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BACTERIOLOGY
Introduction to
Bacteriology
Bacteria are single-celled microorganisms that lack a
nuclear membrane, are metabolically active and divide by
binary fission. Medically they are a major cause of disease.
Superficially, bacteria appear to be relatively simple forms
of life; in fact, they are sophisticated and highly adaptable.
Many bacteria multiply at rapid rates, and different species
can utilize an enormous variety of hydrocarbon substrates,
including phenol, rubber, and petroleum. These organisms exist
widely in both parasitic and free-living forms. Because they
are ubiquitous and have a remarkable capacity to adapt to
changing environments by selection of spontaneous mutants, the
importance of bacteria in every field of medicine cannot be
overstated.
The discipline of bacteriology evolved from the need of
physicians to test and apply the germ theory of disease and
from economic concerns relating to the spoilage of foods and
wine. The initial advances in pathogenic bacteriology were
derived from the identification and characterization of
bacteria associated with specific diseases. During this
period, great emphasis was placed on applying Koch's
postulates to test proposed cause-and-effect relationships
between bacteria and specific diseases. Today, most bacterial
diseases of humans and their etiologic agents have been
identified, although important variants continue to evolve and
sometimes emerge, e.g., Legionnaire's Disease, tuberculosis
and toxic shock syndrome.
Major advances in bacteriology over the last century
resulted in the development of many effective vaccines (e.g.,
pneumococcal polysaccharide vaccine, diphtheria toxoid, and
tetanus toxoid) as well as of other vaccines (e.g., cholera,
typhoid, and plague vaccines) that are less effective or have
side effects. Another major advance was the discovery of
antibiotics. These antimicrobial substances have not
eradicated bacterial diseases, but they are powerful
therapeutic tools. Their efficacy is reduced by the emergence
of antibiotic resistant bacteria (now an important medical
management problem) In reality, improvements in sanitation and
water purification have a greater effect on the incidence of
bacterial infections in a community than does the availability
of antibiotics or bacterial vaccines. Nevertheless, many and
serious bacterial diseases remain.
Most diseases now known to have a bacteriologic etiology
have been recognized for hundreds of years. Some were
described as contagious in the writings of the ancient
Chinese, centuries prior to the first descriptions of bacteria
by Anton van Leeuwenhoek in 1677. There remain a few diseases
(such as chronic ulcerative colitis) that are thought by some
investigators to be caused by bacteria but for which no
pathogen has been identified. Occasionally, a previously
unrecognized diseases is associated with a new group of
bacteria. An example is Legionnaire's disease, an acute
respiratory infection caused by the previously unrecognized
genus, Legionella. Also, a newly recognized pathogen, Helicobacter,
plays an important role in peptic disease. Another important
example, in understanding the etiologies of venereal diseases,
was the association of at least 50 percent of the cases of
urethritis in male patients with Ureaplasma urealyticum
or Chlamydia trachomatis.
Recombinant bacteria produced by genetic engineering are
enormously useful in bacteriologic research and are being
employed to manufacture scarce biomolecules (e.g. interferons)
needed for research and patient care. The antibiotic
resistance genes, while a problem to the physician,
paradoxically are indispensable markers in performing genetic
engineering. Genetic probes and the polymerase chain reaction
(PCR) are useful in the rapid identification of microbial
pathogens in patient specimens. Genetic manipulation of
pathogenic bacteria continues to be indispensable in defining
virulence mechanisms. As more protective protein antigens are
identified, cloned, and sequenced, recombinant bacterial
vaccines will be constructed that should be much better than
the ones presently available. In this regard, a
recombinant-based and safer pertussis vaccine is already
available in some European countries. Also, direct DNA
vaccines hold considerable promise.
In developed countries, 90 percent of documented infections
in hospitalized patients are caused by bacteria. These cases
probably reflect only a small percentage of the actual number
of bacterial infections occurring in the general population,
and usually represent the most severe cases. In developing
countries, a variety of bacterial infections often exert a
devastating effect on the health of the inhabitants.
Malnutrition, parasitic infections, and poor sanitation are a
few of the factors contributing to the increased
susceptibility of these individuals to bacterial pathogens.
The World Health Organization has estimated that each year, 3
million people die of tuberculosis, 0.5 million die of
pertussis, and 25,000 die of typhoid. Diarrheal diseases, many
of which are bacterial, are the second leading cause of death
in the world (after cardiovascular diseases), killing 5
million people annually.
Many bacterial diseases can be viewed as a failure of the
bacterium to adapt, since a well-adapted parasite ideally
thrives in its host without causing significant damage.
Relatively nonvirulent (i.e., well-adapted) microorganisms can
cause disease under special conditions - for example, if they
are present in unusually large numbers, if the host's defenses
are impaired, (e.g., AIDS and chemotherapy) or if anaerobic
conditions exist. Pathogenic bacteria constitute only a small
proportion of bacterial species; many nonpathogenic bacteria
are beneficial to humans (i.e. intestinal flora produce
vitamin K) and participate in essential processes such as
nitrogen fixation, waste breakdown, food production, drug
preparation, and environmental bioremediation. This textbook
emphasizes bacteria that have direct medical relevance.
In recent years, medical scientists have
concentrated on the study of pathogenic mechanisms and host
defenses. Understanding host-parasite relationships involving
specific pathogens requires familiarity with the fundamental
characteristics of the bacterium, the host, and their
interactions. Therefore, this section first presents with the
basic concepts of the immune response, bacterial structure,
taxonomy, metabolism, and genetics. Subsequent chapters
emphasize normal relationships among bacteria on external
surfaces; mechanisms by which microorganisms damage the host;
host defense mechanisms; source and distribution of pathogens
(epidemiology); principles of diagnosis; and mechanisms of
action of antimicrobial drugs. These chapters provide the
basis for the next chapters devoted to specific bacterial
pathogens and the diseases they cause. The bacteria in these
chapters are grouped on the basis of physical, chemical, and
biologic characteristics. These similarities do not
necessarily indicate that their diseases are similar; widely
divergent diseases may be caused by bacteria in the same
group.
Charles
P. Davis
Gail
Woods
David
Niesel
Structure
Milton
R.J. Salton
Kwang-Shin
Kim
General Concepts
Gross Morphology
Bacteria have characteristic shapes (cocci, rods, spirals,
etc.) and often occur in characteristic aggregates (pairs,
chains, tetrads, clusters, etc.). These traits are usually
typical for a genus and are diagnostically useful.
Cell Structure
Prokaryotes have a nucleoid (nuclear body) rather than an
enveloped nucleus and lack membrane-bound cytoplasmic
organelles. The plasma membrane in prokaryotes performs many
of the functions carried out by membranous organelles in
eukaryotes. Multiplication is by binary fission.
Surface Structures
Flagella: The flagella of motile bacteria
differ in structure from eukaryotic flagella. A basal body
anchored in the plasma membrane and cell wall gives rise to a
cylindrical protein filament. The flagellum moves by whirling
about its long axis. The number and arrangement of flagella on
the cell are diagnostically useful.
Pili (Fimbriae): Pili are slender,
hairlike, proteinaceous appendages on the surface of many
(particularly Gram-negative) bacteria. They are important in
adhesion to host surfaces.
Capsules: Some bacteria form a thick outer
capsule of high-molecular-weight, viscous polysaccharide gel;
others have more amorphous slime layers. Capsules confer
resistance to phagocytosis.
Important Chemical Components of Surface Structures
Cell Wall Peptidoglycans: Both
Gram-positive and Gram-negative bacteria possess cell wall
peptidoglycans, which confer the characteristic cell shape and
provide the cell with mechanical protection. Peptidoglycans
are unique to prokaryotic organisms and consist of a glycan
backbone of muramic acid and glucosamine (both N-acetylated),
and peptide chains highly cross-linked with bridges in
Gram-positive bacteria (e.g., Staphylococcus aureus) or
partially cross-linked in Gram-negative bacteria (e.g., ).
The cross-linking transpeptidase enzymes are some of the
targets for b-lactam antibiotics.
Teichoic Acids: Teichoic acids are polyol
phosphate polymers bearing a strong negative charge. They are
covalently linked to the peptidoglycan in some Gram-positive
bacteria. They are strongly antigenic, but are generally
absent in Gram-negative bacteria.
Lipoteichoic Acids: Lipoteichoic acids as
membrane teichoic acids are polymers of amphiphitic
glycophosphates with the lipophilic glycolipid and anchored in
the cytoplasmic membrane. They are antigenic, cytotoxic and
adhesins (e.g., Streptococcus pyogenes).
Lipopolysaccharides: One of the major
components of the outer membrane of Gram-negative bacteria is
lipopolysaccharide (endotoxin), a complex molecule consisting
of a lipid A anchor, a polysaccharide core, and chains of
carbohydrates. Sugars in the polysaccharide chains confer
serologic specificity.
Wall-Less Forms: Two groups of bacteria
devoid of cell wall peptidoglycans are the Mycoplasma species,
which possess a surface membrane structure, and the L-forms
that arise from either Gram-positive or Gram-negative
bacterial cells that have lost their ability to produce the
peptidoglycan structures.
Cytoplasmic Structures
Plasma Membrane: The bacterial plasma
membrane is composed primarily of protein and phospholipid
(about 3:1). It performs many functions, including transport,
biosynthesis, and energy transduction.
Organelles: The bacterial cytoplasm is
densely packed with 70S ribosomes. Other granules represent
metabolic reserves (e.g., poly-b-hydroxybutyrate,
polysaccharide, polymetaphosphate, and metachromatic
granules).
Endospores: Bacillus and Clostridium
species can produce endospores: heat-resistant, dehydrated
resting cells that are formed intracellularly and contain a
genome and all essential metabolic machinery. The endospore is
encased in a complex protective spore coat.
INTRODUCTION
All bacteria, both pathogenic and saprophytic, are
unicellular organisms that reproduce by binary fission. Most
bacteria are capable of independent metabolic existence and
growth, but species of Chlamydia and Rickettsia are obligately
intracellular organisms. Bacterial cells are extremely small
and are most conveniently measured in microns (10-6 m). They
range in size from large cells such as Bacillus anthracis (1.0
to 1.3 µm X 3 to 10 µm) to very small cells such as
Pasteurella tularensis (0.2 X 0.2 to 0.7 µm) Mycoplasmas
(atypical pneumonia group) are even smaller, measuring 0.1 to
0.2 µm in diameter. Bacteria therefore have a
surface-to-volume ratio that is very high: about 100,000.
Bacteria have characteristic shapes. The common microscopic
morphologies are cocci (round or ellipsoidal cells, such as
Staphylococcus aureus or Streptococcus respectively); rods,
such as Bacillus and Clostridium species; long, filamentous
branched cells, such as Actinomyces species; and comma-shaped
and spiral cells, such as Vibrio cholerae and Treponema
pallidum, respectively. The arrangement of cells is also
typical of various species or groups of bacteria (Fig. 2-1 ) .
Some rods or cocci characteristically grow in chains; some,
such as Staphylococcus aureus, form grapelike clusters of
spherical cells; some round cocci form cubic packets.
Bacterial cells of other species grow separately. The
microscopic appearance is therefore valuable in classification
and diagnosis.
FIGURE 2-1 Typical shapes and arrangements of
bacterial cells.
The higher resolving power of the electron microscope not
only magnifies the typical shape of a bacterial cell but also
clearly resolves its prokaryotic organization (Fig. 2-2).
FIGURE 2-2 Electron micrograph of a thin section of
Neisseria gonorrhoeae showing the organizational features of
prokaryotic cells. Note the electron-transparent
nuclear region (n) packed with DNA fibrils, the dense
distribution of ribosomal particles in the cytoplasm, and the
absence of intracellular membranous organelles.
The Nucleoid
Prokaryotic and eukaryotic cells were initially
distinguished on the basis of structure: the prokaryotic
nucleoidthe equivalent of the eukaryotic nucleusis
structurally simpler than the true eukaryotic nucleus, which
has a complex mitotic apparatus and surrounding nuclear
membrane. As the electron micrograph in Fig. 2-2 shows, the
bacterial nucleoid, which contains the DNA fibrils, lacks a
limiting membrane. Under the light microscope, the nucleoid of
the bacterial cell can be visualized with the aid of Feulgen
staining, which stains DNA. Gentle lysis can be used to
isolate the nucleoid of most bacterial cells. The DNA is then
seen to be a single, continuous, "giant" circular
molecule with a molecular weight of approximately 3 X 109 (see
Ch. 5). The unfolded nuclear DNA would be about 1 mm long
(compared with an average length of 1 to 2 µm for bacterial
cells). The bacterial nucleoid, then, is a structure
containing a single chromosome. The number of copies of this
chromosome in a cell depends on the stage of the cell cycle
(chromosome replication, cell enlargement, chromosome
segregation, etc). Although the mechanism of segregation of
the two sister chromosomes following replication is not fully
understood, all of the models proposed require that the
chromosome be permanently attached to the cell membrane
throughout the various stages of the cell cycle.
Bacterial chromatin does not contain basic histone
proteins, but low-molecular-weight polyamines and magnesium
ions may fulfill a function similar to that of eukaryotic
histones. Despite the differences between prokaryotic and
eukaryotic DNA, prokaryotic DNA from cells infected with
bacteriophage g, when visualized by electron microscopy, has a
beaded, condensed appearance not unlike that of eukaryotic
chromatin.
Surface Appendages
Two types of surface appendage can be recognized on certain
bacterial species: the flagella, which are organs of
locomotion, and pili (Latin hairs), which are also known as
fimbriae (Latin fringes). Flagella occur on both Gram-positive
and Gram-negative bacteria, and their presence can be useful
in identification. For example, they are found on many species
of bacilli but rarely on cocci. In contrast, pili occur almost
exclusively on Gram-negative bacteria and are found on only a
few Gram-positive organisms (e.g., Corynebacterium renale).
Some bacteria have both flagella and pili. The electron
micrograph in Fig. 2-3 shows the characteristic wavy
appearance of flagella and two types of pili on the surface of
Escherichia coli.
 
FIGURE 2-3 (A) Electron micrograph of negatively
stained E coli showing wavy flagella and numerous short,
thinner, and more rigid hairlike structures, the pili.
(B) The long sex pilus can be distinguished from the shorter
common pili by mixing E coli cells with a male bacteriophage
that binds specifically to sex pili.
Flagella
Structurally, bacterial flagella are long (3 to 12 µm),
filamentous surface appendages about 12 to 30 nm in diameter.
The protein subunits of a flagellum are assembled to form a
cylindrical structure with a hollow core. A flagellum consists
of three parts: (1) the long filament, which lies external to
the cell surface; (2) the hook structure at the end of the
filament; and (3) the basal body, to which the hook is
anchored and which imparts motion to the flagellum. The basal
body traverses the outer wall and membrane structures. It
consists of a rod and one or two pairs of discs. The thrust
that propels the bacterial cell is provided by
counterclockwise rotation of the basal body, which causes the
helically twisted filament to whirl. The movement of the basal
body is driven by a proton motive force rather than by ATP
directly. The ability of bacteria to swim by means of the
propeller-like action of the flagella provides them with the
mechanical means to perform chemotaxis (movement in response
to attractant and repellent substances in the environment).
Response to chemical stimuli involves a sophisticated sensory
system of receptors that are located in the cell surface
and/or periplasm and that transmit information to
methyl-accepting chemotaxis proteins that control the
flagellar motor. Genetic studies have revealed the existence
of mutants with altered biochemical pathways for flagellar
motility and chemotaxis.
Chemically, flagella are constructed of a class of proteins
called flagellins. The hook and basal-body structures consist
of numerous proteins. Mutations affecting any of these gene
products may result in loss or impairment of motility.
Flagellins are immunogenic and constitute a group of protein
antigens called the H antigens, which are characteristic of a
given species, strain, or variant of an organism. The species
specificity of the flagellins reflects differences in the
primary structures of the proteins. Antigenic changes of the
flagella known as the phase variation of H1 and H2 occurs in
Salmonella typhimurium (see Ch. 21 and Ref. Seifert and So).
The number and distribution of flagella on the bacterial
surface are characteristic for a given species and hence are
useful in identifying and classifying bacteria. Figure 2-4
illustrates typical arrangements of flagella on or around the
bacterial surface. For example, V cholerae has a single
flagellum at one pole of the cell (i.e., it is monotrichous),
whereas Proteus vulgaris and E coli have many flagella
distributed over the entire cell surface (i.e., they are
peritrichous). The flagella of a peritrichous bacterium must
aggregate as a posterior bundle to propel the cell in a
forward direction.
Flagella can be sheared from the cell surface without
affecting the viability of the cell. The cell then becomes
temporarily nonmotile. In time it synthesizes new flagella and
regains motility. The protein synthesis inhibitor
chloramphenicol, however, blocks regeneration of flagella.
FIGURE 2-4 Typical arrangements of bacterial
flagella.
Pili
The terms pili and fimbriae are usually used
interchangeably to describe the thin, hairlike appendages on
the surface of many Gram-negative bacteria and proteins of
pili are referred to as pilins. Pili are more rigid in
appearance than flagella (Fig. 2-3). In some organisms, such
as Shigella species and E coli, pili are distributed profusely
over the cell surface, with as many as 200 per cell. As is
easily recognized in strains of E coli, pili can come in two
types: short, abundant common pili, and a small number (one to
six) of very long pili known as sex pili. Sex pili can be
distinguished by their ability to bind male-specific
bacteriophages (the sex pilus acts as a specific receptor for
these bacteriophages) (Fig. 2-3B). The sex pili attach male to
female bacteria during conjugation.
Pili in many enteric bacteria confer adhesive properties on
the bacterial cells, enabling them to adhere to various
epithelial surfaces, to red blood cells (causing
hemagglutination), and to surfaces of yeast and fungal cells.
These adhesive properties of piliated cells play an important
role in bacterial colonization of epithelial surfaces and are
therefore referred to as colonization factors. The common pili
found on E coli exhibit a sugar specificity analogous to that
of phytohemagglutinins and lectins, in that adhesion and
hemagglutinating capacities of the organism are inhibited
specifically by mannose. Organisms possessing this type of
hemagglutination are called mannose-sensitive organisms. Other
piliated organisms, such as gonococci, are adhesive and
hemagglutinating, but are insensitive to the inhibitory
effects of mannose. Extensive antigenic variations in pilins
of gonococci are well known (see Ref. Seifert and So).
Surface Layers
The surface layers of the bacterial cell have been
identified by various techniques: light microscopy and
staining; electron microscopy of thin-sectioned,
freeze-fractured, and negatively stained cells; and isolation
and biochemical characterization of individual morphologic
components of the cell. The principal surface layers are
capsules and loose slime, the cell wall of Gram-positive
bacteria and the complex cell envelope of Gram-negative
bacteria, plasma (cytoplasmic) membranes, and mesosomal
membrane vesicles, which arise from invaginations of the
plasma membrane. In bacteria, the cell wall forms a rigid
structure of uniform thickness around the cell and is
responsible for the characteristic shape of the cell (rod,
coccus, or spiral). Inside the cell wall (or rigid
peptidoglycan layer) is the plasma (cytoplasmic) membrane;
this is usually closely apposed to the wall layer. The
topographic relationships of the cell wall and envelope layers
to the plasma membrane are indicated in the thin section of a
Gram-positive organism (Micrococcus lysodeikticus) in Figure
2-5A and in the freeze-fractured cell of a Gram-negative
organism (Bacteroides melaninogenicus) in Figure 2-5B. The
latter shows the typical fracture planes seen in most
Gram-negative bacteria, which are weak cleavage planes through
the outer membrane of the envelope and extensive fracture
planes through the bilayer region of the underlying plasma
membrane.
 
FIGURE 2-5 (A) Electron micrograph of a thin
section of the Gram-positive M lysodeikticus showing the thick
peptidoglycan cell wall (cw), underlying cytoplasmic (plasma)
membrane (cm), mesome (m), and nucleus (n). (B)
Freeze-fractured Bacteriodes cell showing typical major convex
fracture faces through the inner (im) and outer (om)
membranes. Bars = 1 µm; circled arrow in Fig. B indicates
direction of shadowing.
Capsules and Loose Slime
Some bacteria form capsules, which constitute the outermost
layer of the bacterial cell and surround it with a relatively
thick layer of viscous gel. Capsules may be up to 10 µm
thick. Some organisms lack a well-defined capsule but have
loose, amorphous slime layers external to the cell wall or
cell envelope. The a hemolytic Streptococcus mutans, the
primary organism found in dental plaque is able to synthesis a
large extracellular mucoid glucans from sucrose. Not all
bacterial species produce capsules; however, the capsules of
encapsulated pathogens are often important determinants of
virulence. Encapsulated species are found among both
Gram-positive and Gram-negative bacteria. In both groups, most
capsules are composed of highmolecular-weight viscous
polysaccharides that are retained as a thick gel outside the
cell wall or envelope. The capsule of Bacillus anthracis (the
causal agent of anthrax) is unusual in that it is composed of
a g-glutamyl polypeptide. Table 2-1 presents the various
capsular substances formed by a selection of Gram-positive and
Gram-negative bacteria. A plasma membrane stage is involved in
the biosynthesis and assembly of the capsular substances,
which are extruded or secreted through the outer wall or
envelope structures. Mutational loss of enzymes involved in
the biosynthesis of the capsular polysaccharides can result in
the smooth-to-rough variation seen in the pneumococci.
The capsule is not essential for viability. Viability is
not affected when capsular polysaccharides are removed
enzymatically from the cell surface. The exact functions of
capsules are not fully understood, but they do confer
resistance to phagocytosis and hence provide the bacterial
cell with protection against host defenses to invasion.
Cell Wall and Gram-Negative Cell Envelope
The Gram stain broadly differentiates bacteria into
Gram-positive and Gram-negative groups; a few organisms are
consistently Gram-variable. Gram-positive and Gram-negative
organisms differ drastically in the organization of the
structures outside the plasma membrane but below the capsule
(Fig. 2-6): in Gram-negative organisms these structures
constitute the cell envelope, whereas in Gram-positive
organisms they are called a cell wall.
FIGURE 2-6 Comparison of the thick cell wall of
Gram-positive bacteria with the comparatively thin cell wall
of Gram-negative bacteria. Note the complexity of the
Gram-negative cell envelope (outer membrane, its hydrophobic
lipoprotein anchor; periplasmic space).
Most Gram-positive bacteria have a relatively thick (about
20 to 80 nm), continuous cell wall (often called the sacculus),
which is composed largely of peptidoglycan (also known as
mucopeptide or murein). In thick cell walls, other cell wall
polymers (such as the teichoic acids, polysaccharides, and
peptidoglycolipids) are covalently attached to the
peptidoglycan. In contrast, the peptidoglycan layer in
Gram-negative bacteria is thin (about 5 to 10 nm thick); in E
coli, the peptidoglycan is probably only a monolayer thick.
Outside the peptidoglycan layer in the Gram-negative envelope
is an outer membrane structure (about 7.5 to 10 nm thick). In
most Gram-negative bacteria, this membrane structure is
anchored noncovalently to lipoprotein molecules (Braun's
lipoprotein), which, in turn, are covalently linked to the
peptidoglycan. The lipopolysaccharides of the Gram-negative
cell envelope form part of the outer leaflet of the outer
membrane structure.
The organization and overall dimensions of the outer
membrane of the Gram-negative cell envelope are similar to
those of the plasma membrane (about 7.5 nm thick). Moreover,
in Gram-negative bacteria such as E coli, the outer and inner
membranes adhere to each other at several hundred sites (Bayer
patches); these sites can break up the continuity of the
peptidoglycan layer. Table 2-2 summarizes the major classes of
chemical constituents in the walls and envelopes of
Gram-positive and Gram-negative bacteria.
The basic differences in surface structures of
Gram-positive and Gram-negative bacteria explain the results
of Gram staining. Both Gram-positive and Gram-negative
bacteria take up the same amounts of crystal violet (CV) and
iodine (I). The CV-I complex, however, is trapped inside the
Gram-positive cell by the dehydration and reduced porosity of
the thick cell wall as a result of the differential washing
step with 95 percent ethanol or other solvent mixture. In
contrast, the thin peptidoglycan layer and probable
discontinuities at the membrane adhesion sites do not impede
solvent extraction of the CV-I complex from the Gram-negative
cell. The above mechanism of the Gram stain based on the
structural differences between the two groups has been
confirmed by sophisticated methods of electron microscopy (see
Ref. Bereridge and Daries). The sequence of steps in the Gram
stain differentiation is illustrated diagrammatically in
Figure 2-7. Moreover, mechanical disruption of the cell wall
of Gram-positive organisms or its enzymatic removal with
lysozyme results in complete extraction of the CV-I complex
and conversion to a Gram-negative reaction. Therefore,
autolytic wall-degrading enzymes that cause cell wall breakage
may account for Gram-negative or variable reactions in
cultures of Gram-positive organisms (such as Staphylococcus
aureus, Clostridium perfringens, Corynebacterium diphtheriae,
and some Bacillus spp).
FIGURE 2-7 General sequence of steps in the Gram
stain procedure and the resultant staining of Gram-positive
and Gram-negative bacteria.
Peptidoglycan
Unique features of almost all prokaryotic cells (except for
Halobacterium halobium and mycoplasmas) are cell wall
peptidoglycan and the specific enzymes involved in its
biosynthesis. These enzymes are target sites for inhibition of
peptidoglycan synthesis by specific antibiotics. The primary
chemical structures of peptidoglycans of both Gram-positive
and Gram-negative bacteria have been established; they consist
of a glycan backbone of repeating groups of b1, 4-linked
disaccharides of b1,4-N-acetylmuramyl-N-acetylglucosamine.
Tetrapeptides of L-alanine-D-isoglutamic acid-L-lysine (or
diaminopimelic acid)-n-alanine are linked through the carboxyl
group by amide linkage of muramic acid residues of the glycan
chains; the D-alanine residues are directly cross-linked to
the e-amino group of lysine or diaminopimelic acid on a
neighboring tetrapeptide, or they are linked by a peptide
bridge. In S aureus peptidoglycan, a glycine pentapeptide
bridge links the two adjacent peptide structures. The extent
of direct or peptide-bridge cross-linking varies from one
peptidoglycan to another. The staphylococcal peptidoglycan is
highly cross-linked, whereas that of E coli is much less so,
and has a more open peptidoglycan mesh. The diamino acid
providing the e-amino group for cross-linking is lysine or
diaminopimelic acid, the latter being uniformly present in
Gram-negative peptidoglycans. The structure of the
peptidoglycan is illustrated in Figure 2-8. A peptidoglycan
with a chemical structure substantially different from that of
all eubacteria has been discovered in certain archaebacteria.
Instead of muramic acid, this peptidoglycan contains
talosaminuronic acid and lacks the D-amino acids found in the
eubacterial peptidoglycans. Interestingly, organisms
containing this wall polymer (referred to as pseudomurein) are
insensitive to penicillin, an inhibitor of the transpeptidases
involved in peptidoglycan biosynthesis in eubacteria.
FIGURE 2-8 Diagrammatic representation of
peptidoglycan structures with adjacent glycan strands
cross-linked directly from the carboxyterminal D-alanine to
the e-amino group of an adjacent tetrapeptide or through a
peptide cross bridge ,N-acetylmuramic acid; N-acetylglucosamine.
The ß-1,4 glycosidic bond between N-acetylmuramic acid and
N-acetylglucosamine is specifically cleaved by the
bacteriolytic enzyme lysozyme. Widely distributed in nature,
this enzyme is present in human tissues and secretions and can
cause complete digestion of the peptidoglycan walls of
sensitive organisms. When lysozyme is allowed to digest the
cell wall of Gram-positive bacteria suspended in an osmotic
stabilizer (such as sucrose), protoplasts are formed. These
protoplasts are able to survive and continue to grow on
suitable media in the wall-less state. Gram-negative bacteria
treated similarly produce spheroplasts, which retain much of
the outer membrane structure. The dependence of bacterial
shape on the peptidoglycan is shown by the transformation of
rod-shaped bacteria to spherical protoplasts (spheroplasts)
after enzymatic breakdown of the peptidoglycan. The mechanical
protection afforded by the wall peptidoglycan layer is evident
in the osmotic fragility of both protoplasts and spheroplasts.
There are two groups of bacteria that lack the protective cell
wall peptidoglycan structure, the Mycoplasma species, one of
which causes atypical pneumonia and some genitourinary tract
infections and the L-forms, which originate from Gram-positive
or Gram-negative bacteria and are so designated because of
their discovery and description at the Lister Institute,
London. The mycoplasmas and L-forms are all Gram-negative and
insensitive to penicillin and are bounded by a surface
membrane structure. L-forms arising "spontaneously"
in cultures or isolated from infections are structurally
related to protoplasts and spheroplasts; all three forms
(protoplasts, spheroplasts, and L-forms) revert infrequently
and only under special conditions.
Teichoic Acids
Wall teichoic acids are found only in certain Gram-positive
bacteria (such as staphylococci, streptococci, lactobacilli,
and Bacillus spp); so far, they have not been found in gram-
negative organisms. Teichoic acids are polyol phosphate
polymers, with either ribitol or glycerol linked by
phosphodiester bonds; their structures are illustrated in
Figure 2-9. Substituent groups on the polyol chains can
include D-alanine (ester linked), N-acetylglucosamine, N-acetylgalactosamine,
and glucose; the substituent is characteristic for the
teichoic acid from a particular bacterial species and can act
as a specific antigenic determinant. Teichoic acids are
covalently linked to the peptidoglycan. These highly
negatively charged polymers of the bacterial wall can serve as
a cation-sequestering mechanism.
FIGURE 2-9 Structures of cell wall teichoic acids.
(A) Ribitol teichoic acid with repeating units of
1,5-phosphodiester linkages of D-ribitol and D-alanyl ester on
position 2 and glycosyl substituents (R) on position 4. The
glycosyl groups may abe N-acetylglucosaminyl (a or b) as in S
aureus or a-glucosyl as in B subtilis W23. (B) Glycerol
teichoic acid with 1,3-phosphodiester linkages of glycerol
repeating units (1,2-linkages in some species). In the
glycerol teichoic acid structure shown, the polymer may be
unsubstituted (R - H) or substituted (R - D-alanyl or glycosyl).
Accessory Wall Polymers
In addition to the principal cell wall polymers, the walls
of certain Gram-positive bacteria possess polysaccharide
molecules linked to the peptidoglycan. For example, the C
polysaccharide of streptococci confers group specificity.
Acidic polysaccharides attached to the peptidoglycan are
called teichuronic acids. Mycobacteria have peptidoglycolipids,
glycolipids, and waxes associated with the cell wall.
Lipopolysaccharides
A characteristic feature of Gram-negative bacteria is
possession of various types of complex macromolecular
lipopolysaccharide (LPS). So far, only one Gram-positive
organism, Listeria monocytogenes, has been found to contain an
authentic LPS. The LPS of this bacterium and those of all
Gram-negative species are also called endotoxins, thereby
distinguishing these cell-bound, heat-stable toxins from
heat-labile, protein exotoxins secreted into culture media.
Endotoxins possess an array of powerful biologic activities
and play an important role in the pathogenesis of many
Gram-negative bacterial infections. In addition to causing
endotoxic shock, LPS is pyrogenic, can activate macrophages
and complement, is mitogenic for B lymphocytes, induces
interferon production, causes tissue necrosis and tumor
regression, and has adjuvant properties. The endotoxic
properties of LPS reside largely in the lipid A components.
Usually, the LPS molecules have three regions: the lipid A
structure required for insertion in the outer leaflet of the
outer membrane bilayer; a covalently attached core composed of
2-keto-3deoxyoctonic acid (KDO), heptose, ethanolamine, N-acetylglucosamine,
glucose, and galactose; and polysaccharide chains linked to
the core. The polysaccharide chains constitute the O-antigens
of the Gram-negative bacteria, and the individual
monosaccharide constituents confer serologic specificity on
these components. Figure 2-10 depicts the structure of LPS.
Although it has been known that lipid A is composed of
b1,6-linked D-glucosamine disaccharide substituted with
phosphomonester groups at positions 4' and 1, uncertainties
have existed about the attachment positions of the six fatty
acid acyl and KDO groups on the disaccharide. The
demonstration of the structure of lipid A of LPS of a
heptoseless mutant of Salmonella typhimurium has established
that amide-linked hydroxymyristoyl and lauroxymyristoyl groups
are attached to the nitrogen of the 2- and 2'-carbons,
respectively, and that hydroxymyristoyl and myristoxymyristoyl
groups are attached to the oxygen of the 3- and 3'-carbons of
the disaccharide, respectively. Therefore, only position 6' is
left for attachment of KDO units.
FIGURE 2-10 The three major, covalently linked
regions that form the typical LPS.
LPS and phospholipids help confer asymmetry to the outer
membrane of the Gram-negative bacteria, with the hydrophilic
polysaccharide chains outermost. Each LPS is held in the outer
membrane by relatively weak cohesive forces (ionic and
hydrophobic interactions) and can be dissociated from the cell
surface with surface-active agents.
As in peptidoglycan biosynthesis, LPS molecules are
assembled at the plasma or inner membrane. These newly formed
molecules are initially inserted into the outer-inner membrane
adhesion sites.
Outer Membrane of Gram-Negative Bacteria
In thin sections, the outer membranes of Gram-negative
bacteria appear broadly similar to the plasma or inner
membranes; however, they differ from the inner membranes and
walls of Gram-positive bacteria in numerous respects. The
lipid A of LPS is inserted with phospholipids to create the
outer leaflet of the bilayer structure; the lipid portion of
the lipoprotein and phospholipid form the inner leaflet of the
outer membrane bilayer of most Gram-negative bacteria (Fig.
2-6).
In addition to these components, the outer membrane
possesses several major outer membrane proteins; the most
abundant is called porin. The assembled subunits of porin form
a channel that limits the passage of hydrophilic molecules
across the outer membrane barrier to those having molecular
weights that are usually less than 600 to 700. Evidence also
suggests that hydrophobic pathways exist across the outer
membrane and are partly responsible for the differential
penetration and effectiveness of certain b-lactam antibiotics
(ampicillin, cephalosporins) that are active against various
Gram-negative bacteria. Although the outer membranes act as a
permeability barrier or molecular sieve, they do not appear to
possess energy-transducing systems to drive active transport.
Several outer membrane proteins, however, are involved in the
specific uptake of metabolites (maltose, vitamin B12,
nucleosides) and iron from the medium. Thus, outer membranes
of the Gram-negative bacteria provide a selective barrier to
external molecules and thereby prevent the loss of
metabolite-binding proteins and hydrolytic enzymes (nucleases,
alkaline phosphatase) found in the periplasmic space. The
periplasmic space is the region between the outer surface of
the inner (plasma) membrane and the inner surface of the outer
membrane (Figure 2-6). Thus, Gram-negative bacteria have a
cellular compartment that has no equivalent in Gram-positive
organisms. In addition to the hydrolytic enzymes, the
periplasmic space holds binding proteins (proteins that
specifically bind sugars, amino acids, and inorganic ions)
involved in membrane transport and chemotactic receptor
activities. Moreover, plasmid-encoded b-lactamases and
aminoglycoside-modifying enzymes (phosphorylation or
adenylation) in the periplasmic space produce antibiotic
resistance by degrading or modifying an antibiotic in transit
to its target sites on the membrane (penicillin-binding
proteins) or on the ribosomes (aminoglycosides). These
periplasmic proteins can be released by subjecting the cells
to osmotic shock and after treatment with the chelating agent
ethylenediaminetetraacetic acid.
Intracellular Components
Plasma (Cytoplasmic) Membranes
Bacterial plasma membranes, the functional equivalents of
eukaryotic plasma membranes, are referred to variously as
cytoplasmic, protoplast, or (in Gram-negative organisms) inner
membranes. Similar in overall dimensions and appearance in
thin sections to biomembranes from eukaryotic cells, they are
composed primarily of proteins and lipids (principally
phospholipids). Protein-to-lipid ratios of bacterial plasma
membranes are approximately 3: 1, close to those for
mitochondrial membranes. Unlike eukaryotic cell membranes, the
bacterial membrane (except for Mycoplasma species and certain
methylotrophic bacteria) has no sterols, and bacteria lack the
enzymes required for sterol biosynthesis.
Although their composition is similar to that of inner
membranes of Gram-negative species, cytoplasmic membranes from
Gram-positive bacteria possess a class of macromolecules not
present in the Gram-negative membranes. Many Gram-positive
bacterial membranes contain membrane-bound lipoteichoic acid,
and species lacking this component (such as Micrococcus and
Sarcina spp) contain an analogous membrane-bound succinylated
lipomannan. Lipoteichoic acids are structurally similar to the
cell wall glycerol teichoic acids in that they have basal
polyglycerol phosphodiester 1-3 linked chains (Fig. 2-9).
These chains terminate with the phosphomonoester end of the
polymer, which is linked covalently to either a glycolipid or
a phosphatidyl glycolipid moiety. Thus, a hydrophobic tail is
provided for anchoring in the membrane lipid layers (Fig.
2-6A). As in the cell wall glycerol teichoic acid, the
lipoteichoic acids can have glycosidic and D-alanyl ester
substituents on the C-2 position of the glycerol.
Both membrane-bound lipoteichoic acid and membrane-bound
succinylated lipomannan can be detected as antigens on the
cell surface, and the glycerol-phosphate and succinylated
mannan chains appear to extend through the cell wall structure
(Fig. 2-6). This class of polymer has not yet been found in
the cytoplasmic membranes of Gram-negative organisms. In both
instances, the lipoteichoic acids and the lipomannans are
negatively charged components and can sequester positively
charged substances. They have been implicated in adhesion to
host cells, but their functions remain to be elucidated.
Multiple functions are performed by the plasma membranes of
both Gram-positive and Gram-negative bacteria. Plasma
membranes are the site of active transport, respiratory chain
components, energy-transducing systems, the H+-ATPase of the
proton pump (see Chapter 4), and membrane stages in the
biosynthesis of phospholipids, peptidoglycan, LPS, and
capsular polysaccharides. In essence, the bacterial
cytoplasmic membrane is a multifunction structure that
combines the mitochondrial transport and biosynthetic
functions that are usually compartmentalized in discrete
membranous organelles in eukaryotic cells. The plasma membrane
is also the anchoring site for DNA and provides the cell with
a mechanism (as yet unknown) for separation of sister
chromosomes.
Mesosomes
Thin sections of Gram-positive bacteria reveal the presence
of vesicular or tubular-vesicular membrane structures called
mesosomes, which are apparently formed by an invagination of
the plasma membrane. These structures are much more prominent
in Gram-positive than in Gram-negative organisms. At one time,
the mesosomal vesicles were thought to be equivalent to
bacterial mitochondria; however, many other membrane functions
have also been attributed to the mesosomes. At present, there
is no satisfactory evidence to suggest that they have a unique
biochemical or physiologic function. Indeed,
electron-microscopic studies have suggested that the mesosomes,
as usually seen in thin sections, may arise from membrane
perturbation and fixation artifacts. No general agreement
exists about this theory, however, and some evidence indicates
that mesosomes may be related to events in the cell division
cycle.
Other Intracellular Components
In addition to the nucleoid and cytoplasm (cytosol), the
intracellular compartment of the bacterial cell is densely
packed with ribosomes of the 70S type (Fig. 2-2). These
ribonucleoprotein particles, which have a diameter of 18 nm,
are not arranged on a membranous rough endoplasmic reticulum
as they are in eukaryotic cells. Other granular inclusions
randomly distributed in the cytoplasm of various species
include metabolic reserve particles such as poly-b-hydroxybutyrate
(PHB), polysaccharide and glycogen-like granules, and
polymetaphosphate or metachromatic granules.
Endospores are highly heat-resistant, dehydrated resting
cells formed intracellularly in members of the genera Bacillus
and Clostridium. Sporulation, the process of forming
endospores, is an unusual property of certain bacteria. The
series of biochemical and morphologic changes that occur
during sporulation represent true differentiation within the
cycle of the bacterial cell. The process, which usually begins
in the stationary phase of the vegetative cell cycle, is
initiated by depletion of nutrients (usually readily
utilizable sources of carbon or nitrogen, or both). The cell
then undergoes a highly complex, well-defined sequence of
morphologic and biochemical events that ultimately lead to the
formation of mature endospores. As many as seven distinct
stages have been recognized by morphologic and biochemical
studies of sporulating Bacillus species: stage 0, vegetative
cells with two chromosomes at the end of exponential growth;
stage I, formation of axial chromatin filament and excretion
of exoenzymes, including proteases; stage II, forespore septum
formation and segregation of nuclear material into two
compartments; stage III, spore protoplast formation and
elevation of tricarboxylic acid and glyoxylate cycle enzyme
levels; stage IV, cortex formation and refractile appearance
of spore; stage V, spore coat protein formation; stage VI,
spore maturation, modification of cortical peptidoglycan,
uptake of dipicolinic acid (a unique endospore product) and
calcium, and development of resistance to heat and organic
solvents; and stage VII, final maturation and liberation of
endospores from mother cells (in some species).
When newly formed, endospores appear as round, highly
refractile cells within the vegetative cell wall, or
sporangium. Some strains produce autolysins that digest the
walls and liberate free endospores. The spore protoplast, or
core, contains a complete nucleus, ribosomes, and energy
generating components that are enclosed within a modified
cytoplasmic membrane. The peptidoglycan spore wall surrounds
the spore membrane; on germination, this wall becomes the
vegetative cell wall. Surrounding the spore wall is a thick
cortex that contains an unusual type of peptidoglycan, which
is rapidly released on germination. A spore coat of
keratinlike protein encases the spore contained within a
membrane (the exosporium). During maturation, the spore
protoplast dehydrates and the spore becomes refractile and
resistant to heat, radiation, pressure, desiccation, and
chemicals; these properties correlate with the cortical
peptidoglycan and the presence of large amounts of calcium
dipicolinate.
Recent evidence indicated that the spores of Bacillus
spharicus were revived which had been preserved in amber for
more than 25 million years. Their claims need to be
reevaluated. Figure 2-11 illustrates the principal structural
features of a typical endospore (Bacillus megaterium) on
initiation of the germination process. The thin section of the
spore shows the ruptured, thick spore coat and the cortex
surrounding the spore protoplast with the germinal cell wall
that becomes the vegetative wall on outgrowth.
FIGURE 2-11 Electron micrograph of a thin section
of a Bacillus megaterium spore showing the thick
spore coat (SC), germinal groove (G) in the spore coat, outer
cortex layer (OCL) and cortex (Cx) germinal cell wall layer (GCW),
underlying spore protoplast membrane (PM), and regions where
the nucleoid (n) is visible. (Courtesy of John H Freer,
University of Glasgow, Scotland.)
REFERENCES
Beveridge TJ, Davies JA: Cellular responses of Bacillus
subtilis and Escherichia coli to the Gram stain. J Bacteriol,
156:846,1983
Costerton JW, Ingram JM, Cheng KJ: Structure and function
of the cell envelope of gram-negative bacteria. Bacteriol Rev,
38:87, 1974
Ghuysen J-M, Hakenbeck R: Bacterial cell wall. Elsevier,
1994
Gould GW, Hurst A (eds): The Bacterial Spore. Academic
Press, San Diego, 1969
Jawetz E, Melnick JL, Adelberg EA: Medical Microbiology.
Appleton & Lange, East Norwalk, CT, 1989
Rogers HJ: Bacterial Cell Structure. American Society for
Microbiology, Washington, D.C., 1983
Seifert HS, So M: Genetic mechanisms of bacterial antigenic
variation. Microbiological Reviews, Vol. 52:327, 1988
Wright A, Tipper DJ: The outer membrane of gram-negative
bacteria. p. 427. In Sokatch JR, Ornston LN (eds): The
bacteria. Vol. 7. Academic Press, San Diego, 1979
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