Micro 503 -Microbial physiology and
metabolism
Topic - Synthesis And Assembly Of Cell Wall
Component
Submitted by:
Tiwari Jyoti Bhaiyalal
M.sc(Agri) Microbiology
The Procaryotic Cell Wall
• The cell wall is the layer, usually fairly rigid, that lies just outside the
plasma membrane. It is one of the most important parts of a procaryotic
cell for several reasons.
• Except for the mycoplasmas and some Archaea, most bacteria have
strong walls that give them shape and protect them from osmotic lysis ;
wall shape and strength is primarily due to peptidoglycan.
• The cell walls of many pathogens have components that contribute to
their pathogenicity. The wall can protect a cell from toxic substances
and is the site of action of several antibiotics.
• After Christian Gram developed the Gram stain in 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 colored
pink or red by the technique.
• The gram-positive cell wall consists of a single 20 to 80 nm thick homogeneous
peptidoglycan or murein layer lying outside the plasma membrane.
• In contrast, the gram-negative cell wall is quite complex. It has a 2 to 7 nm
peptidoglycan layer surrounded by a 7 to 8 nm thick outer membrane.
• Because of the thicker peptidoglycan layer, the walls of gram-positive cells are stronger
than those of gram-negative bacteria.
• Microbiologists often call all the structures from the plasma membrane outward the
envelope or cell 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 may be observed between the plasma
membrane and wall in gram positive bacteria. This space is called the periplasmic space.
• Gram-positive cells may have periplasm even if they lack a discrete, obvious periplasmic space. Size estimates
of the periplasmic space in gram-negative bacteria range from 1 nm to as great as 71 nm.
• 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.
• Denitrifying and chemolithoautotrophic bacteria often have electron transport proteins in their periplasm. The
periplasmic space also contains enzymes involved in peptidoglycan synthesis and the modification of toxic
compounds that could harm the cell.
• Gram positive bacteria may not have a visible periplasmic space and do not appear to have as many periplasmic
proteins; rather, they secrete several enzymes that ordinarily would be periplasmic in gram-negative bacteria.
Such secreted enzymes are often called exoenzymes. Some enzymes remain in the periplasm and are attached to
the plasma membrane.
Peptidoglycan Structure
• Peptidoglycan Subunit Composition. The peptidoglycan
subunit of Escherichia coli, most other gram-negative
bacteria, and many gram-positive bacteria. NAG is N-
acetylglucosamine. NAM is N-acetylmuramic acid (NAG
with lactic acid attached by an ether linkage). The
tetrapeptide side chain is composed of alternating D- and
L-amino acids since meso-diaminopimelic acid is
connected through its L-carbon. NAM and the tetrapeptide
chain attached to it are shown in different shades of color
for clarity
Gram-Positive Cell Walls
• Normally the thick, homogeneous cell wall of gram-positive bacteria is composed
primarily of peptidoglycan, which often contains a peptide interbridge.
• 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 by a covalent bond
with the six hydroxyl of N-acetylmuramic acid 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 its 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 Gram-Positive Envelope. Teichoic Acid Structure. The segment of a
teichoic acid made of phosphate, glycerol, and
a side chain, R. R may represent D-alanine,
glucose, or other molecules
Gram-Negative Cell Walls
• Gram-negative cell walls are much more complex than gram-positive walls. The thin peptidoglycan layer next to
the plasma membrane may constitute not more than 5 to 10% of the wall weight.
• In E. coli it is about 2 nm thick and contains only one or two layers or sheets of peptidoglycan. The outer
membrane lies outside the thin peptidoglycan layer.
• Another structure that may strengthen the gram-negative wall and hold the outer membrane in place is the adhesion
site. The outer membrane and plasma membrane appear to be in direct contact at many locations in the gram-
negative wall.
• Possibly the most unusual constituents of the outer membrane are its lipopolysaccharides (LPSs). These large,
complex molecules contain both lipid and carbohydrate, and consist of three parts: (1) lipid A, (2) the core
polysaccharide, and (3) the O side chain.
• The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or
pyrophosphate attached.
• It is buried in the outer membrane and the remainder of the LPS molecule projects from the surface. The core
polysaccharide is joined to lipid A.
• The O side chain or O antigen is a polysaccharide chain extending outward from the core. 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 host 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 Gram-Negative Envelope
Lipopolysaccharide Structure.
Cells That Lack Cell Walls
• Although most prokaryotes cannot survive in nature without their cell walls,
some do so naturally.
• These include the mycoplasmas, a group of pathogenic bacteria that causes
several infectious diseases of humans and other animals, and the Thermoplasma
group, species of Archaea that naturally lack cell walls.
• These bacteria are able to survive without cell walls because they either contain
unusually tough cytoplasmic membranes or because they live in osmotically
protected habitats such as the animal body.
• Most mycoplasmas have sterols in their cytoplasmic membranes, and these
probably function to add strength and rigidity to the membrane as they do in the
cytoplasmic membranes of eukaryotic cells.
Peptidoglycan Synthesis and Cell Division
• In most cocci, cell walls grow in opposite directions outward from the FtsZ ring, whereas the walls of rod-
shaped cells grow at several locations along the length of the cell.
• However, in both cases preexisting peptidoglycan has to be severed to allow newly synthesized
peptidoglycan to be inserted. How does this occur?
• Beginning at the FtsZ ring, small gaps in the wall are made by enzymes called autolysins, enzymes that
function like lysozyme to hydrolyze the β-1,4 glycosidic bonds that connect N-acetylglucosamine and N-
acetylmuramic acid in the peptidoglycan backbone.
• New cell wall material is then added across the gaps. The junction between new and old peptidoglycan
forms a ridge on the cell surface of gram-positive bacteria called a wall bandanalogous to a scar. It is of
course essential in peptidoglycan synthesis that new cell wall precursors (N-acetylmuramic acid/N-
acetylglucosamine/tetrapeptide units) be spliced into existing peptidoglycan in a coordinated and consistent
manner in order to prevent a breach in peptidoglycan integrity at the splice point; a breach could cause
spontaneous cell lysis, called autolysis
Biosynthesis of Peptidoglycan
• The peptidoglycan layer can be thought of as a stress-bearing fabric, much like a thin
sheet of rubber. Synthesis of new peptidoglycan during growth requires the controlled
cutting of preexisting peptidoglycan by autolysins along with the simultaneous insertion
of peptidoglycan precursors.
• A lipid carrier molecule called bactoprenol plays a major role in this process.
• Bactoprenol is a hydrophobic C55 alcohol that bonds to a N-acetylglucosamine/N-
acetylmuramic acid/pentapeptide peptidoglycan precursor.
• Bactoprenol transports peptidoglycan precursors across the cytoplasmic membrane by
rendering them sufficiently hydrophobic to pass through the membrane interior.
• Once in the periplasm, bactoprenol interacts with enzymes called transglycosylases that
insert cell wall precursors into the growing point of the cell wall and catalyze glycosidic
bond formation.
Peptidoglycan synthesis
Transpeptidation
• The final step in cell wall synthesis is transpeptidation. Transpeptidation forms the peptide cross-
links between muramic acid residues in adjacent glycan chains.
• In gram-negative bacteria such as Escherichia coli, cross-links form between diaminopimelic acid
(DAP) on one peptide and D-alanine on the adjacent peptide.
• Initially, there are two D-alanine residues at the end of the peptidoglycan precursor, but only one
remains in the final molecule as the other D-alanine molecule is removed during the
transpeptidation reaction.
• This reaction, which is exergonic, supplies the energy necessary to drive the reaction forward
(transpeptidation occurs outside the cytoplasmic membrane, where ATP is unavailable). In E. coli,
the protein FtsI is the key protein in transpeptidation at the division septum, while a separate
transpeptidase enzyme cross-links peptidoglycan elsewhere in the growing cell.
• In gram-positive bacteria, where a glycine interbridge is common, cross-links occur across the
interbridge, typically from an L-lysine of one peptide to a D-alanine on the other.
Transpeptidation and Penicillin
• Transpeptidation is medically noteworthy because it is the reaction inhibited by the antibiotic penicillin.
Several penicillin binding proteins have been identified in bacteria, including the previously mentioned FtsI.
• When penicillin is bound to penicillin-binding proteins the proteins lose their catalytic activity. In the
absence of transpeptidation, the continued activity of autolysins so weakens the cell wall that the cell
eventually bursts.
• Penicillin has been a successful drug in clinical medicine for at least two reasons. First, humans are Eukarya
and therefore lack peptidoglycan; the drug can thus be administered in high doses and is typically nontoxic.
• And second, most pathogenic bacteria contain peptidoglycan and are thus potential targets of the drug.
Nevertheless, the continual and excessive use of penicillin since it became commercially available following
World War II has selected for resistant mutants of many common pathogens previously susceptible to this
drug.
• Many of these are widespread in human and other animal populations because they make variants of
penicillin-binding proteins that are catalytically active but no longer bind penicillin. In these cases, cell wall
synthesis occurs uninterrupted in the presence of the drug, and other drugs need to be used to thwart the
infection
Structure and Function of Membrane
Transport Proteins
• At least three transport systems exist in prokaryotes: simple transport, group
translocation, and ABC transport.
• Simple transport consists only of a membrane-spanning transport protein, group
translocation involves a series of proteins in the transport event, and the ABC system
consists of three components: a substrate-binding protein, a membrane-integrated
transporter, and an ATP-hydrolyzing protein.
• All transport systems require energy in some form, either from the proton motive
force, or ATP, or some other energy-rich organic compound.
Periplasmic Binding Proteins and the
ABC System
• Gram-negative bacteria contain a region called the periplasm that lies between the cytoplasmic
membrane and a second membrane layer called the outer membrane, part of the gram-negative
cell wall.
• The periplasm contains many different proteins, several of which function in transport and are
called periplasmic binding proteins.
• Transport systems that employ periplasmic binding proteins along with a membrane transporter
and ATP-hydrolyzing proteins are called ABC transport systems, the “ABC” standing for
ATPbinding cassette, a structural feature of proteins that bind ATP.
• More than 200 different ABC transport systems have been identified in prokaryotes. ABC
transporters exist for the uptake of organic compounds such as sugars and amino acids,
inorganic nutrients such as sulfate and phosphate, and trace metals. A characteristic property of
periplasmic binding proteins is their high substrate affinity.
Mechanism of an ABC transporter. The periplasmic binding protein has high
affinity for substrate, the membrane-spanning proteins form the transport channel,
and the cytoplasmic ATP-hydrolyzing proteins supply the energy for the transport
event
• These proteins can bind their substrate(s) even when they are at extremely low
concentration; for example, less than 1 micromolar (1026 M).
• Once its substrate is bound, the periplasmic binding protein interacts with its
respective membrane transporter to transport the substrate into the cell driven by
ATP hydrolysis.
• Even though gram-positive bacteria lack a periplasm, they have ABC transport
systems. In gram-positive bacteria, however, substrate-binding proteins are
anchored to the external surface of the cytoplasmic membrane.
• Nevertheless, once these proteins bind substrate, they interact with a membrane
transporter to catalyze uptake of the substrate at the expense of ATP hydrolysis,
just as they do in gram-negative bacteria
