Glycocalyx

Many prokaryotes secrete on their surface a substance called Glycocalyx.

Glycocalyx (meaning sugar coat) is the general term used for substances that
surround cells.
The bacterial glycocalyx is a viscous (sticky), gelatinous polymer that is external to
the cell wall and composed of polysaccharide, polypeptide, or both. Its chemical
composition varies widely with the species. For the most part, it is made inside the
cell and secreted to the cell surface.
If the substance is organized and is firmly attached to the cell wall, the glycocalyx is
described as a capsule. The presence of a capsule can be determined by using
negative staining
If the substance is unorganized and only loosely attached to the cell wall, the
glycocalyx is described as a slime layer.
e.g. Bacillus anthracis produces a capsule of d-glutamic acid. (Recall
The glycocalyx is a very important component of biofilms (see page 160). A
glycocalyx that helps cells in a biofilm attach to their target environment and to each
other is called an extracellular polymeric substance (EPS). The EPS protects the
cells within it, facilitates communication among them, and enables the cells to
survive by attaching to various surfaces in their natural environment.
A glycocalyx also can protect a cell against dehydration, and its viscosity may inhibit
the movement of nutrients out of the cell.

Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around
the cell wall called capsule. Capsule consist of a mesh or network of fine strands. This capsule only helps
in disease causing ability of a few types of bacteria. This capsule is divided into two
groups:
(a) Macrocapsule: It is about O.21lm thick and can be seen under light microscope.
(b) Microcapsule: It can't be seen under light microscope but can be demonstrated immunologically.
Chemically the capsules are made up of di or polysaccharide or polypeptide. The
polysaccharide may be homo polysaccharide (composed of single kind of sugar) e.g.
Streptococcus mutans ot it may be heteropolysaccharide (composed of several kind of
sugars) e.g. Klebsiella pneumoniae.

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 is unusual in that it is composed of a g-glutamyl polypeptide.
A plasma membrane stage is involved in the biosynthesis and assembly of the capsularsubstances,
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.

Fimbriae and Pili

Many gram-negative bacteria contain hair-like appendages that are shorter,
straighter, and thinner than flagella
Used for attachment and transfer of DNA rather than for motility.
These structures, which consist of a protein called pilin arranged helically around a
central core, are divided into two types, fimbriae and pili, having very different
functions.
Fimbriae (singular: fimbria) can occur at the poles of the bacterial cell or can be
evenly distributed over the entire surface of the cell. They can number anywhere
from a few to several hundred per cell Fimbriae have a tendency to adhere to each
other and to surfaces. As a result, they are involved in forming biofilms and other
aggregations on the surfaces of liquids, glass, and rocks. Fimbriae can also help
bacteria adhere to epithelial surfaces in the body. For example, fimbriae on the
bacterium Neisseria gonorrhoeae (n-se r-a go-nor-r ), the causative agent of
gonorrhea, help the microbe colonize mucous membranes. Once colonization occurs,
the bacteria can cause disease.
The fimbriae of E. coli O157 enable this bacterium to adhere to the lining of the small
intestine, where it causes a severe watery diarrhea. When fimbriae are absent
(because of genetic mutation), colonization cannot happen, and no disease ensues.
Pili (singular: pilus) are usually longer than fimbriae and number only one or two
per cell. Pili are involved in motility and DNA transfer. In one type of motility, called
twitching motility, a pilus extends by the addition of subunits of pilin, makes
contact with a surface or another cell, and then retracts (powerstroke) as the pilin
subunits are disassembled. This is called the grappling hook model of twitching
motility and results in short, jerky, intermittent movements. Twitching motility has
been observed in Pseudomonas aeruginosa, Neisseria gonorrhoeae, and some
strains of E. coli. The other type of motility associated with pili is gliding motility,
the smooth gliding movement of myxobacteria. Although the exact mechanism is
unknown for most myxobacteria, some utilize pilus retraction. Gliding motility
provides a means for microbes to travel in environments with a low water content,
such as biofilms and soil.
PILI OR FIMBRAE
These are hair like appendages present on the surface of most of the gram negative
bacteria (Enterobacteriaceae, Pseudomondaceae and Caulobacter). They are smaller than
flagella, have no role in the motility of bacteria.
They measure 0.2-20J.! in length and 30-140Ao in width.
A single bacterial cells bears about 100-500 pili which are arranged
peritrichously.
There origin is from cytoplasm and penetrate through the peptidoglycan
layers of the cell wall.
Chemically they are composed of 100% protein named fimbrilin
with a molecular weight of about 16,000. Fimbrilin consist of about 163 amino acids.
Following two types of pili are found in bacteria viz.

(a) Somatic pili

(b) Sex pili or conjugate pili
(a) Somatic Pili : Each bacterial cell bears about 100 somatic pili whose main function is to help the
bacterium for attachment to a substratum.
(b) Sex Pili or Conjugate Pili : They are also known as F pili and are controlled by sex factors. These pili
are comparatively long (20 ll) and broad in width (65-135AO).
There number ranges from 1-10 in male or donor bacterium, but in some bacterium it is found in both viz.
Male donor (+ factors) or female receptor/ receiver (- factor).
At the time of conjugation the sex pili of male donor recognize the receptor protein on the surface of
female or recipient and get attached with the help of conjugation tube.
The DNA from the donor to recipient is transferred through this conjugation tube. There are two types of
sex pili in [Link]. They are F. pili and [Link].
In certain pathogenic bacteria these pili help the bacteria in the attachment of pathogenic bacterial cell to
the host cells. There are generally four types of pili classified on the basis of their attachment ability to the
host cell :
(a) Type I : There diameter is about 90A and found in [Link], Serratia & Salmonella.
(b) Type II : They lack the attachment ability e.g. some species of Salmonella.
(c) Type III : Their attachment ability is effected by mannose sugar. They are about
50 A in diameter e.g. Klebsiella and some spp. of Salmonella.
(d) Type IV : They are found in Proteus bacteria.
Functions of Pili
(i) They help the bacteria to attach themselves to the natural substrate or to other organism due to its
adhesive properties.
(ii) They bear antigenic properties.
(iii) Sex pili are helpful in chromosome transfer during conjugation by acting as conjugation tube.
(iv) They act as bacteriophage receptor.

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.

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 recognised in strains of E coli, pili can come in
two types: short, abundant common pili, and a small number of very long pili known as sex pili.
Sex pili can be distinguished by their ability to bind male-specific bacteriophages.
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, and to surfaces of yeast and fungal cells.
These adhesive properties of piliated cells play an important role in bacterial colonisation of
epithelial surfaces and are therefore referred to as ~colonisation 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
Functions
(i) They provide protection against temporary drying by binding water molecules.
(ii) They may be antiphagocytic i.e. they inhibit the engulfment of pathogenic bacteria by W.B.C. and
contribute to invasive ability.

Axial Filaments
Spirochetes are a group of bacteria that have unique structure and motility. One of
the best-known spirochetes is Treponema pallidum (tre-p-n ma pal li-dum), the
causative agent of
syphilis. Another spirochete is Borrelia burgdorferi (bor-rel--a burg-dor fer-), the
causative agent of Lyme disease. Spirochetes move by means of axial filaments, or
endoflagella, bundles of fibrils that arise at the ends of the cell beneath an outer
sheath andspiral around the cell (Figure 4.10).
Axial filaments, which are anchored at one end of the spirochete, have a structure
similar to that of flagella. The rotation of the filaments produces a movement of the
outer sheath that propels the spirochetes in a spiral motion. This type of movement
is similar to the way a corkscrew moves through a cork.
This corkscrew motion probably enables a bacterium such as T. pallidum to move
effectively through body fluids.

Sheaths
Some species of bacteria of freshwater and marine environment form chains or
trichomes which are enclosed by a hollow tube called sheath. Sheaths may be sometimes
impregnated with ferric or manganese hydroxides which strengthen them.

Prostheceae and Stalks

These are semirigid extensions of cell wall and cytoplasmic membrane. They are
characteristic of a number of aerobic bacteria from freshwater and marine environment.
These prosthecae may be single (ex. Caulobacter) or several (Ancalomicrobrillm)
The main function of prostheceae is that they increase the surface area of the cells
for nutrient absorption in the dilute environment.
Stalk are non living ribbon like or tubular appendages that are excreted by the
cell. The~ stalk aid in attachment of the cells to surfaces e.g. Plallctomyces.

The Cell Wall:

General characteristics:

The cell wall of the bacterial cell is a complex, semi-rigid structure responsible for
the shape of the cell. The cell wall surrounds the underlying, fragile plasma
(cytoplasmic) membrane and protects it and the interior of the cell from adverse
changes in the outside environment. Almost all prokaryotes have cell walls.
The major function of the cell wall is to prevent bacterial cells from rupturing when
the water pressure inside the cell is greater than that outside the cell. It also helps
maintain the shape of a bacterium and serves as a point of anchorage for flagella. As
the volume of a bacterial cell increases, its plasma membrane and cell wall extend
as needed.
The cell wall is resistant to extremely high pressure. The cell wall constitutes a significant portion of the
dry weight of the cell, it may account for as such 10-40% of the dry weight of bacterial cell
Clinically, the cell wall is important because it contributes to the ability of some
species to cause disease and is the site of action of some antibiotics. In addition, the
chemical composition of the cell wall is used to differentiate major types of bacteria.
Although the cells of some eukaryotes, including plants, algae, and fungi, have cell
walls, their walls differ chemically from those of prokaryotes, are simpler in structure,
and are less rigid.

The thickness of these different layers varies both in gram +ve and gram -ve bacteria. The walls of gram
-ve species are generally thinner (10-15 nm) than those of gram +ve species (20-25 nm).

Composition and Characteristics

The bacterial cell wall is composed of a macromolecular network called
peptidoglycan (also known as murein), which is present either alone or in
combination with other substances. which is an insoluble, porous, cross-linked polymer of
enormous strength and rigidity. This peptidoglycan is only found in prokaryotes.
Peptidoglycan consists of a repeating disaccharide attached by polypeptides to form
a lattice that surrounds and protects the entire cell. The disaccharide portion is made
up of monosaccharides called N acetylglucosamine (NAG) and N-acetylmuramic acid
(NAM) (from murus, meaning wall), which are related to glucose.
It is basically a polymer of N-acetyl glucosamine (NAG), N acetylmuramic acid (NAM), and 4 amino

acids (L-alanine, D-alanine, D-glutamate and a diamino acids).

The structural formulas for NAG and NAM Figure 4.12.
The various components of peptidoglycan are assembled in the cell wall ( Figure
4.13a).
Alternating NAM and NAG molecules are linked in rows of 10 to 65 sugars to form a
carbohydrate backbone (the glycan portion of peptidoglycan). Adjacent rows are
linked by polypeptides (the peptide portion of peptidoglycan).
Although the structure of the polypeptide link varies, it always includes tetrapeptide
side chains, which consist of four amino acids attached to NAMs in the backbone.

The amino acids occur in an alternating pattern of d and l forms (see Figure 2.13,
page 43).
The tetrapeptide of one peptidoglycan layer is cross linked with the other peptidoglycan layer and as a
result a strong framework is formed around the cell and impart great rigidity to the total structure. Some
antibiotics viz. penicillin inhibit the synthesis of this framework thus the cell wall synthesis is stopped.

This is unique because the amino acids found in other proteins are l forms. Parallel
tetrapeptide side chains may be directly bonded to each other or linked by a peptide
cross-bridge, consisting of a short chain of amino acids.
NOTE: Penicillin interferes with the final linking of the peptidoglycan rows by peptide
cross-bridges (see Figure 4.13a). As a result, the cell wall is greatly weakened and
the cell undergoes lysis, destruction caused by rupture of the plasma membrane
and the loss of cytoplasm.

Gram-Positive Cell Walls

In most gram-positive bacteria, the cell wall consists of many layers of
peptidoglycan, forming a thick, rigid structure (Figure 4.13b). The cell wall of these bacteria
consist of about 40- 80% of peptidoglycan of the dry weight of cell wall.
By contrast, gram-negative cell walls contain only a thin layer of peptidoglycan
In addition, the cell walls of gram-positive bacteria contain teichoic acids, which
consist primarily of an alcohol (such as glycerol or ribitol) and phosphate. There are
two classes of teichoic acids: lipoteichoic
acid, which spans the peptidoglycan layer and is linked to the plasma membrane,
and wall teichoic acid, which is linked to the peptidoglycan layer.
Because of their negative charge (from the phosphate groups), teichoic acids may
bind and regulate the movement of cations (positive ions) into and out of the cell.
They may also assume a role in cell growth, preventing extensive wall breakdown
and possible cell lysis.
Finally, teichoic acids provide much of the walls antigenic specificity and thus make
it possible to identify gram-positive bacteria by certain laboratory tests (see Chapter
10).
Similarly, the cell walls of gram-positive streptococci are covered with various
polysaccharides that allow them to be grouped into medically significant types.
Gram + ve bacteria have a much greater amount of peptidoglycan in their cell walls than do gram - ve
bacteria.
This peptidoglycan is of about 40 or more layers in gram + ve bacteria. The cell wall measures about 30-80
nm in thickness. Teichoic acid or acidic polysaccharide are mainly present in gram positive bacteria and
are found associated with peptidoglycan by a single terminal covalent bond.

Teichoic acid is a negatively charged substituted polysaccharide polymer made up of ribitol and glycerol
residues joined through diphosphoester linkages. It constitute a major surface antigen. It is hydrophilic and
its main function is to transport positively charged substances to the bacterial cell in the storage of phosphorous.

Gram-Negative Cell Walls

The cell walls of gram-negative bacteria consist of one or a very few layers of
peptidoglycan and an outer membrane (see Figure 4.13c).
The peptidoglycan is bonded to lipoproteins (lipids covalently linked to proteins) in
the outer membrane and is in the periplasm-a gel-like fluid between the outer
membrane and the plasma membrane.
The periplasm contains a high concentration of degradative enzymes and transport
proteins. Gram-negative cell walls do not contain teichoic acids. Because the cell
walls of gram-negative bacteria contain only a small amount of peptidoglycan, they
are more susceptible to mechanical breakage.
The outer membrane of the gram-negative cell consists of lipopolysaccharides (LPS),
lipoproteins, and phospholipids (see Figure 4.13c). The outer membrane has several
specialized functions.
Its strong negative charge is an important factor in evading phagocytosis and the
actions of complement (lyses cells and promotes phagocytosis), two components of
the defenses of the host
(discussed in detail in Chapter 16).
The outer membrane also provides a barrier to certain antibiotics (for example,
penicillin), digestive enzymes such as lysozyme, detergents, heavy metals, bile salts,
and certain dyes.
However, the outer membrane does not provide a barrier to all substances in the
environment because nutrients must pass through to sustain the metabolism of the
cell. Part of the permeability of the outer membrane is due to proteins in the
membrane, called porins that form channels. Porins permit the passage of
molecules such as nucleotides, disaccharides, peptides, amino acids, vitamin B 12,
and iron.
The lipopolysaccharide (LPS) of the outer membrane is a large complex molecule
that contains lipids and carbohydrates and consists of three components: (1) lipid A,
(2) a core polysaccharide, and (3) an O polysaccharide. Lipid A is the lipid portion of
the LPS and is embedded in the top layer of the outer membrane. When gramnegative bacteria die, they release lipid A, which functions as an endotoxin (Chapter
15). Lipid A is responsible for the symptoms associated with infections by gramnegative bacteria such as fever, dilation of blood vessels, shock, and blood clotting.
The core polysaccharide is attached to lipid A and contains unusual sugars. Its role
is structuralto provide stability. The O polysaccharide extends outward from the
core polysaccharide and is composed of sugar molecules. The O polysaccharide
functions as an antigen and is useful for distinguishing species of gram-negative
bacteria.
For example, the foodborne pathogen E. coli O157:H7 is distinguished from other
serovars by certain laboratory tests that test for these specific antigens. This role is
comparable to that of teichoic acids in gram-positive cells.

The wall of gram - ve bacteria are more complex than those of gram + ve bacteria. The envelop of this kind of
bacteria is made up of two unit membranes and are separated by 100Ao space known as periplasmic region and
contains a peptidoglycan layer.
The outermost membrane is known as cell wall while the inner one is referred as cytoplasmic membrane.
Peptidoglycan is only about 5-10% of the dry weight of cell wall. The outer membrane serve as selective barrier
for various external chemicals and enzymes that could damage the cells. Its structure is similar to plasma
membrane or cell membrane.
The outer membrane is anchored to the underlying peptidoglycan by means of Braun's lipoprotein. The
membrane is bilayered structure consisting mainly of phospholipids protein and lipopolysaccharide (L.P.S.). The
phospholipids are bilayered consisting of both hydrophilic and hydrophobic ends. The wall contains 4 types of
protein components along with other major types of protein called lipoprotein.
The Lipopolysaccharide (LPS) has toxic properties and is also known as endotoxin.
It occurs only in the outer layer of the membrane and is composed of three covalently linked parts :
(i) Lipid A = firmly embedded in the membrane.
(ii) Core polysaccharide = located at the membrane surface.
(iii) O-antigens = which extend like whiskers from the membrane surface into the surrounding medium. Many
antigenic properties of gram - ve bacteria are attributable to O-antigens.
The outer membrane is although impermeable to large molecules but can allow smaller molecules such as
nucleosides, oligosaccharide, monosaccharides, pep tides and amino acids. This is accomplished by means of
channels in special proteins called porins.

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 Gramnegative organisms differ drastically in the organisation of the structures
outside the plasma membrane but below the capsule: in Gram-negative organisms these structures
constitute the cell envelope, whereas in Gram-positive organisms they are called a cell walL
Most Gram-positive bacteria have a relatively thick (about 20 to 80 nm), continuous
cell wall, which is composed largely of peptidoglycan. In thick cell walls, other cell wall
polymers are covalently attached to the peptidoglycan. In contrast, the peptidoglycan
layer in Gram-negative bacteria is thin; 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. In most Gram-negative bacteria, this membrane structure is
anchored noncovalently to lipoprotein molecules, 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 organisation and overall dimensions of the outer membrane of the
Gram-negative cell envelope are similar to those of the plasma membrane. Moreover, in Gramnegative bacteria such as E coli, the
outer and inner membranes adhere to each other at several hundred sites; these sites
can break up the continuity of the peptidoglycan layer.
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 [Link] between the two groups has been confirmed by
sophisticated methods of electron miorost'oPY. Moreover, mechanical disruption of the
Bacteria: Structure and Functions 43

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.
Peptidoglycan

Unique features of almost all prokaryotic cells 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 bt 4linked disaccharides of b1,4-N-acetylmuramyl-N-acetylglucosamine.
Tetrapeptides of L-alanine-D-isoglutamic acid-L-Iysine -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 lysin"e 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. 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 are insensitive to penicillin, an inhibitor of the
transpeptidases involved in peptidoglycan biosynthesis in eubacteria.
The 15-1,4 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,
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
44 Introductory Microbiology

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
senitourinary 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 revert infrequently and only under
special conditions.
Teichoic Acids

Wall teichoic acids are found only in certain Gram-positive bacteria; so far, they have
not been found in gram- negative organisms. Substituent groups on the polyol chains
can include D-alanine, 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.
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
Bacteria: Structure and Functions 45

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 ~omposed of 2-keto3deoxyoctonic 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. 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.
LPS and phospholipids help confer asymmetry to the outer membrane of the Gramnegative
bacteria, with the hydrophilic polysaccharide chains outermost. Each LPS is
held in the outer membrane by relatively weak cohesive forces and can be dissociated
from the cell surface with surface-active agents. As in peptidoglycan b~osynthesis, LPS '

molecules are assembled at the plasma or inner membrane. These newly formed
molecules are initially inserted into the outer-inner membrane adhesion sites.
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 Grampositive
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 Gramnegative
bacteria. 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-Iactam antibiotics 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
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
46 Introductory Microbiology

metabolite-binding proteins and hydrolytic enzymes found in the periplasmic space. The
periplasmic space is the region between the outer surface of the inner membrane and
the inner surface of the outer membrane. 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 involved in membrane
transport and chemotactic receptor activities. Moreover, plasmid-encoded b-Iactamases
and aminoglycoside-modifying enzymes in the periplasmic space produce antibiotic
resistance by degrading or modifying an antibiotic in transit to its target sites on the
membr ane or on the ribosomes.

The Nucleoid
The nucleoid of a bacterial cell (see Figure 4.6) usually contains a
single long, continuous, and frequently circularly arranged thread
of double-stranded DNA called the bacterial chromosome. This
is the cells genetic information, which carries all the information
required for the cells structures and functions. Unlike the
chromosomes of eukaryotic cells, bacterial chromosomes are not
surrounded by a nuclear envelope (membrane) and do not include
histones. The nucleoid can be spherical, elongated, or dumbbellshaped.
In actively growing bacteria, as much as 20% of the cell
volume is occupied by DNA because such cells presynthesize nuclear
material for future cells. The chromosome is attached to the
plasma membrane. Proteins in the plasma membrane are believed
to be responsible for replication of the DNA and segregation of
the new chromosomes to daughter cells during cell division.
In addition to the bacterial chromosome, bacteria often contain
small usually circular, double-stranded DNA molecules
called plasmids (see the F factor in Figure 8.26a, page 234).
These molecules are extrachromosomal genetic elements; that is,
they are not connected to the main bacterial chromosome, and

they replicate independently of chromosomal DNA. Research

indicates that plasmids are associated with plasma membrane
proteins. Plasmids usually contain from 5 to 100 genes that are
generally not crucial for the survival of the bacterium under normal
environmental conditions; plasmids may be gained or lost
without harming the cell. Under certain conditions, however,
plasmids are an advantage to cells. Plasmids may carry genes for
such activities as antibiotic resistance, tolerance to toxic metals,
the production of toxins, and the synthesis of enzymes. Plasmids
can be transferred from one bacterium to another. In fact, plasmid
DNA is used for gene manipulation in biotechnology.

Ribosomes
All eukaryotic and prokaryotic cells contain ribosomes, which
function as the sites of protein synthesis. Cells that have high
rates of protein synthesis, such as those that are actively growing,
have a large number of ribosomes. The cytoplasm of a prokaryotic
cell contains tens of thousands of these very small structures,
which give the cytoplasm a granular appearance (see Figure 4.6).
Ribosomes are composed of two subunits, each of which consists
of protein and a type of RNA called ribosomal RNA (rRNA).
Prokaryotic ribosomes differ from eukaryotic ribosomes in the
number of proteins and rRNA molecules they contain; they
are also somewhat smaller and less dense than ribosomes of
eukaryotic cells. Accordingly, prokaryotic ribosomes are called
70S ribosomes (Figure 4.19), and those of eukaryotic cells are
known as 80S ribosomes. The letter S refers to Svedberg units, which
indicate the relative rate of sedimentation during ultra-high-speed
centrifugation. Sedimentation rate is a function of the size,
weight, and shape of a particle. The subunits of a 70S ribosome
are a small 30S subunit containing one molecule of rRNA and a
larger 50S subunit containing two molecules of rRNA.
Several antibiotics work by inhibiting protein synthesis on
prokaryotic ribosomes. Antibiotics such as streptomycin and
gentamicin attach to the 30S subunit and interfere with protein
synthesis. Other antibiotics, such as erythromycin and
chloramphenicol, interfere with protein synthesis by attaching
to the 50S subunit. Because of differences in prokaryotic and
eukaryotic ribosomes, the microbial cell can be killed by the antibiotic
while the eukaryotic host cell remains unaffected.

What properties make endospores resistant to processes that normally kill

vegetative cells?
Cell wall
Plasma
membrane
Bacterial
chromosome
(DNA)
Cytoplasm Spore septum begins to isolate
newly replicated DNA and a
small portion of cytoplasm.
(a) Sporulation, the process of endospore formation
(b) An endospore of Bacillus subtilis
Plasma membrane starts to surround DNA,
cytoplasm, and membrane isolated in step 1.
Peptidoglycan layer forms between membranes.
Spore coat forms.
1
2

3
4
5
6 Endospore is freed from cell.
Spore septum surrounds isolated portion,
forming forespore.
Two membranes
TEM 0.5 _ m

Endospore
Chapter 4 Functional Anatomy of Prokaryotic and Eukaryotic Cells 97

nitrogen source, becomes scarce or unavailable. In the first

observable stage of sporulation, a newly replicated bacterial
chromosome and a small portion of cytoplasm are isolated by
an ingrowth of the plasma membrane called a spore septum. The
spore septum becomes a double-layered membrane that surrounds
the chromosome and cytoplasm. This structure, entirely
enclosed within the original cell, is called a forespore. Thick
layers of peptidoglycan are laid down between the two membrane
layers. Then a thick spore coat of protein forms around
the outside membrane; this coat is responsible for the resistance
of endospores to many harsh chemicals. The original cell is degraded,
and the endospore is released.
The diameter of the endospore may be the same as, smaller
than, or larger than the diameter of the vegetative cell. Depending
on the species, the endospore might be located terminally (at
one end), subterminally (near one end; Figure 4.21b), or centrally
inside the vegetative cell. When the endospore matures, the vegetative
cell wall ruptures (lyses), killing the cell, and the endospore
is freed.
Most of the water present in the forespore cytoplasm is
eliminated by the time sporulation is complete, and endospores
do not carry out metabolic reactions. The endospore contains a
large amount of an organic acid called dipicolinic acid (DPA),
which is accompanied by a large number of calcium ions. Evidence
indicates that DPA protects the endospore DNA against
damage. The highly dehydrated endospore core contains only
DNA, small amounts of RNA, ribosomes, enzymes, and a few
important small molecules. These cellular components are essential
for resuming metabolism later.
Endospores can remain dormant for thousands of years.
An endospore returns to its vegetative state by a process called
germination. Germination is triggered by physical or chemical
damage to the endospores coat. The endospores enzymes then
break down the extra layers surrounding the endospore, water enters,
and metabolism resumes. Because one vegetative cell forms a
single endospore, which, after germination, remains one cell, sporulation
in bacteria is not a means of reproduction. This process does
not increase the number of cells. Bacterial endospores differ from
spores formed by (prokaryotic) actinomycetes and the eukaryotic

Clinical Case Resolved

It is the glycocalyx that enables bacteria in water to stick
inside a pipe. The bacteria grow slowly in the nutrient-poor
tap water but do not get washed away by the flowing
water. A slimy layer of bacteria can accumulate in a pipe.
Irene discovers that the disinfectant in the hospitals water
supply was inadequate to prevent bacterial growth. Some
bacteria can get dislodged by flowing water, and even
normally harmless bacteria can infect a surgical incision
or weakened host.

76 86 88 95 97


principal differences between prokaryotic and eukaryotic cells
are summarized in Table 4.2 page 100.
The following discussion of eukaryotic cells will parallel our
discussion of prokaryotic cells by starting with structures that
extend to the outside of the cell.
As mentioned earlier, eukaryotic organisms include algae, protozoa,
fungi, plants, and animals. The eukaryotic cell is typically
larger and structurally more complex than the prokaryotic
cell (Figure 4.22). When the structure of the prokaryotic cell in
Figure 4.6 is compared with that of the eukaryotic cell, the differences
between the two types of cells become apparent. The

The Eukaryotic Cell

fungi and algae, which detach from the parent and develop into another
organism and, therefore, represent reproduction.
Endospores are important from a clinical viewpoint and in the
food industry because they are resistant to processes that normally
kill vegetative cells. Such processes include heating, freezing, desiccation,
use of chemicals, and radiation. Whereas most vegetative
cells are killed by temperatures above 70C, endospores can
survive in boiling water for several hours or more. Endospores of
thermophilic (heat-loving) bacteria can survive in boiling water
for 19 hours. Endospore-forming bacteria are a problem in the
food industry because they are likely to survive underprocessing,
and, if conditions for growth occur, some species produce toxins
and disease. Special methods for controlling organisms that produce
endospores are discussed in Chapter 7.
Chec k Your Understanding

Where is the DNA located in a prokaryotic cell? 4-10

What is the general function of inclusions? 4-11
Under what conditions do endospores form? 4-12

***
Having examined the functional anatomy of the prokaryotic cell,
we will now look at the functional anatomy of the eukaryotic cell.