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Preface
As all the prior editions of this textbook before, the twenty- Chapter 48 was specifically updated to reflect clinically impor-
eighth edition of Jawetz, Melnick, & Adelberg’s Medical tant and currently emerging infectious disease cases.
Microbiology remains true to the goals of the first edition New to this edition are Peter Hotez, MD, PhD, Rojelio
published in 1954, which is to “to provide a brief, accurate Mejia, MD, and Stefan Riedel, MD, PhD, D(ABMM).
and up-to-date presentation of those aspects of medical Dr. Hotez is the Dean of the National School of Tropical Medi-
microbiology that are of particular significance to the fields cine at Baylor College of Medicine in Houston, TX, and is a
of clinical infections and chemotherapy.” Professor of Pediatrics, Molecular Virology and Microbiology;
For the twenty-seventh edition, under the authorship of he brings extensive expertise in parasitology. Dr. Mejia is an
Dr. Karen Carroll, all chapters had been extensively revised, Assistant Professor in the Department of Pediatrics, Section
reflecting the tremendous expansion of medical knowledge of Tropical Medicine, at the National School of Tropical Medi-
afforded by molecular mechanisms and diagnostics, advances cine, Baylor College of Medicine in Houston, TX. Dr. Riedel
in our understanding of microbial pathogenesis, and the dis- is the Associate Medical Director of the Clinical Microbiol-
covery of novel pathogens. While Dr. Carroll decided to step ogy Laboratories at Beth Israel Deaconess Medical Center in
down as an author and contributor for this new edition, the Boston, MA, and holds the academic rank of Associate Pro-
remaining authors would like to express their gratitude for her fessor of Pathology at Harvard Medical School. Following
leadership and contributions to the previous, greatly expanded Dr. Carroll’s departure as an author and contributor to this
edition. For the 28th edition, Chapter 47, “Principles of Diag- textbook, Dr. Riedel assumed the role as Editor-in-Chief for
nostic Medical Microbiology,” and Chapter 48, “Cases and this revised, 28th edition of the textbook.
Clinical Correlations,” were again updated to reflect the con- The authors hope that the changes to this current edition
tinued expansion in laboratory diagnostics as well as new will continue to be helpful to the student of microbiology and
antimicrobial therapies in the treatment of infectious diseases. infectious diseases.
xi
1
C H A P T E R
Alga
Fungus
Fungal Cortex
hyphae
Alga
layer
Cortex
FIGURE 1-1 Diagram of a lichen, consisting of cells of a phototroph, either an alga or a cyanobacterium, entwined within the hyphae of
the fungal partner. (Reproduced with permission from Nester EW, Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective, 6th ed.
McGraw-Hill, 2009, p. 293. © McGraw-Hill Education.)
selection operating on a vast array of genetically diverse plant and animal hosts as well as protists, fungi, and bacteria.
organisms. It is useful to keep the complexity of natural his- However, most viruses are restricted to infecting specific
tory in mind before generalizing about microorganisms, the types of cells of only one host species, a property known as
most heterogeneous subset of all living creatures. “tropism”. Recently, viruses called virophages have been
A major biologic division separates the eukaryotes, discovered that infect other viruses. Host–virus interactions
organisms containing a membrane-bound nucleus from pro- tend to be highly specific, and the biologic range of viruses
karyotes, organisms in which DNA is not physically sepa- mirrors the diversity of potential host cells. Further diversity
rated from the cytoplasm. As described in this chapter and in of viruses is exhibited by their broad array of strategies for
Chapter 2, further major distinctions can be made between replication and survival.
eukaryotes and prokaryotes. Eukaryotes, for example, are Viral particles are generally small (eg, adenovirus has a
distinguished by their relatively large size and by the pres- diameter of 90 nm) and consist of a nucleic acid molecule,
ence of specialized membrane-bound organelles such as either DNA or RNA, enclosed in a protein coat, or capsid (some-
mitochondria. times itself surrounded by an envelope of lipids, proteins,
As described more fully later in this chapter, eukary- and carbohydrates). Proteins—frequently glycoproteins—
otic microorganisms—or, phylogenetically speaking, the comprising the capsid and/or making up part of the lipid
Eukarya—are unified by their distinct cell structure and phy- envelope (e.g., HIV gp120) determine the specificity of inter-
logenetic history. Among the groups of eukaryotic microor- action of a virus with its host cell. The capsid protects the
ganisms are the algae, the protozoa, the fungi, and the slime nucleic acid cargo. The surface proteins, whether they are
molds. A class of microorganisms that share characteristics externally exposed on the capsid or associated with the enve-
common to both prokaryotes and eukaryotes are the archae- lope facilitates attachment and penetration of the host cell
bacteria and are described in Chapter 3. by the virus. Once inside the cell, viral nucleic acid redirects
the host’s enzymatic machinery to functions associated with
replication and assembly of the virus. In some cases, genetic
VIRUSES information from the virus can be incorporated as DNA into
a host chromosome (a provirus). In other instances, the viral
The unique properties of viruses set them apart from liv- genetic information can serve as a basis for cellular manufac-
ing creatures. Viruses lack many of the attributes of cells, ture and release of copies of the virus. This process calls for
including the ability to self-replicate. Only when it infects a replication of the viral nucleic acid and production of spe-
cell does a virus acquire the key attribute of a living system— cific viral proteins. Maturation consists of assembling newly
reproduction. Viruses are known to infect a wide variety of synthesized nucleic acid and protein subunits into mature
Several transmissible plant diseases are caused by FIGURE 1-2 Prion. Prions isolated from the brain of a scrapie-
viroids—small, single-stranded, covalently closed circu- infected hamster. This neurodegenerative disease is caused by a
lar RNA molecules existing as highly base-paired rod-like prion. (Reproduced with permission from Stanley B. Prusiner.)
structures. They range in size from 246 to 375 nucleotides in
length. The extracellular form of the viroid is naked RNA—
there is no capsid of any kind. The RNA molecule contains protein that prions are made of (PrP) is found through-
no protein-encoding genes, and the viroid is therefore totally out the body, even in healthy people and in animals, and is
dependent on host functions for its replication. Viroid RNA encoded by the host’s chromosomal DNA. The normal form
is replicated by the DNA-dependent RNA polymerase of the of the prion protein is called PrPc. PrPc is a sialoglycoprotein
plant host; preemption of this enzyme may contribute to with a molecular mass of 35,000–36,000 Da and a mainly
viroid pathogenicity. α-helical secondary structure that is sensitive to proteases
The RNAs of viroids have been shown to contain inverted and soluble in detergent. Several topological forms exist:
repeated base sequences (also known as insertion sequences) one cell surface form anchored by a glycolipid, and two
at their 3′ and 5′ ends, a characteristic of transposable ele- transmembrane forms. The disease scrapie manifests itself
ments (see Chapter 7) and retroviruses. Thus, it is likely that when a conformational change occurs in the prion protein,
they have evolved from transposable elements or retroviruses changing it from its normal or cellular form PrPc to the
by the deletion of internal sequences. infectious disease-causing isoform, PrPSc (Figure 1-3); this
The general properties of animal viruses pathogenic in turn alters the way the proteins interconnect. The exact
for humans are described in Chapter 29. Bacterial viruses, three-dimensional structure of PrPSc is unknown; however,
known as bacterial phages, are described in Chapter 7. it has a higher proportion of β-sheet structures in place of
the normal α-helix structures. Aggregations of PrPSc form
highly structured amyloid fibers, which accumulate to form
PRIONS plaques. It is unclear if these aggregates are the cause of the
cell damage or are simply a side effect of the underlying dis-
A number of remarkable discoveries in the past three ease process. One model of prion replication suggests that
decades have led to the molecular and genetic characteriza- PrPc exists only as fibrils, and that the fibril ends bind PrPc
tion of the transmissible agent causing scrapie, a degenera- and convert it to PrPSc.
tive central nervous system disease of sheep. Studies have There are several prion diseases of importance (Table 1-1
identified a specific protein in preparations from scrapie- and see Chapter 42). Kuru, Creutzfeldt-Jakob disease (CJD),
infected brains of sheep that can reproduce the symptoms Gerstmann-Sträussler-Scheinker disease, and fatal familial
of scrapie in previously uninfected sheep (Figure 1-2). insomnia affect humans. Bovine spongiform encephalopa-
Attempts to identify additional components, such as nucleic thy (BSE), which is thought to result from the ingestion of
acid, have been unsuccessful. To distinguish this agent feeds and bone meal prepared from rendered sheep offal, has
from viruses and viroids, the term prion was introduced been responsible for the deaths of more than 184,000 cattle
to emphasize its proteinaceous and infectious nature. The in Great Britain since its discovery in 1985. A new variant
Acquired Variant Creutzfeldt-Jakob diseasea Associated with ingestion or inoculation of prion-infected material
Kuru
Familial Gerstmann-Sträussler-Scheinker Associated with specific mutations within the gene encoding PrP
Creutzfeldt-Jakob disease
Cattle Bovine spongiform encephalopathy Exposure to prion-contaminated meat and bone meal
When few cells are present, the When many cells are present, the
concentration of the signaling concentration of the AHL is high.
molecule acylated homoserine High concentrations of AHL induce
lactone (AHL) is low. expression of specific genes.
FIGURE 1-4 Quorum sensing. (Reproduced with permission from Nester EW, Anderson DG, Roberts CE, et al: Microbiology: A Human
Perspective, 6th ed. McGraw-Hill, 2009, p. 181. © McGraw-Hill Education.)
are drug resistance plasmids that may render diverse bacteria allows a scientist to choose characteristics that allow swift
resistant to antibiotic treatment (Chapter 7). and accurate categorization of a newly encountered organ-
The survival strategy of a single prokaryotic cell line may ism. This categorical organization allows prediction of
lead to a range of interactions with other organisms. These many additional traits shared by other members of the
may include symbiotic relationships illustrated by complex category. In a hospital setting, successful classification of
nutritional exchanges among organisms within the human a pathogenic organism may provide the most direct route
gut. These exchanges benefit both the microorganisms and to its elimination. Classification may also provide a broad
their human host. Parasitic interactions can be quite delete- understanding of relationships among different organ-
rious to the host. Advanced symbiosis or parasitism can lead isms, and such information may have great practical value.
to loss of functions that may not allow growth of the symbi- For example, elimination of a pathogenic organism will be
ont or parasite independent of its host. relatively long-lasting if its habitat is occupied by a non-
The mycoplasmas, for example, are parasitic prokaryotes pathogenic variant.
that have lost the ability to form a cell wall. Adaptation of these The principles of prokaryotic classification are discussed
organisms to their parasitic environment has resulted in incor- in Chapter 3. At the outset, it should be recognized that any
poration of a substantial quantity of cholesterol into their cell prokaryotic characteristic might serve as a potential criterion
membranes. Cholesterol, not found in other prokaryotes, is for classification. However, not all criteria are equally effec-
assimilated from the metabolic environment provided by the tive in grouping organisms. Possession of DNA, for example,
host. Loss of function is exemplified also by obligate intracel- is a useless criterion for distinguishing organisms because all
lular parasites, the chlamydiae and rickettsiae. These bacteria cells contain DNA. The presence of a broad host range plas-
are extremely small (0.2–0.5 µm in diameter) and depend on mid is not a useful criterion because such plasmids may be
the host cell for many essential metabolites and coenzymes. found in diverse hosts and need not be present all of the time.
This loss of function is reflected by the presence of a smaller Useful criteria may be structural, physiologic, biochemical,
genome with fewer genes (see Table 7-1). or genetic. Spores—specialized cell structures that may allow
The most widely distributed examples of bacterial survival in extreme environments—are useful structural cri-
symbionts appear to be chloroplasts and mitochondria, the teria for classification because well-characterized subsets of
energy-yielding organelles of eukaryotes. Evidence points to bacteria form spores. Some bacterial groups can be effectively
the conclusion that ancestors of these chloroplasts and mito- subdivided based upon their ability to ferment specified car-
chondria were endosymbionts, essentially “domesticated bohydrates. Such criteria may be ineffective when applied to
bacteria” that established symbiosis within the cell mem- other bacterial groups that may lack any fermentative capa-
brane of the ancestral eukaryotic host. The presence of mul- bility. A biochemical test, the Gram-stain, is an effective cri-
tiple copies of these organelles may have contributed to the terion for classification because response to the stain reflects
relatively large size of eukaryotic cells and to their capacity fundamental differences in the bacterial cell envelope that
for specialization, a trait ultimately reflected in the evolution divide most bacteria into two major groups.
of differentiated multicellular organisms. Genetic criteria are increasingly used in bacterial clas-
sification, and many of these advances are made possible
by the development of DNA-based technologies. It is now
Classification of the Prokaryotes possible to design DNA probe or DNA amplification assays
An understanding of any group of organisms requires (eg, polymerase chain reaction [PCR] assays) that swiftly
their classification. An appropriate classification system identify organisms carrying specified genetic regions with
common ancestry. Comparison of DNA sequences for some through a series of events achieving physiologic integration
genes has led to the elucidation of phylogenetic relation- of the nucleus with the endoplasmic reticulum, a structure
ships among prokaryotes. Ancestral cell lines can be traced, that has no counterpart in prokaryotes. Eukaryotes are set
and organisms can be grouped based on their evolution- apart by the organization of their cellular DNA in chromo-
ary affinities. These investigations have led to some strik- somes separated by a distinctive mitotic apparatus during cell
ing conclusions. For example, comparison of cytochrome c division.
sequences suggests that all eukaryotes, including humans, In general, genetic transfer among eukaryotes depends
arose from one of three different groups of purple photo- on fusion of haploid gametes to form a diploid cell con-
synthetic bacteria. This conclusion in part explains the evo- taining a full set of genes derived from each gamete. The
lutionary origin of eukaryotes, but it does not fully take into life cycle of many eukaryotes is almost entirely in the dip-
account the generally accepted view that the eukaryotic cell loid state, a form not encountered in prokaryotes. Fusion
was derived from the evolutionary merger of different pro- of gametes to form reproductive progeny is a highly spe-
karyotic cell lines. cific event and establishes the basis for eukaryotic species.
This term can be applied only metaphorically to the pro-
karyotes, which exchange fragments of DNA through
Bacteria and Archaebacteria: The Major recombination. Currently, the term protist is used infor-
Subdivisions Within the Prokaryotes mally as a catch-all term for unicellular eukaryotic micro-
A major success in molecular phylogeny has been the dem- organisms. Because protists as a whole are paraphyletic,
onstration that prokaryotes fall into two major groups. newer classification systems often split up traditional sub-
Most investigations have been directed to one group, the divisions or groups based on morphological or biochemi-
bacteria. The other group, the archaebacteria, has received cal characteristics.
relatively little attention until recently, partly because many Traditionally, microbial eukaryotes—protists—are
of its representatives are difficult to study in the laboratory. placed in one of the four following major groups: algae,
Some archaebacteria, for example, are killed by contact with protozoa, fungi, and slime molds. These traditional sub-
oxygen, and others grow at temperatures exceeding that of divisions, largely based on superficial commonalities,
boiling water. Before molecular evidence became available, have been largely replaced by classification schemes based
the major subgroupings of archaebacteria had seemed dis- on phylogenetics. Molecular methods used by modern
parate. The methanogens carry out an anaerobic respiration taxonomists have been used to generate data support-
that gives rise to methane, the halophiles demand extremely ing the redistribution of some members of these groups
high salt concentrations for growth, and the thermoacido- into diverse and sometimes distantly related phyla. For
philes require high temperature and acidity for growth. It has example, the water molds are now considered to be closely
now been established that these prokaryotes share biochemi- related to photosynthetic organisms such as brown algae
cal traits such as cell wall or membrane components that and diatoms.
set the group entirely apart from all other living organisms.
An intriguing trait shared by archaebacteria and eukary-
otes is the presence of introns within genes. The function
Algae
of introns—segments of DNA that interrupts informational The term algae has long been used to denote all organisms
DNA within genes—is not established. What is known is that produce O2 as a product of photosynthesis. One for-
that introns represent a fundamental characteristic shared mer subgroup of these organisms—the blue-green algae, or
by the DNA of archaebacteria and eukaryotes. This common cyanobacteria—are prokaryotic and no longer are termed
trait has led to the suggestion that—just as mitochondria algae. This classification is reserved exclusively for a large
and chloroplasts appear to be evolutionary derivatives of the diverse group of photosynthetic eukaryotic organisms. For-
bacteria—the eukaryotic nucleus may have arisen from an merly, all algae were thought to contain chlorophyll in the
archaebacterial ancestor. photosynthetic membrane of their chloroplast, a subcellu-
lar organelle that is similar in structure to cyanobacteria.
Modern taxonomic approaches have recognized that some
PROTISTS algae lack chlorophyll and have a free-living heterotrophic
or parasitic life style. Many algal species are unicellular
The “true nucleus” of eukaryotes (from Gr karyon, “nucleus”) microorganisms. Other algae may form extremely large
is only one of their distinguishing features. The membrane- multicellular structures. Kelps of brown algae sometimes
bound organelles, the microtubules, and the microfilaments are several hundred meters in length. Several algae produce
of eukaryotes form a complex intracellular structure unlike toxins that are poisonous to humans and other animals.
that found in prokaryotes. The organelles responsible for Dinoflagellates, a unicellular alga, are responsible for algal
the motility of eukaryotic cells are flagella or cilia—complex blooms, or red tides, in the ocean (Figure 1-5). Red tides
multistranded structures that do not resemble the flagella caused by the dinoflagellate Gonyaulax species are serious
of prokaryotes. Gene expression in eukaryotes takes place because this organism produces potent neurotoxins such as
are known that have flagella at one stage in their life cycle
and pseudopodia at another stage. A fourth major group of
protozoa, the sporozoa, are strict parasites that are usually
nonmotile; most of these reproduce sexually and asexually
in alternate generations by means of spores. Recent taxo-
nomic studies have shown that only the ciliates are mono-
phyletic, that is, a distinct lineage of organisms sharing
common ancestry. The other classes of protozoa are all
polyphyletic groups made up of organisms that, despite
similarities in appearance (eg, flagellates) or way of life
(eg, endoparasitic), are not necessarily closely related to
one another. Protozoan parasites of humans are discussed
in Chapter 46.
Fungi
The fungi are nonphotosynthetic protists that may or may not
grow as a mass of branching, interlacing filaments (“hyphae”)
known as a mycelium. If a fungus grows simply as a single
cell it is called a yeast. If mycelial growth occurs, it is called
FIGURE 1-5 The dinoflagellate Gymnodinium scanning electron a mold. Most fungi of medical importance grow dimorphi-
micrograph (4000×). (Reproduced with permission from Dr. David cally, that is, they exist as a mold at room temperature but as
Phillips/Visuals Unlimited.) a yeast at body temperature. Remarkably, the largest known
contiguous fungal mycelium covered an area of 2400 acres
(9.7 km2) at a site in eastern Oregon. Although the hyphae
saxitoxin and gonyautoxins, which accumulate in shellfish exhibit cross walls, the cross walls are perforated and allow
(eg, clams, mussels, scallops, and oysters) that feed on this free passage of nuclei and cytoplasm. The entire organism is
organism. Ingestion of these shellfish by humans results in thus a coenocyte (a multinucleated mass of continuous cyto-
symptoms of paralytic shellfish poisoning and can lead to plasm) confined within a series of branching tubes. These
death. Some algae (eg, Prototheca and Helicosporidium) are tubes, made of polysaccharides such as chitin, are homolo-
parasites of metazoans or plants. Protothecosis is a disease gous with cell walls.
of dogs, cats, cattle, and rarely humans caused by a type of The fungi probably represent an evolutionary offshoot
algae, Prototheca, that lacks chlorophyll. The two most com- of the protozoa; they are unrelated to the actinomycetes,
mon species are P. wickerhamii and P. zopfii; most human mycelial bacteria that they superficially resemble. The
cases, which are associated with a defective immune system, major subdivisions (phyla) of fungi are Chytridiomycota,
are caused by P. wickerhamii. Zygomycota (the zygomycetes), Ascomycota (the asco-
mycetes), Basidiomycota (the basidiomycetes), and the
“deuteromycetes” (or imperfect fungi). The evolution of the
Protozoa ascomycetes from the phycomycetes is seen in a transitional
Protozoa is an informal term for single-celled nonphoto- group, whose members form a zygote but then transform
synthetic eukaryotes that are either free-living or para- this directly into an ascus. The basidiomycetes are believed
sitic. Protozoa are abundant in aqueous environments to have evolved in turn from the ascomycetes. The classifi-
and soil. They range in size from as little as 1µm to sev- cation of fungi and their medical significance are discussed
eral millimeters, or more. All protozoa are heterotrophic, further in Chapter 45.
deriving nutrients from other organisms, either by ingest-
ing them whole or by consuming their organic tissue or
waste products. Some protozoans take in food by phago- Slime Molds
cytosis, engulfing organic particles with pseudopodia (eg, These organisms are characterized by the presence, as a
amoeba), or taking in food through a mouth-like aperture stage in their life cycle, of an ameboid multinucleate mass
called a cytostome. Other protozoans absorb dissolved of cytoplasm called a plasmodium. The plasmodium of a
nutrients through their cell membranes, a process called slime mold is analogous to the mycelium of a true fungus.
osmotrophy. Both are coenocytic. Whereas in the latter, cytoplasmic flow
Historically, the major groups of protozoa included: is confined to the branching network of chitinous tubes, in
flagellates, motile cells possessing whip-like organelles of the former, the cytoplasm can flow in all directions. This
locomotion; amoebae, cells that move by extending pseu- flow causes the plasmodium to migrate in the direction of
dopodia; and ciliates, cells possessing large numbers of its food source, frequently bacteria. In response to a chemi-
short hair-like organelles of motility. Intermediate forms cal signal, 3′, 5′-cyclic AMP, the plasmodium, which reaches
Spores
Fruiting bodies
Germination
release spores
Myxamoebae
Fruiting body
Plasmodium
A B
FIGURE 1-6 Slime molds. A: Life cycle of an acellular slime mold. B: Fruiting body of a cellular slime mold. (Reproduced with permission
from Carolina Biological Supply/DIOMEDIA.)
macroscopic size, differentiates into a stalked body that can REVIEW QUESTIONS
produce individual motile cells. These cells, flagellated or
1. Which one of the following terms characterizes the interaction
ameboid, initiate a new round in the life cycle of the slime
between herpes simplex virus and a human?
mold (Figure 1-6). The cycle frequently is initiated by sexual
(A) Parasitism
fusion of single cells.
(B) Symbiosis
The growth of slime molds depends on nutrients pro-
(C) Endosymbiosis
vided by bacterial or, in some cases, plant cells. Reproduction (D) Endoparasitism
of the slime molds via plasmodia can depend on intercellular (E) Consortia
recognition and fusion of cells from the same species. The 2. Which one of the following agents lacks nucleic acid?
life cycle of the slime molds illustrates a central theme of this
(A) Bacteria
chapter—the interdependency of living forms. Full under- (B) Viruses
standing of any microorganism requires both knowledge of (C) Viroids
the other organisms with which it coevolved and an apprecia- (D) Prions
tion of the range of physiologic responses that may contribute (E) Protozoa
to survival. 3. Which one of the following is a prokaryote?
(A) Bacteria
(B) Algae
CHAPTER SUMMARY (C) Protozoa
(D) Fungi
• Microorganisms are a large and diverse group of organisms (E) Slime molds
existing as single cells or clusters; they also include viruses, 4. Which one of the following agents simultaneously contains
which are microscopic but not cellular. both DNA and RNA?
• A virus consists of a nucleic acid molecule, either DNA (A) Bacteria
or RNA, enclosed in a protein coat, or capsid, sometimes (B) Viruses
enclosed by an envelope composed of lipids, proteins, and (C) Viroids
carbohydrates. (D) Prions
• A prion is an infectious protein, which is capable of causing (E) Plasmids
chronic neurologic diseases. 5. Which of the following cannot be infected by viruses?
• Prokaryotes consist of bacteria and archaebacteria. (A) Bacteria
• Prokaryotes are haploid. (B) Protozoa
• Microbial eukaryotes, or protists, are members of four (C) Human cells
major groups: algae, protozoa, fungi, and slime molds. (D) Viruses
• Eukaryotes have a true nucleus and are diploid. (E) None of the above
Cell Structure
This chapter discusses the basic structure and function of the B. Phase-Contrast Microscope
components that make up eukaryotic and prokaryotic cells. It The phase-contrast microscope was developed to improve
begins with a discussion of the microscope. Historically, the contrast differences between cells and the surrounding
microscope first revealed the presence of bacteria and later medium, making it possible to see living cells without stain-
the secrets of cell structure. Today, it remains a powerful tool ing them; with bright-field microscopes, killed and stained
in cell biology. preparations must be used. The phase-contrast microscope
takes advantage of the fact that light waves passing through
transparent objects, such as cells, emerge in different phases
OPTICAL METHODS depending on the properties of the materials through which
they pass. This effect is amplified by a special ring in the
The Light Microscope objective lens of a phase-contrast microscope, leading to the
The resolving power of the light microscope under ideal con- formation of a dark image on a light background (Figure 2-1).
ditions is about half the wavelength of the light being used.
(Resolving power is the distance that must separate two C. Dark-Field Microscope
point sources of light if they are to be seen as two distinct The dark-field microscope is a light microscope in which
images.) With yellow light of a wavelength of 0.4 µm, the the lighting system has been modified to reach the speci-
smallest separable diameters are thus about 0.2 µm (ie, one- men from the sides only. This is accomplished through the
third the width of a typical prokaryotic cell). The useful mag- use of a special condenser that both blocks direct light rays
nification of a microscope is the magnification that makes and deflects light off a mirror on the side of the condenser
visible the smallest resolvable particles. Several types of light at an oblique angle. This creates a “dark field” that contrasts
microscopes, which are commonly used in microbiology, are against the highlighted edge of the specimens and results
discussed as follows. when the oblique rays are reflected from the edge of the spec-
imen upward into the objective of the microscope. Resolution
A. Bright-Field Microscope by dark-field microscopy is quite high. Thus, this technique
The bright-field microscope is the most commonly used in has been particularly useful for observing organisms such as
microbiology courses and consists of two series of lenses Treponema pallidum, a spirochete that is smaller than 0.2 µm
(objective and ocular lens), which function together to in diameter and therefore cannot be observed with a bright-
resolve the image. These microscopes generally employ a field or phase-contrast microscope (Figure 2-2A).
100-power objective lens with a 10-power ocular lens, thus
magnifying the specimen 1000 times. Particles 0.2 µm in D. Fluorescence Microscope
diameter are therefore magnified to about 0.2 mm and so The fluorescence microscope is used to visualize specimens
become clearly visible. Further magnification would give no that fluoresce, which is the ability to absorb short wave-
greater resolution of detail and would reduce the visible area lengths of light (ultraviolet) and give off light at a longer wave-
(field). length (visible). Some organisms fluoresce naturally because
With this microscope, specimens are rendered visible of the presence within the cells of naturally fluorescent sub-
because of the differences in contrast between them and stances such as chlorophyll. Those that do not naturally fluo-
the surrounding medium. Many bacteria are difficult to resce may be stained with a group of fluorescent dyes called
see well because of their lack of contrast with the surround- fluorochromes. Fluorescence microscopy is widely used in
ing medium. Dyes (stains) can be used to stain cells or their clinical diagnostic microbiology. For example, the fluoro-
organelles and increase their contrast so that they can be chrome auramine O, which glows yellow when exposed to
more easily seen in the bright-field microscope. ultraviolet light, is strongly absorbed by the cell envelope of
11
Rough
endoplasmic
Mitochondrion
reticulum
Peroxisome
Cell
Cytoskeleton Nuclear envelope membrane
Smooth
Actin endoplasmic
filament reticulum Nucleus Nucleolus
Nucleus
Microtubule Rough
endoplasmic
Nuclear
Intermediate reticulum
membrane
filament
Plasma Ribosomes
membrane
Mitochondrion
Golgi Central
complex vacuole
Cell wall
Lysosome Mitochondrion C
1 µm
Cytoplasm
Adjacent
Peroxisome
cell wall
Chloroplast (opened to
B show thylakoids)
FIGURE 2-5 Eukaryotic cells. A: Diagrammatic representation of an animal cell. B: Diagrammatic representation of a plant cell.
C: Micrograph of an animal cell shows several membrane-bound structures, including mitochondria and a nucleus. (Fig. 2-3(A) and (B)
Reproduced with permission from Nester EW, Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective, 6th ed. McGraw-Hill, 2009.
© McGraw-Hill Education. Fig. 2-3(C) Reproduced with permission from Thomas Fritsche, MD, PhD.)
elaborate cytoskeleton composed of microtubules, microfila- are intermediate and appear to be evolving from a type II
ments, and intermediate filaments. to type I. Some hydrogenosomes have been identified that
The endoplasmic reticulum (ER) is a network of contain DNA and ribosomes. The hydrogenosome, although
membrane-bound channels continuous with the nuclear mem- similar in size to mitochondria, lacks cristae and the enzymes
brane. Two types of ER are recognized: rough, to which 80S of the tricarboxylic acid cycle. Pyruvate is taken up by the
ribosomes are attached; and smooth, which does not have hydrogenosome, and H2, CO2, acetate, and ATP are pro-
attached ribosomes (see Figure 2-5). Rough ER is a major pro- duced. The mitosome has only recently been discovered and
ducer of glycoproteins as well as new membrane material that is named, and its function has not been well characterized.
transported throughout the cell; smooth ER participates in the Lysosomes are membrane-enclosed vesicles that contain
synthesis of lipids and in some aspects of carbohydrate metabo- various digestive enzymes that the cell uses to digest macromol-
lism. The Golgi complex consists of a stack of membranes that ecules such as proteins, fats, and polysaccharides. The lysosome
function in concert with the ER to chemically modify and sort allows these enzymes to be partitioned away from the cytoplasm
products of the ER into those destined to be secreted and those proper, where they could destroy key cellular macromolecules if
that function in other membranous structures of the cell. not contained. After the hydrolysis of macromolecules in the
The plastids include mitochondria and chloroplasts. lysosome, the resulting monomers pass from the lysosome into
Several lines of evidence suggest that mitochondria and the cytoplasm, where they serve as nutrients.
chloroplasts arose from the engulfment of a prokaryotic cell The peroxisome is a membrane-enclosed structure
by a larger cell (endosymbiosis). Current hypotheses, mak- whose function is to produce H2O2 from the reduction of
ing use of mitochondrial genome and proteome data, sug- O2 by various hydrogen donors. The H2O2 produced in the
gest that the mitochondrial ancestor was most closely related peroxisome is subsequently degraded to H2O and O2 by the
to Alphaproteobacteria and that chloroplasts are related to enzyme catalase. Peroxisomes are believed to be of evolution-
nitrogen-fixing cyanobacteria. Mitochondria are of prokary- ary origin unrelated to mitochondria.
otic size (Figure 2-5), and its membrane, which lacks sterols, The cytoskeleton is a three-dimensional structure that
is much less rigid than the eukaryotic cell’s cytoplasmic mem- fills the cytoplasm. Eukaryotic cells contain three main
brane, which does contain sterols. Mitochondria contain two kinds of cytoskeletal filaments: microfilaments, intermedi-
sets of membranes. The outermost membrane is rather perme- ate filaments, and microtubules. Each cytoskeletal filament
able, having numerous minute channels that allow passage of type is formed by polymerization of a distinct type of pro-
ions and small molecules (eg, adenosine triphosphate [ATP]). tein subunit and has its own shape and intracellular distri-
Invagination of the outer membrane forms a system of inner bution. Microfilaments are about 7 nm in diameter and are
folded membranes called cristae. The cristae are the sites of polymers composed of the protein actin. These fibers form
enzymes involved in respiration and ATP production. Cristae scaffolds throughout the cell, defining and maintaining the
also contain specific transport proteins that regulate passage shape of the cell. Microfilaments can also carry out intracel-
of metabolites into and out of the mitochondrial matrix. The lular transport/trafficking, and cellular movements, includ-
matrix contains a number of enzymes, particularly those of ing gliding, contraction, and cytokinesis.
the citric acid cycle. Chloroplasts are the photosynthetic cell Microtubules are hollow cylinders about 23 nm in diameter
organelles that can convert the energy of sunlight into chemi- (lumen is approximately 15 nm in diameter) most commonly
cal energy through photosynthesis. Chlorophyll and all other comprising 13 protofilaments that, in turn, are polymers of
components needed for photosynthesis are located in a series alpha and beta tubulin. Microtubules assist microfilaments
of flattened membrane discs called thylakoids. The size, shape, in maintaining cell structure, form the spindle fibers for sepa-
and number of chloroplasts per cell vary markedly; in contrast rating chromosomes during mitosis, and play an important
to mitochondria, chloroplasts are generally much larger than role in cell motility. Intermediate filaments are composed of
prokaryotes. Mitochondria and chloroplasts contain their various proteins (eg, keratin, lamin, and desmin) depending
own DNA, which exists in a covalently closed circular form on the type of cell in which they are found. They are normally
and codes for some (not all) of their constituent proteins and 8–12 nm in diameter and provide tensile strength for the cell.
transfer RNAs. Mitochondria and chloroplasts also contain They are most commonly known as the support system or
70S ribosomes, the same as those of prokaryotes. “scaffolding” for the cell and nucleus. All filaments react with
Eukaryotic microorganisms that were previously thought accessory proteins (eg, Rho and dynein) that regulate and link
to lack mitochondria (amitochondriate eukaryotes) have the filaments to other cell components and each other.
been recently shown to contain some mitochondrial rem-
nants either through the maintenance of membrane-enclosed
respiratory organelles called hydrogenosomes, mitosomes, Surface Layers
or nuclear genes of mitochondrial origin. There are two types The cytoplasm is enclosed within a plasma membrane com-
of amitochondriate eukaryotes: type II (eg, Trichomonas posed of protein and phospholipid similar to the prokaryotic
vaginalis) harbors a hydrogenosome, while type I (eg, Giardia cell membrane illustrated later (see Figure 2-13). Most animal
lamblia) lacks organelles involved in core energy metabolism. cells have no other surface layers; however, plant cells have
Some amitochondrial parasites (eg, Entamoeba histolytica) an outer cell wall composed of cellulose (Figure 2-5B). Many
Both the flagella and the cilia of eukaryotic cells have the same
basic structure and biochemical composition. Both consist of
a series of microtubules, hollow protein cylinders composed
of a protein called tubulin surrounded by a membrane. The
arrangement of the microtubules is commonly referred to as
the “9 + 2 arrangement” because it consists of nine doublets
of microtubules surrounding two single central microtubules
(Figure 2-7). Each doublet is connected to another by the protein
dynein. The dynein arms attached to the microtubule function
as the molecular motors.
The Nucleoid
eukaryotic microorganisms also have an outer cell wall, Prokaryotes have no true nuclei; instead they package their
which may be composed of a polysaccharide such as cellulose DNA in a structure known as the nucleoid. The negatively
or chitin or may be inorganic (eg, the silica wall of diatoms). charged DNA is at least partially neutralized by small poly-
amines and magnesium ions. Nucleoid-associated proteins
exist in bacteria and are distinct from histones in eukaryotic
Motility Organelles chromatin.
Many eukaryotic microorganisms have organelles called Electron micrographs of a typical prokaryotic cell reveal
flagella (eg, T. vaginalis) or cilia (eg, Paramecium) that move the absence of a nuclear membrane and a mitotic apparatus.
with a wavelike motion to propel the cell through water. Eukary- The exception to this rule is the planctomycetes, a diver-
otic flagella emanate from the polar region of the cell, and cilia, gent group of aquatic bacteria, which have a nucleoid sur-
which are shorter than flagella, surround the cell (Figure 2-6). rounded by a nuclear envelope consisting of two membranes.
Outer
dynein arm
Inner
dynein arm
Central
microtubule
Spoke head
Radial spoke
Doublet
microtubule
Nexin link
Central sheath
Subtubule A
Subtubule B
A B
FIGURE 2-7 Cilia and flagella structure. A: An electron micrograph of a cilium cross section. Note the two central microtubles surrounded
by nine microtubule doublets (160,000×). (Reproduced with permission. © Kallista Images/Visuals Unlimited, Inc.) B: A diagram of cilia and
flagella structure. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed.
McGraw-Hill; 2008. © McGraw-Hill Education.)
Cytoplasmic Structures
Prokaryotic cells lack autonomous plastids, such as mito-
chondria and chloroplasts; the electron transport enzymes
are localized instead in the cytoplasmic membrane. The
photosynthetic pigments (carotenoids, bacteriochlorophyll)
of photosynthetic bacteria are contained in intracytoplas-
mic membrane systems of various morphologies. Mem-
brane vesicles (chromatophores) or lamellae are commonly
observed membrane types. Some photosynthetic bacteria
have specialized nonunit membrane-enclosed structures
called chlorosomes. In some cyanobacteria (formerly
known as blue-green algae), the photosynthetic membranes
often form multilayered structures known as thylakoids
A (Figure 2-9). The major accessory pigments used for light
0.5 µm harvesting are the phycobilins found on the outer surface of
the thylakoid membranes.
Bacteria often store reserve materials in the form of
insoluble granules, which appear as refractile bodies in the
cytoplasm when viewed by phase-contrast microscopy.
DNA fibers
These so-called inclusion bodies almost always function in
the storage of energy or as a reservoir of structural building
Membrane blocks. Most cellular inclusions are bounded by a thin non-
unit membrane consisting of lipid, which serves to separate
Ruptured cell
the inclusion from the cytoplasm proper. One of the most
common inclusion bodies consists of poly-β-hydroxybutyric
acid (PHB), a lipid-like compound consisting of chains of
B
Plasma membrane
FIGURE 2-8 The nucleoid. A: Color-enhanced transmission Cell wall
electron micrograph of E. coli with the DNA shown in red.
(Reproduced with permission. © CNRI/SPL/Photo Researchers, Inc.)
B: Chromosome released from a gently lysed cell of E. coli. Note how
tightly packaged the DNA must be inside the bacterium. (Reproduced
with permission. © Dr. Gopal Murti/SPL/Photo Researchers Inc.)
β-hydroxybutyric acid units connected through ester link- limiting, the sulfur in the granules is oxidized, usually to sul-
ages. PHB is produced when the source of nitrogen, sulfur, fate, and the granules slowly disappear. Many bacteria accu-
or phosphorous is limited and there is excess carbon in the mulate large reserves of inorganic phosphate in the form of
medium (Figure 2-10A). Another storage product formed granules of polyphosphate. These granules can be degraded
by prokaryotes when carbon is in excess is glycogen, which and used as sources of phosphate for nucleic acid and phos-
is a polymer of glucose. PHB and glycogen are used as car- pholipid synthesis to support growth. These granules are
bon sources when protein and nucleic acid synthesis are sometimes termed volutin granules or metachromatic gran-
resumed. A variety of prokaryotes are capable of oxidizing ules because they stain red with a blue dye. They are charac-
reduced sulfur compounds, such as hydrogen sulfide and teristic features of Corynebacterium (see Chapter 13).
thiosulfate, producing intracellular granules of elemental Certain groups of autotrophic bacteria that fix carbon
sulfur (Figure 2-10B). As the reduced sulfur source becomes dioxide to make their biochemical building blocks contain poly-
hedral bodies surrounded by a protein shell (carboxysomes)
containing the key enzyme of CO2 fixation, ribulosebisphos-
PM PHB phate carboxylase (see Figure 2-9). Magnetosomes are intra-
cellular crystal particles of the iron mineral magnetite (Fe3O4)
that allow certain aquatic bacteria to exhibit magnetotaxis (ie,
R migration or orientation of the cell with respect to the earth’s
magnetic field). Magnetosomes are surrounded by a nonunit
membrane containing phospholipids, proteins, and glycopro-
teins. Gas vesicles are found almost exclusively in microorgan-
isms from aquatic habitats, where they provide buoyancy. The
gas vesicle membrane is a 2-nm-thick layer of protein, imper-
meable to water and solutes but permeable to gases; thus, gas
vesicles exist as gas-filled structures surrounded by the con-
N stituents of the cytoplasm (Figure 2-11).
The most numerous intracellular structure in most
bacteria is the ribosome, the site of protein synthesis in all
M
CW
A
Oligosaccharide
Integral Glycolipid
Hydrophobic protein
α helix
Hopanoid
Peripheral Phospholipid
protein
FIGURE 2-13 Bacterial plasma membrane structure. This diagram of the fluid mosaic model of bacterial membrane structure shown
the integral proteins (green and red) floating in a lipid bilayer. Peripheral proteins (yellow) are associated loosely with the inner membrane
surface. Small spheres represent the hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains. Other
membrane lipids such as hopanoids (purple) may be present. For the sake of clarity, phospholipids are shown proportionately much larger size
than in real membranes. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology,
7th ed. McGraw-Hill; 2008. © McGraw-Hill Education.)
likely fulfill the same function. Unlike eukaryotes, bacte- the cell. However, facilitated diffusion is selective. Chan-
ria can have a wide variety of fatty acids within their mem- nel proteins form selective channels that facilitate the pas-
branes. Along with the typical saturated and unsaturated sage of specific molecules. Facilitated diffusion is common
fatty acids, bacterial membranes can contain fatty acids with in eukaryotic microorganisms (eg, yeast) but is rare in pro-
additional methyl, hydroxy, or cyclic groups. The relative karyotes. Glycerol is one of the few compounds that enters
proportions of these fatty acids can be modulated by the bac- prokaryotic cells by facilitated diffusion.
terium to maintain the optimum fluidity of the membrane.
b. Active transport—Many nutrients are concentrated more
For example, at least 50% of the cytoplasmic membrane
than a thousandfold as a result of active transport. There
must be in the semifluid state for cell growth to occur. At
are two types of active transport mechanisms depending on
low temperatures, this is achieved by greatly increased syn-
the source of energy used: ion-coupled transport and ATP-
thesis and incorporation of unsaturated fatty acids into the
binding cassette (ABC) transport.
phospholipids of the cell membrane.
The cell membranes of the Archaea (see Chapter 1) dif- 1) Ion-coupled transport—These systems move a molecule
fer from those of the Bacteria. Some Archaeal cell membranes across the cell membrane at the expense of a previously estab-
contain unique lipids, isoprenoids, rather than fatty acids, lished ion gradient such as proton- or sodium-motive force.
linked to glycerol by ether rather than an ester linkage. Some There are three basic types: uniport, symport, and antiport
of these lipids have no phosphate groups, and therefore, they (Figure 2-14). Ion-coupled transport is particularly common
are not phospholipids. In other species, the cell membrane is in aerobic organisms, which have an easier time generating
made up of a lipid monolayer consisting of long lipids (about an ion-motive force than do anaerobes. Uniporters catalyze
twice as long as a phospholipid) with glycerol ethers at both the transport of a substrate independent of any coupled ion.
ends (diglycerol tetraethers). The molecules orient themselves Symporters catalyze the simultaneous transport of two sub-
with the polar glycerol groups on the surfaces and the non- strates in the same direction by a single carrier; for example, an
polar hydrocarbon chain in the interior. These unusual lipids H+ gradient can permit symport of an oppositely charged ion
contribute to the ability of many Archaea to grow under envi- (eg, glycine) or a neutral molecule (eg, galactose). Antiport-
ronmental conditions such as high salt, low pH, or very high ers catalyze the simultaneous transport of two like-charged
temperature. compounds in opposite directions by a common carrier (eg,
H+:Na+). Approximately, 40% of the substrates transported by
B. Function E. coli use this mechanism.
The major functions of the cytoplasmic membrane are (1) 2) ABC transport—This mechanism uses ATP directly to
selective permeability and transport of solutes; (2) electron transport solutes into the cell. In Gram-negative bacte-
transport and oxidative phosphorylation in aerobic spe- ria, the transport of many nutrients is facilitated by spe-
cies; (3) excretion of hydrolytic exoenzymes; (4) contain the cific binding proteins located in the periplasmic space; in
enzymes and carrier molecules that function in the biosyn- Gram-positive cells, the binding proteins are attached to the
thesis of DNA, cell wall polymers, and membrane lipids; and outer surface of the cell membrane. These proteins function
(5) bear the receptors and other proteins of the chemotactic by transferring the bound substrate to a membrane-bound
and other sensory transduction systems. protein complex. Hydrolysis of ATP is then triggered, and
the energy is used to open the membrane pore and allow
1. Permeability and transport—The cytoplasmic the unidirectional movement of the substrate into the cell.
membrane forms a hydrophobic barrier impermeable to Approximately 40% of the substrates transported by E. coli
most hydrophilic molecules. However, several mechanisms use this mechanism.
(transport systems) exist that enable the cell to transport
c. Group translocation—In addition to true transport, in
nutrients into and waste products out of the cell. These trans-
which a solute is moved across the membrane without change
port systems work against a concentration gradient to increase
in structure, bacteria use a process called group translocation
the nutrient concentrations inside the cell, a function that
(vectorial metabolism) to effect the net uptake of certain
requires energy in some form. There are three general trans-
sugars (eg, glucose and mannose), the substrate becom-
port mechanisms involved in membrane transport: passive
ing phosphorylated during the transport process. In a strict
transport, active transport, and group translocation.
sense, group translocation is not active transport because
a. Passive transport—This mechanism relies on diffusion, no concentration gradient is involved. This process allows
uses no energy, and operates only when the solute is at higher bacteria to use their energy resources efficiently by coupling
concentration outside than inside the cell. Simple diffusion transport with metabolism. In this process, a membrane car-
accounts for the entry of very few nutrients, including dis- rier protein is first phosphorylated in the cytoplasm at the
solved oxygen, carbon dioxide, and water itself. Simple dif- expense of phosphoenolpyruvate; the phosphorylated car-
fusion provides neither speed nor selectivity. Facilitated rier protein then binds the free sugar at the exterior mem-
diffusion also uses no energy, so the solute never achieves brane face and transports it into the cytoplasm, releasing it as
an internal concentration greater than what exists outside a sugar phosphate. Such systems of sugar transport are called
Cell exterior
TolC
Outer
Yop membrane
PulS
Periplasmic
YscJ space
SecD
Tat
EFGY
Plasma
Sec
ADP membrane
+ Pi
ATP ADP ADP ADP ADP
+ Pi + Pi ATP + Pi + Pi
ATP ATP ATP
ATP
Cytoplasm
Chaperone Chaperone
Protein
FIGURE 2-15 The protein secretion systems of Gram-negative bacteria. Five secretion systems of Gram-negative bacteria are shown.
The Sec-dependent and Tat pathways deliver proteins from the cytoplasm to the periplasmic space. The type II, type V, and sometimes
type IV systems complete the secretion process begun by the Sec-dependent pathway. The Tat system appears to deliver proteins only to
the type II pathway. The type I and III systems bypass the Sec-dependent and Tat pathways, moving proteins directly from the cytoplasm,
through the outer membrane, to the extracellular space. The type IV system can work either with the Sec-dependent pathway or can work
alone to transport proteins to the extracellular space. Proteins translocated by the Sec-dependent pathway and the type III pathway are
delivered to those systems by chaperone proteins. ADP, adenosine diphosphate; ATP, adenosine triphosphate; EFGY; PuIS; SecD; TolC; Yop.
(Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008.
© McGraw-Hill Education.)
synthesized on cytoplasmic ribosomes as preproteins con- targeting translocase (tat pathway) can move proteins across
taining an extra leader or signal sequence of 15–40 amino the IM. In Gram-negative bacteria, these proteins are then
acids—most commonly about 30 amino acids—at the amino delivered to the type II system (Figure 2-15). The tat pathway
terminal and require the sec system for transport across the is distinct from the sec system in that it translocates already
IM. In E. coli, the sec pathway comprises a number of IM folded proteins.
proteins (SecD to SecF, SecY), a cell membrane-associated Although proteins secreted by the type II and V systems
ATPase (SecA) that provides energy for export, a chaperone are similar in the mechanism by which they cross the IM, dif-
(SecB) that binds to the preprotein, and the periplasmic ferences exist in how they traverse the OM. Proteins secreted
signal peptidase. After translocation, the leader sequence is by the type II system are transported across the OM by a mul-
cleaved off by the membrane-bound signal peptidase, and the tiprotein complex (see Figure 2-15). This is the primary path-
mature protein is released into the periplasmic space. In con- way for the secretion of extracellular degradative enzymes
trast, proteins secreted by the type I and III systems do not by Gram-negative bacteria. Elastase, phospholipase C, and
have a leader sequence and are exported intact. exotoxin A are secreted by this system in Pseudomonas
In Gram-negative and Gram-positive bacteria, another aeruginosa. However, proteins secreted by the type V system
cytoplasmic membrane system that uses the twin-arginine autotransport across the outer membrane by virtue of a
Cytoplasmic
membrane Periplasm
Cytoplasmic
membrane
Gram-Positive Gram-Negative
Distinguishing Structures/Components
Porin proteins Absent (unnecessary because there is no Present; allow passage of molecules through
outer membrane) outer membrane
General Characteristics
Sensitivity to penicillin Generally more susceptible (with notable Generally less susceptible (with notable
exceptions) exceptions)
connected by β1→4 linkages; a set of identical tetrapeptide when given l-lysine alone, however, they lyse, because they
side chains attached to N-acetylmuramic acid; and a set of continue to grow but are specifically unable to make new cell
identical peptide cross-bridges (Figure 2-17). The backbone wall peptidoglycan.
is the same in all bacterial species; the tetrapeptide side The fact that all peptidoglycan chains are cross-linked
chains and the peptide cross-bridges vary from species to means that each peptidoglycan layer is a single giant mol-
species. In many Gram-negative cell walls, the cross-bridge ecule. In Gram-positive bacteria, there are as many as 40
consists of a direct peptide linkage between the diaminopi- sheets of peptidoglycan, comprising up to 50% of the cell wall
melic acid (DAP) amino group of one side chain and the car- material; in Gram-negative bacteria, there appears to be only
boxyl group of the terminal d-alanine of a second side chain. one or two sheets, comprising 5–10% of the wall material.
The tetrapeptide side chains of all species, however, have Bacteria owe their shapes, which are characteristic of particu-
certain notable features in common. Most have l-alanine at lar species, to their cell wall structure.
position 1 (attached to N-acetylmuramic acid), d-glutamate
or substituted d-glutamate at position 2, and d-alanine at B. Special Components of Gram-Positive Cell Walls
position 4. Position 3 is the most variable one: Most Gram- Most Gram-positive cell walls contain considerable amounts
negative bacteria have diaminopimelic acid at this position, to of teichoic and teichuronic acids, which may account for up
which is linked the lipoprotein cell wall component discussed to 50% of the dry weight of the wall and 10% of the dry weight
as follows. Gram-positive bacteria usually have l-lysine at of the total cell. In addition, some Gram-positive walls may
position 3; however, some may have diaminopimelic acid or contain polysaccharide molecules.
another amino acid at this position.
Diaminopimelic acid is a unique element of bacterial 1. Teichoic and teichuronic acids—The term teichoic
cell walls. It is never found in the cell walls of Archaea or acids encompass all wall, membrane, or capsular polymers
eukaryotes. Diaminopimelic acid is the immediate precursor containing glycerophosphate or ribitol phosphate residues.
of lysine in the bacterial biosynthesis of that amino acid (see These polyalcohols are connected by phosphodiester link-
Figure 6-19). Bacterial mutants that are blocked before diami- ages and usually have other sugars and d-alanine attached
nopimelic acid in the biosynthetic pathway grow normally (Figure 2-18A). Because they are negatively charged, tei-
when provided with diaminopimelic acid in the medium; choic acids are partially responsible for the net negative
CH2OH CH2OH
O O
H H
O O O
H H OH H H
H NH H NH
O
C O C O
HC CH3
CH3 CH3
C O
OH
Glycan
NAG NAM NAG NAM Tetrapeptide chain Peptide interbridge
chain
(amino acids)
B
Peptidoglycan
Tetra-
peptide Peptide interbridge
chain (Gram-positive cells)
(amino acids)
Tetrapeptide
chain
Tetrapeptide (amino acids) FIGURE 2-17 Components and structure of peptidoglycan.
chains A: Chemical structure of N-acetylglucosamine (NAG) and
N-acetylmuramic acid (NAM); the ring structures of the two molecules
are glucose. Glycan chains are composed of alternating subunits of
NAG and NAM joined by covalent bonds. Adjacent glycan chains are
cross-linked via their tetrapeptide chains to create peptidoglycan.
B: Interconnected glycan chains form a very large three-dimensional
Glycan
chain NAM NAG NAM NAG molecule of peptidoglycan. The β1→4 linkages in the backbone are
cleaved by lysozyme. (Reproduced with permission from Nester EW,
Anderson DG, Roberts CE, et al: Microbiology: A Human Perspective,
A 6th ed. McGraw-Hill, 2009. © McGraw-Hill Education.)
charge of the cell surface. There are two types of teichoic position 3 or 4 of ribitol. In some of the more complex tei-
acids: wall teichoic acid (WTA), covalently linked to pepti- choic acids, however, d-alanine is attached to one of the
doglycan; and membrane teichoic acid, covalently linked to sugar residues. In addition to d-alanine, other substituents
membrane glycolipid. Because the latter are intimately asso- may be attached to the free hydroxyl groups of glycerol
ciated with lipids, they have been called lipoteichoic acids and ribitol (eg, glucose, galactose, N-acetylglucosamine,
(LTA). Together with peptidoglycan, WTA and LTA make N-acetylgalactosamine, or succinate). A given species may
up a polyanionic network or matrix that provides functions have more than one type of sugar substituent in addition to
relating to the elasticity, porosity, tensile strength, and elec- d-alanine; in such cases, it is not certain whether the different
trostatic properties of the envelope. Although not all Gram- sugars occur on the same or on separate teichoic acid mole-
positive bacteria have conventional LTA and WTA, those cules. The composition of the teichoic acid formed by a given
that lack these polymers generally have functionally similar bacterial species can vary with the composition of the growth
ones. medium.
Most teichoic acids contain substantial amounts of The teichoic acids constitute major surface antigens of
d-alanine, usually attached to position 2 or 3 of glycerol or those Gram-positive species that possess them, and their
Peptidoglycan
H C O R
CH2
O
O P O–
O
CH2 Periplasmic
space
H C O R
CH2
membrane
O
Plasma
O P O–
O
A B
FIGURE 2-18 A: Teichoic acid structure. The segment of a teichoic acid made of phosphate, glycerol, and a side chain, R. R may represent
d-alanine, glucose, or other molecules. B: Teichoic and lipoteichoic acids of the Gram-positive envelope. (Reproduced with permission from
Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008. © McGraw-Hill Education.)
accessibility to antibodies has been taken as evidence that C. Special Components of Gram-Negative Cell Walls
they lie on the outside surface of the peptidoglycan. Their Gram-negative cell walls contain three components that lie
activity is often increased, however, by partial digestion of outside of the peptidoglycan layer: outer membrane, lipo-
the peptidoglycan; thus, much of the teichoic acid may lie polysaccharide, and lipoprotein (Figure 2-19).
between the cytoplasmic membrane and the peptidogly-
can layer, possibly extending upward through pores in the 1. Outer membrane—The outer membrane is chemi-
latter (Figure 2-18B). In the pneumococcus (Streptococcus cally distinct from all other biological membranes. It is a
pneumoniae), the teichoic acids bear the antigenic determi- bilayered structure; its inner leaflet resembles in composi-
nants called Forssman antigen. In Streptococcus pyogenes, tion that of the cytoplasmic membrane, and its outer leaf-
LTA is associated with the M protein that protrudes from the let contains a distinctive component, a lipopolysaccharide
cell membrane through the peptidoglycan layer. The long M (LPS) (see next). As a result, this is an asymmetrical mem-
protein molecules together with the LTA form microfibrils brane, and the properties of this bilayer differ considerably
that facilitate the attachment of S. pyogenes to animal cells from those of a symmetrical biologic membrane such as the
(see Chapter 14). cell membrane.
The teichuronic acids are similar polymers, but the The ability of the outer membrane to exclude hydropho-
repeat units include sugar acids (eg, N-acetylmannosuronic bic molecules is an unusual feature among biologic mem-
or d-glucosuronic acid) instead of phosphoric acids. They branes and serves to protect the cell (in the case of enteric
are synthesized in place of teichoic acids when phosphate is bacteria) from deleterious substances such as bile salts.
limiting. Because of its lipid nature, the outer membrane would be
expected to exclude hydrophilic molecules as well. However,
2. Polysaccharides—The hydrolysis of Gram-positive the outer membrane has special channels, consisting of pro-
walls has yielded, from certain species, neutral sugars, such tein molecules called porins that permit the passive diffusion
as mannose, arabinose, rhamnose, and glucosamine, and of low-molecular-weight hydrophilic compounds, such as
acidic sugars, such as glucuronic acid and mannuronic acid. sugars, amino acids, and certain ions. Large antibiotic mol-
It has been proposed that these sugars exist as subunits of ecules penetrate the outer membrane relatively slowly, which
polysaccharides in the cell wall; the discovery, however, that accounts for the relatively high resistance of Gram-negative
teichoic and teichuronic acids may contain a variety of sug- bacteria to some antibiotics. The permeability of the outer
ars (see Figure 2-18A) leaves the true origin of these sugars membrane varies widely from one Gram-negative species to
uncertain. another; in P. aeruginosa, for example, which is extremely
O-antigen
repeat
GlcNAc
Lipopoly- Glucose Outer
saccharide core
Galactose
Heptose
Inner
Porin core
KDO
Lipid A
Outer membrane
Lipoprotein Peptidoglycan
Periplasm
MDO
Phospholipids
Inner membrane
Proteins
Cytoplasm
FIGURE 2-19 Molecular representation of the envelope of a Gram-negative bacterium. Ovals and rectangles represent sugar residues,
and circles depict the polar head groups of the glycerophospholipids (phosphatidylethanolamine and phosphatidylglycerol). The core region
shown is that of E. coli K-12, a strain that does not normally contain an O-antigen repeat unless transformed with an appropriate plasmid. MDO,
membrane-derived oligosaccharides. (Reproduced with permission from Raetz CRH: Bacterial endotoxins: Extraordinary lipids that activate
eucaryotic signal transduction. J Bacteriol 1993;175:5745.)
resistant to antibacterial agents, the outer membrane is 100 receptor for lambda bacteriophage, is responsible for most of
times less permeable than that of E. coli. the transmembrane diffusion of maltose and maltodextrins;
The major proteins of the outer membrane, named Tsx, the receptor for T6 bacteriophage, is responsible for the
according to the genes that code for them, have been placed transmembrane diffusion of nucleosides and some amino
into several functional categories on the basis of mutants in acids. LamB allows some passage of other solutes; however,
which they are lacking and on the basis of experiments in its relative specificity may reflect weak interactions of solutes
which purified proteins have been reconstituted into artifi- with configuration-specific sites within the channel.
cial membranes. Porins, exemplified by OmpC, D, and F and The OmpA protein is an abundant protein in the outer
PhoE of E. coli and Salmonella typhimurium, are trimeric membrane. The OmpA protein participates in the anchoring
proteins that penetrate both the inner and outer leaflets of the of the outer membrane to the peptidoglycan layer; it is also
outer membrane (Figure 2-20). They form relatively nonspe- the sex pilus receptor in F-mediated bacterial conjugation
cific pores that permit the free diffusion of small hydrophilic (see Chapter 7).
solutes across the outer membrane. The porins of different The outer membrane also contains a set of less abun-
species have different exclusion limits, ranging from molecu- dant proteins that are involved in the transport of specific
lar weights of about 600 in E. coli and S. typhimurium to more molecules, such as vitamin B12 and iron–siderophore com-
than 3000 in P. aeruginosa. plexes. They show high affinity for their substrates and prob-
Members of a second group of outer membrane pro- ably function like the classic carrier transport systems of the
teins, which resemble porins in many ways, are exemplified cytoplasmic membrane. The proper function of these pro-
by LamB and Tsx. LamB, an inducible porin that is also the teins requires energy coupled through a protein called TonB.
A B
FIGURE 2-20 A: General fold of a porin monomer (OmpF porin from Escherichia coli). The large hollow β-barrel structure is formed by
antiparallel arrangement of 16 β-strands. The strands are connected by short loops or regular turns on the periplasmic rim (bottom), and long
irregular loops face the cell exterior (top). The internal loop, which connects β-strands 5 and 6 and extends inside the barrel, is highlighted
in dark. The chain terminals are marked. The surface closest to the viewer is involved in subunit contacts. B: Schematic representation of
the OmpF trimer. The view is from the extracellular space along the molecular threefold symmetry axis. (Reproduced with permission from
Schirmer T: General and specific porins from bacterial outer membranes. J Struct Biol 1998;121:101.)
Additional minor proteins include a limited number of presence of LPS is required for the function of many outer
enzymes, among them phospholipases and proteases. membrane proteins.
The topology of the major proteins of the outer mem- Lipid A consists of phosphorylated glucosamine disac-
brane, based on cross-linking studies and analyses of func- charide units to which are attached several long-chain fatty
tional relationships, is shown in Figure 2-19. The outer acids (Figure 2-21). β-Hydroxymyristic acid, a C14 fatty acid,
membrane is connected to both the peptidoglycan layer and is always present and is unique to this lipid; the other fatty
the cytoplasmic membrane. The connection with the pepti- acids, along with substituent groups on the phosphates, vary
doglycan layer is primarily mediated by the outer membrane according to the bacterial species.
lipoprotein (see next). About one-third of the lipoprotein The polysaccharide core, shown in Figure 2-21A and B,
molecules are covalently linked to peptidoglycan and help is similar in all Gram-negative species that have LPS and
hold the two structures together. A noncovalent association includes two characteristic sugars, ketodeoxyoctanoic
of some of the porins with the peptidoglycan layer plays a acid (KDO) and a heptose. Each species, however, con-
lesser role in connecting the outer membrane with this struc- tains a unique repeat unit, that of Salmonella being shown
ture. Outer membrane proteins are synthesized on ribosomes in Figure 2-21A. The repeat units are usually linear trisac-
bound to the cytoplasmic surface of the cell membrane. They charides or branched tetra- or pentasaccharides. The repeat
are translocated into the periplasm via the Sec translocase. unit is referred to as the O antigen. The hydrophilic carbohy-
They then fold in the periplasm before being inserted into the drate chains of the O antigen cover the bacterial surface and
outer membrane. In E. coli, YaeT appears to function primar- exclude hydrophobic compounds.
ily in outer membrane protein insertion. The negatively charged LPS molecules are noncovalently
cross-bridged by divalent cations (ie, Ca2+ and Mg2+); this
2. Lipopolysaccharide (LPS)—The LPS of Gram- stabilizes the membrane and provides a barrier to hydropho-
negative cell walls consists of a complex glycolipid, called bic molecules. Removal of the divalent cations with chelating
lipid A, to which is attached a polysaccharide made up of agents or their displacement by polycationic antibiotics, such
a core and a terminal series of repeat units (Figure 2-21A). as polymyxins and aminoglycosides, renders the outer mem-
The lipid A component is embedded in the outer leaflet of brane permeable to large hydrophobic molecules.
the membrane anchoring the LPS. LPS is synthesized on the LPS, which is extremely toxic to animals, has been called
cytoplasmic membrane and transported to its final exterior the endotoxin of Gram-negative bacteria because it is firmly
position. In E. coli, LPS insertion is mediated by OstA. The bound to the cell surface and is released only when the cells
Man Abe
Rha
Gal n
O side chain
Man Abe
Rha
Gal
Glc NAG
Gal
Glc Gal
Hep Core polysaccharide
Hep P P ethanolamine
KDO
KDO KDO P ethanolamine
P GlcN GlcN P
A B
FIGURE 2-21 Lipopolysaccharide structure. A: The lipopolysaccharide from Salmonella. This slightly simplified diagram illustrates
one form of the LPS. Abe, abequose; Gal, galactose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose;
NAG, N-acetylglucosamine; P, phosphate; Rha, l-rhamnose. Lipid A is buried in the outer membrane. B: Molecular model of an E. coli
lipopolysaccharide. The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in this model. (Reproduced with
permission from Willey JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008.
© McGraw-Hill Education.)
are lysed. When LPS is split into lipid A and polysaccharide, important virulence factor. Epitopes have been identified on
all the toxicity is associated with the former. The O antigen is LOS that mimic host structures and may enable these organ-
highly immunogenic in a vertebrate animal. Antigenic speci- isms to evade the immune response of the host. Some LOS (eg,
ficity is conferred by the O antigen because this antigen is those from N. gonorrhoeae, N. meningitidis, and H. ducreyi)
highly variable among species and even in strains within a have a terminal N-acetyllactosamine (Galβ-1→4-GlcNAc)
species. The number of possible antigenic types is very great: residue that is immunochemically similar to the precursor of
Over 1000 have been recognized in Salmonella alone. Not the human erythrocyte I antigen. In the presence of a bacte-
all Gram-negative bacteria have outer membrane LPS com- rial enzyme called sialyltransferase and a host or bacterial
posed of a variable number of repeated oligosaccharide units substrate (cytidine monophospho-N-acetylneuraminic acid,
(see Figure 2-21); the outer membrane glycolipids of bacteria CMP-NANA), the N-acetyllactosamine residue is sialylated.
that colonize mucosal surfaces (eg, Neisseria meningitidis, This sialylation, which occurs in vivo, provides the organism
N. gonorrhoeae, Haemophilus influenzae, and Haemophilus with the environmental advantages of molecular mimicry of
ducreyi) have relatively short, multiantennary (ie, branched) a host antigen and the biologic masking thought to be pro-
glycans. These smaller glycolipids have been compared with vided by sialic acids.
the “R-type” truncated LPS structures, which lack O antigens
and are produced by rough mutants of enteric bacteria such as 3. Lipoprotein—Molecules of an unusual lipoprotein
E. coli. However, the structures of these glycolipids more closely cross-link the outer membrane and peptidoglycan layers (see
resemble those of the glycosphingolipids of mammalian cell Figure 2-19). The lipoprotein contains 57 amino acids, rep-
membranes, and they are more properly termed lipooligosac- resenting repeats of a 15-amino-acid sequence; it is peptide-
charides (LOS). These molecules exhibit extensive antigenic linked to DAP residues of the peptidoglycan tetrapeptide
and structural diversity even within a single strain. LOS is an side chains. The lipid component, consisting of a diglyceride
thioether linked to a terminal cysteine, is noncovalently l-amino acids rather than d-amino acids and disaccharide
inserted in the outer membrane. Lipoprotein is numerically units with an α-1→3 rather than a β1→4 linkage. Archaea
the most abundant protein of Gram-negative cells (ca 700,000 that have a pseudomurein cell wall are Gram-positive.
molecules per cell). Its function (inferred from the behavior
of mutants that lack it) is to stabilize the outer membrane and F. Crystalline Surface Layers
anchor it to the peptidoglycan layer. Many bacteria, both Gram-positive and Gram-negative bac-
teria as well as Archaebacteria, possess a two-dimensional
4. The periplasmic space—The space between the crystalline, subunit-type layer lattice of protein or glycoprotein
inner and outer membranes, called the periplasmic space, molecules (S-layer) as the outermost component of the cell
contains the peptidoglycan layer and a gel-like solution of envelope. In both Gram-positive and Gram-negative bacteria,
proteins. The periplasmic space is approximately 20–40% of this structure is sometimes several molecules thick. In some
the cell volume, which is far from insignificant. The periplas- Archaea, it is the only layer external to the cell membrane.
mic proteins include binding proteins for specific substrates S-layers are generally composed of a single kind of pro-
(eg, amino acids, sugars, vitamins, and ions), hydrolytic tein molecule, sometimes with carbohydrates attached. The
enzymes (eg, alkaline phosphatase and 5′-nucleotidase) isolated molecules are capable of self-assembly (ie, they make
that break down nontransportable substrates into trans- sheets similar or identical to those present on the cells). S-layer
portable ones, and detoxifying enzymes (eg, β-lactamase proteins are resistant to proteolytic enzymes and protein-
and aminoglycoside-phosphorylase) that inactivate certain denaturing agents. The function of the S-layer is uncertain
antibiotics. The periplasm also contains high concentrations but is probably protective. In some cases, it has been shown to
of highly branched polymers of d-glucose, 8 to 10 residues protect the cell from wall-degrading enzymes, from invasion
long, which are variously substituted with glycerol phos- by Bdellovibrio bacteriovorus (a predatory bacterium), and
phate and phosphatidylethanolamine residues; some contain from bacteriophages. It also plays a role in the maintenance
O-succinyl esters. These so-called membrane-derived oligo- of cell shape in some species of Archaebacteria, and it may be
saccharides appear to play a role in osmoregulation because involved in cell adhesion to host epidermal surfaces.
cells grown in media of low osmolarity increase their synthe-
sis of these compounds 16-fold.
G. Enzymes That Attack Cell Walls
The β1→4 glycan linkage of the peptidoglycan backbone
D. The Acid-Fast Cell Wall is hydrolyzed by the enzyme lysozyme (see Figure 2-17),
Some bacteria, notably the tubercle bacillus (M. tuberculosis) which is found in animal secretions (tears, saliva, nasal
and its relatives, have cell walls that contain substan- secretions) as well as in egg white. Gram-positive bacteria
tial amounts of waxes, complex branched hydrocarbons treated with lysozyme in low-osmotic-strength media lyse;
(70–90 carbons long) known as mycolic acids. The cell wall is if the osmotic strength of the medium is raised to balance
composed of peptidoglycan and an external asymmetric lipid the internal osmotic pressure of the cell, free spherical bodies
bilayer; the inner leaflet contains mycolic acids linked to an called protoplasts are liberated. The outer membrane of the
arabinoglycan, and the outer leaflet contains other extract- Gram-negative cell wall prevents access of lysozyme unless
able lipids. This is a highly ordered lipid bilayer in which disrupted by an agent such as ethylene-diaminetetraacetic
proteins are embedded, forming water-filled pores through acid (EDTA), a compound that chelates divalent cations; in
which nutrients and certain drugs can pass slowly. Some osmotically protected media, cells treated with EDTA-lyso-
compounds can also penetrate the lipid domains of the cell zyme form spheroplasts that still possess remnants of the
wall albeit slowly. This hydrophobic structure renders these complex Gram-negative wall, including the outer membrane.
bacteria resistant to many harsh chemicals, including deter- Bacteria themselves possess a number of autolysins,
gents and strong acids. If a dye is introduced into these cells hydrolytic enzymes that attack peptidoglycan, including
by brief heating or treatment with detergents, the dye cannot muramidases, glucosaminidases, endopeptidases, and car-
be removed by dilute hydrochloric acid, as in other bacteria. boxypeptidases. These enzymes catalyze the turnover or
These organisms are therefore called acid fast. The permea- degradation of peptidoglycan in bacteria. These enzymes
bility of the cell wall to hydrophilic molecules is 100- to 1000- presumably participate in cell wall growth and turnover and
fold lower than for E. coli and may be responsible for the slow in cell separation, but their activity is most apparent during
growth rate of mycobacteria. the dissolution of dead cells (autolysis).
Enzymes that degrade bacterial cell walls are also found
E. Cell Walls of the Archaea in cells that digest whole bacteria (eg, protozoa and the
The Archaea do not have cell walls like the Bacteria. Some have phagocytic cells of higher animals).
a simple S-layer (see next) often composed of glycoproteins.
Some Archaea have a rigid cell wall composed of polysaccha- H. Cell Wall Growth
rides or a macromolecule called pseudomurein. The pseudo- Cell wall synthesis is necessary for cell division; however,
murein differs from the peptidoglycan of bacteria by having the incorporation of new cell wall material varies with the
shape of the bacterium. Rod-shaped bacteria (eg, E. coli and Some L forms can revert to the normal bacillary form
Bacillus subtilis) have two modes of cell wall synthesis; new upon removal of the inducing stimulus. Thus, they can
peptidoglycan is inserted along a helical path leading to elon- resume normal cell wall synthesis. Others are stable and
gation of the cell and is inserted in a closing ring around the never revert. The factor that determines their capacity to
future division site, leading to the formation of the division revert may again be the presence of residual peptidoglycan,
septum. Coccoid cells such as S. aureus do not seem to have which normally acts as a primer in its own biosynthesis.
an elongation mode of cell wall synthesis. Instead, new pepti- Some bacterial species produce L forms spontaneously.
doglycan is inserted only at the division site. A third form of The spontaneous or antibiotic-induced formation of L forms
cell wall growth is exemplified by S. pneumoniae, which are in the host may produce chronic infections, the organisms
not true cocci, because their shape is not totally round but persisting by becoming sequestered in protective regions of
instead have the shape of a rugby ball. S. pneumoniae synthe- the body. Because L-form infections are relatively resistant
sizes cell wall not only at the septum but also at the so-called to antibiotic treatment, they present special problems in che-
equatorial rings (Figure 2-22). motherapy. Their reversion to the bacillary form can produce
relapses of the overt infection.
I. Protoplasts, Spheroplasts, and L Forms
Removal of the bacterial wall may be accomplished by hydro- J. The Mycoplasmas
lysis with lysozyme (as described above) or by blocking The mycoplasmas are cell wall-lacking bacteria containing
peptidoglycan synthesis with an antibiotic such as penicil- no peptidoglycan (see Figure 25-1). There are also wall-less
lin. In osmotically protective media, such treatments liber- Archaea, but they have been less well studied. Genomic anal-
ate protoplasts from Gram-positive cells and spheroplasts ysis places the mycoplasmas close to the Gram-positive bac-
(which retain outer membrane and entrapped peptidoglycan) teria from which they may have been derived. Mycoplasmas
from Gram-negative cells. lack a target for cell wall-inhibiting antimicrobial agents (eg,
If such cells are able to grow and divide, they are called penicillins and cephalosporins) and are therefore resistant
L forms. L forms are difficult to cultivate and usually require to these drugs. Some, such as Mycoplasma pneumoniae, an
a medium that is solidified with agar as well as having the agent of pneumonia, contain sterols in their membranes. The
right osmotic strength. L forms are produced more readily difference between L forms and mycoplasmas is that when
with penicillin than with lysozyme, suggesting the need for the murein is allowed to reform, L forms revert to their origi-
residual peptidoglycan. nal bacteria shape, but mycoplasmas never do.
FIGURE 2-22 Incorporation of new cell wall in differently shaped bacteria. Rod-shaped bacteria such as B. subtilis or E. coli have two
modes of cell wall synthesis: New peptidoglycan is inserted along a helical path (A), leading to elongation of the lateral wall and is inserted in
a closing ring around the future division site, leading to the formation of the division septum (B). S. pneumoniae cells have the shape of a rugby
ball and elongate by inserting new cell wall material at the so-called equatorial rings (A), which correspond to an outgrowth of the cell wall
that encircles the cell. An initial ring is duplicated, and the two resultant rings are progressively separated, marking the future division sites of
the daughter cells. The division septum is then synthesized in the middle of the cell (B). Round cells such as S. aureus do not seem to have an
elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division septum (B). (Reproduced with permission
from Scheffers DJ, Pinho MG: Bacterial cell wall synthesis: new insights from localization studies. Microbiol Mol Biol Rev 2005;69:585.)
A B
FIGURE 2-23 Bacterial capsules. A: B. anthracis M’Faydean capsule stain, grown at 35°C, in defibrinated horse blood. B: Demonstration of
the presence of a capsule in B. anthracis by negative staining with India ink. This method is useful for improving visualization of encapsulated
bacteria in clinical samples such as blood, blood culture bottles, or cerebrospinal fluid. (CDC, courtesy of Larry Stauffer, Oregon State Public
Health Laboratory.)
A bacterial flagellum is made up of several protofila- of bacteria, new flagella are rapidly formed by the synthesis,
ments, each made up of thousands of molecules of a protein aggregation, and extrusion of flagellin subunits; motility is
subunit called flagellin. In a few organisms (eg, Caulobacter restored within 3–6 minutes. The flagellins of different bac-
species), flagella are composed of two types of flagellin, but terial species presumably differ from one another in primary
in most, only a single type is found. The flagellum is formed structure. They are highly antigenic (H antigens), and some
by the aggregation of subunits to form a helical structure. If of the immune responses to infection are directed against
flagella are removed by mechanically agitating a suspension these proteins.
A B C
FIGURE 2-24 Bacterial flagellation. A: Vibrio metschnikovii, a monotrichous bacterium (7500×). (Reproduced with permission from
van Iterson W: Biochim Biophys Acta 1947;1:527.) B: Electron micrograph of Spirillum serpens, showing lophotrichous flagellation (9000×).
(Reproduced with permission from van Iterson W: Biochim Biophys Acta 1947;1:527.) C: Electron micrograph of Proteus vulgaris, showing
peritrichous flagellation (9000×). Note basal granules. (Reproduced with permission from Houwink A, van Iterson W: Electron microscopical
observations on bacterial cytology; a study on flagellation. Biochim Biophys Acta 1950;5:10.)
Filament Filament
(FliC) cap (FliD)
Filament
Hook-filament
20 nm junction Propellor
(FlgK FlgL)
10 µm
Hook
Hook
(FlgE)
L ring (FlgH)
Bushing
Outer membrane P ring (Flgl)
Basal
body Periplasmic space
Distal rod (Flgl)
Cell membrane Transmission
Proximal rod (FliE, shaft
FlgB, FlgC, FlgF)
Switch Motor
Switch
Export (FliG, FliM, FliN) Mounting
MS ring (FliF)
apparatus plate
Export
(FlhA, FliH, Flil) ? Motor (MotA, MotB)
A B
FIGURE 2-25 A: General structure of the flagellum of a Gram-negative bacterium, such as E. coli or S. typhimurium. The filament-hook-
basal body complex has been isolated and extensively characterized. The location of the export apparatus has not been demonstrated. B: An
exploded diagram of the flagellum showing the substructures and the proteins from which they are constructed. The FliF protein is responsible
for the M-ring feature, S-ring feature, and collar feature of the substructure shown, which is collectively termed the MS ring. The location of
FliE with respect to the MS ring and the rod—and the order of the FlgB, FlgC, and FlgF proteins within the proximal rod—is not known. (From
Macnab RM: Genetics and biogenesis of bacterial flagella. Annu Rev Genet 1992;26:131. Reproduced with permission from Annual Review of
Genetics, Volume 26, © 1992 by Annual Reviews.)
The flagellum is attached to the bacterial cell body by sodium ion gradient—rather than the proton gradient—to
a complex structure consisting of a hook and a basal body. drive the flagellar motor (Figure 2-26).
The hook is a short curved structure that appears to act as the All the components of the flagellar motor are located
universal joint between the motor in the basal structure and in the cell envelope. Flagella attached to isolated, sealed cell
the flagellum. The basal body bears a set of rings, one pair in envelopes rotate normally when the medium contains a suit-
Gram-positive bacteria and two pairs in Gram-negative bacte- able substrate for respiration or when a proton gradient is
ria. An interpretative diagram of the Gram-negative structure artificially established.
is shown in Figure 2-25; the rings labeled L and P are absent in When a peritrichous bacterium swims, its flagella associ-
Gram-positive cells. The complexity of the bacterial flagellum ate to form a posterior bundle that drives the cell forward in a
is revealed by genetic studies, which show that over 40 gene straight line by counterclockwise rotation. At intervals, the fla-
products are involved in its assembly and function. gella reverse their direction of rotation and momentarily dis-
Flagella are made stepwise (see Figure 2-25). First, the sociate, causing the cell to tumble until swimming resumes in
basal body is assembled and inserted into the cell envelope. a new, randomly determined direction. This behavior makes
Then the hook is added, and finally, the filament is assembled possible the property of chemotaxis: A cell that is moving away
progressively by the addition of flagellin subunits to its grow- from the source of a chemical attractant tumbles and reori-
ing tip. The flagellin subunits are extruded through a hollow ents itself more frequently than one that is moving toward the
central channel in the flagella; when it reaches the tip, it con- attractant, the result being the net movement of the cell toward
denses with its predecessors, and thus the filament elongates. the source. The presence of a chemical attractant (eg, a sugar
or an amino acid) is sensed by specific receptors located in the
B. Motility cell membrane (in many cases, the same receptor also partici-
Bacterial flagella are semirigid helical rotors to which the pates in membrane transport of that molecule). The bacterial
cell imparts a spinning movement. Rotation is driven by the cell is too small to be able to detect the existence of a spatial
flow of protons into the cell down the gradient produced chemical gradient (ie, a gradient between its two poles); rather,
by the primary proton pump (see earlier discussion); in the experiments show that it detects temporal gradients, that is,
absence of a metabolic energy source, it can be driven by a concentrations that decrease with time during which the cell
proton motive force generated by ionophores. Bacteria living is moving away from the attractant source and increase with
in alkaline environments (alkalophiles) use the energy of the time during which the cell is moving toward it.
Filament
Hook
Outer membrane
Murein
Basal +
body H+
H H+
H+ H+
+ + + + + + +
Cell membrane
– – – – – –
Switch Motor
H+
FIGURE 2-26 Structural components within the basal body of the flagellum allow the inner portion of this structure, the rods of the basal
body, and the attached hook–filament complex to rotate. The outer rings remain statically in contact with the inner and outer cell membranes
and cell wall (murein), anchoring the flagellum complex to the bacterial cell envelope. Rotation is driven by the flow of protons through the
motor from the periplasmic space, outside the cell membrane, into the cytoplasm in response to the electric field and proton gradient across
the membrane, which together constitute the proton motive force. A switch determines the direction of rotation, which in turn determines
whether the bacteria swim forward (by counterclockwise rotation of the flagellum) or tumble (caused by clockwise rotation of the flagellum).
(Reproduced with permission from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.)
Some compounds act as repellants rather than attrac- are composed of structural protein subunits termed pilins.
tants. One mechanism by which cells respond to attractants Some pili contain a single type of pilin, others more than
and repellents involves a cGMP-mediated methylation and one. Minor proteins termed adhesins are located at the tips
demethylation of specific proteins in the membrane. Whereas of pili and are responsible for the attachment properties. Two
attractants cause a transient inhibition of demethylation of classes can be distinguished: ordinary pili, which play a role
these proteins, repellents stimulate their demethylation. in the adherence of symbiotic and pathogenic bacteria to
The mechanism by which a change in cell behavior is host cells; and sex pili, which are responsible for the attach-
brought about in response to a change in the environment ment of donor and recipient cells in bacterial conjugation (see
is called sensory transduction. Sensory transduction is Chapter 7). Pili are illustrated in Figure 2-27, in which the
responsible not only for chemotaxis but also for aerotaxis sex pili have been coated with phage particles for which they
(movement toward the optimal oxygen concentration), serve as specific receptors.
phototaxis (movement of photosynthetic bacteria toward the Motility via pili is completely different from flagel-
light), and electron acceptor taxis (movement of respiratory lar motion. Pilin molecules are arranged helically to form
bacteria toward alternative electron acceptors, such as nitrate a straight cylinder that does not rotate and lacks a complete
and fumarate). In these three responses, as in chemotaxis, basal body. Their tips strongly adhere to surfaces at a distance
net movement is determined by regulation of the tumbling from the cells. Pili then depolymerize from the inner end,
response. thus retracting inside the cell. The result is that the bacte-
rium moves in the direction of the adhering tip. This kind of
surface motility is called twitching and is widespread among
Pili (Fimbriae) piliated bacteria. Unlike flagella, pili grow from the inside of
Many Gram-negative bacteria possess rigid surface append- the cell outward.
ages called pili (L “hairs”) or fimbriae (L “fringes”). They The virulence of certain pathogenic bacteria depends on
are shorter and thinner than flagella; similar to flagella, they the production not only of toxins but also of “colonization
Endospores
Sex pilus Members of several bacterial genera can form endospores
(Figure 2-28). The two most common are Gram-positive
rods: the obligately aerobic genus Bacillus and the obligately
anaerobic genus Clostridium. The other bacteria known to
form endospores are Thermoactinomyces, Sporolactobacillus,
Sporosarcina, Sporotomaculum, Sporomusa, and Sporohalo-
Other Flagellum bacter spp. These organisms undergo a cycle of differentia-
pili tion in response to environmental conditions: The process,
sporulation, is triggered by near depletion of any of several
nutrients (carbon, nitrogen, or phosphorous). Each cell forms
a single internal spore that is liberated when the mother cell
1 µm undergoes autolysis. The spore is a resting cell, highly resis-
tant to desiccation, heat, and chemical agents; when returned
FIGURE 2-27 Pili. Pili on an E. coli cell. The short pili (fimbriae) to favorable nutritional conditions and activated (see below),
mediate adherence; the sex pilus is involved in DNA transfer. the spore germinates to produce a single vegetative cell. The
(Courtesy of Dr. Charles Brinton, Jr.) location of an endospore within a cell is species-specific and
can be used to determine the identity of a bacterium.
antigens,” which are ordinary pili that provide the cells with
adherent properties. In enteropathogenic E. coli strains, both A. Sporulation
the enterotoxins and the colonization antigens (pili) are The sporulation process begins when nutritional conditions
genetically determined by transmissible plasmids, as dis- become unfavorable, near depletion of the nitrogen or carbon
cussed in Chapter 7. source (or both) being the most significant factor. Sporula-
Pili of different bacteria are antigenically distinct and tion occurs massively in cultures that have terminated expo-
elicit the formation of antibodies by the host. Antibodies nential growth because of this near depletion.
against the pili of one bacterial species will not prevent the Sporulation involves the production of many new struc-
attachment of another species. Some bacteria (see Chapter 21), tures, enzymes, and metabolites along with the disappearance
such as N. gonorrhoeae, can make pili of different antigenic of many vegetative cell components. These changes represent
types (antigenic variation) and thus can still adhere to cells a true process of differentiation: A series of genes whose
in the presence of antibodies to their original type of pili. Like products determine the formation and final composition of
capsules, pili inhibit the phagocytic ability of leukocytes. the spore are activated. These changes involve alterations in
A B C
FIGURE 2-28 Sporulating cells of bacillus species. A: Unidentified bacillus from soil. B: B. cereus. C: B. megaterium. (Reproduced with
permission from Robinow CF: Structure. In Gunsalus IC, Stanier RY [editors]. The Bacteria: A Treatise on Structure and Function, Vol 1. Academic
Press, 1960.)
the transcriptional specificity of RNA polymerase, which is Different morphologic and chemical events occur at sequen-
determined by the association of the polymerase core protein tial stages of the process. Seven distinct stages have been
with one or another promoter-specific proteins called sigma identified.
factors. During vegetative growth, a sigma factor desig- Morphologically, sporulation begins with the formation
nated σA predominates. Then, during sporulation, five other of an axial filament (Figure 2-29). The process continues with
sigma factors are formed that cause various spore genes to be an infolding of the membrane to produce a double-membrane
expressed at various times in specific locations. structure whose facing surfaces correspond to the cell wall-
The sequence of events in sporulation is highly com- synthesizing surface of the cell envelope. The growing points
plex: Differentiation of a vegetative cell of B. subtilis into an move progressively toward the pole of the cell to engulf the
endospore takes about 7 hours under laboratory conditions. developing spore.
Cell wall
0 1 Axial filament
formation
DNA
3 Engulfment of forespore
Spore
mother
cell
4 Cortex synthesis
5 Coat deposition
6 Maturation
Cortex
Germ Spore
cell wall coats
7 Lysis of
mother
cell
Spore
FIGURE 2-29 The stages of endospore formation. (Reproduced with permission from Merrick MJ: Streptomyces. In: Parish JH [editor].
Developmental Biology of Procaryotes. Univ California Press, 1979.)
The two spore membranes now engage in the active syn- 1. Activation—Most endospores cannot germinate imme-
thesis of special layers that will form the cell envelope: the diately after they have formed. But they can germinate after
spore wall and the cortex, lying outside the facing mem- they have rested for several days or are first activated in a
branes. In the newly isolated cytoplasm, or core, many veg- nutritionally rich medium by one or another agent that dam-
etative cell enzymes are degraded and are replaced by a set of ages the spore coat. Among the agents that can overcome
unique spore constituents. spore dormancy are heat, abrasion, acidity, and compounds
containing free sulfhydryl groups.
B. Properties of Endospores
2. Initiation—After activation, a spore will initiate germi-
1. Core—The core is the spore protoplast. It contains a
nation if the environmental conditions are favorable. Differ-
complete chromosome, all the components of the protein-
ent species have evolved receptors that recognize different
synthesizing apparatus, and an energy-generating system
effectors (ie, germinants) as signaling a rich medium: Thus,
based on glycolysis. Cytochromes are lacking even in aero-
initiation is triggered by l-alanine in one species and by ade-
bic species, the spores of which rely on a shortened electron
nosine in another. Binding of the effector activates an autoly-
transport pathway involving flavoproteins. A number of
sin that rapidly degrades the cortex peptidoglycan. Water is
vegetative cell enzymes are increased in amount (eg, alanine
taken up, calcium dipicolinate is released, and a variety of
racemase), and a number of unique enzymes are formed (eg,
spore constituents are degraded by hydrolytic enzymes.
dipicolinic acid synthetase). Spores contain no reduced pyri-
dine nucleotides or ATP. The energy for germination is stored
3. Outgrowth—Degradation of the cortex and outer layers
as 3-phosphoglycerate rather than as ATP.
results in the emergence of a new vegetative cell consisting of
The heat resistance of spores is partly attributable to
the spore protoplast with its surrounding wall. A period of
their dehydrated state and in part to the presence in the core
active biosynthesis follows; this period, which terminates in
of substantial amounts (5–15% of the spore dry weight) of
cell division, is called outgrowth. Outgrowth requires a sup-
calcium dipicolinate, which is formed from an intermediate
ply of all nutrients essential for cell growth.
of the lysine biosynthetic pathway (see Figure 6-19). In some
way not yet understood, these properties result in the stabi-
lization of the spore enzymes, most of which exhibit normal
heat lability when isolated in soluble form. STAINING
Stains combine chemically with the bacterial protoplasm; if
2. Spore wall—The innermost layer surrounding the inner the cell is not already dead, the staining process itself will kill
spore membrane is called the spore wall. It contains normal it. The process is thus a drastic one and may produce artifacts.
peptidoglycan and becomes the cell wall of the germinating The commonly used stains are salts. Basic stains con-
vegetative cell. sist of a colored cation with a colorless anion (eg, methylene
blue+ chloride−); acidic stains are the reverse (eg, sodium+
3. Cortex—The cortex is the thickest layer of the spore eosinate−). Bacterial cells are rich in nucleic acid, bearing neg-
envelope. It contains an unusual type of peptidoglycan, atively charged phosphate groups. These combine with the
with many fewer cross-links than are found in cell wall positively charged basic dyes. Acidic dyes do not stain bacte-
peptidoglycan. Cortex peptidoglycan is extremely sen- rial cells and hence can be used to stain background material
sitive to lysozyme, and its autolysis plays a role in spore a contrasting color (see Negative Staining).
germination. The basic dyes stain bacterial cells uniformly unless the
cytoplasmic RNA is destroyed first. Special staining tech-
4. Coat—The coat is composed of a keratin-like protein con- niques can be used, however, to differentiate flagella, cap-
taining many intramolecular disulfide bonds. The imperme- sules, cell walls, cell membranes, granules, nucleoids, and
ability of this layer confers on spores their relative resistance spores.
to antibacterial chemical agents.
Negative Staining
This procedure involves staining the background with an the result that the cell and background appear dark blue and
acidic dye, leaving the cells contrastingly colorless. The black the capsule a much paler blue.
dye nigrosin is commonly used. This method is used for cells or
structures that are difficult to stain directly (see Figure 2-23B).
Staining of Nucleoids
Nucleoids are stainable with the Feulgen stain, which is
The Flagella Stain specific for DNA. The DNA-intercalating stains DAPI and
Flagella are too fine ( ca 20 nm in diameter) to be visible in ethidium bromide are widely used for fluorescence micros-
the light microscope. However, their presence and arrange- copy of nucleoids.
ment can be demonstrated by treating the cells with an unsta-
ble colloidal suspension of tannic acid salts, causing a heavy
The Spore Stain
precipitate to form on the cell walls and flagella. In this man-
ner, the apparent diameter of the flagella is increased to such Spores are most simply observed as intracellular refractile bod-
an extent that subsequent staining with basic fuchsin makes ies (see Figure 2-28) in unstained cell suspensions or as color-
the flagella visible in the light microscope. Figure 2-30 shows less areas in cells stained by conventional methods. The spore
cells stained by this method. wall is relatively impermeable, but dyes can be made to pen-
In peritrichous bacteria, the flagella form into bundles etrate it by heating the preparation. The same impermeability
during movement, and such bundles may be thick enough then serves to prevent decolorization of the spore by a period
to be observed on living cells by dark-field or phase-contrast of alcohol treatment sufficient to decolorize vegetative cells.
microscopy. The latter can finally be counterstained. Spores are commonly
stained with malachite green or carbolfuchsin (Figure 2-31).
Fig. 109.
(1) Begin by making a square prism which shall have the same
dimensions for its width and thickness as is desired for the diameter
of the cylinder. (2) Change this square prism to a regular octagonal
or eight-sided prism by planing off the four arrises. The gage lines
which indicate the amount to be taken off of each arris are made by
holding the gage block against each of the surfaces and gaging from
each arris each way, two lines on each surface. These lines must be
made lightly. The distance at which to set the spur of the gage from
the head is equal to one-half the diagonal of the square end of the
prism. Fig. 109. Since the ends are less likely to be accurate than
any other part, it is advisable to get this distance as follows: Lay off
two lines on the working face a distance apart equal to the width of
the prism. These lines with the two arrises form a square the
diagonal of which can be measured and one-half of it computed.
Fig. 110.
Fig. 111.
Fig. 112.
Fig. 113.
With the spokeshave, Fig. 114, carefully cut off the two arrises to
the pencil lines so as to form two bevels. This gives three surfaces to
the edge of the board. Estimating the amount with the eye, cut off
the two arrises formed by these three surfaces until five equal
surfaces are formed in their place. This process may be repeated
until the surface of the edge is practically a curved surface. With a
piece of sandpaper held as shown in Fig. 115, rub until the surface is
smooth and evenly curved.
61. Modeling.—This term is used to apply to the method of
making objects of such irregular form that the
judgment of the worker must be depended upon to give the correct
result without the aid of gage and knife marks. The forming of a
canoe paddle or a hammer handle are good illustrations.
Fig. 116.
Fig. 118.
Fig. 119.
When a scraper becomes dull (1) each edge is drawfiled, Fig. 121,
so as to make it square and straight, with the corners slightly
rounded. Sometimes the edges are rounded slightly from end to end
to prevent digging. Frequently the scraper has its edges and
surfaces ground square on an oilstone after the drawfiling that the
arrises may be formed into smoother burs. (2) After filing, the
scraper is laid flat on the bench and the arrises forced over as in Fig.
122. The tool used is called a burnisher; any smooth piece of steel
would do. (3) Next, turn these arrises back over the side of the
scraper. Fig. 123. Great pressure is not necessary to form the burs
properly.
Fig. 124.
Fig. 126.
Common wire nails are thick and have large flat heads. They are
used in rough work where strength is desired. Fig. 126 A. Finishing
nails, Fig. 126 B, are used for fine work such as inside woodwork,
cabinet work, etc. Casing nails, Fig. 126 C, are somewhat thicker
and stronger than finishing nails; they have small heads.
67. Nailing.—Especial care is necessary in starting cut nails. Fig.
127 shows two views of a cut nail. From these it will be
seen that the sides of the nail form a wedge in one of the views while
in the other they are parallel. The nail should be so started that the
wedging action shall take place along, not across the grain.
Fig. 127. Fig. 128.