Bacterial Cell Wall

Beneath the external structure as capsules, sheaths, and Flagella and external to the
cytoplasmic membrane is the cell wall, a very rigid structure that gives shape to the cell. Its main
function is to prevent the cell from expanding and eventually bursting because of uptake of
water, since most bacteria live in hypotonic environments (i.e., environments having a lower
osmotic pressure than exists within the bacterial cells). The rigidity of the wall can be readily
demonstrated by subjecting bacteria to very high pressure or other severe physical conditions:
most bacterial cells retain their original shapes during and after such treatments. To obtain
isolated cell walls for analysis, bacteria usually must be mechanically disintegrated by drastic
means, as by sonic or ultrasonic treatment or by exposure to extremely high pressures with
subsequent release of pressure. The broken cell walls are then separated from the rest of the
components of the disintegrated cells by differential centrifugation. Isolated cell walls, devoid of
other cellular constituents, retain the original contour of the cells from which they were derived.
Among the ordinary or typical bacteria (which are sometimes called eubacteria to distinguish
them from the phylogenetically district group known as the archeabacteria), the wall of gram-
negative species are generally thinner (10 to 15 nm) than those of gram-positive species (20 to 25
nm). The walls of gram-negative archeabacteria are also thinner than those of gram-positive
arecheabacteria. Since the chemical composition of the walls of archeabacteria is quite different
from that of eubacteria, wall thickness rather than chemical composition may be the major factor
in the gram reaction.

The cell wall constitutes a significant portion of the dry weight of the cell; depending on
the species and culture condition, it may account for as much as 10 to 40 present. Bacterial cell
walls are usually essential for bacterial growth and division. Cells whose walls have been
completely removed (i.e., protoplasts) are incapable of normal growth and division.

The cell wall is one of the most important parts of a prokaryotic cell for several reasons.
Except for the mycoplasmas and some archeabacteria most bacteria have strong walls that give
them shape and protect them from osmotic lysis. The cell walls of many pathogens have
components that contribute to their pathogenicity. The wall can protect a cell from toxicity and is
the site of action of several antibiotics.

After Christian gram developed the gram stain is 1884, it soon became evident that
bacteria could be divided into two major groups based on their response to the gram-stain
procedure. Gram-positive bacteria stained purple whereas gram-negative bacteria were coloured
pink or red by the technique. The true structural difference between these two groups become
clear with the advent of the transmission electron microscope. The gram-positive cell wall
consists of a single 20 to 80nm thick homogeneous peptidoglycan or murein layer lying outside
the plasma membrane. In contrast, the gram-negative cell wall is quite complex. It has a 1 to 3nm
peptidoglycan layer coated by a 7 to 8 nm thick outer membrane. Microbiologists often call all
the structures outside the plasma membrane the envelope. This includes the wall and structures
like capsules when present.
 Frequently a space is seen between the plasma membrane and the outer membrane in
electron micrographs of gram-negative bacteria, and sometimes a similar but smaller gap is
observed between the plasma membrane and wall in gram-positive bacteria, this space is called
the periplasmic space or periplasm. Recent evidence indicates that the periplasmic space may be
filled with a loose network of peptidoglycan. Possibly it is more a gel than a fluid –filled space.
Size estimates of the periplasmic space in gram-negative bacteria range from 1nm to as great as
71 nm. Some recent studies indicate that it may constitute about 20 to 40% of the total cell
volume ( around 30 to 70nm), but more research is required to establish an accurate value. When
cell walls are disrupted carefully or removed without disturbing the underlying plasma
membrane, periplasmic enzymes and other proteins are released and may be easily studied. The
periplasmic space of gram-negative bacteria contains many proteins that participate in nutrient
acquisition-for example, hydrolytic enzymes attacking nucleic acids and phosphorylated
molecules, and binding proteins involved in transport of materials into the cell. Gram-positive
bacteria do not appear to have as many periplasmic proteins; rather, they recreate several
enzymes that ordinarily would he periplasmic in gram- negative bacteria. Such recreated
enzymes are often called exoenzymes.

The recently discovered archaeabacteria differ from other bacteria in many respects.
Although they may be either gram positive or gram negative, their cell walls are distinctive in
structure and chemical composition. The walls lack peptidoglycan and are composed of proteins,
glycoproteins or polysaccharides.

Following this one view of the envelope, peptidoglycan structure and the organisation of
gram-positive and gram-negative cell walls are discussed in more detail.

Function

1. It gives shape to the cell which is the characteristic of the bacterial species. The shape is
genetically determined and given by the cell wall.
2. It gives protection to the bacterial cells against the destruction or lysis by osmotic shock.
The structure without cell wall and the contents are cover by cell membrane then they are
spherical and has no shape at all. Then it is called protoplast. Cell wall is a rigid cross-
linked structure which is resistant to the effect of the huge osmotic pressure difference
between the external and inside of the cell. So, they have no higher osmotic shock.

Structure and chemical composition

Peptidoglycan
For eubacteria, the shape-determining part of the cell wall is largely peptidoglycan (sometimes
called murein), an insobluble, proteins, cross-linked polymer of enormous strength and rigidity.
Peptidoglycan is found only in prokaryotes; it occurs in the from of a “bag-shaped
macromolecule” outside the cytoplasmic membrane. Peptidoglycan differs somewhat in
composition and structure from one species to another but it is basically a polymer of N-
acetylglucosamine and N-acetylmuramic acid. L-alamine,(humans have only this L-form), D-
alamine, D-glutamate, and a diamino acid LL-or mero-diaminopimlic acid an important/unique
feature of peptidoglycan, L-Lysine, L-osmithine, or L-diaminobutyric acid. It is important to
realize that as though as peptidoglycan is, it is also in a dynamic state. That is, in order for the
cell to grow and divide, portions of the peptidoglycan must continually be degraded by well-
associated hydrolytic enzymes so that new polymer can be added.
Peptidoglycan or murein is an enormous polymer composed of many identical subunits.
The polymer contains two sugar derivatives N-acetylglucosamine and N-acetylmuramic acid (the
lactyl ether of N-acetylglucosamine), and several different amino acids, three of which –D-
glutamic acid, D-alanine and meso-diaminopimelic acid – are not found in proteins. The
peptidoglycan submit present in most gram-negative bacteria and many gram-positive ones as
the name comprises is composed of peptides and glycan (a polysaccharide of glucose). The
backbone of this polymer is composed of alternating N-acetylglucosamine and N-acetylmuramic
acid residues. A peptide chain of four alternating D and L-amino acids is connected to the
carboxyl group of N-acetylmuramic acid. Many bacteria substitute another diamino acid, usually
L-lysine, in the third position for meso-diaminopimelic acid.
Chains of linked peptidoglycan submits are joined by cross-links between the peptides. Often the
carboxyl group of the terminal D-alanine is connected directly to the amino group of
diaminopimelic acid, but a peptide interbridge may be used instead. Most gram-negative cell
wall peptidoglycan lacks the peptide interbridge. This cross linking results in an enourmous
peptidoglycan sac that is actually one dense, interconnected network. These sacs have been
isolated from gram-positive bacteria and are strong enough to retain their shape and integrity, yet
they are elastic and somewhat stretchable, unlike cellulose. They also must be porous, as
molecules can penetrate them.
Synthesis of Macromolecules : The Structure and Bio-synthesis of a cell-wall peptidoglycan
In all cells the major end products of biosynthesis are proteins and nucleic acids. However, there
are other macromolecules peculiar to the prokaryotes which requires specialized biosynthetic
processes. The utilization of energy in one of these processes is illustrated by the biosynthesis of
bacterial cell-wall peptidoglycan. This particular biosynthetic process also serves as an example
of how polymers are synthesized outside the membrane. Synthesis of cell-wall components is of
interest because polymerization takes place outside the cell membrane by enzymes located on the
membrane outer surface.
Structure of peptidoglycan
As discussed the rigid portion of a bacterial cell wall is a polymeric structure known as a murein,
peptidoglycan, or mucopeptide. The walls of gram-positive bacteria contain a large proportion of
peptidoglycan; those of gram-negative bacteria have a much smaller proportion.
Fig :- General structure of peptidoglycans.
Peptidoglycans vary in their chemical composition and structure from species to species, but
there are basic similarities. Peptidoglycans are very large polymers composed of three kinds of
building blocks: 1)Acetylglycosamine (AGA Glc NAc),2) Acetylmuramic acid (AMA or Mur
NAc), and 3)A peptide consisting of four or five amino acids of limited variety. Several of the
amino acids exist in the D configuration, not usually found elsewhere in nature. A peptidoglycan
can best be thought of as consisting of polysaccharide backbone chains composed of alternating
units of AGA and AMA linked by β(1→4) bonds, with the short peptide chains projecting from
the AMA units. Many of these peptide chains are cross-linked with each other, imparting great
rigidity to the total structure. Some peptidoglycans differ in that the peptide chains may not be
directly cross-linked to each other,being linked instead by another kind of peptide which forms a
bridge between the terminal carboxyl group of one side chain with the free amino group of lysine
or diaminopimelic acid (DPM or DAP) on the other side chain;,e.g.; in Staphylococcus aureus a
bridge composed of five glycine molecules can link two muramic acid peptides together.
Activation of a peptidoglycan precursor
Escherichia coli can synthesize cell wall peptidoglycan when grown in a simple medium
of glucose, ammonium sulfate, and mineral salts. One of the early steps in this synthesis is the
formation of an activated derivative of AMA. This process requires energy at several paints and
occurs in the cytoplasm. The activation of sugars, such as acetyl glucosamine, by the attachment
of a uridine diphosphate (UDP) to form a sugar-UDP precursor is not peculiar to AMA but is a
general method involved in the biosynthesis of many kinds of polysaccharides.
Fig :- Biosynthesis of acetylglucosamine-UDP and acetyl-muramic acid-UDP, key
precursors in the synthesis of peptidoglycans.
This process takes place in cytoplasm. The high energy compounds are ATP, ADP, Pi, Acetyl
CoA, CoA, UTP, PEP, NADPH and NADP+.
Synthesis of Peptidoglycan
After formation of the activated AMA, the synthesis of peptidoglycan proceeds as
follows:
1) Amino acids are sequentially to the AMA portion of the activated precursors to form a
short pentapeptide chain. Ribosomes are not involved, but each amino acid addition
required energy from the breakdown of ATP and the presence of Mg +2 or Mn+2 and a
specific enzyme. These reactions occur in the cytoplasm.
2) The AMA-UDP processor is coupled to a membrane phospholipid called bactoprenol
(indecaprenol phosphate).
3) The AGA couples with AMA of the AMA-UDP precursor. This reaction requires the
activated form of AGA, that is the AGA-UDP derivative. In some organisms, the addition
of bridging peptides takes place at this step. Reactions of steps 2 and 3 occur in the cell
membrane.
4) The precursor, still linked to bactoprenol, is carried out of the cell though the cell
membrane and is linked to a growing peptidoglycan chain in the cell wall. Peptide cross-
linking may now occur, and the incorporation of the precursor into the growing
peptidoglycan is thus completed. These reactions occur in the periplasm.
The synthesis of peptidoglycan illustrates the utilization of energy in joining together smaller
molecules into larger ones. Note that all the energy needed for polymerization is used in the
cytosolic (cytoplasmic) reactions in synthesizing the activated precursors. Other macromolecule
synthesis requires a template which, acting like a tape provides information about the order in
which the smaller pieces are assemble into larger ones, such processes include DNA synthesis
(another piece of DNA is the template) and protein synthesis (a molecule of RNA serves as
template).
The Mechanism of Gram-Staining
Although several explanations have been given for the gram stain reaction results, it
seems likely that the difference between gram-positive and gram-negative bacteria is due to the
physical nature of their cell walls. If the cell wall is removed from gram-positive bacteria they
become gram negative. The peptidoglycan itself is not stained; instead, it seems to act as a
permeability barrier preventing loss of crystal violet. During the procedure, the bacteria are first
stained with crystal violet and next treated with iodine to promote dye retention. When gram
positive bacteria then are decolorized with ethanol, the alcohol (or alcohol – acetone mixture) is
thought to shrink the pores of the thick peptidoglycan. Thus, the dye-iodine complex is retained
during the short decolorization step and the bacteria remain purple. In contrast, gram-negative
peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol treatment
also may extract enough lipid from the gram-negative wall to increase its porosity further. For
these reasons, alcohol more readily removes the purple crystal violet-iodine complex from gram-
negative bacteria.
Wall of Gram-Positive Eubacteria
Gram-Positive bacteria usually have a much greater amount of peptidoglycan in their cell walls
than do gram-negative bacteria; it may account for 50 percent or more of the dry weight of the
wall of some gram-positive species, but only about 10 percent of the wall of gram-negative
bacteria. Other substances may occur in addition to peptidoglycan.
1) For instance the walls of Streptococcus pyogens contain polysaccharides that are
covalently linked to the peptidoglycan and which can be extracted without dilute
hydrochloric acid.
2) Normally, the thick, homogeneous cell wall of gram-positive bacteria is composed
primarily of peptidoglycan which often contains a peptide inter-bridge. However, gram-
positive cell walls usually also contain large amounts of teichoic acids, polymers of
glycerol or ribitol joined by phosphate groups. Amino acids such as D-alanine or sugars
like glucose are attached to the glycerol and ribitol groups. The teichoic acids are
connected to either the peptidoglycan itself or to plasma membrane lipids; in the latter
case. They are called lipoteichoic acids. Teichoic acids appear to extend to the surface of
the peptidoglycan, and because they are negatively charged, help give the gram-positive
cell wall into negative charge. The functions of these molecules are still unclear, but they
may be important in maintaining the structure of the wall. Teichoic acids are not present
in gram-negative bacteria.
The walls of Staphlylococcus aureus and Streptococcus faecalis contain teichoic
acids –Acidic polymers of ribitol phosphate or glycerol phosphate which are covalently
linked to peptidoglycan and which can be extracted with cold dilute acid. Teichoic acids
bind magnesium ions, and there is some evidence that they help to protect bacteria from
 thermal injury by providing an accessible pool of these cations for stabilization of the
cytoplasmic membrane.
3) The walls of most gram-positive bacteria contain very little lipid, but share of
Mycobacterium, Cornybacterium, and certain other genera are exceptions, being rich in
lipids. There compounds have the following general structure :
R1-CH-CH-COOH
OH R2
Where R1 and R2 are long hydrocarbon chains. The ability of mycobacteria to exhibit acid-fast
staining (i.e.; when stained, the cells cannot be decolorized easily despite treatment with dilute
acids) is correlated with the presence of cell wall mycolic acids. A mycolic acid derivative called
cord factor (trehalose dimycolate) is toxic and plays an important role in the disease caused by
[Link] and M. tulerculosis.

Fig: - The Gram-positive Envelope

4) Several protein structure also remain attached to this peptidoglycan layer eg. is M
proteins as is present in case of Streptococcus pyogens. This is important for the
pathogenesis of the particular bacteria. As the afforesaid one causes rheumatic fever,
throat infections etc. All these structures give pathogenic potential for bacteria.
Several antibiotics like Penicillin, is very important for the destruction of peptidoglycan.
Thus such antibiotics that which are like penicillin like streptomycin etc. are used against
gram-positive bacteria. It destroys the crossbridging in the peptidoglycan layer. But such
antibiotics have no effect on human cells as they do not have peptidoglycan layer.
Different enzymes like lysozyme which is present in many secretions like saliva, tears and thus
forms the first line defense mechanism it too destroys the peptidoglycan layer. But it should be
kept in mind that no enzyme or antibiotics can restrict the transport across the gram-positive
bacterial cell wall as it is very porous. It only dehydrating agents like alcohol which cannot
dissolve it, but dries it reducing the pore size thus, the porosity is decreased. This is the reason
why in gram-staining the dye-iodine complex is not washed out of the cell and it is stained
purple.
 But the defect of this process is that, as these agents destroy the peptidoglycan layer only,
it is changed to protoplast, i.e.; no cell wall and it behaves like the gram-negative bacteria.
Gram-negative cell walls
The walls of the gram-negative bacteria are more complex than those of gram-positive
bacteria. The walls of gram-negative bacteria have a comparatively low peptidoglycan content,
seldom exceeding 5 to 10 % of the weight of the wall. The location of the peptidoglycan layer in
this type of wall was first established by W. Weidel and his collaborators, for walls of
Escherichia coli. In E. coli, it is about 1nm thick and contains only one or two layers or sheets
of peptidoglycan. As mentioned earlier, the peptidoglycan may be in the form of a gel matter
than a compact layer. They showed that the peptidoglycan constitutes the inner most layer of the
multi layered wall and can be isolated as a very thin sac that retains the form and shape of the
original cell, other wall components have been stripped off it by appropriate treatments.
The peptidoglycans of gram-negative bacteria characteristically display a rather low
degree of cross-linkage between the glycan strands: many of the peptide chains are not cross-
linked. The thickness of the peptidoglycan layer of the wall varies somewhat in different groups
of gram-negative bacteria. Calculation suggest that in many gram-negative organisms it is a
monomolecular (or at most bimolecular) layer.
There are several other structure present in gram- negative cell wall than is present in
gram positive one. These additional structures are important for the additional stability of the
gram-negative bacteria. They confer together with the peptidoglycan, protection, stability and
strength to the cell wall. The most interesting difference is the presence of an outer membrane
that surround a thin underlying layer of peptidoglycan.
The outer membrane
Superimposed on the thin murein sac characteristic of gram-negative bacteria is an outer layer
that has the width and fine structure typical of a unit membrane. This layer, the outer membrane,
has some chemical and physical proteins in common with the cell membrane, and others that are
quite different. Like the cell membrane it is a lipid bilayer containing phospholipids and proteins,
but in addition it contains large amounts of a unique lipid, lipopolysaccharide (LPS), which
replaces, probably completely phospholipids in the outer leaf of this unique structure. Although
chemically quite different from a phospholipids, LPS has physical properties that are sufficiently
similar so that it can participate in forming a membrane; one end of the molecule is hydrophobic
and the other is hydrophilic; the hydrophobic end becomes inserted in the membranes
hydrophobic core and the hydrophilic end is on the outer surface.
The lipopolysaccharides are complex molecules with MW over 10,000 that vary widely
in chemical composition, both within and between gram-negative groups. Most work on their
structure has been conducted on the forms present in the Salmonella group. (Salmonella
typhimurium mainly).
LPS is composed of three distinct regions; lipid A, the R core region/ core
polyvaccharide, and the O side chain/ O antigen. Lipid A, the hydrophobic membrane-anchoring
region of LPS, rather than carrying the two fatty acid residence typical of a phospholipid has six
or seven attached to a phosphorylated glucosamine dimer. Unlike those in phospholipids, all the
fatty acids in lipid A are saturated. Some are attached directly to the glucosamine dimer and
others are esterified to the 3-hydroxy fatty acids that are characteristically present. Attached to
the 6 position of one glucosamine residue in lipid A is the R core oligosaccharide a short chain of
sugars, which include two unusual ones, 2-keto-3- dcoxyoctonoic acid (KDO) and heptose. In
Salmonella, it is constructed of ten sugars, many of them unusual in structure. The R core in turn
bears the hydrophilic O side chain, likewise composed of sugars. It is much longer than the R
core, being composed of many repeating tetra-or pentasaccharide units. The elucidation of this
structure depended heavily on the availability of mutants, each blocked at a particular point in
LPS biosynthesis. Biosynthesis of LPS is strictly sequential, starting with lipid A from which the
ohigosaccharide is built by successive sugar additions, the O side chain being added last. The O
side chain extend like whisks from the membrane surface into the surrounding medium. Many of
the serological properties of gram-negative bacteria are attributable to O antigens; they can also
serve as receptors for bacteriophage attachment. It has several peculiar sugars and varies in
composition between bacterial strains. Although O side chains are readily recognized by host
antibodies, gram-negative bacteria may thwart lost defenses by rapidly changing the nature of
their O side chains to avoid detection. Antibody interaction with the LPS before reaching the
outer membrane proper may also protect the cell wall from direct attack. The O antigen can be
divided into different serological groups according to the type of Ab formation triggered in the
serum. Thus it is important for its virulence. The LPS has tonic properties and is also known as
endotoxin. The innermost region, consisting of lipid A and three residues of KDO, appears to be
essential, but the rest of the molecule is dispensable.
The peptidoglycan layer of the wall bears a small (MW=7,200) specific type of
lipoprotein, termed murein lipoprotein, which forms an anchoring bridge to the outer membrane.
The C-terminus of this protein is a lysine residue which is peptide bonded to an amino group of a
meso-diamino-pimelic acid residue that is not cross-linked in the peptidoglycan layer. At the
other end (the N-terminus) of the protein is a cysteine residue to which fatty acids are attached :
one is attached in an amide linkage to the terminal amino group, and two more are esterified to a
glycerol residue which is attached by sulfur ether linkage to the cysteine.

The resulting brush like structure composed of the hydrophobic chains of these fatty
acids becomes inserted into the inner leaf of the outer membrane, there by anchoring it to the
peptidoglycan layer.

The physiological significance of the outer membrane is threefold: (1) it forms the outer
limit of the periplasm, the region between the two membranes that contain a set of digestive
enzymes. (2) It presents an outer surface with strong negative charge which is important in
evading phagocytosis and the action of complement and (3) It provides a permeability barrier
and there by increased resistance to a number of toxic agents. Among these are host defense
agents like lysozyme, beta-lysin, and leucocyte protein, which are quite toxic to gram-positive
bacteria which lack an outer membrane. Destructive agents in the mammalian digestive tract,
such as bile salts and digestive enzymes and a variety of antibiotics, are also excluded by the
outer membrane.

The LPS is important for several reasons other than the avoidance of host defences. Since
the core polysaccharide, usually contains charged sugars and phosphate, LPS contributes to the
negative charge on the bacterial surface. Lipid A is a major constituents of the outer membrane,
and the LPS helps stabilize membrane structure. Further-more lipid A often is toxic; as a result ,
the LPS can act as an endo toxin and cause some of the symptoms that arise in gram-negative
bacterial injections.

Thus the outer membrane is the outermost layer of the cell wall. Capsule [Link] be
present above it but they are not a part of cell wall structure. Because of this membrane, the walls
of gram negative bacteria are rich in lipids (11 to 22 percent of the dry weight of the wall), in
contrast to those of gram-positive bacteria. This outer membrane serves as an impermeable
barrier to prevent the escape of important enzymes, such as those involved in cell wall growth,
from the space between the cytoplasmic membrane and the outer membrane (periplasmic space).
The outer membrane also serves as a barrier to various external chemicals and enzymes that
could damage the cell. As most of the enzymes are protein in nature thus it is hydrophilic, or
water soluble or polar. Whereas the outer membrane is rich in lipid and thus is hydrophobic, thus
it has a repelling tendency towards the polar substance. But the lipid nature substance can easily
pass through it which water and hydrophilic substances cannot. For example, the walls of many
gram-positive bacteria can be easily destroyed by treatment with an enzyme called lysozyme.
Which selectively dissolves peptidoglycan; however gram-negative bacteria are refractory to this
enzyme because large protein molecules cannot penetrate the outer membrane. Only if the outer
membrane is first damaged, as by removal of stabilizing magnesium ions by a chelating agent,
can the enzyme penetrate and attack the underlying peptidoglycan layer.

A most important outer membrane function is to serve as a protective barrier. It prevents

or slows the entry of bile salts, antibiotics, and other toxic substance that might kill or injure the
bacterium. Even so, the outer membrane is more permeable than the plasma membrane and
permits the passage of small molecules like glucose and other monosaccharides. This is due to
the presence of special porin proteins. Three porin molecules cluster together and span the outer
membrane to form a narrow channel though which molecules smaller than about 600 to 700
daltons can pass. Larger molecules such as vitamin B12 must be transported across the outer
membrane by specific carriers. The outer membrane also prevents the loss of constituents like
periplasmic enzymes.

Although impermeable to large molecules such as proteins, the outer membrane can
allow smaller molecules, such as nucleosides, oligasaccharides, peptides and amino acids, to pass
across. This is accomplished by means of channels in special proteins called porins, which span
the membrane. The various porins are specific for different kinds or classes of small molecules,
and some can even allow certain essential large molecules to penetrate, such as vitamin B 12.
Many porins also serve as receptors for attachment of bactteriophages and bacteriocins. The
porins are thus a type of integrated protein which is specialized for various ions and water. The
ions channels formed by the porins have their open state and closed state regulated by the
pressure gradient or difference in the osmotic pressure on both the sides of the cell wall, i.e.;
inside and outside the cell and by electrical gradient.

Of course the outer membrane cannot present a barrier to all substance in the
environment because all cellular nutrients most pass through it. Permeability of the outer
membrane to nutrients is provided in part by proteins collectively termed porins which, in
aggregates generally of three, form cross-membrane channels through which certain small
molecules can diffuse. A variety of different porins are present in the outer membrane. They vary
with respect to the size of the channel they form and the environmental conditions that stimulate
their synthesis. For example, in E. coli the pore formed by Omp F (outer membrane protein F) is
slightly larger than the one formed by Omp C. The synthesis of Omp F is repressed by elevated
temperature (>37°c) and by growth in a medium of elevated osmotic pressure. The physiological
rationale for this regulation of the synthesis of Omp F is presumed to be a mechanism of reusing
whether the cell finds itself within a eucaryotic host or in an external environment. In the latter,
usually cooler environment, the concentration of substrate is typically quite low; this necessitates
the presence of larger pores formed by Omp F to allow diffusion of substrate molecules to occur
at a greater rate, because rate of diffusion is proportional to the product of concentration
difference across the membrane and cross-sectional area of the pore. Within a host where the
concentration of substrates is typically much higher, the larger pore is unnecessary and even
detrimental because antibacterial substance present in the host can enter more readily through
these larger pores.

In addition to the nonspecific channels formed by porins, the outer membrane contains a
variety of channels formed by other proteins that exhibit a remarkable specificity. For example,
the channel sometimes called the maltoporin, formed by the inducible Lam B protein,
specifically allow the diffusional entrance of the disaccharide maltose and maltodextrans into the
cell. Maltotriose diffuses through these channels at 100 times the rate of the similar-sized
trisaccharide, raffinose. Presumably, proteins that bind tightly to a specific substrate are
associated with these channels, thereby conferring specificity on them.

In addition to the channel- forming proteins, a protein termed Qmp A is quite abundant in
the outer membrane. Its specific role has not been clearly defined, but mutant strains that lack it
produce a more fragile outer membrane, so we assume that Qmp A contributes in some way to
the membranes structural integrity.

Although proteins constitute about half the mass of the outer membrane, until recently it
was assumed that the member of different types of proteins located there was quite limited. Now
it is clear that a large variety of different proteins are present in small quantities. With few
exceptions, proteins in the outer membrane are not found in the cytoplasmic membrane.

The molecular basis of a remarkable property of the outer membrane that distinguishes it
from other membrane, namely its impermeability to hydrophobic molecules is not yet
understood, but this property accounts for resistance to certain dyes (eg. eosin, methylene blue
and brilliant green) that are used in certain selective media.

The most abundant membrane protein is Braun’s lipoprotein, a small lipoprotein

covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its
hydrophobic end. The outer membrane and peptidoglycan are so firmly linked by this lipoprotein
that they can be isolated as one unit.
 One of the questions posed by the structure of gram-negative cell walls is; How can
water- insoluble, liphophilic substances such as LPS pass from their place of synthesis within the
cytoplasm and cytoplasmic membrane across a waterly periplasmic space to be inserted into the
outer membrane? A likely explanation has been provided by the discovery of numerous
adhesions or points of direct contact between the two membranes. These adhesions seem to be
the export sites for newly synthesized LPS and porins, and they are also the sites at which pili
and flagella are made.

Macromolecular Surface Array

The cell walls of some bacteria, both gram-negative and gram-positive are covered by a mosaic
layer of protein subunits. The functions of these mosaic layers are not well understood, but at
least one function is to protect gram-negative bacteria against attack and penetration by other
small, predatory bacteria known as bdellovibrios.

Fig: - The gram-negative Envelope

The cell wall and Osmotic protection

The cell wall is usually required to protect bacteria against destruction by osmotic pressure.
Solutes are much more concentrated in bacterial cytoplasm than in most microbial habitats,
which are hypotonic. During osmosis, water moves across selectively permeable membranes
such as the plasma membrane from dilute solutions (higher water concentration) to more
concentrated solutions (lower water concentration). Thus, water normally enters bacterial cells
and the osmotic pressure may reach 20 atmospheres or 300 pounds/square inch. The plasma
membrane cannot withstand such pressures and the cell will swell and lyse, or be physically
disrupted and destroyed, without the wall that protects it by resisting cell swelling. Solutes are
more concentrated in hypertonic habitats than in the cell. Thus, water flows outward and the
cytoplasm shrivels up and pulls away from the cell wall. This phenomenon is known as
plasmolysis and is useful in food preservation because many microorganisms cannot grow in
dried foods and jellies as they cannot avoid plamolysis.

The importance of the cell wall in protecting bacteria against osmotic lysis is demonstrated by
treatment with lysozyme or penicillin. The enzyme lysozyme attacks peptidoglycan by
hydrolyzing the bond that connects N-acetylmuramic acid with carbon four of N-
acetylglucosamic. Penicillin inhibits peptidoglycan synthesis. If bacteria are incubated with
penicillin in an isotonic solution, gram positive bacteria are converted to protoplasts that
continue to grow normally when isotonicity is maintained even though they completely lack a
wall. Gram-negative cells retain their outer membrane after penicillin treatment and are
classified as spheroplasts because some of their cell wall remains. Protoplasts and spheroplasts
are osmotically sensitive. If they are transferred to a dilute solution, they will lyse due to
uncontrolled water influx.

Although most bacteria require an intact cell wall for survival, some have none at all. For
example, the mycoplasmas lack a cell wall, yet often can grow in dilute media or terrestrial
environments because their plasma membranes are stronger than normal. The precise reason for
this not known, although the presence of sterols in the membranes of many species may provide
added strength. Without a rigid cell wall, mycoplasmas tend to be pleomorphic or variable in
shape.

The L forms (named after the Lister Institute in London where they were discovered) also
lack cell walls. The loss may be complete or partial (some have a defective wall), and the parent
organism may be either gram positive or gram negative. They are pleomorphic like mycoplasmas
and continue to reproduce. These organism can arise through spontaneous mutations or from
treatments such as growth in isotonic or hypertonic media containing penicillin. If all traces of the
peptidoglycan disappear, bacteria cannot re-synthesize it because preexisting wall is necessary to
construct new peptidoglycan. The L forms which cannot revert back to normal are bacteria in which
the primer for peptidoglycan synthesis has been either eliminated or modified by penicillin
treatment. In this case, the L form may be stable; (stable L form), that is, it may continue to grow
and reproduce after the penicillin treatment has ceased. All unstable L forms, and certain stable
ones, still contain muramic, but the concentration is relatively low (about 10-15% of its
concentration in normal cells). Furthermore, the muramic acid is in an unusual chemical state, being
readily extractable with dilute acid, whereas the muramic acid in a normal cell is not. Other L forms
sometimes synthesize a wall again. L forms retain O antigens and are still susceptible to infection
by phages for which the receptors are contained in the outer wall layer. These L forms were first
identified in Streptobacillus moniliforms who normally are rod shaped. L forms are not closely
related to mycoplasmas and should not be confused with them.
Fig: - Protoplast formation