoinducer synthesis
leads to high-level expression
of lux operon.
Low cell densities:
Basal level
High cell densities:
AI concentration

learning.
transcription of rises; lux operon
lux operon transcription rises.
AI diffuses in
and out of cells.
32.7 Antibodies Bind Specific 3-D Antigens 681

RNA polymerase
V1 V2 V3 Vn J1 J2 J3 J4 C During the process of joining either VJ or
Germ line DNA VDJ sequences in light- and heavy-chainand
Transcription rear-translation
luxR rangement,
luxC respectively,Dthere is an additionalA op- B E G
portunity to introduce genetic diversity. Because
Deletion of
intervening DNA combinatorial joining occurs between different
LuxR-AI LuxI
nucleotides different codons can result in a pro-
binds promoter SAM
V2 J3 J4 C cess known as splice site variability. For exam-
B-cell DNA ple, one VJ splicing event can join the V sequence O
Acyl-ACP O H
CCTCCC with the J sequence TGGTGG in two O
LuxR Transcription
ways: CCTCCC + TGGTGG = CCGTGG, which N
(inactive) codes for proline and tryptophan. Alternatively, H
mRNA (intron in black) the VJ AI AI (autoinducer)
splicing event can eliminate nucleotides, O
Splicing and giving rise to the sequence CCTCGG, which
translation
codes for proline and arginine. Thus the same VJ
Figure 14.23 Quorum Sensing in V. fischeri. The AHL signalingjoining could (AI)
molecule produce polypeptides
diffuses differing
out of the in a cell density is high, AI diffuses back into the cell,
cell; when
Light chain where it binds to and activates the transcriptional regulator LuxR. Active single amino acid. Importantly, because combina-
LuxR then stimulates transcription of the gene coding for AHL synthase (luxl), as well as
V2 J3 C torial joining, tdt-dependent nucleotide addition,
the genes encoding proteins needed for light production. and splice site variability involve changing DNA
Figure 32.16 Light-Chain Production. One V segment is randomly joined with one J region by
deletion of the intervening DNA. The remaining J segments are eliminated from the RNA transcript
sequences (as opposed to RNA splicing), each
during RNA processing.
B cell produced has a new and different genotype
and its replication will give rise to genetically
distinct clones.
region that encodes the constant portion of the gene. RNA splic- Initially, all heavy chains have the μ type of constant region
ing subsequently joins the V, J, and C regions, creating mRNA. (figure 32.17b). This explains why IgM is produced upon initial
Combinatorial joining of the heavy-chain gene is quite simi- antigen stimulation (figure 32.15). Upon T-cell-dependent
lar except three different parts of the antibody locus V, J, and D B-cell activation in lymphoid tissue, antibody class switching
must be joined. Therefore heavy-chain development involves occurs when the VDJ region is joined with a new constant re-
cutting and joining the heavy-chain counterparts of V and J gion that encodes a different class of antibody, usually IgG or
DNA, as well as the D (diversity) sequences (figure 32.17). IgA (figure 32.17c).

Annotated Figures—All key metabolic pathways and ­molecular (a) Germ line DNA V1 V2 V3 Vn D1 D2 D3 Dn J1 J2 J3 J4 Cμ Cδ Cγ

processes are annotated, so each step is clearly illustrated and

explained. (b) B-cell DNA after first DNA splice
(lgM)
Cμ Cδ Cγ

RNA transcript
(intron in black)
Antigen exposure
D2 J3
V2 Cμ

lgM heavy chain

(c) B-cell DNA after second DNA splice Cγ

(lgG)

mRNA transcript
(intron in black) Class switching

Figure 32.17 The Formation of a Gene for the Heavy Chain D2 J3

V2 Cγ
of an Antibody Molecule. A similar mechanism is used for the
creation of T-cell receptor diversity. lgG heavy chain

42 CHAPTER 2 | Microscopy

Key Concepts
2.1 Lenses Create Images by Bending Light
∙ A light ray moving from air to glass or vice versa is bent in
a process known as refraction (figure 2.1).
∙ Lenses focus light rays at a focal point and magnify images
(figure 2.2).
2.2 There Are Several Types of Light Microscopes
∙ In a compound microscope such as the bright-field
microscope, the primary image is an enlarged image formed
2.4 Electron Microscopes Use Beams of Electrons
by the objective lens. The primary image is further enlarged
by the ocular lens to yield the final image (figure 2.3).
∙ Microscope resolution increases as the wavelength of
radiation used to illuminate the specimen decreases and as
the numerical aperture increases. The maximum resolution
∙ In simple staining, a single dye is used to stain
microorganisms (figure 2.16).
∙ Differential staining procedures such as Gram and acid-fast
staining distinguish between microbial groups by staining
them differently (figures 2.17 and 2.18a, b). Other differential
staining techniques are specific for particular structures such
as bacterial capsules and flagella (figure 2.18c, d).
Key Concepts—At the end of each chapter, organized by
to Create Highly Magnified Images
∙ The transmission electron microscope (TEM) uses magnetic numbered headings, this feature distills the content to its
lenses to form an image from electrons that have passed
through a very thin section of a specimen (figure 2.21).
Resolution is high because the wavelength of a beam of
essential components with cross-references to f­ igures and

∙
of a light microscope is about 0.2 μm (figure 2.4).
The dark-field microscope uses only refracted light to form
an image, and objects appear light against a black
background (figure 2.6).
∙ The phase-contrast microscope converts variations in
the refractive index into changes in light intensity and
thus makes colorless, unstained, live cells visible
(figures 2.8–2.10).
∙ The differential interference contrast microscope uses
two beams of light to create high-contrast images of live
specimens (figure 2.11).
∙ The fluorescence microscope illuminates a fluorochrome-
labeled specimen and forms an image from its
fluorescence (figures 2.12–2.14).
∙ The confocal microscope is used to study thick, complex
specimens. It creates an image by using only the light
emanating from the plane of focus, while blocking out light
from above and below the plane of focus (figure 2.15).
2.3 Staining Helps to Visualize and Identify Microbes
∙ Specimens are often fixed and stained before viewing in
the bright-field microscope. There are two fixation
methods: heat fixation and chemical fixation.
∙ Most dyes are either positively charged basic dyes or negatively
charged acidic dyes that bind to ionized parts of cells.
∙
electrons is very short.
Specimens for TEM are usually prepared by methods
tables.
that increase contrast. Specimens can be stained by
treatment with solutions of heavy metals such as
osmium, uranium, and lead. They can also be prepared
for TEM by negative staining, shadowing with metal, or
freeze-etching (figures 2.23 and 2.24).
∙ The scanning electron microscope is used to study external
surface features of microorganisms (figures 2.25 and 2.26).
∙ Cryo-EM enables visualization of single molecules and
complex molecular structures. Samples are flash frozen
and when examined, a series of images are captured that
when combined and processed form a three-dimensional
reconstruction of the specimen (figure 2.27).
2.5 Scanning Probe Microscopy Can Visualize
Molecules and Atoms
∙ Scanning probe microscopes reach very high
magnifications that allow scientists to observe biological
molecules (figures 2.28 and 2.30).
∙ Scanning tunneling microscopy enables the visualization of
molecular surfaces using electron interaction between the
probe and the specimen, whereas atomic force microscopy
can scan the surface of molecules that do not conduct
electricity well (figure 2.29).

Active Learning—Includes questions taken from current

Active Learning literature; designed to stimulate analytical problem-solving
1. You have prepared a specimen for light microscopy,
stained it using the Gram-staining procedure, but failed to
2. Which type of microscopy and stain (if appropriate) would
you use to visualize each of the following? (There may be
skills.
see anything when you looked through your light more than one correct answer.) Be sure to explain your
microscope. Discuss the things you may have done answer. Mycobacterium tuberculosis (which causes
incorrectly. tuberculosis), microbes in pond scum, Staphylococcus

xi

wil88396_fm_i-xxiv.indd 11 06/12/21 3:02 PM

 List of Content Changes
Each chapter has been thoroughly reviewed.
Part One
Chapter 1—We open the text with a new emphasis on the funda-
mentals of microbial evolution, thereby setting the stage for
weaving this theme throughout the text.
Chapter 2—A new section has been added that describes excit-
ing advances in cryo-electron microscopy and the visualization
of biomolecules.
Chapter 3—In this discussion of the bacterial cell, the two main
types of cell walls have been reframed as monoderm and diderm,
reflecting their structural differences. A new section describes the
structure and function of extracellular vesicles. Membraneless
organelles and liquid-liquid phase separation are introduced in a
Microbial Diversity & Ecology box. New figures complement an
expanded discussion of nucleoid-associated proteins and nucleoid
structure.
Chapter 4—The discussion of the archaeal cell features an en-
hanced comparison of bacterial and archaeal cells, and an ex-
panded diagram of archaeal lipids. Extracellular vesicles,
nanotubes, and nanopods are described and illustrated.
Chapter 5—An updated discussion of endocytic pathways and
extracellular vesicles has been added.
Chapter 6—This introduction to the morphological, physiologi-
cal, and genetic elements of viruses has been streamlined with
new images and figures and an updated discussion of prions.
Part Two
Chapter 7—This discussion of microbial growth highlights re-
cent advances in Z ring and divisome formation. New figures
complement discussions of the archaeal cell cycle, biofilm devel-
opment, and quorum sensing. A new Microbial Diversity &
Ecology box illustrates how some microbes can form bioconcrete.
Chapter 8—Microbial control is reorganized into physical,
chemical, and biological methods, with information on destruc-
tion of the SARS-CoV-2 virus.
Chapter 9—In addition to reviewing the structure and mecha-
nism of action of antimicrobial classes, the growing threat of
antimicrobial resistance is emphasized.
Part Three
Chapter 10—This chapter provides the foundation for under-
standing energy conservation and biosynthesis.
Chapter 11—Catabolic pathways and energy conservation
have been refocused in this chapter to emphasize bacterial and
archaeal processes. A new art program uses concept maps to
provide overviews of microbial catabolic strategies such as aer-
obic versus anaerobic respiration.
xii
wil88396_fm_i-xxiv.indd 12
Chapter 12—Biosynthetic pathways are illustrated in detail in
this chapter, and several have been expanded to include archaeal
variations. Lipopolysaccharide biosynthesis is elaborated and il-
lustrated in a new figure.
Part Four
Chapter 13—Revisions to this chapter on the basic molecular
biology of the cell include expanded discussion and images for
the origin of replication and the replisome. A new section de-
scribes the physical constraints on DNA and RNA polymerases
acting on the same chromosomal template.
Chapter 14—The regulation of cellular processes has been ex-
panded to include control by RNA secondary structures such as
RNA thermometers and T box riboswitches. The importance of
secondary messengers is highlighted in the updated discussion of
cyclic-di-GMP regulation.
Chapter 15—This chapter focuses on a discussion of eukary-
otic and archaeal molecular biology, including an introduction
to biomolecular condensates for eukaryotic processes. Recent
research on gene regulation in archaea is presented, as well as an
updated discussion of transcription from a chromatin template.
Chapter 16—This focus on mutation and repair features new
figures and an updated description of DNA repair mechanisms.
Recent research on mobile genetic elements and mechanisms of
gene transfer is included.
Chapter 17—This chapter introduces students to the common
laboratory techniques for manipulating DNA, including gene
cloning, PCR, heterologous gene expression, and CRISPR/Cas9
gene editing. A section on synthetic biology is now included.
Chapter 18—Essential genomic techniques, including single-
cell genomic sequencing and metagenomics, are introduced with
real-world applications, including those related to SARS-CoV-2.
Part Five
Chapter 19—Archaeal taxonomy now reflects the formalism
established in the Genome Taxonomy Database. Microbial dark
matter is described, as many archaea are known only from
metagenomic sequences. The discussion of archaeal carbon path-
ways has been streamlined, and the Wolfe cycle of hydrogeno-
trophic methanogenesis has been carefully annotated.
Chapter 20—Bacterial taxonomy now also reflects the formalism
derived in the Genome Taxonomy Database. As a consequence,
organisms previously classified as delta- and epsilonproteobacteria
are now included in this chapter. Variations on the diderm cell
envelope are discussed. Cable bacteria and extracellular electron
transport are introduced, and discussions of radiation resistance in
Deinococcus and chromatic acclimation in cyanobacteria have
been updated.
06/12/21 3:02 PM
 List of Content Changes
Chapter 21—This chapter on the proteobacteria contains a new
section describing Acinetobacter. In addition, the β-hydroxy­
aspartate cycle linking autotrophs and heterotrophs in the open
ocean is included.
Chapter 22—This chapter surveying the Gram-positive bacteria
now includes Mycoplasma spp. The section on Streptomyces
presents a discussion of biosynthetic gene clusters.
Chapter 23—Updated protist classification based on recent phy-
logenomic analysis is provided as clades of protists of medical
and environmental importance are reviewed.
Chapter 24—This chapter has been reorganized based on recent
phylogenomic evidence. The six major fungal groups are presented.
Chapter 25—Viral taxonomy has been revised, and this chapter
reflects the classification used by the International Committee on
the Taxonomy of Viruses. The detailed life cycle of a coronavirus
serves as an example of positive-strand RNA viruses, thereby
presenting the replication cycle of SARS-CoV-2. This is accom-
panied by new figures.
Part Six
Chapter 26—This chapter presents a discussion of key tech-
niques used for assessing microbial populations and communities
and includes an expanded discussion on metagenomics. Applica-
tions to environmental and microbiome research are included.
Chapter 27—This chapter on microbial interactions has been
extensively revised, grouping interactions as mutualism, cooper-
ation, or antagonism. Multiple new examples are detailed, with
emphasis on metabolic interdependence.
Chapter 28—An expanded introduction to nutrient cycling and
biogeochemical cycling precedes the review of major elemental
cycles. New art brings these cycles to life. The chapter builds
upon these concepts to explain the role of microbes in an updated
discussion of climate change.
Chapter 29—Discussions of microbial adaptation to the marine
environment and the importance of the oceans in global climate
change have been updated. Coverage of freshwater microbiology
has also been revised, emphasizing anthropogenic impacts.
Chapter 30—This chapter complements chapter 27 with discus-
sions of mycorrhizal fungi and nitrogen-fixing bacteria. The role
of metagenomics in advancing our understanding of soil microbi-
ology is stressed. Coverage of plant pathogens has been expanded.
Part Seven
Chapter 31—This chapter has been updated and its organization
refined to provide a concise introduction of innate immunity,
including advances in our understanding of the role of the in-
flammasome and innate lymphoid cells.
wil88396_fm_i-xxiv.indd 13
Chapter 32—Revised and updated, this discussion of adaptive
immunity and immunopathologies provides a current overview
to introduce students to the dynamics of human immunity. Many
figures have been revised for clarity.
Chapter 33—The rapidly expanding field of the human micro-
biome is introduced. This chapter follows those on immunology
for a complete discussion of the role of human microbiota in
immune function, as well as their role in maintaining system
homeostasis.
Chapter 34—This chapter provides a broad overview of infec-
tious disease from pathogen transmission to pathogenicity.
Part Eight
Chapter 35—This chapter has been revised to reflect recent
epidemiological data, a discussion of R0 and herd immunity, and
an updated vaccine section to include mRNA vaccines. The epi-
demiology of SARS-CoV-2 is highlighted, as well as pandemic
management.
Chapter 36—This chapter provides students with an overview of
key microbiological and immunological techniques enabling the
identification of clinical samples.
Chapter 37—This chapter now includes a complete discussion of
our current understanding of the pathobiology of SARS-CoV-2.
The genomics and evolution of the virus is emphasized, as well as
the clinical manifestations of COVID-19.
Chapter 38—Students are introduced to bacterial diseases, in-
cluding pathogenesis, prevalence, and clinical presentation. Where
applicable, the importance of vaccine prevention is stressed.
Chapter 39—This chapter provides an overview of fungal and
protozoan diseases of local and global significance. The global
burden of key diseases such as Chagas and malaria is emphasized.
Part Nine
Chapter 40—The essentials of food safety now include a discus-
sion of hazards and safety measures at all stages from farm to
market. Methods for food testing have been updated to reflect the
use of molecular methods and whole-genome sequencing.
Chapter 41—The growing reach of biotechnology is illustrated in
several examples, including an expanded discussion of industrial
enzymes derived from microbes, rational vaccine design strategies,
microbial biosensors, and diatoms as nanotechnology platforms.
Chapter 42—The discussion of water safety has been expanded
to include a discussion of microbial source tracking, and a COVID
box notes the importance of monitoring sewage for SARS-CoV-2
as an aspect of public health. The section on biodegradation has
been expanded to include petroleum hydrocarbons, halogenated
organic molecules, and a description of the plastisphere.
xiii
06/12/21 3:02 PM
 About the Authors
Joanne Willey
Joanne M. Willey has been a
professor at Hofstra University on Long
Island, New York, since 1993, where she is
the Leo A. Guthart Professor of
Biomedical Science and Chair of the
Department of Science Education at the
Donald and Barbara Zucker School of
Medicine at Hofstra/Northwell. Dr. Willey
received her B.A. in Biology from the
University of Pennsylvania, where her
interest in microbiology began with work
on cyanobacterial growth in eutrophic
streams. She earned her Ph.D. in
biological oceanography (specializing
in marine microbiology) from the
Massachusetts Institute of Technology–
Woods Hole Oceanographic Institution
Joint Program in 1987. She then went to
Harvard University, where she spent her
postdoctoral fellowship studying the
filamentous soil bacterium Streptomyces
coelicolor. Dr. Willey has coauthored a
number of publications that focus on its
complex developmental cycle. She is an
active member of the American Society
for Microbiology (ASM), and served on
the editorial board of the journal Applied
and Environmental Microbiology for nine
years and as Chair of the Division of
General Microbiology. Dr. Willey taught
microbiology to biology majors for 20 years
and now teaches microbiology and
infectious disease to medical students. She
has taught courses in cell biology, marine
microbiology, and laboratory techniques in
molecular genetics. Dr. Willey lives on the
north shore of Long Island and has two
grown sons. She is an avid runner and
enjoys skiing, hiking, rock climbing,
and reading. She can be reached at
joanne.m.willey@hofstra.edu.
xiv
wil88396_fm_i-xxiv.indd 14
Kim Tyndall
Kathleen M. Sandman received her
B.A. in Biology from La Salle University
and her Ph.D. in Cellular and Developmental
Biology from Harvard University. She was
inspired to a career in science by her older
brother’s experience as an organic chemist
and by the developing technology in
recombinant DNA in the 1970s. Her
graduate work used a transposable element
as a mutagen in Bacillus subtilis to study
gene expression during endospore formation.
She continued in the genetics of Gram-
positive bacteria with a postdoctoral year
studying Bacillus thuringiensis at the
University of Cambridge in the United
Kingdom. Another postdoctoral opportunity
at The Ohio State University provided an
introduction to the emerging field of
archaeal molecular biology, where
Dr. Sandman discovered archaeal histones
and continued research in the structural
biology of archaeal chromatin for about
20 years. She served the National Science
Foundation as a research grant reviewer
and panelist for the Life in Extreme
Environments program, and has organized
conference sessions on archaeal molecular
biology and proteins from extremophiles.
Dr. Sandman has taught microbiology to
hundreds of students, at both the
introductory level and in an advanced
molecular microbiology laboratory.
Dr. Sandman has worked as a consultant
in a variety of industries, including
industrial microbiology, environmental
geomicrobiology, and technical publishing.
She lives with her husband in Columbus,
Ohio, and has two grown daughters. She
enjoys biking, fabric arts, reading, and
genealogy, and can be reached at
kathleenmsandman@gmail.com.
Dorothy Wood
Dorothy H. Wood taught
microbiology and general biology at
Durham Technical Community College in
North Carolina for 17 years. Dr. Wood
received her B.A. in Biology from Rhode
Island College where her love of microbes
began, nurtured by Dr. Charles Owens.
She earned her Ph.D. in Cell and
Molecular Pathology from the University
of North Carolina at Chapel Hill, focusing
on pancreatic damage caused by
antimicrobial drugs, and investigated
alternative therapies based on receptor
binding by novel compounds. After three
years as Assistant Professor at NC Central
University, Dr. Wood made the move to
the NC Community College System to
focus her attention on her primary interest
of teaching. Throughout her career she
developed additional courses, including
graduate bacteriology, pathophysiology,
and biotechnology. Dr. Wood is a digital
faculty consultant for McGraw Hill and
has worked on several textbooks in a
variety of disciplines, developing and
editing digital content to accompany the
texts. Dr. Wood is a curriculum design
consultant for Education & Training
Systems International based in Chapel
Hill, NC. She is a fitness professional,
leads health and wellness seminars, and
has been the treasurer of a nonprofit
organization for the past 10 years. She
enjoys life in North Carolina with her
husband and two grown children,
and can be reached at
dorothywood241@gmail.com.
06/12/21 3:02 PM

In the preparation of each edition, we are guided by the collective
wisdom of reviewers who are expert microbiologists and
excellent teachers. They represent experience in community
colleges, liberal arts colleges, comprehensive institutions, and
research universities. We have followed their recommendations,
while remaining true to our overriding goal of writing readable,
student-centered content. Each feature incorporated into this
edition has been carefully considered in terms of how it may be
used to support student learning in both the traditional and the
flipped learning environment.
Also in this edition, we are very excited to incorporate
real student data points and input, derived from thousands of
We offer our sincere thanks to the following reviewers and/or focus group participants who provided valuable input. Your suggestions
and recommendations helped shape and guide this revision.
Chris Allen, Lone Star College-University Park
Daniel Aruscavage, Kutztown University
Paul Babitzke, Pennsylvania State University
R. Berlemont, California State University, Long Beach
Linda D. Bruslind, Oregon State University
Becky Buckley, Metropolitan State University of Denver
Hildamarie Caceres-Velazquez, Coastal Carolina
Community College
Kathleen L. Campbell, Emory University
Daniel N. Clark, Weber State University
Laszlo Csonka, Purdue University
Deborah Dardis, Southeastern Louisiana University
Ellen B. Duffy, Siena College
Dale Emeagwali, Excelsior College
Jed A. Fuhrman, University of Southern California
Melanie C. Griffin, Kennesaw State University
Judyth Gulden, Northeastern State University
Isaac M. Hagenbuch, University of South Carolina, Columbia
Cecily L. Haley, San Jacinto College North Campus
Andrew F. Herbig, Washburn University
Timothy Hoover, University of Georgia
Amy G. Hurst, Rose State College
Mark Kainz, Ripon College
wil88396_fm_i-xxiv.indd 15
Acknowledgements
our LearnSmart users, to help guide our revision. With this
information, we were able to hone both book and digital
content.
The authors wish to extend their gratitude to our team at
McGraw Hill, including Lauren Vondra, Darlene Schueller,
Tami Hodge, Vicki Krug, David Hash, Beth Cray, and Tammy
Juran. Thanks to Rebecca E. Steiner, Shonteria L. Johnson, Rita
Mary King, Jonathan A. Miller, Brittany Gasper, and Mary
Colavito for your assistance with this edition. Finally, we thank
our spouses and children, who provided support and tolerated
our absences (mental, if not physical) while we completed this
demanding project.
Bessie W. Kebaara, Baylor University
Juliana Kelley, Laredo College
Peter J. King, University of Texas, Austin
Yong Jin Lee, Albany State University
Cynthia Littlejohn, The University of Southern Mississippi
Danielle M. McGrath, San Jacinto College
Daniel P. Moore, Southern Maine Community College
Roderick M. Morgan, Grand Valley State University
Kathryn Morris, Xavier University
Florence Okafor, Alabama A&M University
Hyun-Woo Park, California Baptist University
Kaustubha Qanungo, Clemson University
Shawna Reed, Quinnipiac University
Rebecca D. Riggs, Auburn University
Janet Rinehart-Kim, Old Dominion University
Paul G. Rudenberg, Southern Maine Community College
Pratibha Saxena, University of Texas at Austin
Sarah Sidiropoulos, Oakland Community College
Gehan Soliman, Campbell University
Henry G. Spratt, Jr., University of Tennessee at Chattanooga
Helene C. Ver Eecke, Metropolitan State University of Denver
Lisa Waidner, University of West Florida
Fanxiu Zhu, Florida State University
xv
06/12/21 3:02 PM
 Contents

3.7 The Bacterial Cytoplasm Is More Complex

Part One Introduction to Microbiology than Once Thought 62
1 The Evolution of Microorganisms and Microbial Diversity & Ecology 3.1
Organelles Without Membranes? 65
Microbiology 1
Micro Focus: 3.8 External Structures Are Used for
Microbiology’s Reach 1 Attachment and Motility 67
1.1 Members of the Microbial World 2 3.9 Bacteria Move in Response to
Environmental Conditions 70
1.2 Microbes Have Evolved and Diversified
for Billions of Years 4 3.10 Bacterial Endospores Are a Survival
Strategy 74
Microbial Diversity & Ecology 1.1
Hydrothermal Vents: Did Life Begin
Under the Sea? 7
4 Archaeal Cell Structure 80
1.3 Microbiology Advanced as New Tools for Micro Focus:
Studying Microbes Were Developed 14 Methane—The Other Greenhouse Gas 80
1.4 Microbiology Encompasses 4.1 Archaea Are Diverse but Share Some
Many Subdisciplines 19 Common Features 81
4.2 Archaeal Cell Envelopes Are Structurally
2 Microscopy 23 Diverse 83
Micro Focus: 4.3 Archaeal Cytoplasm Is Similar to Bacterial
Anthrax Bioterrorism Attack 23 Cytoplasm 86
2.1 Lenses Create Images by Bending Light 24 4.4 Many Archaea Have External Structures
2.2 There Are Several Types of Light Used for Attachment and Motility 88
Microscopes 24
5 Eukaryotic Cell Structure 91
2.3 Staining Helps to Visualize and Identify
Microbes 32 Micro Focus:
Red Means Dead 91
2.4 Electron Microscopes Use Beams of
Electrons to Create Highly Magnified 5.1 Eukaryotic Cells Are Diverse but Share
Images 35 Some Common Features 92
2.5 Scanning Probe Microscopy Can Visualize 5.2 Eukaryotic Cell Envelopes 93
Molecules and Atoms 40 5.3 The Eukaryotic Cytoplasm Contains
a Cytoskeleton and Organelles 94
3 Bacterial Cell Structure 44 5.4 Several Organelles Function in the
Micro Focus: Secretory and Endocytic Pathways 96
Bacteria Use Rapid Transport 44 5.5 The Nucleus and Ribosomes Are Involved
3.1 Use of the Term Prokaryote Is Controversial 45 in Genetic Control of the Cell 100
3.2 Bacteria Are Diverse but Share Some 5.6 Mitochondria, Related Organelles, and
Common Features 45 Chloroplasts Are Involved in Energy
3.3 Bacterial Plasma Membranes Control Conservation 101
What Enters and Leaves the Cell 48 Microbial Diversity & Ecology 5.1
There Was an Old Woman Who
3.4 Cell Walls Have Many Functions 54 Swallowed a Fly 104
3.5 Extracellular Vesicles Emerge from
5.7 Many Eukaryotic Microbes Have External
Bacterial Membranes 60
Structures Used for Motility 105
3.6 The Cell Envelope Often Includes Layers
Outside the Cell Wall 61

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 Contents

6 Viruses and Other Acellular Infectious Agents 109 8.2 Microbes Can Be Controlled by
Micro Focus: Physical Means 166
Viruses to the Rescue 109 Techniques & Applications 8.1
6.1 Viruses Are Acellular 110 Come Fly with Me? 167

6.2 Virion Structure Is Defined by Capsid 8.3 Microorganisms Are Controlled with
Symmetry and Presence or Absence of an Chemical Agents 170
Envelope 110 8.4 Antimicrobial Agents Must Be
6.3 Viral Life Cycles Have Five Steps 114 Evaluated for Effectiveness 174
6.4 There Are Several Types of Viral Infections 119 8.5 Microorganisms Can Be Controlled
by Biological Methods 176
6.5 Virus Cultivation and Enumeration 120
6.6 Viroids and Satellites: Nucleic 9 Antimicrobial Chemotherapy 179
Acid-Based Subviral Agents 123
Micro Focus:
6.7 Prions Are Composed Only of Protein 124 A Gift from Traditional Chinese Medicine 179
9.1 Antimicrobial Chemotherapy Evolved from
Antisepsis Efforts 180
Part Two Microbial Nutrition, Growth, and Control
9.2 Antimicrobial Drugs Have Selective
7 Bacterial and Archaeal Growth 127 Toxicity 181
Micro Focus: 9.3 Antimicrobial Activity Can Be Measured
How Low Can You Go? 127 by Specific Tests 181
7.1 Most Bacteria and Archaea Reproduce 9.4 Antibacterial Drugs 185
by Binary Fission 128 9.5 Antiviral Drugs 190
7.2 Bacterial Cell Cycles Are Divided into 9.6 Antifungal Drugs 193
Three Phases 129 9.7 Antiprotozoan Drugs 193
7.3 Archaeal Cell Cycles Are Unique 133 Disease 9.1
7.4 Growth Curves Consist of Five Phases 134 Chloroquine and COVID-19:
A Cautionary Tale 195
7.5 Environmental Factors Affect Microbial
Growth 138 9.8 Antimicrobial Drug Resistance Is a Public
Health Threat 196
Microbial Diversity & Ecology 7.1
Microbial Sculptors 141
7.6 Microbial Growth in Natural Environments 145
Part Three Microbial Metabolism
7.7 Laboratory Culture of Microbes Requires
Conditions that Mimic Their Normal 10 Introduction to Metabolism 201
Habitats 150 Micro Focus:
7.8 Microbial Population Size Can Be Flushed Away 201
Measured Directly or Indirectly 155 10.1 Metabolism: Important Principles
7.9 Chemostats and Turbidostats Are Used for and Concepts 202
Continuous Culture of Microorganisms 157 10.2 ATP: The Major Energy Currency
of Cells 204
8 Control of Microorganisms in the Environment 162
10.3 Redox Reactions: Reactions of Central
Micro Focus: Importance in Metabolism 205
To Wipe or Not to Wipe? That Is the Question. 162
10.4 Electron Transport Chains: Sets
8.1 Microbial Growth and Replication: of Sequential Redox Reactions 207
Targets for Control 163

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 Contents

10.5 Biochemical Pathways: Sets of Linked

Chemical Reactions 209
Part Four M
 icrobial Molecular Biology
10.6 Enzymes and Ribozymes Speed
and Genetics
Up Cellular Chemical Reactions 210 13 Bacterial Genome Replication and Expression 277
10.7 Metabolism Must Be Regulated to Micro Focus:
Maintain Homeostasis 214 Making Code 277
11 Catabolism: Energy Release and 13.1 Experiments Using Bacteria and Viruses
Conservation 219 Demonstrated that DNA Is the Genetic
Material 278
Micro Focus:
13.2 Nucleic Acid and Protein Structure 280
The Richest Hill on Earth 219
13.3 DNA Replication in Bacteria 283
11.1 Metabolic Diversity and Nutritional
Types 220 13.4 Bacterial Genes Consist of Coding
Regions and Other Sequences Important
11.2 There Are Two Chemoorganotrophic
for Gene Function 289
Fueling Processes 222
13.5 Transcription in Bacteria 291
11.3 Aerobic Respiration Starts with Glucose
Oxidation 223 13.6 The Genetic Code Consists of
Three-Letter “Words” 294
11.4 Electron Transport and Oxidative
Phosphorylation Generate the 13.7 Translation in Bacteria 297
Most ATP 230 13.8 Coordination of Gene Expression
11.5 Anaerobic Respiration Uses the Same Processes 302
Steps as Aerobic Respiration 235 13.9 Protein Maturation and Secretion 304
11.6 Fermentation Does Not Involve
an Electron Transport Chain 236 14 Regulation of Cellular Processes 310
11.7 Catabolism of Organic Molecules Other Micro Focus:
than Glucose 239 Promoting Expression 310
11.8 Chemolithotrophy: “Eating Rocks” 241 14.1 Bacteria Use Many Regulatory Strategies 311
11.9 Flavin-Based Electron Bifurcation 244 14.2 Regulation of Transcription Initiation Saves
Considerable Energy and Materials 311
11.10 Phototrophy 245
14.3 Attenuation and Riboswitches Stop
12 Anabolism: The Use of Energy in Transcription Prematurely 316
Biosynthesis 255 14.4 RNA Secondary Structures Control
Micro Focus: Translation 319
Building Penicillin 255 14.5 Mechanisms Used for Global Regulation 320
12.1 Principles Governing Biosynthesis 256 14.6 Bacteria Combine Several Regulatory
12.2 Precursor Metabolites: Starting Molecules Mechanisms to Control Complex Cellular
for Biosynthesis 257 Processes 327
12.3 CO2 Fixation: Reduction and Assimilation
15 Eukaryotic and Archaeal Genome Replication
of CO2 Carbon 257
and Expression 336
12.4 Synthesis of Carbohydrates 260
Micro Focus:
12.5 Synthesis of Amino Acids Consumes Many Pharming 336
Precursor Metabolites 262
15.1 Genetic Processes in the Three Domains 337
12.6 Synthesis of Purines, Pyrimidines, and
15.2 DNA Replication: Similar Overall, but with
Nucleotides 268
Different Replisome Proteins 337
12.7 Lipid Synthesis 270
15.3 Transcription 341

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 Contents

15.4 Translation and Protein Maturation and 18.2 Genome Sequencing 402
Localization 344 18.3 Metagenomics Provides Access to
15.5 Regulation of Cellular Processes 349 Uncultured Microbes 404
18.4 Bioinformatics: What Does the Sequence
16 Mechanisms of Genetic Variation 353 Mean? 406
Micro Focus: 18.5 Functional Genomics Links Genes
Manure Happens 353 to Phenotype 407
16.1 Mutations: Heritable Changes in 18.6 Systems Biology: Making and Testing
a Genome 354 Complex Predictions 413
16.2 Detection and Isolation of Mutants 358 18.7 Comparative Genomics 413
16.3 DNA Repair Maintains Genome Stability 359
16.4 Microbes Use Mechanisms Other than
Mutation to Create Genetic Variability 362
Part Five The Diversity of the Microbial World
16.5 Mobile Genetic Elements Move Genes
Within and Between DNA Molecules 364 19 Archaea 419
16.6 Conjugation Requires Cell-Cell Contact 365 Micro Focus:
16.7 Transformation Is the Uptake of Free DNA 368 Methanogens Fuel Domestic Energy Debate 419
16.8 Transduction Is Virus-Mediated 19.1 Overview of Archaea 420
DNA Transfer 370 19.2 Phyla Asgardarchaeota and Nanoarchaeota
16.9 Evolution in Action: The Development of Are Known Primarily from Metagenomics 423
Antibiotic Resistance in Bacteria 373 19.3 Phylum Thermoproteota: Sulfur-
Dependent Thermophiles 424
17 Microbial DNA Technologies 377
19.4 Phylum Nitrosphaeria: Mesophilic
Micro Focus: Ammonia Oxidizers 426
Spinning Stronger Silk 377
19.5 Phyla Methanobacteriota, Halobacteriota,
17.1 Key Discoveries Led to the Development and Thermoplasmatota: Methanogens,
of DNA Cloning Technology 378 Haloarchaea, and Others 426
Techniques & Applications 17.1
Gel Electrophoresis 379
20 Nonproteobacterial Gram-Negative Bacteria 433
17.2 Polymerase Chain Reaction Amplifies
Targeted DNA 383 Micro Focus:
From Food Waste to Fuel 433
17.3 Genomic and Metagenomic Libraries:
Cloning Genomes in Pieces 386 20.1 Diderm Cell Envelopes Are Not Uniform 434
17.4 Expressing Foreign Genes in Host Cells 387 20.2 Aquificota and Thermotogota Are
Hyperthermophiles 434
17.5 Cas9 Nuclease Is a Programmable
Tool for Genome Editing 389 20.3 Deinococcota Includes Radiation-
Resistant Bacteria 434
17.6 Biotechnology Develops Custom
Microbes for Industrial Use 391 20.4 Photosynthetic Bacteria Are Diverse 435
Techniques & Applications 17.2 20.5 PVC Superphylum (Planctomycetota
How to Build a Microorganism 394 and Verrucomicrobiota): Atypical
Cell Division 442
18 Microbial Genomics 397 20.6 Phylum Spirochaetota: Bacteria with a
Micro Focus: Corkscrew Morphology 444
What’s in a Genome? 397 20.7 Phylum Bacteroidota Includes Important
18.1 DNA Sequencing Methods 398 Gut Microbiota 446

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20.8 Phylum Fusobacteriota: Commensal 24 Fungi 518

Anaerobes 447 Micro Focus:
20.9 Phylum Desulfobacterota: Anaerobic The Complex Story of Caterpillar Fungus 518
Sulfate/Sulfur Reducers 447 24.1 Fungal Biology Reflects Vast Diversity 519
20.10 Phyla Bdellovibrionota and Myxococcota: 24.2 Zoosporic Fungi Produce Motile Spores 522
Bacterial Predators 449 24.3 Zygomycetous Fungi Have Coenocytic
20.11 Phylum Campylobacterota: Human and Hyphae 523
Animal Commensals 451 24.4 Dikarya Is the Most Diverse Fungal
Group 525
21 Proteobacteria 455
Disease 24.1
Micro Focus: White-Nose Syndrome Is Decimating
Bison and Brucellosis Spark Controversy 455 North American Bat Populations 528
21.1 Class Alphaproteobacteria Includes
Many Oligotrophs 456 25 Viruses 532
21.2 Gammaproteobacteria Is the Largest Micro Focus:
Bacterial Class 464 Disrupting the Viral Life Cycle 532
Microbial Diversity & Ecology 21.1 25.1 Virus Phylogeny Relies on Genomics 533
Acid Mine Drainage 469 25.2 Double-Stranded DNA Viruses Infect
All Cell Types 534
22 Gram-Positive Bacteria 479
25.3 Single-Stranded DNA Viruses Use a
Micro Focus: Double-Stranded Intermediate in
Antibiotic Production: Is It Actually Their Life Cycles 542
Bacterial Chitchat? 479 25.4 Double-Stranded RNA Viruses: RNA-
22.1 Phylum Actinobacteriota 480 Dependent RNA Polymerase Replicates
22.2 Phylum Firmicutes, Class Bacilli: Aerobic the Genome and Synthesizes mRNA 543
Endospore-Forming Bacteria 487 25.5 Positive-Strand RNA Viruses: Genomes
22.3 Phylum Firmicutes, Class Clostridia: that Are Translated upon Entry 545
Anaerobic Endospore-Forming Bacteria 494 25.6 Negative-Strand RNA Viruses:
22.4 Phylum Firmicutes, Classes Negativicutes RNA-Dependent RNA Polymerase
and Halanaerobiia: Gram-Positive Bacteria Is Part of the Virion 549
with Outer Membranes 495 25.7 Retroviruses: Positive-Strand Viruses
that Use Reverse Transcriptase in
23 Protists 498 Their Life Cycles 551
Micro Focus: 25.8 Reverse Transcribing DNA Viruses 552
Setting the Record Straight 498
23.1 Protist Diversity Reflects Broad
Phylogeny 499 Part Six Ecology and Symbiosis
23.2 Discoba-Metamonada Clade 501
23.3 Amoebozoa Clade Includes Protists 26 Exploring Microbes in Ecosystems 556
with Pseudopodia 503 Micro Focus:
23.4 TSAR Clade: Protists of Global Scientists Search for Intraterrestrial
Importance 505 Life—and Find It 556
23.5 Haptista Clade 514 26.1 Microbial Biology Relies on Cultures 557
23.6 Archaeplastida Clade Includes Green Microbial Diversity & Ecology 26.1
and Red Algae 514 Patience, Hard Work, Luck, and the
Evolution of Eukaryotes 559

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26.2 Microbial Identification Is Largely Based 30.3 Microbe-Plant Interactions Can Be

on Molecular Characterization 560 Positive, Negative, or Neutral 621
26.3 Assessing Microbial Populations 563 Disease 30.1
26.4 Assessing Microbial Community Activity 566 Citrus Greening and the Power of “Why?” 633
30.4 The Subsurface Biosphere Is Vast 633
27 Microbial Interactions 571
Micro Focus:
Microbes in Community 571 Part Seven Pathogenicity and Host Response
27.1 Many Types of Microbial Interactions Exist 572
31 Innate Host Resistance 636
27.2 Mutualism: Obligatory Positive Interaction 573
Micro Focus:
27.3 Cooperation: Nonobligatory Positive
The Hygiene Hypothesis 636
Interaction 577
31.1 Immunity Arises from Innate Resistance
27.4 Antagonistic Interactions Prompt Microbial
and Adaptive Defenses 637
Responses 579
31.2 Innate Resistance Starts with Barriers 637
Microbial Diversity & Ecology 27.1
Wolbachia: The World’s Most Infectious 31.3 Innate Resistance Relies on Chemical
Microbe? 581 Mediators 640
31.4 Each Type of Innate Immune Cell Has a
28 Biogeochemical Cycling and Global Specific Function 646
Climate Change 584 31.5 Organs and Tissues of the Immune
Micro Focus: System Are Sites of Host Defense 651
Global Climate Change; Infectious 31.6 Phagocytosis Destroys Invaders 654
Disease Change 584 31.7 Inflammation Unites All Components
28.1 Biogeochemical Cycling Sustains of Immunity 659
Life on Earth 585
28.2 Microbes Mediate Nutrient Cycling 587 32 Adaptive Immunity 663
28.3 Global Climate Change: Infectious Micro Focus:
Disease Change 594 Killing Cancer, Immunologically 663
32.1 Adaptive Immunity Relies on Recognition
29 Microorganisms in Marine and Freshwater and Memory 664
Ecosystems 599 32.2 Antigens Elicit Immunity 664
Micro Focus: 32.3 Adaptive Immunity Can Be Earned or
Ocean Death Coming Soon to a Coast Near Borrowed 665
You 599
32.4 Recognition of Foreignness Is Critical
29.1 Water Is the Largest Microbial Habitat 600 for a Strong Defense 666
29.2 Microorganisms in Marine Ecosystems 601 32.5 T Cells Are Critical for Immune Function 669
29.3 Microorganisms in Freshwater Ecosystems 610 32.6 B Cells Make Antibodies 673
Microbial Diversity & Ecology 29.1 32.7 Antibodies Bind Specific 3-D Antigens 676
Attention All Dog Owners! 614
Techniques & Applications 32.1
Monoclonal Antibody Therapy 683
30 Microorganisms in Terrestrial Ecosystems 617
32.8 Antibodies Doom Antigens 684
Micro Focus:
Historical Highlights 32.2
Bread for a Hungry World 617
Convalescent Plasma: An Old Treatment
30.1 Soils Are an Important Microbial Habitat 618 for a New Disease 684
30.2 Diverse Microorganisms Inhabit Soil 620 32.9 The Immune System Can Malfunction 686

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 Contents

33 The Microbe-Human Ecosystem 696 35.6 Coordinated Efforts Are Required to

Micro Focus: Prevent and Control Epidemics 741
Embrace Your Gut Flora 696 Historical Highlights 35.3
33.1 Humans Are Holobionts 697 The First Immunizations 744

33.2 The Microbiome Develops from Birth to 35.7 Bioterrorism Readiness Is an Integral
Adulthood 697 Component of Public Health
Microbiology 746
33.3 A Functional Core Microbiome Is
Required for Host Homeostasis 702 Historical Highlights 35.4
1346—Early Biological Warfare Attack 747
33.4 Many Diseases Have a Connection
with Dysbiosis 708
36 Clinical Microbiology and Immunology 750
33.5 Microbiome Manipulation Can Be
Micro Focus:
Therapeutic 711
Ebola and Global Health Security 750
34 Infection and Pathogenicity 714 36.1 The Clinical Microbiology Laboratory
Detects Infectious Agents and Protects
Micro Focus:
Its Workers 751
The Unlikely Tale of Miasmas, Bras, and Masks 714
36.2 Identification of Microorganisms from
34.1 The Process of Infection 715
Specimens 753
34.2 Transmission and Entry into the Host 716
36.3 Immune Responses Can Be Exploited to
Historical Highlights 34.1
Detect Infections 760
The First Indications of Person-to-Person
Spread of an Infectious Disease 717
37 Human Diseases Caused by Viruses
34.3 Surviving the Host Defenses 722 and Prions 769
34.4 Damage to the Host 724 Micro Focus:
Remembering HIV/AIDS 769
37.1 Viruses Can Be Transmitted by Airborne
Part Eight M
 icrobial Diseases, Detection, and Routes 770
Their Control 37.2 Arthropods Can Transmit Viral Diseases 780
35 Epidemiology and Public Health Microbiology 730 37.3 Direct Contact Diseases Can Be Caused
by Viruses 782
Micro Focus:
Protecting the Herd 730 37.4 Food and Water Are Vehicles for Viral
Diseases 792
35.1 Epidemiology Is an Evidence-Based
Science 731 Historical Highlights 37.1
A Brief History of Polio 795
Historical Highlights 35.1
John Snow, the First Epidemiologist 732 37.5 Zoonotic Diseases Arise from
Human-Animal Interactions 795
35.2 Epidemiology Is Rooted in Well-Tested
Methods 732 37.6 Prion Proteins Transmit Disease 798
35.3 Infectious Disease Is Revealed Through
38 Human Diseases Caused by Bacteria 801
Patterns Within a Population 735
Micro Focus:
Historical Highlights 35.2
“Typhoid Mary” 736 The Plague Family Tree 801
38.1 Bacteria Can Be Transmitted by Airborne
35.4 Infectious Diseases and Pathogens
Routes 802
Are Emerging and Reemerging 738
38.2 Arthropods Can Transmit Bacterial
35.5 Healthcare Facilities Harbor Infectious
Diseases 811
Agents 740

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 Contents

38.3 Direct Contact Diseases Can Be Caused 40.3 Food-Borne Disease Outbreaks 875
by Bacteria 814 40.4 Detection of Food-Borne Pathogens
Disease 38.1 Requires Government-Industry
Syphilis and the Tuskegee Study 821 Cooperation 877
Disease 38.2 40.5 Microbiology of Fermented Foods:
Biofilms 822 Beer, Cheese, and Much More 879
38.4 Food and Water Are Vehicles for Bacterial Techniques & Applications 40.1
Diseases 827 Chocolate: The Sweet Side of Fermentation 880
Techniques & Applications 38.3
Clostridial Toxins as Therapeutic Agents: 41 Biotechnology and Industrial Microbiology 887
Benefits of Nature’s Most Toxic Proteins 831 Micro Focus:
38.5 Zoonotic Diseases Arise from Where Are the New Antibiotics? 887
Human-Animal Interactions 835 41.1 Microbes Are the Source of Many
38.6 Opportunistic Diseases Can Be Caused Products of Industrial Importance 888
by Bacteria 837 41.2 Biofuel Production Is a Dynamic Field 890
41.3 Growing Microbes in Industrial Settings
39 Human Diseases Caused by Fungi Presents Challenges 892
and Protists 845
41.4 Agricultural Biotechnology Relies on
Micro Focus: a Plant Pathogen 893
Mushrooms of Death 845
41.5 Some Microbes Are Products 894
39.1 Relatively Few Fungi and Protists
Are Human Pathogens 846 42 Applied Environmental Microbiology 898
39.2 Fungi Can Be Transmitted by Airborne Micro Focus:
Routes 847 Deepwater Horizon Oil Consumed by
39.3 Arthropods Can Transmit Protozoal Microbes 898
Disease 849 42.1 Purification and Sanitary Analysis
Disease 39.1 Ensure Safe Drinking Water 899
A Brief History of Malaria 850
42.2 Wastewater Treatment Maintains Human
39.4 Direct Contact Diseases Can Be Caused and Environmental Health 901
by Fungi and Protists 857 42.3 Microbial Fuel Cells: Batteries Powered
39.5 Food and Water Are Vehicles of Protozoal by Microbes 906
Diseases 860 42.4 Biodegradation and Bioremediation
39.6 Opportunistic Diseases Can Be Caused Harness Microbes to Clean the
by Fungi and Protists 865 Environment 907

Appendix 1  Review of the Chemistry of

A
Part Nine Applied Microbiology Biological Molecules A-1

40 Microbiology of Food 871 Appendix 2 Common Metabolic Pathways A-9

Micro Focus: Appendix 3 Microorganism Pronunciation Guide A-17
The Art, Science, and Genetics of
Brewing Beer 871 Glossary G-1
40.1 Microbial Growth Can Cause Index I-1
Food Spoilage 872
40.2 Environmental Factors Control
Food Spoilage 873

xxiii

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 twelfth edition

Prescott’s Microbiology

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 CHAPTER
1
The Evolution of
Microorganisms and
Microbiology
Microbiology’s Reach
H ow does it feel to witness history? The COVID-19
pandemic will be studied for years to come by scientists,
clinicians, and politicians. However, as the COVID-19 pandemic
exploded, we had the tools to address many of the questions that
needed answers in real time. Each of these questions also illustrates
the reach of microbiology. Let’s explore five of them:
■ What is the nature of the virus that causes COVID-19, SARS-CoV-2?
It is easy to see that virologists—those who study viruses—helped
answer this question. But they were supported by many others. For
example, electron microscopists were needed to visualize the virus,
and the work of molecular biologists and geneticists was critical. The
ability to rapidly sequence the first isolated SARS-CoV-2 genome,
followed by new many new isolates cultured from patients, illustrates
the importance of bioinformaticists (people who analyze large
biological data sets), computer scientists, and clinical microbiologists.
■ How does SARS-CoV-2 cause disease? This turned out to be much
more complicated than anyone initially anticipated. To answer
this question, immunologists, physiologists, infectious disease
specialists, pathologists, and every manner of clinician-scientist
conducted studies.
■ How do we best treat patients with COVID-19? The process of
repurposing existing drugs and developing new drugs required
the coordinated efforts of virologists, molecular biologists,
biochemists, chemists, and immunologists to identify and
design new drugs. Meanwhile, clinicians—including physicians,
nurses, pharmacists, and public health officials—tested new
therapies on patients. Data scientists and statisticians were
needed to interpret the outcomes of trials.
wil88396_ch01_001-022.indd 1
Raman Tyukin/Shutterstock
■ How do we prevent the spread of COVID-19? The world got a
crash course on the role of epidemiologists and disease modelers
in tracking, tracing, and predicting the spread of disease.
Geographic information scientists helped figure out where the
virus was spreading. As it became clear that vaccines take time
for microbiologists, biochemists, and immunologists to develop,
the design and deployment of cheaper and easier testing by
industrial microbiologists and bioengineers was critical.
These are only some of the questions wrought by COVID-19. The
goal of this textbook is to introduce you to the microbial world—the
magnitude of its diversity, the elegance of its biology, and the many
subdisciplines within microbiology. Unfortunately, COVID-19 has
probably already convinced you of microbiology’s importance.
Our goal in this chapter is to introduce you to this amazing world
of microorganisms and outline their evolution and history of discov-
ery. Much of microbiology is similar to what you have learned in other
biology classes that focus on large organisms. But microbes have
unique properties that often require unique approaches to under-
stand them. But before you delve into this chapter, check to see if you
have the background needed to get the most from it.
Readiness Check:
Based on what you have learned previously, you should be able to:
✓ List the features of eukaryotic cells that distinguish them from other cell
types
✓ Understand the basic structure of the macromolecules, nucleic acids,
proteins, carbohydrates, and lipids (see appendix I)
✓ Explain the terms genome, genotype, and mutation
06/12/21 5:46 PM
 2 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

The diversity of microorganisms has always presented a

1.1 Members of the Microbial World
After reading this section, you should be able to:
a. Define the term microbiology
b. Explain Carl Woese’s contributions in establishing the three-domain
system for classifying cellular life
c. Determine the type of microbe (bacterium, fungus, etc.) when given
a description of a microorganism
d. Provide an example of the importance to humans of each of the
major types of microbes
Microorganisms—those organisms too small to be seen clearly
by the unaided eye (figure 1.1) are fabulously diverse and
unimaginably abundant. It is difficult to count the number of
microbes on Earth, but estimates are about 1030 microbial cells
in habitats as diverse as termite guts and sediments deep be-
neath the seafloor (figure 1.2). There are more microbes on
Earth than stars in the known universe.
Although microbes are generally 1 millimeter or less in di-
ameter, some, such as bread molds, are visible without micro-
scopes. Some macroscopic microorganisms are multicellular.
They are distinguished from other multicellular life forms such as
plants and animals by their lack of highly differentiated tissues.
In addition, a variety of acellular biological entities, including
viruses and subviral agents, are also called microorganisms and
microbes. This is not without controversy because these agents
cannot reproduce independently.
challenge to microbial taxonomists. Early descriptions of cellu-
lar microbes as either plants or animals were too simple. For in-
stance, some microbes are motile like animals but also have cell
walls and are photosynthetic like plants. An important break-
through in microbial taxonomy arose from studies of their cel-
lular architecture, when it was discovered that cells exhibited one
of two possible “floor plans.” Cells that came to be called
prokaryotic cells (Greek pro, before; karyon, nut or kernel) have
an open floor plan. That is, their contents are not divided into
compartments by membranes. Only eukaryotic cells (Greek eu,
true) have a nucleus and other membrane-bound organelles
(e.g., mitochondria, chloroplasts) that separate some cellular ma-
terials and processes from others.
These observations eventually led to the development of a
classification scheme that divided organisms into five kingdoms:
Monera, Protista, Fungi, Animalia, and Plantae. Microorgan-
isms (except for viruses and other acellular infectious agents)
were placed in the first three kingdoms. In this scheme, all or-
ganisms with prokaryotic cell structure were placed in Monera.
However, the five-kingdom system is no longer accepted by mi-
crobiologists. This is because prokaryotes are too diverse to be
grouped together in a single kingdom. Use of the term prokar-
yote is controversial (section 3.1)
Classifying microbes has benefited from progress in three
areas. First, the development of electron microscopy tech-
niques reveals the detailed structure of microbial cells. Sec-
ond, methods that measure the biochemical and physiological
characteristics of many different microorganisms demonstrate

Organisms and
biological entities
studied by
microbiologists

can be

Cellular Acellular

includes includes

Fungi Protists Bacteria Archaea Viruses Viroids Satellites Prions

e.g. e.g. e.g. e.g. composed of composed of composed of composed of

Yeasts Algae Escherichia Methanogens Protein and RNA Nucleic acid Protein
Molds Protozoa coli nucleic acid enclosed in a
Slime molds protein shell

Figure 1.1 Concept Map Showing the Types of Biological Entities Studied by Microbiologists.
MICRO INQUIRY How would you alter this concept map so that cellular organisms are differentiated by their key features?

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 1.1 Members of the Microbial World 3

Major habitats body ­digest food and produce vitamins. In

these and many other ways, the human mi-
crobiome helps maintain our health and
Atmosphere:
5 X 1023
well-being. Overview of bacterial cell
wall structure (section 3.4); Human micro-
biome and host interactions (chapter 33)
Unfortunately some bacteria do cause
Soil: 3 X 1029 disease, and some of these diseases can have
a huge impact. In 1347 the plague (Black
Death), a disease caused by bacteria living
Deep continental in fleas, struck Europe with brutal force,
subsurface: 3 X 1029 killing one-third of the population within
Humans: 4 X 1023
4 years. Over the next 80 years, the disease
struck repeatedly, eventually wiping out
roughly half of the European population.
Oceans: 1 X 1029 The resulting labor shortage gave workers
Other habitats more power, ultimately eliminating serf-
Upper ocean Groundwater 5 X 1027 dom, and preparing the way for the
sediment: 5 X 1028 Phyllosphere 2 X 1026 Renaissance.
23 Cattle 4 X 1024
Deep ocean Sea surface: 2 X 10 Termites 6 X 10 23 Members of domain Archaea are dis-
sediment: 4 X 1029 Pigs 7 X 1023 tinguished from bacteria by many fea-
tures, most notably their distinctive rRNA
Figure 1.2 Bacterial and Archaeal Habitats and Abundance. Numbers indicate the number of sequences, cell walls, and membrane li-
microbial cells in each habitat. The majority of bacteria and archaea live in oceans and sediments, either pids. Some have unique metabolic charac-
within the Earth’s crust or deep below the crust (subsurface). The discovery of viable microbes so deep teristics, such as the ability to generate
within our planet is a recent and exciting development. Other habitats include the phyllopshere (above methane (natural) gas. Some archaea are
ground portions of plants), livestock, and humans. found in extreme environments, including
Fotout/Shutterstock those with high temperatures (thermo-
philes) and high concentrations of salt (ex-
many similarities and differences. Third, the genomic revolu- treme halophiles). Archaea do not appear to directly cause disease
tion enabled the analysis of nucleic acid and protein sequences in humans.
from a wide variety of organisms. The comparison of riboso- Domain Eukarya includes plants, animals, and microor-
mal RNA (rRNA) nucleic acid sequences, begun by Carl Wo- ganisms classified as protists or fungi. Protists are generally
ese (1928–2012) in the 1970s, transformed our understanding unicellular but larger than most bacteria and archaea. They
of the term prokaryote. It was discovered that there are two have traditionally been divided into protozoa, which have an
very different groups of organisms with prokaryotic cell mor- animal-like metabolism, and algae, which are photosynthetic.
phology: Bacteria and Archaea. Among eukaryotic microbes, However, these terms lack taxonomic value because protists,
later studies showed that Protista is not a cohesive taxonomic algae, and protozoa do not form three groups, each with a sin-
unit (i.e., taxon) and that it should be ­divided into three or gle evolutionary history. Nonetheless, for convenience, we use
more kingdoms. These studies and others led many taxono- these terms here. Protist diversity reflects broad phylogeny
mists to reject the five-kingdom system in favor of one that (section 23.1)
divides cellular organisms into three d­omains: Bacteria, Fungi are a diverse group of microorganisms that range
Archaea, and Eukarya (all eukaryotic organisms) ­(figure 1.3). from ­unicellular forms (yeasts) to multicellular molds and mush-
Nucleic acids (appendix I); Proteins (appendix I) rooms. Because of their metabolic capabilities, many fungi play
Members of domain Bacteria are usually single-celled beneficial roles, including making bread dough rise, producing
­organisms.1 Most have cell walls that contain the structural mole- antibiotics, and decomposing dead organisms. Some fungi as-
cule peptidoglycan. Despite popular belief, most bacteria do not sociate with plant roots to form mycorrhizae. Mycorrhizal fungi
cause disease. In fact, bacteria are major inhabitants of our bodies, transfer nutrients to the roots, improving growth of the plants,
forming the human microbiome. Indeed, at least as many micro- especially in poor soils. Other fungi cause plant diseases
bial cells are found in and on the human body as there are human (e.g., rusts, powdery mildews, and smuts) and diseases in
cells. These microbes contribute to the development of the body’s humans and other animals. Fungal biology reflects vast
immune system. Microbes that inhabit the large intestine help the diversity (section 24.1)

1 In this text, the term bacteria (s., bacterium) is used to refer to those microbes belonging to domain Bacteria, and the term archaea (s., archaeon) is used to refer to those that belong to domain Archaea. In
some publications, the term bacteria is used to refer to all cells having prokaryotic cell structure. That is not the case in this text.

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 4 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

Bacteria Comprehension Check

Actinobacteria
1. How did the methods used to classify microbes change,

Acidob

ia
Bac micut particularly in the last half of the twentieth century? What was the

ro cter
tero
Fir

ts
acteria

a
result of these technological advances?

as
ch anob

ia
Th

pl

er
idot s
Ve
er

ct
r 2. Identify one characteristic for each of these types of microbes that
m

dria

lo
ru

ba
Cy
ot

co
a

hon
e

eo
og

mi oc distinguishes it from the other types: bacteria, archaea, protists,

mit

ot
Ba cro
ae

Pr
cte
roid bia fungi, viruses, viroids, satellites, and prions.
ete
Chlo s
rofle
xi s 3. Describe one interaction with microbes you had yesterday.
te
lla

i
e

als
ng
fl ag

Fu

im
no e

An
h oa lga
Origin C dA
Re ts
Plan
Nanoarchaeota
eota
Stramenopiles 1.2 Microbes Have Evolved and
archa
Asgard teo
ta
pro eria
Alveolates
Acanth
Diversified for Billions of Years
o amoe
rm ha bae
The trosp Eu
ta

Ni gle
rio

ato a

After reading this section, you should be able to:

ta

no
pla teriot

zoa
cte

Archaea
ba

He
sm

a. Explain the RNA world hypothesis and the evidence that supports it
ac
o

te
an

lob

ro
nads
th

b. Design a set of experiments that could be used to place a newly

lo
Ha
rmo
Me

bo
onads

Eukarya discovered cellular microbe on a phylogenetic tree based on small

s
omo
The

ea

subunit (SSU) rRNA sequences

Diplom
Trich

rRNA sequence change

c. Compare and contrast the evolution of mitochondria and chloroplasts
Unresolved branching order

Figure 1.3 Universal Phylogenetic Tree. Only representative

lineages have been identified. A review of figure 1.3 reminds us that microbes are the dominant
organisms on Earth. How has microbial life been able to radiate
MICRO INQUIRY How many of the taxa listed in the figure include
to such an astonishing level of diversity? To answer this question,
microbes?
we must consider microbial evolution. The field of microbial
evolution, like any other scientific endeavor, is based on the
formulation of hypotheses, the gathering and analysis of data,
The microbial world also includes numerous acellular and the reformation of hypotheses based on newly acquired
infectious agents. Viruses are acellular entities that must evidence. That is to say, the study of microbial evolution is based
invade a host cell to multiply. The simplest virus particles on the scientific method. To be sure, it is difficult to amass
(also called virions) are composed only of proteins and a evidence when considering events that occurred millions, and
nucleic acid, and can be extremely small (the smallest is often billions, of years ago, but the application of molecular
10,000 times smaller than a typical bacterium). However, their methods has revealed a living record of life’s ancient history.
small size belies their power. They cause many animal and This section describes the outcome of this scientific research.
plant diseases and, as we saw most recently with COVID-19,
can trigger epidemics and pandemics that shape human his-
tory. In addition to COVID-19, viral diseases include rabies, Theories of the Origin of Life Depend
influenza, AIDS, the common cold, and some cancers. Vi- Primarily on Indirect Evidence
ruses are also important in aquatic environments, where they Dating meteorites through the use of radioisotopes places our
play a critical role in shaping microbial communities. Viroids planet at an estimated 4.5 to 4.6 billion years old. However, con-
are infectious agents composed only of ribonucleic acid ditions on Earth for the first 100 million years or so were far too
(RNA). They cause numerous plant diseases. Satellites are harsh to sustain any type of life. Eventually bombardment by
composed of a nucleic acid enclosed in a protein shell. They meteorites decreased, water appeared on the planet in liquid
must coinfect a host cell with a virus, called a helper virus, to form, and gases were released by geological activity to form
complete their life cycle. Satellites and their helper viruses Earth’s atmosphere. These conditions were amenable to the ori-
cause both plant and animal diseases. F ­ inally, prions, infec- gin of the first life forms. But how did this occur, and what did
tious agents composed only of protein, are responsible for these life forms look like?
causing neurological diseases such as scrapie and “mad cow To find evidence of life and develop hypotheses about its
disease.” Viruses and other acellular infectious agents origin and subsequent evolution, scientists must be able to define
(chapter 25) life. Although even very young children can examine an object

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and correctly determine whether it is living or not, defining life
succinctly is actually not that easy. Most definitions of life con-
sist of a set of attributes. The attributes of particular importance
to paleobiologists are an orderly structure, the ability to obtain
and use energy (i.e., metabolism), and the ability to reproduce.
Just as NASA scientists are using the characteristics of microbes
on Earth today to search for life elsewhere, so too are scientists
examining extant organisms, those organisms present today, to
explore the origin of life. Some extant organisms have structures
and molecules that represent relics of ancient life forms. These
can provide scientists with ideas about the type of evidence to
seek when testing hypotheses.
The best direct evidence for the nature of primitive life
would be a fossil record. There have been reports of microbial
fossil discoveries since 1977. These have always met with skepti-
cism because some objects that look like cells can be formed by
geological forces that occurred as the rock was formed. The re-
sult is that the fossil record for microbes is sparse and always
open to reinterpretation.
Despite these problems, most scientists agree that life was
present on Earth about 3.5 to 3.8 billion years ago (figure 1.4).
To reach this conclusion, biologists rely on indirect evidence.
Among the indirect evidence used are molecular fossils. These
are chemicals found in rock or sediment that are chemically re-
lated to biological molecules. For instance, the presence of mol-
ecules called hopanes in a rock indicates that bacteria were
present when the rock was formed. This conclusion is reached
because hopanes are formed from hopanoids, which are found in
the plasma membranes of extant bacteria. As you can see, no
single piece of evidence can stand alone. Instead many pieces of
evidence are put together in an attempt to get a coherent picture
to emerge, as with a jigsaw puzzle.
Early Life Was Probably RNA-based
Before there was life, most evidence suggests that Earth was a
very different place: hot and anoxic, with an atmosphere rich
in water vapor, carbon dioxide, and nitrogen. In the oceans,
hydrogen, methane, and carboxylic acids were formed by geo-
logical and chemical processes. Areas near hydrothermal
vents may have provided the conditions that allowed chemi-
cals to react with one another, randomly testing the usefulness
of the reaction and the stability of its products. Some reactions
generated molecules that functioned as catalysts, some
aggregated with other molecules to form the predecessors of
modern cell structures, and others were able to replicate and
act as units of hereditary information (Microbial Diversity &
Ecology 1.1).
How did early cells, sometimes called probionts, arise? In
modern cells, three different molecules fulfill the roles of cata-
lysts, structural molecules, and hereditary molecules. Proteins
have two major roles in modern cells: catalytic and structural.
Catalytic proteins are enzymes and structural proteins serve
myriad functions, such as transport, attachment, and motility.
wil88396_ch01_001-022.indd 5
1.2 Microbes Have Evolved and Diversified for Billions of Years 5
DNA stores hereditary information that is replicated and passed
on to the next generation. RNA converts the information stored
in DNA into protein. Any hypothesis about the origin of life must
account for the evolution of these molecules, but their relation-
ships to each other in modern cells complicates attempts to im-
agine how they evolved. Proteins can do cellular work, but their
synthesis involves other proteins and RNA, and uses information
stored in DNA. DNA cannot do cellular work, and proteins are
needed for its replication. RNA synthesis requires both DNA as
the template and proteins as catalysts.
Based on these considerations, it is hypothesized that at some
time in the evolution of probionts, there must have been a single
molecule that could do both cellular work and replicate. This idea
was supported in 1981 when Thomas Cech discovered an RNA
molecule in a protist (Tetrahymena sp.) that also had catalytic ac-
tivity. Since then, other catalytic RNA molecules have been dis-
covered, including an RNA found in ribosomes that is responsible
for forming peptide bonds—the bonds that hold together amino
acids, the building blocks of proteins. Catalytic RNA molecules
are now called ribozymes.
The discovery of ribozymes suggested that RNA at some
time was capable of storing, copying, and expressing genetic
information, as well as catalyzing other chemical reactions. In
1986 Nobel laureate Walter Gilbert coined the term RNA world
to describe this precellular stage in the evolution of life. However,
for this precellular RNA-based stage to proceed to the evolution
of cellular life forms, a lipid membrane must have formed around
the RNA (figure 1.5). This important evolutionary step is easier
to imagine than other events in the origin of cellular life forms
because lipids, major structural components of the membranes of
modern organisms, spontaneously form liposomes—vesicles
bounded by a lipid bilayer. The notion of an RNA world has
caused some scientists to look for evidence on Mars, where
conditions are thought to have been frozen in the prebiotic era.
Lipids (appendix I)
Back here on Earth, Jack Szostak, also a Noble laureate, is
a leader in experimentally simulating how protobionts con-
taining only RNA may have formed. When his group created
liposomes using simpler fatty acids than those found in mem-
branes today, the liposomes were leaky. These leaky liposomes
allowed single RNA nucleotides to move into the ­l iposome, but
prevented large RNA chains from moving out. Furthermore,
researchers could prod the liposomes into growing and divid-
ing. Dr. Szostak’s group has also been able to create conditions
in which an RNA molecule could serve as a template for syn-
thesis of a complementary RNA strand. These experiments
may have recapitulated early steps in the evolution of cells. As
seen in figure 1.5, several other processes need to occur to
reach the level of complexity found in extant cells.
Apart from its ability to perform catalytic activities, the
function of RNA suggests its ancient origin. Consider that much
of the cellular pool of RNA in modern cells exists in the ribo-
some, a structure that consists largely of ribosomal RNA
(rRNA) and uses messenger RNA (mRNA) and transfer RNA
06/12/21 5:46 PM
 6 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

Millions

Eras
Eons

Periods
of Years
Ago
0
Quaternary
CENOZOIC
1.8
7 mya—Hominids first appear.
Tertiary
65
Cretaceous
MESOZOIC

144
Jurassic
PHANEROZOIC

206
Triassic 225 mya—Dinosaurs and mammals first appear.
248
Permian
290
Carboniferous 300 mya—Reptiles first appear.
354
PALEOZOIC

417
Devonian

443
Silurian
450 mya—Large terrestrial colonization by plants and animals.
Ordovician
490
Cambrian 520 mya—First vertebrates; first land plants.
543

533–525 mya—Cambrian explosion creates diverse animal life.

LATE

900
MIDDLE
PROTEROZOIC

1.5 bya—Multicellular eukaryotic organisms first appear.

1,600
EARLY
PRECAMBRIAN

2,500
2.5–2.0 bya—Eukaryotic cells with mitochondria or chloroplasts first appear.
LATE

3,000
ARCHAEAN

MIDDLE

3,400

3.7 bya—Fossils of primitive microbes.

EARLY

3,800
3.8–3.5 bya—First cells appear.
HADEAN

4,550

Figure 1.4 An Overview of the History of Life on Earth. mya = million years ago; bya = billion years ago.

wil88396_ch01_001-022.indd 6 06/12/21 5:46 PM


MICROBIAL DIVERSITY & ECOLOGY
1.1 Hydrothermal Vents: Did Life Begin Under the Sea?
Whether or not early life was RNA-based, one thing is clear:
the origin of life needed energy to synthesize biomolecules.
So, perhaps the most fundamental evolutionary question is
“Where did biomolecules and the energy needed to build
them come from?” Three hypotheses have been suggested.
First, the panspermia theory speculates that meteorites
bombarded our planet, bringing with them other-worldly bio-
molecules. Second, the more familiar primordial soup theory
suggests that organic molecules were spontaneously assem-
bled by an input of energy, such as lightning strikes. The last
theory, which has gained evidence in recent years, hypothe-
sizes that both the energy and the molecules originated in
hydrothermal vents. Let’s explore the hydrothermal vent
theory.
Hydrothermal vents are geothermally active deep-sea
chasms thousands of meters below the surface of the ocean.
Their discovery in 1977 sparked tremendous excitement as im-
ages of entirely new ecosystems with mysterious organisms cap-
tured the attention of scientists and the public (see section 27.2).
These vents pump 400oC sulfide-rich water into cold ambient
water, causing the sulfide to instantly precipitate, so these
chimneylike structures are dubbed “black smokers.” In
2000, scientists made yet another deep-sea discovery with a
different kind of vent system. These are cooler (45–90oC)
and alkaline (pH 9–11). When these waters mix with the
surrounding seawater (pH about 8.0), calcium carbonate pre-
cipitates, forming white chimneys, as seen in the Lost City
vents (box figure).
This pH gradient is critical to the hypothesis that a vent
system, such as Lost City, could be the origin of biomolecules.
As you may have learned when studying mitochondria or
batteries, the separation of positive and negative charges cap-
tures potential energy (remember that energy can’t be cre-
ated). In Lost City vents, the thin walls of the chimneys serve
(tRNA) to construct proteins. Also rRNA itself catalyzes pep-
tide bond formation during protein synthesis. Thus RNA seems
to be well poised for its importance in the development of pro-
teins (figure 1.5). Because RNA and DNA are structurally simi-
lar, RNA could have given rise to double-stranded DNA. It is
suggested that once DNA evolved, it became the storage facility
for genetic information because it provides a more chemically
stable structure. Two other pieces of evidence support the RNA
world hypothesis: the fact that the energy currency of cells,
ATP, is a ribonucleotide and the discovery that RNA can regu-
late gene expression. ATP: the major energy currency of
cells (section 10.2); Riboswitches: effector-mRNA interactions
wil88396_ch01_001-022.indd 7
1.2 Microbes Have Evolved and Diversified for Billions of Years 7
D.Kelley, University of Washington.
to separate these fluids with as much as a 3-unit pH differ-
ence. The question now being asked is “Was this potential
energy tapped to convert CO2 in seawater to simple carbon-
based molecules, such as amino acids, short hydrocarbons,
and others?”
If the answer is yes, a 2019 study shows that a mixture of
molecules called single-chain amphiphiles (SCAs), which are
simpler versions of more familiar phospholipids, can form
vesicles in hot, alkaline pH seawater that mimics that of Lost
City. Putting this together, we can hypothesize a series of
events that occurred 3.7–4.0 billion years ago. First, the pres-
ence of the pH gradient across geological barriers in the Lost
City drove the formation of random organic molecules, some
of which were SCAs. These SCAs accumulated and formed
vesicles that entrapped fluids preserving the pH gradient.
These vesicles had the energy to test the formation of differ-
ent molecules. Was one of them RNA?
regulate transcription (section 14.3); Translational riboswitches
(section 14.4)
However, the RNA world hypothesis is not without prob-
lems, and more recent experiments suggest the first nucleic ac-
ids may have been a mix of DNA and RNA molecules. Another
area of research also fraught with considerable debate is the
evolution of metabolism. The early Earth was a hot environment
that lacked oxygen. Thus cells that arose there must have been
able to use the available energy sources under these harsh con-
ditions. Today there are heat-loving a­ rchaea capable of using
inorganic molecules such as FeS as a source of energy. Some
suggest that this interesting metabolic capability is a remnant of
06/12/21 5:46 PM
 8 CHAPTER 1 | The Evolution of Microorganisms and Microbiology
Lipid vesicle RNA
Lipid membrane forms
around RNA.
Probiont: RNA only
RNA replicates
and catalyzes
protein synthesis.
Probiont: RNA and proteins
DNA evolves
from RNA as the
information storage
molecule.
Cellular life: RNA, DNA, and proteins
Figure 1.5 The RNA World Hypothesis for the Origin of Life.
MICRO INQUIRY Why are the probionts pictured above not considered
cellular life?
the first form of energy metabolism. Another metabolic strat-
egy, oxygen-releasing photosynthesis (oxygenic photosynthe-
sis), appears to have evolved perhaps as early as 2.7 billion years
ago. This is supported by the discovery of ancient stromatolites
(figure 1.6). Stromatolites are layered rocks formed by the in-
corporation of mineral sediments into layers of cyanobacteria
growing in thick mats on surfaces. The oxygen released by these
early cyanobacteria is thought to have altered Earth’s atmosphere
to its ­current oxygen-rich state, allowing the evolution of addi-
tional energy-capturing strategies such as aerobic respiration,
the oxygen-consuming metabolic process used by many mi-
crobes and animals.
Evolution of the Three Domains of Life
Look closely at figure 1.3 and find a line labeled “Origin.” This
is where data indicate the last universal common ancestor
(LUCA) to all three domains should be placed. LUCA is the
most recent organism from which all three types of life—
bacterial, archaeal, and eukaryotic—arose. On this tree of life,
wil88396_ch01_001-022.indd 8
Figure 1.6 Stromatolites. Modern stromatolites from Western Australia.
Each stromatolite is a rocklike structure, typically 1 m in diameter, containing
layers of ­c yanobacteria.
Horst Mahr/imagebroker/age fotostock
LUCA is on the bacterial branch, which means that Archaea and
Eukarya evolved independently, separate from Bacteria.
The evolutionary relationship of Archaea and Eukarya is
still a matter of considerable debate. According to the universal
phylogenetic tree (figure 1.3), Archaea and Eukarya shared
common ancestry but diverged and became separate domains.
Recent evidence supports the notion that Eukarya evolved from
Archaea (see Microbial Diversity & Ecology 26.1). The close
evolutionary relationship of these two forms of life is still evident
in the manner in which they process genetic information. For
instance, certain protein subunits of archaeal and eukaryotic
RNA polymerases, the enzymes that catalyze RNA synthesis,
resemble each other to the exclusion of those of bacteria. How-
ever, archaea have other features that are most similar to their
counterparts in bacteria (e.g., mechanisms for conserving en-
ergy). This has further complicated and fueled the debate. The
evolution of the nucleus and endoplasmic reticulum is also con-
troversial. However, hypotheses regarding the evolution of other
membrane-bound organelles are more widely accepted and are
considered next.
Mitochondria, Mitochondria-Like Organelles, and
Chloroplasts Evolved from Endosymbionts
The endosymbiotic hypothesis is generally accepted as the ori-
gin of several eukaryotic organelles, including mitochondria,
chloroplasts, and hydrogenosomes. Endosymbiosis is an interac-
tion between two organisms in which one organism lives inside
the other. The original endosymbiotic hypothesis proposed that
over time a bacterial endosymbiont of an ancestral cell in the
eukaryotic lineage lost its ability to live independently. If the in-
tracellular bacterium used aerobic respiration, it became a mito-
chondrion. If the endosymbiont was a cyanobacterium and
therefore performed photosynthesis, it became a chloroplast
(figure 1.7).
06/12/21 5:47 PM

Animals, fungi, and protists
(contain mitochondria)
0
Billions of years ago (bya)
Primordial
Evolution eukaryotic
cells
1
Cyanobacterium
Proteobacterium
2 sexually, whereby each offspring has a mixture of parental genes
(a) Mitochondria originated
from endosymbiotic
proteobacteria.
Figure 1.7 The Endosymbiotic Theory. (a) According to this hypothesis,
mitochondria derived from a bacterium in the phylum Proteobacteria.
(b) A similar phenomenon occurred for chloroplasts, which derived from
cyanobacteria.
Although the mechanism by which the endosymbiotic rela-
tionship was established is unknown, there is considerable evi-
dence to support this hypothesis. Mitochondria and chloroplasts
contain DNA and ribosomes; both are similar to bacterial DNA
and ribosomes. Peptidoglycan, the unique bacterial cell wall
molecule, has even been found between the two membranes that
enclose the chloroplasts of some algae. Indeed, inspection of fig-
ure 1.3 shows that both organelles belong to the bacterial lineage.
More specifically, mitochondria are most closely related to bac-
teria called proteobacteria. The chloroplasts of plants and green
algae are thought to have descended from an ancestor of the
cyanobacterial genus Prochloron, which contains species that
live within marine invertebrates. Phylum Cyanobacteria: oxy-
genic photosynthetic bacteria (section 20.4); The proteobacte-
rial origin of mitochondria (section 21.1)
The endosymbiotic hypothesis for mitochondria has been
refined by the hydrogen hypothesis. This asserts that the
endosymbiont was an anaerobic bacterium that produced H2 and
CO2 as end products of its metabolism. Over time, the host be-
came dependent on the H2 produced by the endosymbiont. Ulti-
mately the endosymbiont evolved into one of several organelles
(see figure 5.13). Some endosymbionts evolved into organelles
such as a hydrogenosome—an organelle found in some extant
wil88396_ch01_001-022.indd 9
Plants and algae
(contain mitochondria
and chloroplasts)
Evolution
(b) Chloroplasts originated
from endosymbiotic
cyanobacteria.
1.2 Microbes Have Evolved and Diversified for Billions of Years 9
protists that produces ATP by a process called fermentation (see
figure 5.15).
Evolution of Cellular Microbes
Although the history of early cellular life forms may never be
known, we know that once microbial cells arose, they were sub-
jected to the same evolutionary processes as modern organisms.
The ancestral bacteria, archaea, and eukaryotes possessed ge-
netic information that could be duplicated, lost, or mutated in
other ways. Mutations could have many outcomes. Some led to
the death of the microbe, but others allowed new functions and
characteristics to evolve. Mutations that allowed the organism to
increase its rate of reproduction or survival were selected and
passed on to subsequent generations. In addition to selective
forces, geographic isolation of populations allowed some groups
to evolve separately from others. Thus selection and isolation led
to the eventual development of new collections of genes (i.e.,
genotypes) and new species.
In addition to mutation, other mechanisms exist for reconfig-
uring genomes and therefore creating genetic diversity. Most eu-
karyotic species increase their genetic diversity by reproducing
and a unique genotype. Bacteria and archaea do not reproduce
sexually. They increase their genetic diversity by mutation and
horizontal gene transfer (HGT). During HGT, genetic information
from a donor organism is transferred to a recipient, creating a new
genotype in the recipient. In this way genetic information is
passed between individuals of the same generation and even be-
tween species found in different domains of life. Genome se-
quencing has revealed that HGT has played an important role in
the evolution of all microbial species. Importantly, HGT still
occurs in bacteria and archaea leading to the rapid evolution of
microorganisms with antibiotic resistance, new virulence proper-
ties, and novel metabolic capabilities. The outcome of HGT is that
most microbes have mosaic genomes composed of bits and pieces
of the genomes of other organisms. Horizontal gene transfer:
creating genetic variation the asexual way (section 16.4)
Phylogenetic or phyletic classification systems compare
organisms on the basis of evolutionary relationships. The term
phylogeny (Greek phylon, tribe or race; genesis, generation or
origin) refers to the evolutionary development of organisms.
As discussed, microbial phylogeny relies on comparisons of
multiple features found in extant organisms. These include
cell wall structure, biomolecules such as fatty acids, and cer-
tain housekeeping proteins (proteins used to maintain cellular
life, therefore found in many different organisms), and nucleo-
tide sequences, particularly of small subunit rRNA molecules
(SSU rRNA) (table 1.1). Bacterial ribosomes (section 3.7);
Archaeal ribosomes (section 4.3); Eukaryotic ribosomes
(section 5.5)
Phylogenetic Trees
Figure 1.3 is an example of a phylogenetic tree. The goal of
phylogenetic tree construction is to display the evolutionary
06/12/21 5:47 PM
 10 CHAPTER 1 | The Evolution of Microorganisms and Microbiology
Table 1.1
Property Bacteria
relationships between different organisms. Trees are built by
comparing the sequences of amino acids or nucleotides from
diverse organisms. Amino acid sequences are often compared
because nucleotide changes may not alter the resulting protein
and therefore have little or no impact on evolution. Historically,
the rRNAs from small ribosomal subunits (16S from bacteria
and archaea and 18S from eukaryotes) were the molecules of
choice for inferring microbial phylogenies and making taxo-
nomic assignments for several reasons. Like housekeeping pro-
teins (proteins with essential functions), SSU rRNAs play the
same role in all microorganisms and are absolutely necessary
for survival. Therefore neither housekeeping proteins nor genes
encoding SSU rRNAs tolerate large changes in sequence. The
utility of SSU rRNAs is extended by the presence of certain
sequences within SSU rRNA genes that vary among organisms
as well as other regions that are quite similar. The variable re-
gions enable comparison between closely related microbes,
whereas the stable sequences allow the comparison of more
distantly related microorganisms. The stability of SSU rRNA
wil88396_ch01_001-022.indd 10
Archaea Eukarya
sequences means that they evolve very slowly, so analysis of
housekeeping genes is more frequently used to infer the evolu-
tion of more closely related organisms.
Comparative analysis of SSU rRNA sequences from thou-
sands of organisms has demonstrated the presence of oligonu-
cleotide signature sequences (figure 1.8). These are short,
conserved nucleotide sequences specific for phylogenetically
defined groups of organisms. Thus the signature sequences
found in bacterial rRNAs are rarely or never found in archaeal
rRNAs, and vice versa. Likewise, the 18S rRNA of eukaryotes
bears signature sequences that are specific to the domain
Eukarya.
Approaches to building a phylogenetic tree can be divided
into two broad categories: a distance-based approach and a char-
acter-based approach. Distance-based approaches are the most
intuitive. Here the differences between the aligned sequences are
counted for each pair and summarized in a single statistic
(figure 1.9). This value serves as a measure of the evolutionary
distance between the organisms; the more differences counted,
06/12/21 5:47 PM

Bacteria
Figure 1.8 Signature rRNA Help Identify Microbes. Representative examples of rRNA secondary structures from the three domains: Bacteria (Escherichia
coli), Archaea (Methanococcus vannielii), and Eukarya (Saccharomyces cerevisiae).
the greater the evolutionary distance. The evolutionary distances
from many comparisons are used by sophisticated computer pro-
grams to construct the tree. The tip of each branch in the tree
(called a node) represents one of the organisms used in the com-
parison. The distance from one node to another is the evolution-
ary distance between the two organisms.
An algorithm analyzes this information to generate a tree.
One algorithm called cluster analysis serially links pairs that are
ever more distantly related (i.e., start with those with the least
number of sequence differences and move to those with the
most). Although this is the easiest to understand, it can generate
trees based on artifact. Neighbor joining is another distance-
based method that uses a different matrix to modify the distance
between each pair of nodes based on the average divergence
from all other nodes.
Character-based approaches for phylogenetic tree building
are more complicated but generate more robust trees. These
methods start with assumptions about the pathway of evolution,
infer the ancestor at each node, and choose the best tree according
to a specific model of evolutionary change. Sometimes called
tree-searching, these methods include maximum parsimony,
which assumes that the fewest number of changes occurred
between ancestor and extant organisms. Another approach is
called maximum likelihood. This requires a large data set,
because for each possible tree that can be built, its probability
(i.e., the likelihood) based on certain evolutionary and molecular
information is determined so that the tree with the greatest
probability based on these criteria is selected. Bayesian inference
is another character-based approach. Rather than looking at a
single tree, Bayesian inference analyzes multiple potential trees
wil88396_ch01_001-022.indd 11
1.2 Microbes Have Evolved and Diversified for Billions of Years 11
Archaea Eukarya
~ 230
bases
and calculates the probability that each branch would appear
based on this comparison.
Once a tree is constructed, it is important to get a sense
of whether the placement of its branches and nodes is legitimate.
There are a variety of methods to assess the strength of a tree, but
the most common is bootstrapping. Bootstrapping involves re-
analyzing a randomly selected subset of the data presented on the
tree. A bootstrap value is the percent of times in which that par-
ticular branch was found. Typically, bootstrap values of 70% or
greater are thought to support a tree. Of note is that Bayesian
inference values are also reported as percentages, but they are
not directly comparable to bootstrap values. Only values greater
than 95% are acceptable when Bayesian inference is used.
Two things should be kept in mind when examining
phylogenetic trees. The first is that they are molecular trees, not
organismal trees. In other words, they represent, as accurately
as possible, the evolutionary history of a molecule (e.g., rRNA).
Second, the distance between nodes is a measure of related-
ness, not of time. If the distance along the lines is very long,
then the two organisms are more evolutionarily diverged (i.e.,
less related). However, we do not know when they diverged
from each other. This concept is analogous to a printed map
that accurately shows the distance between two cities but
because of many factors (traffic, road conditions, etc.) cannot
show the time needed to travel that distance.
Importantly, a tree may be unrooted or rooted. An unrooted
tree (figure 1.10a) represents phylogenetic relationships but does
not indicate which organisms are more primitive relative to the
others. Figure 1.10a shows that A is more closely related to C
than it is to either B or D, but it does not indicate which of the
06/12/21 5:47 PM
 12 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

A B
Branch

Cells from organism 1

Lyse cells to release contents and isolate DNA.

C Nodes D
DNA
(a)
Use polymerase chain reaction to amplify
and purify SSU rRNA genes. B

SSU rRNA genes

A
Sequence genes.
C
ATGCTCAAGTCA D

Repeat process for other organisms.

C
Align sequences to be compared.
B
Organism SSU rRNA sequence D
1 ATGCTCAAGTCA
2 TAGCTCGTGTAA A Z
3 AAGCTCTAGTTA
AACCTCATGTTA (b) (c)
4
Figure 1.10 Phylogenetic Tree Topologies. (a) Unrooted tree joining
Count the number of nucleotide differences between four taxonomic units (A, B, C, D). (b) Rooted tree. (c) The tree can be rooted by
each pair of sequences and calculate evolutionary
distance (ED). adding an outgroup, represented by Z. The arrow indicates a speciation event
that resulted in the development of new species from an ancestral organism.

Pair compared ED Corrected ED For organisms 1 and 2, 5 of the 12

nucleotides are different: four species might be the oldest. In contrast, the rooted tree
1 2 0.42 0.61
ED = 5/12 = 0.42 (figure 1.10b) includes a node (taxonomic unit) that serves as the
1 3 0.25 0.30
The initial ED calculated is corrected common ancestor and shows the development of the four species
1 4 0.33 0.44
using a statistical method that from this root. It is much more difficult to develop a rooted tree.
2 3 0.33 0.44 considers for each site the probability For example, there are 15 possible rooted trees that connect four
2 4 0.33 0.44 of a mutation back to the original
nucleotide or of additional forward
species but only three possible unrooted trees.
3 4 0.25 0.30
mutations. An unrooted tree can be rooted by adding data from an
outgroup—a species known to be very distantly related to all the
Computer analysis is used to construct phylogenetic tree. species in the tree (figure 1.10c). The root is determined by the
point of the tree where the outgroup joins. This provides a point
of reference to identify the oldest node on the tree, which is the
3
node closest to the outgroup. So, for example, in figure 1.10c,
Unrooted phylogenetic tree. Note
0.08 that distance from one tip to
organism Z is the outgroup and the oldest node on the tree is
1 another is proportional to the ED. marked with an arrow.
0.08
0.23
2
0.15 0.30
Microbial Taxonomy
The science of classifying living things is called taxonomy.
Taxonomy consists of three separate but interrelated parts:
4
classification, nomenclature (naming), and identification. A
Figure 1.9 The Construction of a Phylogenetic Tree Using a taxonomic scheme is used to arrange organisms into groups
Distance Method. The polymerase chain reaction is described in called taxa (s., taxon) based on mutual similarity. Microbes
chapter 17. are placed in taxonomic levels arranged in a nonoverlapping
MICRO INQUIRY Why does the branch length indicate amount of hierarchy so that each level includes not only the traits that
evolutionary change but not the time it took for that change to occur? define the rank above it but also a new set of more restrictive

wil88396_ch01_001-022.indd 12 06/12/21 5:47 PM


Domain
Phylum
Class α-Proteobacteria
Order Chromatiales Thiotrichales
Family
Genus Enterobacter Escherichia Klebsiella
Species
Figure 1.11 Hierarchical Arrangement in Taxonomy. In this example, members of the genus Shigella are placed within higher taxonomic ranks. Not all
classification possibilities are given for each rank to simplify the diagram. Note that -ales denotes order and -ceae indicates family.
traits (figure 1.11). Thus within each domain—Bacteria,
Archaea, or Eukarya—each organism is assigned (in
descending order) to a phylum, class, order, family, genus, and
species epithet or name. Some microbes are also given a
subspecies designation. Microbial groups at each level have a
specific suffix that indicates rank or level.
Microbiologists name microbes using the binomial system
of the eighteenth-century biologist and physician Carl Linnaeus.
The Latin, italicized name consists of two parts. The first part,
which is capitalized, is the generic name (i.e., the name of the
genus to which the microbe belongs), and the second is the
uncapitalized species epithet. For example, the bacterium that
causes plague is called Yersinia pestis. Often the name of an
organism will be shortened by abbreviating the genus name with
a single uppercase letter (e.g., Y. pestis).
This straightforward organizational approach is complicated
by the fact that bacteria and archaea do not reproduce sexually.
You may recall from a general biology class that plant and animal
species are defined as a group of interbreeding or potentially
interbreeding natural populations reproductively isolated from
other groups. This definition also is appropriate for the many
eukaryotic microbes that reproduce sexually. However, bacterial
and archaeal species cannot be defined by this criterion because
they do not reproduce sexually. Therefore comparisons of ge-
nome sequences are often used to distinguish one species from
another. An appropriate definition is currently debated. A
common definition is that bacterial and archaeal species are a
collection of strains that share many stable properties and differ
significantly from other groups of strains. A strain consists of
the descendants of a single, pure microbial culture. Strains
within a species may be described in a number of different ways.
Biovars are variant strains characterized by biochemical or
wil88396_ch01_001-022.indd 13
1.2 Microbes Have Evolved and Diversified for Billions of Years 13
Bacteria
Proteobacteria
β-Proteobacteria γ-Proteobacteria
Legionellales Pseudomonadales Enterobacterales
Enterobacteriaceae
Vibrio Proteus Salmonella Serratia Shigella Pasteurella Yersinia
S. boydii S. dysenteriae S. flexneri S. sonnei
S. flexneri serotype 2a strain 2457T
physiological differences, morphovars differ morphologically,
serovars have distinctive properties that can be detected by
antibodies, and pathovars are pathogenic strains distinguished
by the plants in which they cause disease.
Although microbiologists continue to use Linnaeus’s classifica-
tion system, the ongoing explosion in metagenomic analysis has had
an impact on this historically accepted hierarchy. Within the last few
decades, thousands of 16S rRNA genes and protein-coding genes
have been sequenced that do not belong to any previously defined
taxa. These data have led to the recent development of the taxonomic
classification superphylum, below domain and above phylum (e.g.,
in figure 1.11, superphylum would be placed between Bacteria and
Proteobacteria). A superphylum includes organisms of several phyla
that share a number of distinctive characteristics, such as unusual
morphological or metabolic features.
Comprehension Check
1. Describe two reasons why RNA is thought to be the first self-
replicating biomolecule.
2. Explain the endosymbiotic hypothesis of the origin of mitochondria,
hydrogenosomes, and chloroplasts. List two pieces of evidence that
support this hypothesis.
3. What is the goal of phylogenetic tree construction?
4. In general terms, what is the difference between a distance-based
and character-based tree? Does a rooted or unrooted phylogenetic
tree provide more information? Explain your answer.
5. What is the difference between mutation and horizontal gene transfer?
6. What is the correct way to write this microbe’s name: bacillus
subtilis, Bacillus subtilis, Bacillus Subtilis, or Bacillus subtilis?
Identify the genus name and the species epithet.
06/12/21 5:47 PM
 14 CHAPTER 1 | The Evolution of Microorganisms and Microbiology
1.3 Microbiology Advanced as New Tools
for Studying Microbes Were
Developed
After reading this section, you should be able to:
a. Evaluate the importance of the contributions made by Hooke,
Leeuwenhoek, Pasteur, Lister, Koch, Beijerinck, von Behring,
Kitasato, Metchnikoff, and Winogradsky to microbiology
b. Outline a set of experiments that might be used to decide if a
particular microbe is the causative agent of a disease
c. Predict the difficulties that might arise when using Koch’s postulates
to determine if a microbe causes a disease unique to humans
Even before microorganisms were seen, some investigators sus-
pected their existence and role in disease. Among others, the
Roman philosopher Lucretius (about 98–55 bce) and the physi-
cian Girolamo Fracastoro (1478–1553) suggested that disease
was caused by invisible living creatures. However, until mi-
crobes could actually be seen and studied in some other way,
their exis­tence remained a matter of conjecture. Therefore
microbiology is defined not only by the organisms it studies but
also by the tools used to study them. The development of micro-
scopes was the critical first step in the evolution of the disci-
pline. However, microscopy alone is unable to answer the many
questions scientists ask about microbes. A distinct feature of
microbiology is that microorganisms are usually removed from
their normal habitats and grown in isolation, apart from all
other microbes. This is called a pure or axenic culture. Al-
though the development of techniques for isolating microbes in
pure culture was another critical step in microbiology’s history,
it is now recognized as having limitations. Microbes in pure
culture are in some ways like animals in a zoo; just as a zoolo-
gist cannot fully understand animals by studying them in zoos,
microbiologists cannot fully understand microbes by studying
them in pure culture. Today molecular genetic techniques and
genomic analyses are providing new insights into the lives of
microbes.
Here we describe how the tools used by microbiologists have
influenced the development of the field. As microbiology evolved
as a science, it contributed greatly to the well-being of humans.
The historical context of some of the important discoveries in
microbiology is shown in figure 1.12.
Microscopy Led to the Discovery of Microorganisms
The earliest microscopic observations of organisms appear to
have been made between 1625 and 1630 on bees and weevils by
the Italian Francesco Stelluti (1577–1652), using a microscope
probably supplied by Galileo (1564–1642). Robert Hooke
(1635–1703) is credited with publishing the first drawings of
microorganisms in the scientific literature. In 1665 he published
a highly detailed drawing of the fungus Mucor in his book
wil88396_ch01_001-022.indd 14
Micrographia. Micrographia is important not only for its
exquisite drawings but also for the information it provided on
building microscopes. One design discussed in Micrographia
was probably a prototype for the microscopes built and used by
the amateur microscopist Antony van Leeuwenhoek (1632–1723)
of Delft, the Netherlands. Leeuwenhoek earned his living selling
men’s clothing and accessories but spent much of his spare time
constructing simple microscopes composed of double convex
glass lenses held between two silver plates (figure 1.13a). His
microscopes could magnify about 50 to 300 times, and he may
have illuminated his liquid specimens by placing them between
two pieces of glass and shining light on them at a 45-degree angle
to the specimen plane. This would have provided a form of dark-
field illumination whereby organisms appeared as bright objects
against a dark background. Beginning in 1673, Leeuwenhoek
sent detailed letters describing his discoveries to the Royal
­Society of London. It is clear from his descriptions that he
saw both bacteria and “protists” (figure 1.13b). Dark-field
­microscope: bright object, dark background (section 2.2)
Culture-based Methods for Studying
Microorganisms Were a Major Development
As important as Leeuwenhoek’s observations were, the develop-
ment of microbiology essentially languished for the next 200 years
until techniques for isolating and culturing microbes in the labora-
tory were formulated. During this time, scientists grappled with the
conflict over the theory of spontaneous generation. This conflict
and the subsequent ­studies on the role played by microorganisms in
causing disease ultimately led to what is now called the golden age
of microbiology.
Spontaneous Generation
From earliest times, people had believed in spontaneous
­generation—that living organisms could develop from nonliv-
ing matter. This view was challenged by the Italian physician
Francesco Redi (1626–1697), who carried out a series of experi-
ments on decaying meat, which was thought to produce maggots
spontaneously. Using covered and uncovered containers of meat,
Redi clearly demonstrated that maggots on decaying meat re-
sulted from the presence of fly eggs, and meat did not spontane-
ously generate maggots. Other e­ xperiments helped discredit the
theory for larger organisms. However, Leeuwenhoek’s commu-
nications on microorganisms renewed the controversy. Some
proposed that microbes arose by spontaneous generation but
larger organisms did not. They pointed out that boiled extracts of
hay or meat gave rise to microorganisms after sitting for a while.
In 1748, the English priest John Needham (1713–1781) suggested
that the organic ­matter in these extracts contained a “vital force”
that could confer the properties of life on nonliving matter.
A few years after Needham’s experiments, the Italian priest
and naturalist Lazzaro Spallanzani (1729–1799) sealed glass
flasks that contained water and seeds and then placed the flasks
in boiling water for about 45 minutes. He found that no growth
took place as long as the flasks remained sealed. He proposed
06/12/21 5:47 PM
 1.3 Microbiology Advanced as New Tools for Studying Microbes Were Developed 15

1668 Redi refutes

1348 Plague (Black Death) 1665 Hooke publishes spontaneous generation
reaches England. Micrographia. of maggots. 1765–1776 Spallanzani
attacks spontaneous
1674–1676 Leeuwenhoek generation.
discovers “animalcules.”
1798 Jenner introduces
1543 Publication of cowpox vaccination for
Copernicus’s work smallpox.
on heliocentric 1687 Newton’s 1775 American
solar system 1854 Snow traces
1620 Francis Bacon Principia published. Revolution begins. cholera source to water
argues for importance 1859 Darwin’s pump.
of inductive reasoning Origin of Species
in scientific method. 1861–1865 1861 Pasteur disproves
American Civil War spontaneous generation.

1876 Bell invents

1879 Edison’s telephone. 1876 Koch demonstrates
first light bulb that Bacillus anthracis
causes anthrax.

1889 Eiffel Tower 1884 Koch’s postulates

completed. published; Metchnikoff
1887–1890 describes phagocytosis;
Winogradsky studies 1885 Pasteur autoclave developed;
1893 Munsch sulfur and nitrifying develops Gram stain developed.
paints The Scream. bacteria. rabies vaccine.

1888 Beijerinck
1898 Spanish- isolates root
American War nodule bacteria.
1911 Rous discovers a
1899 Beijerinck proves
virus can cause cancer.
virus causes tobacco
1900 Planck mosaic disease. 1915–1917 D’Herelle
develops and Twort discover
quantum theory. bacterial viruses.
1903 Wright brothers’
1914 World 1923 First edition of
first powered aircraft
War I begins. 1917 Russian Bergey’s Manual
1905 Einstein’s Revolution
theory of relativity 1918 Influenza pandemic
1908 First kills over 50 million people. 1927 Lindbergh’s 1928 Griffith discovers
Model T Ford transAtlantic flight bacterial transformation.
1937 Krebs 1933 Hitler
becomes 1929 Stock 1929 Fleming
1939 World discovers market crash
citric acid cycle. chancellor discovers penicillin.
War II begins. of Germany.
1945 Atomic bomb 1932 Knoll and Ruska
dropped on Hiroshima. build first electron
microscope.
1953 Watson and
1950 Korean War Crick propose 1990 First human
begins. DNA double helix. gene therapy
testing begun.
1961 Jacob and Monod
propose lac operon. 1983–1984 HIV
isolated and 2010 First
identified by Gallo bacterium with
1970 Arber and Smith synthetic genome
and Montagnier;
1961 First discover restriction constructed.
Mullis develops
human endonucleases. 1992 First
PCR technique.
in space 1977 Woese divides human trials 2005 Genome of
prokaryotes into of antisense 1918 influenza
Bacteria and Archaea. therapy virus sequenced.
1969 Neil Armstrong
walks on the moon.
1973 Vietnam
War ends.
1991 Soviet 2014 2-year
1980 First home 1981 First space 2003 Second
Union collapses. Ebola
computers shuttle launch war with Iraq; 2019 COVID-19
SARS outbreak outbreak pandemic
2001 World Trade
Figure 1.12 Some Important Events in the Development of Microbiology. Center attack;
in China begins.
2010 H1N1
Milestones in microbiology are marked in red and are discussed within this Anthrax bioterrorism
influenza outbreak
textbook; other historical events are in black. attacks in U.S.

wil88396_ch01_001-022.indd 15 06/12/21 5:47 PM

 16 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

these experiments, the French

naturalist Felix Pouchet
(1800–1872) claimed in 1859
Lens to have carried out experi-
ments conclusively proving
Specimen that microbial growth could
holder
occur without contact with air.
Pouchet’s claim provoked
Louis Pasteur (1822–1895) to
Focus settle the matter of spontane-
screw ous generation. Pasteur (fig­­­­ure
1.14) first filtered air through
cotton and found that objects
resembling plant spores had
been trapped. If a piece of the
Figure 1.14 Louis Pasteur. cotton was placed in sterile
Pixtal/age fotostock medium after air had been fil-
Handle tered through it, microbial
growth occurred. Next he placed nutrient solutions in flasks, heated
their necks in a flame, and pulled them into a variety of curves. The
swan-neck flasks he produced in this way remained open to the at-
(a) mosphere (figure 1.15). Pasteur then boiled the solutions and al-
lowed them to cool. No growth took place even though the contents
of the flasks were exposed to the air. Pasteur inferred that growth did
not occur because dust and germs had been trapped on the walls of
the curved necks. If the necks were broken, growth commenced im-
mediately. Pasteur had not only resolved the controversy by 1861 but
also had shown how to keep solutions sterile.
The English physicist John Tyndall (1820–1893) and the Ger-
man botanist Ferdinand Cohn (1828–1898) dealt the final blow to
spontaneous generation. In 1877 Tyndall demonstrated that dust

(b)
Microbes
Figure 1.13 van Leeuwenhoek’s Microscope and Drawings. (a) A brass being
replica of the Leeuwenhoek microscope. Inset photo shows how it is held. Broth
destroyed
free of
(b) Leeuwenhoek’s drawings of bacteria from the human mouth. live cells (sterile)
Vigorous heat
(a, 1) Kathy Park Talaro/Pasadena City College; (a, 2) Pasadena City College/Kathy Park Talaro;
is applied.
(b) Dr. Jeremy Byrgess/SPL/Getty Images

that air carried germs to the culture medium but also commented
that external air might be required for growth of animals ­already
in the medium. The supporters of spontaneous generation re-
sponded that heating the air in sealed flasks destroyed its ability
to support life, and therefore did not discredit the theory of spon-
taneous generation.
In the mid-1800s, several investigators attempted to counter Neck on second Neck intact; airborne
sterile flask microbes are
such arguments. These experiments involved allowing air to enter is broken; trapped at base,
a flask containing a nutrient solution after boiling. The air was growth occurs. and broth is sterile.
also very hot or it was filtered through sterile cotton wool. In all
cases, no microbial growth occurred in the medium. Despite Figure 1.15 Pasteur’s Experiments with Swan-Neck Flasks.

wil88396_ch01_001-022.indd 16 06/12/21 5:47 PM

 1.3 Microbiology Advanced as New Tools for Studying Microbes Were Developed 17

did indeed carry germs and that if dust was absent, broth remained heating the wines to destroy the undesirable microbes. The pro-
sterile even if directly exposed to air. During the course of his stud- cess is now called pasteurization.
ies, Tyndall provided evidence for the existence of exceptionally Indirect evidence for the germ theory of disease came from
heat-resistant forms of bacteria. Working independently, Cohn the work of the English surgeon Joseph Lister (1827–1912) on
discovered that the heat-­resistant bacteria recognized by Tyndall the prevention of wound infections. Lister, impressed with
were species capable of producing bacterial endospores. Cohn later Pasteur’s studies on fermentation, developed a system of
played an instrumental role in establishing a classification system antiseptic surgery designed to prevent microorganisms from
for bacteria based on their morphology and physiology. Bacterial entering wounds. Instruments were heat sterilized, and phenol
endospores are a survival strategy (section 3.10) was used on surgical dressings and at times sprayed over the
These early microbiologists disproved spontaneous genera- surgical area. The approach was remarkably successful in
tion thereby contributing to the rebirth of microbiology. They reducing the rate of surgical infection. It also provided strong
developed liquid media and the methods for sterilizing it so that indirect evidence for the role of microorganisms in disease.
microbes could be cultured. These techniques were next applied
to understanding the role of microorganisms in disease.
Koch’s Postulates
The first direct demonstration that bacteria cause disease came
Microorganisms and Disease from the study of anthrax by the German physician Robert Koch
For hundreds of years, most people believed that disease was (pronounced “Koke”; 1843–1910). Koch (figure 1.16) used the
caused by supernatural forces, poisonous vapors, and imbal- criteria proposed by his former teacher Jacob Henle (1809–1885)
ances among the four humors thought to be present in the body. and others to establish the relationship between Bacillus anthracis
The role of the four humors (blood, phlegm, yellow bile and anthrax. In these studies, published in 1876, Koch used mice
[choler], and black bile [melancholy]) in ­d isease had been as his model or test organism. Following the steps outlined in
widely accepted since the time of the Greek physician Galen figure 1.17, he next used guinea pigs to show that Mycobacterium
(129–199). Support for the idea that microorganisms cause tuberculosis causes tuberculosis (TB), which at that time was a
disease—that is, the germ theory of disease—began to leading cause of death in Europe. In 1905 Koch was awarded the
­accumulate in the early nineteenth century from diverse fields. Nobel Prize in Physiology or Medicine and his criteria for proving
Agostino Bassi (1773–1856) demonstrated in 1835 that a silk- the causal relationship between a microorganism and a specific
worm disease was due to a fungal infection. In 1845 M. J. disease are known as Koch’s postulates. Koch’s postulates have
Berkeley (1803–1889) proved that the great potato blight of since been used to discover the causative microorganisms for
Ireland was caused by a protozoan (then thought to be a fun- many infectious diseases.
gus), and in 1853 Heinrich de Bary (1831–1888) showed that
fungi caused crop diseases.
Pasteur also contributed to this area of research in what
may seem an unlikely way. Pasteur was trained as a chemist
and spent many years studying the ­fermentations that yield
ethanol and are used in the production of wine and other
alcoholic beverages. When he began his work, leading chemists
were convinced that fermentation was due to a chemical
instability in sugars that resulted in their breakdown into
alcohol. Pasteur did not agree; he believed that fermentations
were carried out by living organisms.
In 1856 M. Bigo, an industrialist in Lille, France, where
Pasteur worked, requested Pasteur’s assistance. His business
produced ethanol from the fermentation of beet sugars, and the
alcohol yields had recently declined and the product had become
sour. Pasteur discovered that the fermentation was failing
because the yeast normally responsible for alcohol formation had
been replaced by bacteria that produced acid rather than ethanol.
In solving this practical problem, Pasteur demonstrated that fer-
mentations were due to the activities of yeasts and bacteria.
Pasteur was also called upon by the wine industry in France
for help. For several years, poor-quality wines had been produced.
Pasteur referred to the wines as diseased and demonstrated that
particular wine diseases were linked to particular microbes Figure 1.16 Robert Koch. Koch examining a specimen in his laboratory.
contaminating the wine. He eventually suggested a method for Bettmann/Getty Images

wil88396_ch01_001-022.indd 17 06/12/21 5:47 PM

 18 CHAPTER 1 | The Evolution of Microorganisms and Microbiology

Postulate Experimentation

1 The microorganism must be Koch developed a staining technique to

present in every case of the examine human tissue. Mycobacterium
disease but absent from tuberculosis could be identified in
healthy organisms. diseased tissue. M. tuberculosis

TB patient
2 The suspected microorganisms Koch grew M. tuberculosis in pure
must be isolated and grown in a culture on coagulated blood serum.
pure culture.
M. tuberculosis
colonies

3 The same disease must result Koch injected cells from the pure
when the isolated microorganism culture of M. tuberculosis into guinea
is inoculated into a healthy host. pigs. The guinea pigs subsequently
died of tuberculosis.

4 The same microorganisms must Koch isolated M. tuberculosis in pure

be isolated again from the culture on coagulated blood serum M. tuberculosis
diseased host. from the dead guinea pigs. colonies

Figure 1.17 Koch’s Postulates Applied to Tuberculosis.

MICRO INQUIRY Why is the fourth postulate necessary?
COVID-19
Because it is a viral infection, Koch’s postulates could not be
used to determine the cause of COVID-19. Instead, lung fluid
from a 41-year-old man with respiratory distress admitted to a
hospital in Wuhan, China on December 26, 2019 was analyzed.
After excluding known pathogens, nucleic acid sequencing re-
vealed a virus with startling nucleotide similarity to other coro-
naviruses previously isolated from bats in China. The virus now
known as SARS-CoV-2, was originally called WH-Human 1’
coronavirus. The same virus was soon isolated from thousands
of other patients and the name COVID-19 was coined.
While Koch’s postulates are still widely used, their
application is at times not feasible. For instance, viruses and
organisms that must live within host cells such as Mycobacterium
leprae, the causative agent of leprosy, cannot be isolated in pure
culture. Some human diseases lack an appropriate animal model
so the postulates cannot be fully met. To avoid some of these
difficulties, microbiologists sometimes use molecular and
genetic evidence. For instance, molecular methods might be used
to detect the nucleic acid of a microorganism in body tissues,
rather than isolating it, or the genes thought to be associated with
the virulence of a disease-causing microbe (pathogen) might be
mutated. In this case, the mutant organism should have decreased
ability to cause disease. Introduction of the normal gene back
into the mutant should restore the pathogen’s virulence.
Our focus thus far has been on the discovery of bacteria,
fungi, and protists. But viral pathogens were also being studied
during this time. The discovery of viruses and their role in disease
was made possible when Charles Chamberland (1851–1908),
one of Pasteur’s associates, constructed a porcelain filter to
remove bacteria. Dimitri Ivanowski (1864–1920) and Martinus
Beijerinck (pronounced “by-a-rink”; 1851–1931) used the filter
to study tobacco mosaic disease. They found that plant extracts
and sap from diseased plants were infectious, even after being
filtered with Chamberland’s filter. Because the infectious agent
passed through a filter that trapped bacterial cells, they reasoned
that the agent must be something smaller than a bacterium.
Beijerinck proposed that the agent was a “filterable virus” (Latin
virus, slimy liquid, poison). Eventually viruses were shown to be
tiny, acellular infectious agents.

wil88396_ch01_001-022.indd 18 06/12/21 5:47 PM


Immunology
The ability to culture microbes also played an important role in early
immunological studies. During studies on the bacterium that causes
chicken cholera, Pasteur and Pierre Roux (1853–1933) discovered
that bacteria incubated in cultures for long periods of time lost their
ability to cause disease. These bacteria were said to be attenuated or
weakened. When chickens were injected with attenuated bacteria,
they remained healthy and were surprisingly able to resist the dis-
ease when exposed to virulent strains of the same bacteria. Pasteur
called the attenuated bacteria a vaccine (Latin vacca, cow) in honor
of Edward Jenner (1749–1823) because, many years earlier, Jenner
had used material from cowpox lesions to protect people against
smallpox (see Historical Highlights 35.3). Shortly after this, Pasteur
and Chamberland developed an a­ttenuated anthrax vaccine.
Vaccines immunize susceptible populations (section 35.6)
Pasteur also prepared a vaccine using an attenuated strain of
rabies virus. During the course of these studies, a nine-year-old boy
named Joseph Meister was bitten by a rabid dog and was brought to
Pasteur. Because Joseph’s death was certain in the absence of
treatment, Pasteur agreed to try vaccination. Joseph was injected
13 times over the next 10 days with increasingly virulent preparations
of the attenuated virus. His survival marked a huge advance in the
use of vaccines. To thank Pasteur, people from around the world
contributed to the construction of the Pasteur Institute in Paris,
France. One of the initial tasks of the institute was vaccine production.
These early advances in immunology were made without un-
derstanding how the immune system works. Immunologists now
know that white blood cells and the chemicals they produce play a
central role in immunity. Among the chemicals are soluble proteins
called antibodies, found in blood, lymph, and other body fluids. The
role of antibodies in preventing disease was recognized by Emil von
Behring (1854–1917) and Shibasaburo Kitasato (1852–1931). After
the discovery that diphtheria was caused by a toxin produced by
bacteria, they injected inactivated diphtheria toxin into rabbits. The
inactivated toxin induced rabbits to produce an ­antitoxin, which
protected against the disease. Antitoxins are now known to be anti-
bodies that specifically bind and neutralize toxins (see Historical
Highlights 32.2). The first immune system cells were discovered
when Eˊlie Metchnikoff (1845–1916) found that some white blood
cells could engulf disease-causing bacteria. He called these cells
phagocytes and the process phagocytosis (Greek phagein, eating).
Microbial Ecology
Early microbial ecologists studied microbial involvement in the
carbon, nitrogen, and sulfur cycles. The Russian microbiologist
Sergei Winogradsky (1856–1953) made many contributions to
soil microbiology, including the discovery that soil bacteria could
oxidize iron, sulfur, and ammonia to obtain energy and that many
of these bacteria could incorporate CO2 into organic matter much
as photosynthetic organisms do. Winogradsky also isolated
anaerobic nitrogen-fixing soil bacteria and studied the
decomposition of cellulose. Martinus Beijerinck made
fundamental contributions to microbial ecology as well as
virology. He isolated several kinds of nitrogen-fixing bacteria
and sulfate-­reducing bacteria. Beijerinck and Winogradsky
wil88396_ch01_001-022.indd 19
1.4 Microbiology Encompasses Many Subdisciplines 19
also developed enrichment culture techniques and selective me-
dia, which have been of great importance in microbiology.
Enrichment cultures (section 7.7); Biogeochemical cycling sus-
tains life on Earth (section 28.1)
Comprehension Check
1. What did Pasteur prove when he showed that a cotton plug that had
filtered air would trigger microbial growth when transferred to a
sterile medium? What argument made previously was he addressing?
2. Discuss the contributions of Lister, Pasteur, and Koch to the germ
theory of disease and the treatment or prevention of diseases.
3. What role did the ability to grow bacteria in pure culture play in the
development of Koch’s postulates?
4. What did Jenner, Pasteur, von Behring, Kitasato, and Metchnikoff
contribute to the development of immunology?
5. How did Winogradsky and Beijerinck contribute to the study of
microbial ecology? What new culturing techniques did they develop
in their studies?
1.4 M
 icrobiology Encompasses
Many Subdisciplines
After reading this section, you should be able to:
a. Construct a concept map, table, or drawing that illustrates the diverse
nature of microbiology and how it has improved human conditions
b. Discuss the opinion held by many microbiologists that microbiology
is experiencing its second golden age
Microbiology today is as diverse as the organisms it studies. It
has both basic and applied aspects. The basic aspects are con-
cerned with the biology of microorganisms themselves. The
­applied aspects are concerned with practical problems such as
disease, water and wastewater treatment, food spoilage and food
production, and industrial uses of microbes. Despite this apparent
dichotomy, the basic and applied aspects of microbiology are
intertwined. Basic research is often conducted in applied fields,
and applications often arise out of basic research.
An important development in microbiology is the increasing
use of molecular and genomic methods to study microbes and their
interactions with other organisms. These methods have led to a time
of rapid advancement that rivals the golden age of microbiology.
Indeed, many feel that microbiology is in its second golden age.
Microbial DNA technologies (chapter 17); Microbial genomics
(chapter 18) Exploring microbes in ecosystems (chapter 26)
Major Fields in Microbiology
Microbiology is commonly divided into subdisciplines based on
the type of microbe studied. Thus microbiology encompasses
bacteriology and virology as well as other microbe-specific
fields. Microbiology can also be divided based on the activities of
06/12/21 5:47 PM
 20 CHAPTER 1 | The Evolution of Microorganisms and Microbiology
microbes—for instance, environmental microbiology and agri-
cultural microbiology. Finally, microbiologists may study only
one aspect of the biology of microbes, leading to subdisciplines
such as microbial genetics and microbial physiology.
One of the most active and important fields in microbiology
is medical microbiology, which deals with diseases of humans.
Medical microbiologists investigate agents causing infectious
diseases and measures for their control and elimination. They are
involved in tracking down new, unidentified pathogens such as
those causing Zika virus and COVID-19. These microbiologists
also study how microorganisms cause disease. Clinical labora-
tory scientists, the microbiologists who work in hospitals and
other clinical laboratories, use culture and molecular techniques
to p­ rovide information needed by physicians to diagnose and
treat infectious disease.
Major epidemics have regularly affected human history. For
example, the human immunodeficiency virus/acquired immune
deficiency syndrome (HIV/AIDS) has infected about 75 million
people since the beginning of the AIDS pandemic in 1981. The
COVID-19 pandemic demonstrated to the world that public
health microbiology is concerned with the control and spread of
such communicable diseases. Public health microbiologists and
epidemiologists monitor the amount of disease in p­ opulations to
detect outbreaks and epidemics as they begin, and implement
appropriate control measures. They also conduct surveillance
for new diseases as well as bioterrorism events. Public health
microbiologists working for local governments monitor commu-
nity food establishments and water supplies to ensure they are
safe and free from pathogens. Epidemiology and public
health microbiology (chapter 35)
To understand, treat, and control infectious disease, it is
important to understand how the immune system protects the
body from pathogens; this question is the concern of immu-
nology. Immunology is one of the fastest growing areas in
science. Many advances have been in response to the discovery
of HIV, which specifically targets cells of the immune system.
Immunology also deals with the nature and treatment of
allergies and autoimmune diseases such as rheumatoid ar-
thritis. Innate host resistance (chapter 31); Adaptive im-
munity (chapter 32)
Microbial ecology is another important field in microbiology.
Microbial ecologists employ a variety of culture and molecular
approaches to describe the vast diversity of microbes in terms of
their morphology, physiology, and relationships with organisms
and the components of their habitats. The importance of microbes
in the local and global cycling of carbon, nitrogen, and sulfur has
been long studied; however, these studies have acquired new
urgency as our climate changes. Of particular interest is the role
of microbes in both the ­production and removal of greenhouse
gases such as carbon dioxide and methane. Microbial ecologists
also are employing microorganisms in bioremediation to reduce
pollution. An exciting frontier in microbial ecology is the study
of the microbes normally associated with the human body—the
human microbiome. Global climate change (section 28.3);
The microbe-human ecosystem (chapter 33); Biodegradation
wil88396_ch01_001-022.indd 20
and bioremediation harness microbes to clean the environment
(section 42.4)
Agricultural microbiology is a field related to both medical
microbiology and microbial ecology. It is concerned with the
impact of microorganisms on food production, such as
nitrogen-fixing bacteria, which affect soil fertility. Other
microbes live in the digestive tracts of ruminants such as cattle
and break down the plant materials these animals ingest. There
are also plant and animal pathogens that have significant eco-
nomic ­impact if not controlled. Furthermore, some pathogens
of domestic animals also cause human disease. Agricultural
micro­biologists work on methods to increase soil fertility and
crop yields, study rumen microorganisms to increase meat and
milk production, and try to combat plant and animal diseases.
Currently many agricultural microbiologists are studying the
use of bacterial and viral insect pathogens as substitutes for
chemical pesticides. Microorganisms in terrestrial ecosys-
tems (chapter 30)
In addition to agricultural microbiology, food microbiology
has contributed to the ready supply of high-quality foods. Food
microbiologists study the microbes used to make food and
beverages (e.g., yogurt, cheese, beer) as well as the microbes that
cause food spoilage or are pathogens that are spread through
food. For example, periodic outbreaks of certain Escherichia
coli strains have led to renal failure and death. To protect the
public, the specific strain must be traced back to the contaminated
food source. Food microbiologists also work to prevent microbial
spoilage of food and conduct research on the use of microorgan-
isms as nutrient sources for livestock and humans. Microbiol-
ogy of food (chapter 40)
Industrial microbiology involves the use of microbes to
make products helpful to humans. An important advance oc-
curred in 1929 when Alexander Fleming rediscovered that the
fungus Penicillium sp. produced what he called penicillin, the
first antibiotic that could successfully control bacterial infec-
tions. Although it took World War II for scientists to learn how to
mass-produce penicillin, microbiologists soon found other mi-
crobes capable of producing additional antibiotics. Today indus-
trial microbiologists also use microorganisms to make products
such as vaccines, steroids, alcohols and other solvents, vitamins,
amino acids, enzymes, and biofuels. These alternative fuels are
renewable and may help reduce fossil fuel dependence. Biofuel
production is a dynamic field (section 41.2); Microbial fuel cells:
batteries powered by microbes (section 42.3)
The advances in medical microbiology, agricultural micro-
biology, food and dairy microbiology, and industrial microbiol-
ogy are outgrowths of basic research in areas such as microbial
physiology, microbial genetics, molecular biology, and bioinfor-
matics. Microbial physiologists study many aspects of the biol-
ogy of microorganisms, including their diverse ­ metabolic
capabilities. They also study the synthesis of antibiotics and
toxins, the ways in which microorganisms survive harsh envi-
ronmental conditions, and the effects of chemical and physical
agents on microbial growth and survival. Microbial geneti-
cists, molecular biologists, and bioinformaticists study the
06/12/21 5:47 PM
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ampui kuitenkin hiukan ohi maalin, ja tätä osasi puolustaja
oivallisesti käyttää hyväkseen. Vastatessaan Grušenjkaa koskeviin
kysymyksiin Rakitin, innostuneena menestyksestään, jonka hän
tietysti jo itse oli huomannut, ja siitä korkeasta ylevyydestä, johon
hänen oli onnistunut kohota, katsoi voivansa lausua Agrafena
Aleksandrovnasta hieman halveksivasti, että tämä oli »kauppias
Samsonovin jalkavaimo». Hän olisi myöhemmin ollut valmis
maksamaan paljon siitä, että tuo sana olisi jäänyt sanomatta, sillä
siitäpä hänet Fetjukovitš heti saikin kiinni. Ja tämä kaikki johtui vain
siitä, että Rakitin ei ollut ensinkään ottanut huomioon, että
Fetjukovitš oli voinut niin lyhyessä ajassa tutustua juttuun niin tarkoin
kaikkein yksityisluontoisimpiakin pikku seikkoja myöten.

— Sallikaa minun kysyä, — alkoi puolustaja hymyillen mitä

ystävällisimmin, jopa kunnioittavastikin, kun oli tullut hänen vuoronsa
tehdä kysymyksiä, — te olette tietysti sama herra Rakitin, jonka
kirjoittaman, hiippakunnan hallituksen julkaiseman kirjasen Herrassa
nukkuneen luostarinvanhimman, isä Zosiman elämä, joka on täynnä
syviä ja uskonnollisia ajatuksia ja jossa on oivallinen ja hurskas
omistus korkea-arvoiselle piispalle, minä äskettäin olen niin suurella
nautinnolla lukenut.

— Minä en kirjoittanut painettavaksi… se on painettu myöhemmin,

— mutisi Rakitin, ikäänkuin häntä jokin olisi ällistyttänyt, ja miltei
häveten.

— Oi, se on oivallista! Sellainen ajattelija kuin te voi ja hänen

täytyykin suhtautua sangen suurpiirteisesti jokaiseen
yhteiskunnalliseen ilmiöön. Korkea-arvoisen piispan suojeluksella on
teidän perin hyödyllinen kirjasenne levinnyt ja tuottanut suhteellisesti
paljon hyötyä… Mutta mieleni tekisi ensi sijassa udella teiltä
seuraavaa: te mainitsitte äsken juuri, että olette ollut rouva Svetlovin
läheinen tuttava? (Nota bene: Grušenjkan sukunimi oli Svetlov. Sen
minä sain ensimmäisen kerran tietää vasta tuona päivänä, jutun
käsittelyn aikana.)

— Minä en voi vastata kaikista tuttavuuksistani… Minä olen nuori

mies… ja kukapa voi vastata kaikista niistä, jotka sattuu
kohtaamaan, — kivahti Rakitin.

— Minä ymmärrän, ymmärrän varsin hyvin! — huudahti

Fetjukovitš, aivan kuin olisi itsekin ollut nolo ja ikäänkuin rientäen
nopeasti pyytämään anteeksi. — Te kuten jokainen muukin saatoitte
vuorostanne pitää mielenkiintoisena tutustumista nuoreen ja
kauniiseen naiseen, joka mielellään otti luonansa vastaan täkäläistä
kukkeinta nuorisoa, mutta… tahdoin vain saada tietää: tehän
tiedätte, että rouva Svetlov noin pari kuukautta sitten suuresti halusi
tutustua nuorimpaan Karamazoviin, Aleksei Fjodorovitšiin, ja vain
siitä, että te toisitte tämän hänen luokseen ja nimenomaan hänen
silloisessa luostariveljen puvussaan, hän lupasi antaa teille
kaksikymmentäviisi ruplaa heti, kun tuotte hänet sinne. Tämä, kuten
tiedetään, tapahtuikin juuri sen päivän iltana, joka päättyi
traagilliseen katastrofiin, siihen, joka on antanut aiheen tähän
oikeusjuttuun. Te veitte Aleksei Karamazovin rouva Svetlovin luo —
ja saitteko te silloin nuo kaksikymmentäviisi ruplaa palkkiota rouva
Svetlovilta, kas, senpä haluaisin nyt saada kuulla teiltä?

— Se oli pilaa… Minä en näe, miksi tämä saattaa olla teistä

mielenkiintoista. Minä otin piloillani… antaakseni myöhemmin
takaisin…

— Siis otitte. Mutta ettehän te vieläkään ole antanut takaisin… vai

oletteko?
 — Se on jonninjoutavaa… — mutisi Rakitin, — minä en voi
vastata sellaisiin kysymyksiin. Tietysti minä annan takaisin.

Puheenjohtaja sekaantui asiaan, mutta puolustaja ilmoitti, ettei

hänellä enää ollut mitään kysyttävää herra Rakitinilta. Herra Rakitin
poistui näyttämöltä hieman nolattuna. Hänen ylevähenkisen
puheensa vaikutus oli tehty tyhjäksi, ja Fetjukovitš seuratessaan
häntä silmillään näytti ikäänkuin osoittavan häntä yleisölle ja
sanovan: »Tuollaisia ovat teidän jalot syyttäjänne!» Muistan, ettei
tämäkään mennyt ilman pientä Mitjan aiheuttamaa välikohtausta:
raivostuneena siitä äänensävystä, jolla Rakitin puhui Grušenjkasta,
hän yhtäkkiä huudahti paikaltaan: »Bernard!» Kun sitten
puheenjohtaja, sen jälkeen kuin Rakitinin kuulustelu oli päättynyt,
kääntyi syytetyn puoleen kysyen, eikö tämä halua huomauttaa
jotakin omasta puolestaan, niin Mitja huudahti raikuvalla äänellä:

— Hän on ottanut minulta, kun jo olin syytteessä, rahoja lainaksi!

Hän on halveksittava Bernard ja onnenonkija eikä usko Jumalaan,
vaan on vetänyt nenästä piispaa!

Mitjaa tietysti taaskin huomautettiin sopimattomasta puhetavasta,

mutta herra Rakitin oli lopullisesti nujerrettu. Ei myöskään alikapteeni
Snegirevin todistuksella ollut menestystä, vaikka aivan toisenlaisesta
syystä. Hän esiintyi aivan repaleisena, likaisessa puvussa ja
likaisissa saappaissa ja kaikista varokeinoista sekä edellä käyneistä
»asianymmärtäjän lausunnoista» huolimatta aivan humalassa. Kun
häneltä kysyttiin sitä pahoinpitelyä koskevia seikkoja, jonka alaiseksi
hän oli joutunut Mitjan puolelta, niin hän yhtäkkiä kieltäytyi
vastaamasta.

— Jumala heidän kanssaan. Iljušetška kielsi. Minulle palkitsee

Jumala siellä.
 — Kuka teitä kielsi puhumasta? Kenestä te puhutte?

— Iljušetška, pikku poikani: »Isäkulta, isäkulta, kuinka hän sinua

häpäisi!» Sen hän sanoi kiven luona. Nyt hän kuolee…

Alikapteeni alkoi yhtäkkiä ääneensä itkeä ja heittäytyi pitkäkseen

puheenjohtajan jalkojen juureen. Hänet vietiin nopeasti pois yleisön
nauraessa. Prokuraattorin valmistelemaa vaikutusta ei ollenkaan
syntynyt.

Mutta puolustaja käytti edelleen kaikkia keinoja ja pani yhä

enemmän ihmettelemään sitä, että hän oli perehtynyt asiaan
kaikkein pienimpiäkin yksityiskohtia myöten. Niinpä esimerkiksi
Trifon Borisovitšin todistus teki erittäin voimakkaan vaikutuksen ja oli
tietysti sangen epäsuotuisa Mitjalle. Hän laski tarkalleen, miltei
sormillaan, että ensimmäisellä käynnillään Mokrojessa kuukausi
ennen katastrofia Mitja ei ollut voinut mitenkään tuhlata vähempää
kuin kolmetuhatta tai »korkeintaan hivenen vähemmän». Miten
paljon menikään yksistään noille mustalaistytöille! Ja meikäläisille,
meikäläisille täisille moukille he eivät ainoastaan »paiskelleet
puoliruplasia pitkin katua», vaan lahjoittelivat vähintään
viidenkolmatta ruplan seteleitä, pienempiä eivät antaneet. Ja miten
paljon heiltä suorastaan varastettiinkaan! Ken varasti, se ei
tietenkään, jättänyt puumerkkiään, ei semmoista saa kiinni, varasta
nimittäin, kun he itse paiskelevat rahoja turhaan menemään! Meidän
rahvashan on rosvojoukkoa, ei niillä ole tuntoa. Entä tytöille, miten
paljon menikään meidän maalaistytöille! Meillä ovat sen jälkeen
rikastuneet, semmoista se on, kun taas aikaisemmin oli pelkkää
köyhyyttä. Sanalla sanoen, hän muisti jokaisen menon, teki kaikesta
tarkan tilin… Näin ollen se otaksuma, että muka oli tuhlattu
ainoastaan puolitoista tuhatta ja loput pantu kaulassa olevaan
pussiin, kävi mahdottomaksi. »Näin itse, heidän käsissään näin
kolmetuhatta yhtä selvästi kuin näkee kopeekan, omin silmin
katselin, mekö emme osaisi rahaa laskea!» huudahteli Trifon
Borisovitš koettaen kaikin voimin olla mieliksi »esivallalle». Mutta kun
tuli puolustajan vuoro kysellä, niin tämä ei juuri koettanutkaan
osoittaa todistusta vääräksi, vaan alkoi yht'äkkiä puhua siitä, että
kyytimies Timofei ja toinen talonpoika Akim olivat Mokrojessa tuon
ensimmäisen, kuukausi ennen vangitsemista tapahtuneen juomingin
aikana löytäneet eteisen lattialta sata ruplaa, jotka Mitja
juovuspäissään oli pudottanut, ja antaneet ne Trifon Borisovitšille, ja
tämä oli antanut heille siitä ruplan mieheen. »No, annoitteko te silloin
nuo sata ruplaa takaisin herra Karamazoville vai ettekö?» Vaikka
Trifon Borisovitš kiemurtelikin, niin hän kuitenkin, sitten kun
mainittuja miehiä oli kuulusteltu, myönsi kertomuksen sadan ruplan
löytämisestä todeksi ja lisäsi vain, että hän oli silloin rehellisesti
antanut kaikki takaisin Dmitri Fjodorovitšille ja palauttanut
»omantunnon mukaisesti, mutta kun he itse silloin olivat aivan
päissään, niin he tuskin voivat sitä muistaa». Koska hän joka
tapauksessa, ennenkuin talonpojat oli tuotu todistamaan, oli kieltänyt
koko jutun sadan ruplan löytämisestä, niin koko hänen
todistuksensa, että hän oli antanut rahat takaisin juopuneelle Mitjalle,
luonnollisesti tuntui hyvin vähän uskottavalta. Näin taaskin yksi
prokuraattorin hankkimista kaikkein vaarallisimmista todistajista
joutui epäluulojen alaiseksi ja oli saanut maineeseensa pahan
tahran. Samoin kävi puolalaisillekin: nämä tulivat esille ylpeinä ja
riippumattomina. Kovalla äänellä he todistivat, että ensiksikin
kumpikin heistä »palveli kruunua» ja että »pan Mitja» oli tarjonnut
heille kolmetuhatta ostaakseen heidän kunniansa ja että he itse
olivat nähneet hänen käsittelevän suuria rahoja. Herra Mussjalovitš
sekoitti hirveän paljon puolalaisia sanoja lauseisiinsa ja nähdessään
sen vain kohottavan hänen arvoaan puheenjohtajan ja prokuraattorin
silmissä hän pöyhistyi lopullisesti ja alkoi puhua kokonaan
puolankieltä. Mutta Fetjukovitš sai kiedotuksi heidätkin verkkoihinsa:
niin paljon kuin taas uudestaan kuultavaksi kutsuttu Trifon Borisovitš
kiemurtelikin, niin hänen sittenkin oli pakko tunnustaa, että herra
Vrublevski oli vaihtanut häneltä saadun korttipakan toiseen ja että
herra Mussjalovitš pankkia pitäessään oli puikeltanut päällimmäisen
kortin. Tämän vahvisti todeksi Kalganov, kun hänen vuoronsa oli
todistaa, ja molemmat puolalaisherrat poistuivat häväistyinä yleisön
nauraessa.

Sen jälkeen kävi aivan samoin melkein kaikille vaarallisemmille

todistajille. Jokaisen heistä Fetjukovitš osasi siveellisesti mustata, ja
he saivat jollakin tavoin pitkän nenän. Asianharrastajat ja lakimiehet
vain nauttivat tästä, mutta eivät sentään oikein ymmärtäneet, mitä
suuria ja lopullisia seurauksia tästä kaikesta oikeastaan saattoi olla,
sillä, toistan sen, kaikki tunsivat, että syyte oli kumoamaton, ja tämä
mahdottomuus saada se torjutuksi kasvoi yhä ja sai yhä
traagillisemman luonteen. Mutta »suuren taikurin» varmuudesta he
näkivät, että hän oli levollinen, ja he odottivat: eihän ollut suotta tullut
Pietarista »sellainen mies», eihän hän ollut niitä miehiä, että palaisi
tyhjin toimin takaisin.

3.

Lääketieteellinen asianymmärtäjäin lausunto ja naula pähkinöitä

Lääketieteellinen asiantuntijain lausunto ei myöskään ollut kovin

edullinen syytetylle. Eikä itse Fetjukovitškaan näyttänyt siitä suuria
toivovan, kuten myöhemmin selvisikin. Se oli alunperin syntynyt
ainoastaan Katerina Ivanovnan vaatimuksesta, tämä kun oli
vartavasten tilannut kuuluisan tohtorin Moskovasta. Puolustuksella ei
tietysti ollut mitään menetettävää tämän kautta, ja parhaassa
tapauksessa se saattoi siitä jotakin voittaakin. Muuten asia sai
hieman koomillisenkin luonteen siitä syystä, että lääkärit olivat
jossakin määrin erimielisiä. Asiantuntijoina olivat — kuuluisa
moskovalainen tohtori, meidän tohtorimme Herzenstube ja nuori
lääkäri Varvinskij. Viimeksimainitut kaksi esiintyivät myös
prokuraattorin kutsumina tavallisina todistajina. Ensimmäisenä
kuulusteltiin asianymmärtäjän ominaisuudessa tohtori Herzenstubea.
Hän oli seitsemänkymmenen vuoden ikäinen ukko, harmaa ja
kaljupäinen, keskikokoinen ja vanttera. Kaikki kaupungissamme
pitivät häntä hyvin suuressa arvossa ja kunnioittivat häntä. Hän oli
tunnollinen lääkäri, hyvä ja jalo ihminen, jonkinmoinen
herrnhutilainen tai »määriläinen veli» — en tiedä sitä varmasti. Hän
oli asunut kaupungissamme hyvin kauan ja esiintyi erittäin
arvokkaasti. Hän oli hyväsydäminen ja ihmisiä rakastava, hoiti
köyhiä potilaita ja talonpoikia ilmaiseksi, kävi itse heidän
hökkeleissään ja mökeissään ja jätti heille rahaa lääkkeitten ostoon,
mutta samalla hän oli itsepäinen kuin muuli. Oli aivan mahdotonta
saada häntä luopumaan siitä, mitä hän kerran oli saanut päähänsä.
Mainittakoon tässä, että melkein kaikki kaupungissa jo tiesivät uuden
kuuluisan lääkärin pari kolme päivää kestäneen olonsa aikana
luonamme jo ennättäneen antaa muutamia sangen loukkaavia
lausuntoja tohtori Herzenstuben kyvystä. Asian laita oli niin, että
vaikka moskovalainen lääkäri ottikin käynnistä vähintään
kaksikymmentäviisi ruplaa, niin jotkut kaupungissamme sentään
ilostuivat hänen tulostaan, eivät säästäneet rahojaan ja riensivät
hänen luokseen saamaan neuvoja. Kaikkia näitä sairaita oli sitä
ennen tietysti hoitanut tohtori Herzenstube, ja nyt kuuluisa lääkäri
kaikissa kohdin arvosteli erittäin ankarasti hänen hoitotapojaan.
Lopulta hän sairaan luo tullessaan suorastaan kysyi: »No, kuka teitä
täällä on puoskaroinut, Herzenstubeko? Hehe!» Tohtori Herzenstube
sai tietysti tietää kaiken tämän. Ja nyt kaikki kolme lääkäriä tulivat
toinen toisensa jälkeen kuulusteltaviksi. Tohtori Herzenstube julisti
suoraan, että »syytetyn henkisten kykyjen epänormaalisuus on
itsestään selvä». Esitettyään tämän jälkeen mietteensä, jotka minä
tässä jätän kertomatta, hän lisäsi, että tämä epänormaalisuus
havaitaan etupäässä ei ainoastaan syytetyn monista aikaisemmista
teoista, vaan nytkin, tällä hetkelläkin, ja kun häntä pyydettiin
selittämään, miten se on havaittavissa nyt, tällä hetkellä, niin vanha
tohtori koko avomielisellä suoruudellaan viittasi siihen, että syytetty
saliin tullessaan »oli asianhaaroihin nähden tavallisuudesta
poikkeavan ja omituisen näköinen, asteli eteenpäin niinkuin sotamies
ja katsoi itsepintaisesti suoraan eteensä, vaikka luonnollisempaa
olisi ollut, että hän olisi katsonut vasemmalle, missä yleisön joukossa
istuivat naiset, sillä hän piti paljon kauniista sukupuolesta ja hänen
on täytynyt ajatella sangen paljon sitä, mitä naiset mahtavat hänestä
sanoa», lopetti ukko puhuen omituista kieltään. On lisättävä, että hän
puhui venäjää paljon ja mielellään, mutta jotenkin hänen jokainen
lauseensa muodostui saksalaisen mallin mukaan, mistä hän ei
kuitenkaan koskaan ollut millänsäkään, sillä koko elämän ajan oli
hänellä ollut se heikkous, että hän piti venäjänkielistä puhettaan
mallikelpoisena, »parempana kuin mitä itse venäläiset puhuivat», ja
hän käytti myös hyvin mielellään venäläisiä sananlaskuja vakuuttaen
joka kerta, että venäläiset sananlaskut ovat parhaat ja nasevimmat
koko maailman kaikista sananlaskuista. Huomautan vielä, että hän
kenties jonkinmoisen hajamielisyyden vaikutuksesta
keskustellessaan usein unohti kaikkein tavallisimmat sanat, jotka
hän tunsi erinomaisen hyvin, mutta jotka jostakin syystä yhtäkkiä
lennähtivät pois hänen päästään. Samaa tapahtui muuten myös
hänen puhuessaan saksaa, ja tällöin hän aina heilutteli kättään
kasvojensa edessä ikäänkuin koettaen saada kiinni kadonneen
sanan, eikä kukaan olisi voinut saada häntä jatkamaan
aloittamaansa puhetta, ennenkuin hän oli löytänyt kadonneen sanan.
Hänen huomautuksensa, että syytetyn olisi pitänyt sisään
astuessaan katsoa naisiin, sai syntymään leikillistä kuiskailua yleisön
keskuudessa. Kaikki naiset meillä pitivät ukostamme hyvin paljon ja
tiesivät myös, että hän, joka oli ollut koko ikänsä naimaton, oli jalosti
ajatteleva ja siveä mies sekä piti naisia korkeampina ja ihanteellisina
olentoina. Senvuoksi hänen odottamaton huomautuksensa oli
kaikista hirveän omituinen.

Moskovalainen tohtori, kun häneltä vuorostaan kysyttiin, vakuutti

jyrkästi ja lujasti, että hän pitää syytetyn sieluntilaa epänormaalisena,
»vieläpä mitä suurimmassa määrässä». Hän puhui paljon ja viisaasti
»affektista» ja »maniasta» ja todisteli, että kaikista kootuista
tosiseikoista päättäen syytetty oli jo useita päiviä ennen
vangitsemista ollut ehdottomasti sairaalloisen affektin vallassa, ja
joskin hän oli tehnyt rikoksen tietoisesti, niin kuitenkin melkein
tahtomattaan, koska hänellä ei ollenkaan ollut voimia vastustaa
häntä vallannutta siveellistä viettymystä. Mutta paitsi affektia tohtori
otti tarkastettavakseen myöskin manian, joka hänen sanojensa
mukaan ennusti jo edeltäpäin suoraa tietä mielenvikaisuuteen, mikä
mielenhäiriö nyt jo oli puhjennut. (Huom.! Minä kerron omilla
sanoillani, tohtori esitti asian käyttäen hyvin oppinutta kieltä ja
spesiaalitermejä.) »Kaikki hänen tekonsa ovat ristiriidassa terveen
järjen ja logiikan kanssa», jatkoi hän. »Minä en edes puhukaan siitä,
mitä en ole nähnyt, nimittäin itse rikoksesta ja koko tuosta
katastrofista, mutta toissa päivänäkin, kun hän puheli kanssani, oli
hänellä selittämättömän liikkumaton katse. Odottamatonta naurua
silloin, kun se ei ole ollenkaan paikallaan. Käsittämätön ainainen
ärtymyksen tila, omituisia sanoja: Bernard, etiikka ynnä muita, jotka
ovat tarpeettomia.» Mutta erikoisesti ilmeni tohtorin mielestä tuo
mania siinä, että syytetty ei voi edes puhua noista
kolmestatuhannesta ruplasta, joihin nähden hän katsoo tulleensa
petetyksi, joutumatta jonkinmoisen tavattoman suuttumuksen
valtaan, kun hän taas kaikista muista kärsimistään vastoinkäymisistä
ja loukkauksista puhuu ja niitä muistelee jokseenkin kevyin mielin.
Lopuksi on myös tiedusteluista käynyt ilmi, että hän on samalla
tavalla ennenkin joka kerta, kun on tullut kysymys noista
kolmestatuhannesta, joutunut jonkinmoisen raivon valtaan, ja
kuitenkin hänestä todistetaan, että hän on omaa voittoa pyytämätön
eikä halua kiistellä toisten kanssa. »Oppineen ammattiveljeni
mielipiteen johdosta taasen», lisäsi moskovalainen tohtori ivallisesti
puheensa lopussa, »että syytetyn, tullessaan saliin, olisi pitänyt
katsoa naisiin eikä suoraan eteensä, sanon vain, että tuommoinen
johtopäätös, paitsi että se on leikillinen, on sen lisäksi myös perin
pohjin virheellinen; sillä vaikka yhdynkin täydellisesti siihen, ettei
syytetyn, astuessaan sisälle saliin, jossa hänen kohtalonsa
ratkaistaan, olisi pitänyt katsella niin jäykästi eteensä ja että tätä
todellakin saatettaisiin pitää todistuksena hänen sieluntilansa
epänormaalisuudesta tällä hetkellä, niin vakuutan kuitenkin samalla,
ettei hänen olisi pitänyt katsoa vasemmalle naisiin, vaan päinvastoin
oikealle hakien silmillään puolustusasianajajaansa, jonka apuun
kaikki hänen toivonsa perustuu ja jonka puolustuksesta nyt koko
hänen kohtalonsa riippuu». Mielipiteensä tohtori lausui päättävästi ja
varmasti. Mutta erityisesti koomillisen valaistuksen molempien
oppineitten asianymmärtäjäin erimielisyydelle antoi viimeiseksi
kuulusteltavaksi otetun lääkäri Varvinskin odottamaton johtopäätös.
Hänen mielestään syytetty oli sekä nyt että ennen täydellisesti
normaalisessa tilassa, ja vaikka hänen tosiaankin oli täytynyt ennen
vangitsemista olla hermostuneessa ja tavattoman kiihtyneessä
mielentilassa, niin se saattoi johtua monista aivan silminnähtävistä
syistä: mustasukkaisuudesta, vihasta, pitkäaikaisesta
juopumuksesta y.m. Mutta tuohon hermostuksen tilaan ei voinut
sisältyä mitään »affektia», jommoisesta äsken oli puhuttu. Siihen
seikkaan nähden taasen, oliko syytetyn, tullessaan saliin, katsottava
vasemmalle vai oikealle, oli hänen »vaatimaton mielipiteensä» se,
että syytetyn oli saliin astuessaan katsottava suoraan eteensä, kuten
hän todella oli katsonutkin, sillä suoraan hänen edessään istuivat
oikeuden puheenjohtaja ja sen jäsenet, joista nyt riippui hänen
kohtalonsa, »niin että katsomalla suoraan eteensä hän juuri todistikin
olevansa tällä hetkellä täysin normaalisessa sieluntilassa», lopetti
nuori lääkäri hieman kiihkeästi »vaatimattoman» todistuksensa.

— Bravo, lääkäri! — huudahti Mitja paikaltaan. — Juuri niin!

Mitjan puhe tietysti keskeytettiin, mutta nuoren lääkärin mielipide

vaikutti aivan ratkaisevasti sekä oikeuteen että yleisöön, sillä
myöhemmin osoittautui, että kaikki olivat samaa mieltä kuin hän.
Muuten tohtori Herzenstube, kun häntä sitten kuultiin todistajana,
aivan odottamatta yhtäkkiä oli hyödyksi Mitjalle. Kaupungin vanhana
asukkaana, joka kauan oli tuntenut Karamazovien perheen, hän
esitti muutamia »syytteen» kannalta varsin mielenkiintoisia asioita ja
lisäsi lopuksi aivan kuin jotakin olisi kypsynyt hänen mielessään:

— Ja kuitenkin onneton nuori mies olisi voinut saada verrattomasti

paremman kohtalon, sillä hänellä on ollut hyvä sydän sekä lapsena
että lapsuusajan jälkeen, minä tiedän sen. Mutta venäläinen
sananlasku sanoo: »Jos jollakulla on yksi äly, niin se on hyvä asia,
mutta jos tulee vieraaksi vielä älykäs ihminen, niin se on sitäkin
parempi, sillä silloin on kaksi älyä, eikä vain yksi…»

— Äly on hyvä, mutta kaksi on parempi, — toisti prokuraattori tuon

sananlaskun kärsimättömästi, hän kun jo vanhastaan tiesi, että
ukolla oli tapana puhua hitaasti ja pitkäpiimäisesti, välittämättä siitä,
minkä vaikutuksen hänen puheensa teki, ja siitä, että toisten täytyi
häntä odottaa, ja päinvastoin pitäen sangen onnistuneena kömpelöä,
hidasjärkistä ja alati riemukkaan itsetyytyväistä saksalaista
sukkeluuttaan. Ukosta oli hauskaa lasketella sukkeluuksia.

— Niin, n-iin, sitä minäkin sanon, — tarttui hän itsepäisesti

puheeseen, — yksi äly on hyvä, mutta kaksi on paljon parempi.
Mutta hänen luokseen ei tullut toinen älyniekka ja hän päästi
omansakin… Kuinka se on, mihin hän sen päästi? Tuo sana — mihin
hän päästi älynsä, minä en muista, — jatkoi hän pyöritellen kättään
silmiensä edessä, — ah niin, spazieren.

— Vapaasti kuljeksimaan?

— No niin, kuljeksimaan, sitähän minäkin sanon. Hänen älynsä

lähti kuljeksimaan ja tuli niin syvään paikkaan, että hukkui sinne. Ja
kuitenkin hän oli kiitollinen ja tunteellinen nuorukainen, oi minä
muistan, kun hän vielä oli tuommoinen pikku poika, jonka isä oli
työntänyt takapihalle, hänen vielä juoksennellessaan ilman
saappaita ja jalassa housut, joissa oli vain yksi nappi…

Tunteellinen ja ymmärtäväinen sävy alkoi äkkiä tuntua rehellisen

vanhuksen äänessä. Fetjukovitš hätkähti kuin aavistaen jotakin ja
kiintyi heti asiaan.
 — Oi, niin, minä olin itsekin silloin vielä nuori mies… Olin… no
niin, olin silloin neljänkymmenen viiden vuoden ikäinen ja olin vasta
saapunut. Minun tuli silloin sääli poikaa, ja minä kysyin itseltäni:
miksi en voisi ostaa hänelle yhtä naulaa… No niin, mitä yhtä naulaa?
Olen unohtanut, miten sitä nimitetään… naula sitä, mistä lapset
hyvin paljon pitävät, mitä se on, — no, mikä se on nimeltään… —
tohtori alkoi taas huitoa käsiään, — se kasvaa puussa ja sitä
kootaan ja lahjoitetaan kaikille…

— Omenia?

— Oi, e-e-ei! Naula, naula, omenia myydään kymmenittäin eikä

nauloittain… ei, niitä on paljon ja ne ovat kaikki pieniä, pannaan
suuhun ja rr-rits!…

— Pähkinöitä?

— No niin, pähkinöitä, sitä minäkin sanon, — vahvisti tohtori aivan

tyynesti aivan kuin ei olisi etsinytkään sanaa, — ja minä toin hänelle
naulan pähkinöitä, sillä pojalle ei ollut vielä kukaan milloinkaan
tuonut naulaa pähkinöitä, ja minä kohotin sormeni ja sanoin hänelle:
»Poika! Gott der Vater», — hän rupesi nauramaan ja sanoo: »Gott
der Vater. — Gott der Sohn». Hän alkoi taaskin nauraa ja lepersi:
»Gott der Sohn. — Gott der Heilige Geist». Silloin hän vieläkin rupesi
nauramaan ja lausui niin hyvin kuin osasi: »Gott der Heilige Geist».
Minä menin pois. Kolmantena päivänä kuljen ohitse, ja hän huutaa
minulle itse: »Setä, Gott der Vater, Gott der Sohn», ja vain Gott der
Heilige Geist oli häneltä unohtunut, mutta minä johdatin sen hänen
mieleensä ja minun tuli taas hyvin sääli häntä. Mutta hänet vietiin
pois enkä minä häntä enää nähnyt. Kului sitten
kaksikymmentäkolme vuotta, istun eräänä aamuna työhuoneessani,
pääni on jo valkoinen, ja yhtäkkiä astuu sisälle kukoistava
nuorukainen, jota en mitenkään voi tuntea, mutta hän kohotti
sormensa ja sanoo nauraen: »Gott der Vater, Gott der Sohn und
Gott der Heilige Geist! Saavuin juuri ja tulin kiittämään teitä naulasta
pähkinöitä, sillä ei kukaan koskaan silloin ostanut minulle naulaa
pähkinöitä, vaan te yksin ostitte minulle naulan pähkinöitä.» Ja silloin
muistui minulle mieleen onnellinen nuoruuteni ja poikaparka, joka oli
ulkona ilman saappaita, ja sydäntäni liikutti ja minä sanoin: »Olet
kiitollinen nuori mies, sillä sinä olet koko elämäsi ajan muistanut sen
naulan pähkinöitä, minkä minä toin sinulle lapsena ollessasi.» Ja
minä syleilin häntä ja siunasin, siunasin. Ja rupesin itkemään. Hän
nauroi, mutta itki myös… sillä venäläinen nauraa hyvin usein siellä,
missä pitäisi itkeä. Mutta hän itki myös, minä näin sen. Ja nyt, voi,
voi!…

— Nytkin itken, saksalainen, nytkin itken, sinä Jumalan mies! —

huudahti yhtäkkiä Mitja paikaltaan.

Oli miten oli, niin tämä pikku kertomus teki yleisöön jossakin
määrin hyvän vaikutuksen. Mutta enimmän vaikutti Mitjan hyväksi
Katerina Ivanovnan todistus, josta kohta kerron. Ja yleensäkin, kun
alkoivat esiintyä todistajat à décharge, se on puolustajan haastamat,
niin kohtalo näytti yhtäkkiä ja oikein tosissaan rupean hymyilemään
Mitjalle ja, — mikä on kaikkein merkillisintä, — se näytti olevan
yllätys itse puolustusasianajajallekin. Mutta ennen Katerina
Ivanovnaa kuulusteltiin vielä Aljošaa, joka oli äkkiä muistanut erään
tosiasian, mikä näytti muodostuvan suoranaiseksi todistukseksi
erästä syytteen varsin tärkeätä kohtaa vastaan.

4.
 Onni hymyilee Mitjalle

Se sattui aivan epähuomiossa itselleen Aljošallekin. Hänet

kutsuttiin esille ilman valaa, ja minä muistan, että kaikki puolet
suhtautuivat häneen aivan kuulustelun ensimmäisistä sanoista asti
tavattoman lempeästi ja myötätuntoisesti. Näkyi, että hän oli hyvässä
maineessa. Aljoša todisti vaatimattomasti ja hillitysti, mutta hänen
todistuksissaan tuntui selvästi lämmin myötätunto onnetonta veljeä
kohtaan. Erääseen kysymykseen vastatessaan hän kuvasi veljensä
luonteeltaan kenties hurjapäiseksi ja intohimojensa vallassa olevaksi
mieheksi, joka kuitenkin myös oli ylevämielinen, ylpeä ja jalo, valmis
uhrautumaankin, jos sitä häneltä vaadittiin. Hän oli muuten sitä
mieltä, että hänen veljensä oli viimeisinä päivinä kiintymyksensä
vuoksi Grušenjkaan ja tietäessään isänsä kilpailevan hänen
kanssaan ollut sietämättömässä asemassa. Mutta hän torjui
paheksuen sen otaksumankin, että hänen veljensä olisi voinut tehdä
murhan ryöstämistarkoituksessa, vaikka hän myönsikin, että nuo
kolmetuhatta olivat Mitjan mielessä kehittyneet miltei jonkinmoiseksi
maniaksi, että hän piti niitä jäännöksenä omasta perinnöstään, jonka
isä oli vääryydellä pidättänyt itsellään, ja että hän, kun itse ei
ollenkaan ollut voitonhimoinen, ei voinut edes puhua noista
kolmestatuhannesta joutumatta raivon ja vimmastuksen valtaan.
Kahden »henkilön», niinkuin prokuraattori lausui, keskinäisestä
kilpailusta, nimittäin Grušenjkan ja Katjan, hän antoi vältteleviä
vastauksia, vieläpä kieltäytyi vastaamastakin yhteen tai kahteen
kysymykseen.

— Puhuiko teille veljenne edes siitä, että hän aikoo tappaa

isänsä? — kysyi prokuraattori. — Te voitte olla vastaamatta, jos
katsotte sen tarpeelliseksi, — lisäsi hän.
 — Ei puhunut siitä suoraan, — vastasi Aljoša.

— Kuinka siis? Epäsuorastiko?

— Hän puhui minulle kerran persoonallisesta

vastenmielisyydestään isää kohtaan ja siitä, että pelkää…
äärimmäisessä tapauksessa… suuren inhon hetkellä… kenties
voivansa vaikka tappaa hänet.

— Ja uskoitteko te häntä, kun kuulitte sen?

— Minua peloittaa sanoa, että uskoin. Mutta minä olin aina

vakuutettu siitä, että jokin korkeampi tunne aina pelastaa hänet
kohtalokkaalla hetkellä, niinkuin todella pelastikin, sillä hän ei ole
tappanut isääni, — lopetti Aljoša kovalla äänellä, joka kuului yli salin.
Prokuraattori vavahti niinkuin sotaratsu, joka kuulee merkkitorven
törähdyksen.

— Olkaa vakuutettu siitä, että minä täydelleen uskon

vakaumuksenne täydelliseen vilpittömyyteen enkä katso sen
ollenkaan johtuvan rakkaudesta onnetonta veljeänne kohtaan enkä
katso näiden kahden asian sulautuneen yhteen. Teidän itsenäinen
mielipiteenne koko tästä teidän perheessänne sattuneesta
traagillisesta episodista on meille tuttu jo valmistavasta tutkinnasta.
En salaa teiltä, että se on mitä suurimmassa määrässä
erikoislaatuinen ja ristiriidassa kaikkien muitten
prokuraattorinviraston saamien todistusten kanssa. Siksipä
katsonkin tarpeelliseksi kysyä teiltä nyt vakavasti: mitkä tosiseikat
nimenomaan ovat vaikuttaneet ajatukseenne ja johtaneet sen siihen
lopulliseen vakaumukseen, että veljenne on syytön ja että syyllinen
on eräs toinen henkilö, johon te suoraan viittasitte jo valmistavassa
tutkinnassa?
 — Valmistavassa tutkinnassa minä ainoastaan vastasin
kysymyksiin, — lausui Aljoša hiljaa ja tyynesti, — enkä itse tehnyt
syytöstä Smerdjakovia vastaan.

— Viittasittehan kuitenkin häneen?

— Viittaukseni perustui veli Dmitrin sanoihin. Minulle oli jo ennen

kuulustelua kerrottu siitä, mitä oli tapahtunut häntä vangittaessa ja
kuinka hän silloin itse oli viitannut Smerdjakoviin. Minä uskon
täydellisesti, että veljeni on viaton. Mutta jos murhaaja ei ole hän,
niin…

— Niin se on Smerdjakov? Mutta miksi juuri Smerdjakov? Ja

minkä tähden te niin lopullisesti olette tullut vakuutetuksi veljenne
viattomuudesta?

— Minä en voinut olla uskomatta veljeäni. Minä tiedän, ettei hän

valehtele minulle. Näin hänen kasvoistaan, ettei hän valehtele
minulle.

— Ainoastaanko kasvoista? Siinäkö ovat kaikki todistuksenne?

— Muita todistuksia minulla ei ole.

— Ettekä perusta käsitystänne Smerdjakovin syyllisyydestäkään

mihinkään muuhun todistuskappaleeseen kuin veljenne sanoihin ja
hänen kasvojensa ilmeeseen?

— Niin, minulla ei ole muita todistuksia.

Tähän prokuraattori lopetti kyselynsä. Aljošan vastaukset tekivät

yleisöön sangen masentavan vaikutuksen. Smerdjakovista oli meillä
puhuttu jo ennen oikeuden istuntoa, joku oli kuullut jotakin, joku oli
viitannut johonkin, oli puhuttu Aljošasta, että hän oli koonnut joitakin
tavattomia todistuskappaleita veljensä hyväksi ja lakeijan syyllisyyttä
osoittamaan, ja nyt — ei ollutkaan mitään, ei minkäänlaisia
todistuksia, paitsi jonkinlaista siveellistä vakaumusta, joka oli perin
luonnollinen, koska hän oli syytetyn oma veli.

Mutta myös Fetjukovitš alkoi kysellä. Vastatessaan kysymykseen:

milloin syytetty oli puhunut hänelle, Aljošalle, vihastaan isää kohtaan
ja siitä, että voisi hänet tappaa, ja oliko hän kuullut tämän häneltä
esimerkiksi silloin, kun tapasi hänet viimeisen kerran ennen
katastrofia, Aljoša yhtäkkiä hätkähti, aivan kuin jotakin vasta nyt olisi
johtunut hänen mieleensä ja hän nyt olisi tullut sitä ajatelleeksi:

— Muistan nyt erään seikan, jonka jo itsekin olin aivan unohtanut,

silloin se oli minulle niin epäselvä, mutta nyt…

Ja Aljoša muisteli nyt innostuneena, ilmeisesti itsekin aivan

sattumalta johdettuaan asiaa muistamaan, miten hänen
kohdatessaan viimeisen kerran Mitjan illalla puun luona luostariin
vievän tien varrella Mitja lyöden rintaansa, »rinnan ylimpään osaan»,
oli useita kertoja sanonut hänelle, että hänellä on keino kunniansa
palauttamiseksi, että se keino on tässä, juuri tässä, hänen
rinnallaan… — Minä luulin silloin, että hän lyödessään rintaansa
puhui omasta sydämestään — jatkoi Aljoša, — siitä, että hän voisi
löytää sydämestään voimaa päästäkseen jostakin kauheasta
häpeästä, joka häntä uhkasi ja jota hän ei uskaltanut tunnustaa edes
minullekaan. Tunnustan luulleeni silloin todellakin hänen puhuvan
isästä ja kauhistuvan aivan kuin häpeätä sitä ajatusta, että menisi
isän luo ja tekisi siellä jotakin väkivaltaa, mutta samalla kertaa hän
ikäänkuin nimenomaan osoitti rintaansa, niin että, minä muistan sen,
mieleeni juuri silloin välähti ajatus, että sydän on aivan toisella
puolen rintaa ja alempana, mutta hän lyö itseään paljon ylemmäksi,
tähän näin, aivan kaulan alapuolelle, ja osoitteli koko ajan sitä
paikkaa. Ajatukseni näytti minusta silloin tyhmältä, mutta ehkäpä hän
juuri osoittelikin minulle silloin sitä pussia, johon oli ommeltu nuo
puolitoista tuhatta!…

— Juuri niin! — huudahti äkkiä Mitja paikaltaan. — Niin se on,

Aljoša, niin, minä löin silloin nyrkilläni siihen!

Fetjukovitš syöksyi kiireesti hänen luokseen rukoillen häntä

rauhoittumaan ja samalla hetkellä hän takertui kovasti kiinni Aljošan
puheeseen. Muistostaan innostuneena Aljoša lausui kiihkeästi sen
otaksumansa, että tuo häpeä luultavimmin oli juuri sitä että Mitja,
vaikka hänellä oli nuo tuhatviisisataa ruplaa, jotka hän olisi voinut
palauttaa Katerina Ivanovnalle ja jotka olivat toinen puoli hänen
velkaansa, kuitenkaan ei päättänyt antaa hänelle tuota puolta, vaan
aikoi käyttää sen muuhun, nimittäin viedäkseen pois Grušenjkan, jos
tämä suostuisi…

— Se on niin, se on juuri niin, — huudahteli Aljoša äkillisesti

innostuen, — veljeni huudahteli nimenomaan silloin minulle, että
puolet, puolet häpeästä (hän lausui useampaan kertaan: puolet!) hän
voisi heti poistaa päältään, mutta että hänellä on niin onnettoman
heikko luonne, ettei hän sitä tee… hän tietää edeltäpäin, ettei hän
kykene sitä tekemään!

— Ja muistatteko te selvästi ja varmasti, että hän löi itseään juuri

tuohon paikkaan rintaa? — kysyi Fetjukovitš halukkaasti.

— Selvästi ja varmasti, koska minä juuri silloin ajattelin: miksi hän

lyö niin ylös, kun sydän on alempana, ja minusta tuntui silloin kohta
tämä ajatukseni tyhmältä… minä muistan sen, että se tuntui minusta
tyhmältä… niin välähti mielessäni. Siksipä minä juuri asian nyt
muistinkin. Ja kuinka olenkaan voinut unohtaa sen aina tähän asti!
Tuota pussia hän juuri osoitti huomauttaen, että hänellä on keino,
mutta että hän ei anna takaisin noita puoltatoista tuhatta! Ja kun
hänet vangittiin Mokrojessa, niin hän nimenomaan huusi, — minä
tiedän sen, minulle on kerrottu, — että hän pitää koko elämänsä
häpeällisimpänä tekona sitä, että vaikka hänellä oli varoja antaa
puolet (nimenomaan puolet!) velasta Katerina Ivanovnalle ja lakata
olemasta tämän silmissä varas, niin hän kuitenkin oli päättänyt olla
antamatta ja mieluummin jäädä hänen edessään varkaaksi kuin
erota rahoista! Ja miten tämä velka kiusasikaan häntä! — huudahti
Aljoša lopuksi.

Tietysti prokuraattorikin tarttui asiaan. Hän pyysi Aljošaa

kuvaamaan vielä kerran, kuinka kaikki tapahtui, ja kysyi useamman
kerran itsepintaisesti: ihanko todella syytetty lyödessään rintaansa
ikäänkuin osoitti jotakin? Kenties hän vain yksinkertaisesti löi
nyrkillään rintaansa?

— Ei edes nyrkillä! — huudahti Aljoša. — Hän nimenomaan osoitti

sormillaan, ja osoitti tähän paikkaan, hyvin korkealle… Mutta kuinka
olenkaan voinut unohtaa tämän niin tyystin aina tähän hetkeen asti!

Puheenjohtaja kääntyi Mitjan puoleen kysyen, mitä tämä voi sanoa

esitetyn todistuksen johdosta. Mitja vahvisti kaiken niin olleen ja
nimenomaan osoittaneensa puoltatoista tuhattansa, jotka hänellä
olivat rinnan päällä, aivan kaulan alapuolella, ja vakuutti, että se
tietysti oli häpeä, »häpeä, jota en kiellä, häpeällisin teko koko minun
elämässäni!» huudahti Mitja. »Minä olisin voinut antaa ne takaisin
enkä antanut. Tahdoin mieluummin jäädä hänen silmissään
varkaaksi, mutta en antanut rahoja takaisin, mutta kaikkein suurin