MOOC 4, Module 5

Bio-Synthesis & Degradation of Bacterial Cell Wall

Wall of bacterial endospore:

The endospore is the structure seen different Gram positive/negative bacteria like Bacillus or

Aquaspirillum. It is formed during unfavourable condition and usually acts as resting spore and

is only activated with the advent of favourable situation. But the wall is distinctly different from

normal bacterial cell.

Fig.1: Different layers of Bacterial Endospore.

1. Exosporium: The spore is sometimes surrounded by a thin covering known as

the exosporium, which overlies the spore coat. The main composition is protein (52%),

amino and neutral polysaccharides (20%), lipids (18%), and ash (4%). The amino acids

including 17 amino acids including very low amount of cystine-cysteine, methionine,

tyrosine, and histidine. Glucosamine was the only amino sugar, and glucose and

rhamnose were the principal neutral sugars. The lipid fraction contained 5.5% cardiolipin

as the only phospholipid, 12.5% neutral lipids, and at least 19 fatty acids, among which

normal C16 and C18 ones predominated. Calcium and phosphorus salts are also present.

2. Spore coat: The spore coat, which acts like a sieve that excludes large toxic molecules

like lysozyme, is resistant to many toxic molecules and may also contain enzymes that

are involved in germination. Composed of layers of spore specific proteins with a

average molecular weight of 13 to 15 kDa. There may be histidine present in the NH

terminus and the other amino acids are mostly hydrophobic in nature.

3. Cortex: The cortex lies beneath the spore coat and consists of loosely linked

peptidoglycan with dipicolinic acid (DPA), which is particular to all bacterial endospores.

The DPA cross links with calcium ions embedded in the spore coat. This cross linkage

greatly contributes to the extreme resistance capabilities of the endospores because it

creates a highly impenetrable barrier. The calcium DPA cross linkages compose up to

20% of the dry weight of the endospores.Calcium directly can also act as a heat resistant

material and also as an oxidizing agent.

4. Core wall: It lies beneath the cortex and surrounds the protoplast or core of the

endospore. The core contains the spore chromosomal DNA which is encased

in chromatin-like proteins known as SASPs (small acid-soluble spore proteins), that

protect the spore DNA from UV radiation and heat. The core also contains normal cell

structures, such as ribosomes and other enzymes, but is not metabolically active.

The endospore can be seen as a thick walled structure under light microscope but in order to be

seen properly, it has to be stained with a penetrative stain like the Schaeffer-Fulton stain.

Fig.2: Different stages of endospore formation

Biosynthesis of Peptidoglycan:

Major events: The biosynthesis of peptidoglycan follows the general pattern of polysaccharide

biosynthesis, in which sugar residues are transferred from nucleoside diphosphate sugars to the

growing polymer chain. The pentapeptide gets attached to the sugar residue and this process is

mediated by the Mur factors. The ribitol and glycerol residue are attached to the sugar residue to

produce the Teichoic acid, which is regulated by the Tag factors.

Fig.3: Synthesis of Teichoic acid and Peptidoglycan in S. aureus The Bar charts represent the

fold changes of respective metabolites from ΔpknB/Δstp deletion mutants compared with wild-

type cells (red line) at different growth stages (exponential, mid-, and late exponential growth

phase; OD540 1, 2, and 3). Significant alterations (Student's two-tailed t test p < 0.1) are marked

with an asterisk under the bar chart (error bars representing SD of results from three independent

cultivations).

Stages of peptidoglycan biosynthesis:

1. The first stage concerns the assembly of the disaccharide-peptide monomer unit via a

series of UDP precursors and lipid intermediates is the participation of the isoprenoid

lipid undecaprenol as its phosphate.

2. Six cytoplasmic steps (mediated by MurA to MurF) lead to the formation of the UDP-

MurNAc-pentapeptide precursor from UDP-GlcNAc.

The factors MurA–F perform the six cytoplasmic steps in peptidoglycan biosynthesis.

a)The process of bacteria cell wall biosynthesis starts with the MurA transferase, which

transfers the addition of an enolpyruvyl moiety to 3′-hydroxyl-UDP-NAG.

b) Subsequently, the MurB reductase reduces the enol ether to a lactyl ether, utilizing one

equivalent of NADPH and a solvent proton to form UDP-NAM.

c) Next, a series of ATP-dependent amino acid ligases (MurC, MurD, MurE and MurF)

catalyze the stepwise synthesis of the pentapeptide side-chain using the newly

synthesized carboxylate as the first acceptor site. Each enzyme is responsible for the

addition of one residue except for MurF, which catalyzes the addition of the dipeptide d-

Ala-d-Ala. MurE in Gram-negative bacteria catalyzes the meso-2,6-diaminopimelate

(DAP) addition, whereas in Gram-positive bacteria MurE catalyzes l-lysine addition.

3. The product of MurF, UDP-NAM-pentapeptide (NAM now abbreviated to M), is then

catalyzed at the plasma membrane by the translocase MraY to transfer the phospho-UDP-

NAM-pentapeptide moiety to the membrane acceptor undecaprenyl phosphate to form

Lipid I.

Fig.4: Synthesis of UDPNAG to UDPNAM pentapeptide and its transport outside the cell

wall by Lipid I and Lipid II

4. Then the MurG transferase catalyzes the addition of UDP-NAG (NAG now

abbreviated to G) yielding UDP-NAM-(pentapeptide)-pyrophosphoryl-undecaprenol

(Lipid II). Lipid II is then moved across the membrane into periplasm and catalyzed by

the transglycosylase (TG) and the transpeptidase (TP) to form the extensively cross-

linked polymer peptidoglycan essential for the integrity of the bacterial cell wall. The

large penicillin binding proteins (PBPs) contain both TG and TP domains.

5. The next stage of peptidoglycan synthesis concerns the polymerization of the monomer

unit on the outside surface of the cytoplasmic membrane and the binding of newly made

material to the pre-existing cell wall.

6. The final stage of peptidoglycan synthesis is the cross linking of peptide chains: the

additional D-alanine residue is displaced from the carboxyl terminus of a peptide chain

by a glycine from a neighbouring chain. The result is the linking together of adjacent

peptide chains with the elimination of D-alanine.

Two major types of membrane-bound activities are involved: glycosyltransferases that

catalyze the formation of the linear glycan chains and transpeptidases that catalyze the

formation of the peptide cross-bridges.

Additional Change: In the course of the formation of the lipid intermediates the peptide subunit

can undergo various modifications (amidation, addition of extra amino acids, etc.). The final

lipid intermediate is transferred by an unknown mechanism through the hydrophobic

environment of the membrane to the externally located sites of incorporation of the monomer

unit into growing peptidoglycan.

Fig.5: Summary of events of Peptidoglycan biosynthsis

In growing cells, polymerization reactions are accompanied by concomitant or subsequent

structural modifications of peptidoglycan. Its closed covalent structure must continuously adjust

to the requirements of surface growth and cell division.

Teichoic acid formation:

Teichoic acids are synthesized from cytidine diphosphate glycerol and cytidine diphosphate

ribitol chain extension occurs through the transfer of polyol phosphate by transphosphorylation.

The sugar residues occur as appendages on the main chain; these can be transferred from the

appropriate nucleoside diphosphate sugar after the chain has been formed, whereas when the

sugar residues form a part of the chain, then the transfer occurs alternately to the transfer of

polyol phosphate.

Types of Teichoic acid:

a) LTA

The lipoteichoic acid is a lipid-linked, membrane-bound form of the teichoic acid (TA), a

carbohydrate backbone formed by repeating glycerophosphate or ribitol phosphate. The cell wall

is a network of peptidoglycan decorated by covalently linked teichoic acid. The peptidoglycan is

a continuous backbone of repeating N-acetyl glucosamine (G) and N-acetyl muramic acid (M)

that is cross-linked to neighboring backbones by stem peptides.

Fig.6: Structure of LTA

b) WTA

Structure: The WTA is linked via a phosphodiester bond to the 6 OH-group of Muramic acid

residue in the glycan strand. 1,3-ManNAc-GlcNAc-PP-prenol serves as substrate. The 2 OH-

group of ManNAc is phosphodiester linked with three units of D-alanylated glycerol-phosphates

to which approximately 40 units of ribitol-phosphates (substituted with D-alanine and/or

GlcNAc) are linked. The process is actually catalyzed by Tag factors, which include the Tag O –

L factors.

Fig.7 Position of WTA and LTA, Structure of WTA and synthesis of WTA

Role of transglycosylation and transpeptidation in cell wall biosynthesis:

In normally growing bacteria, transglycosylation catalyzing the formation of the glycan chains

and transpeptidation catalyzing the cross-linking between peptide subunits are continuous,

tightly coupled reactions. The problems of their correlation and of the attachment of newly

synthesized material to preexisting peptidoglycan have been investigated with numerous in vivo

and in vitro systems. The conclusions drawn from these studies can be briefly summarized as

follows:

1. Transglycosylation is the primary process: It can proceed independently from

transpeptidation as exemplified by the formation of uncross-linked or low cross-linked

soluble peptidoglycan in various cell-free systems and in protoplasts. Furthermore, the

treatment of growing cells or cell-free peptidoglycan-synthesizing systems with β-lactam

antibiotics, which are specific inhibitors of the trans-peptidation reactions, results in the

formation of soluble uncross-linked peptidoglycan material. In a few cases it has been

shown that the soluble material can function as an intermediate in the synthesis of cell

wall–linked peptidoglycan.

Fig.8: PGT mediated glycosyl transferase activity showing building of disaccharide

with a C35 pyrophosphate residue.

2. Peptidoglycan is formed on pre existing peptidoglycan: It has been proposed that perhaps

small amounts of nascent uncross-linked peptidoglycan are transiently synthesized prior to cross-

linking. Though it should be kept in mind that in low levels of soluble peptidoglycan material, it

is difficult to distinguish between true nascent material and possible autolytic degradation

products.

3. Simultaneous synthesis: The concomitant formation of glycan chains along with peptide

synthesis has been reported only in very special cases. In peptidoglycan the glycan chains are

generally quite longer than the peptide chains, and only some of the monomer units are involved

in cross-bridges. This makes the addition of monomer units to pre-existing peptidoglycan by

transpeptidation and their subsequent interlinking by transglycosylation is not a very probable

process.

Fig.9: Site of Transglycosylation and transpeptidation

Taken together, these facts suggest that in growing cells the polymerization of the monomer unit

proceeds essentially by tranglycosylation, which precedes cross-linking to the cell wall.

Transpeptidation is responsible not only for the formation of cross-bridges in the new material

but also for the transfer of newly made material to preexisting peptidoglycan. It follows glycan

chain formation or is at best concomitant with it. The average length of the glycan chains

synthesized in intact cells or in cell-free systems can vary from 10 to over 100 disaccharide units

as was determined in E. coli.

Role of PBP:

Nearly all eubacteria possess a set of minor membrane proteins designated as penicillin-binding

proteins (PBPs) that are the specific targets of the β-lactam antibiotics and that are involved in

the late steps of peptidoglycan synthesis. They are detected by their ability to covalently bind

radio labeled penicillin and the stability of the penicillin-protein complexes has greatly facilitated

their study.

PBPs vary from species to species in number, size, amount, and affinity for β-lactam antibiotics.

High-molecular-mass PBPs (HMM-PBPs) are essentially two-domain proteins that belong either

to class A or class B, depending on the structure and the catalytic activity of their N-terminal

domain. The C-terminal domain of both classes is responsible for transpeptidation activity and β-

lactam antibiotics covalently bind to its catalytic centre. In class A HMM-PBPs, the N-terminal

domain is responsible for their glycosyl transferase activity, whereas in class B the N-terminal is

presumably involved in interactions with other membrane proteins.

Fig.10: GT domain of PBP draws the TP domain helping in polymerization.

Role of Mgt:

A number of membrane-bound, non-penicillin-binding, monofunctional glycosyltransferases

(Mgt) capable of catalyzing only the formation of uncross-linked peptidoglycan were found in E.

coli, Micrococcus luteus, S. aureus and S. pneumoniae. They were solubilized, partially purified

and characterized. They accounted for a large part of the in vitro measurable glycosyltransferase

activity determined with lipid II as substrate. More recently, in a variety of bacterial strains (E.

coli, Haemophilus influenzae, Klebsiella pneumoniae, Neisseria gonorrhoeae, Ralstonia

eutropha, and S. aureus) genes encoding monofunctional glycosyltransferases were detected on

the basis of sequence homology with genes encoding class A HMM-PBPs.

Fig.11: Structure of Mgt

Degradation of Bacterial Cell Wall:

There are mainly four different ways:

1. Degradation by lysozyme

2. Degradation by the bacteriophage hydrolases

3. Degradation of cell wall by antibiotics

4. Auto degradation by bacterial enzymes.

1. Lysozyme:

The enzyme found in the tears, saliva, breast milk, nasal secretion, a protein composed of

129 amino acids and it is chemically 1,4-β-N- acetylmuramidase, denoted by A.Fleming

(1921).

Fig.12: Structure of lysozyme

Lysozyme is known for damaging bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-

linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in

peptidoglycan, and between N-acetyl-D-glucosamine residues in chitodextrins.

Fig.13: Site of lysozyme action

2. Cell Wall Degradation by bacteriophage hydrolase:

This takes place in different ways, viz. one gene system, two gene system and multigene system.

One gene system: Lysis in one-gene system is observed in the phage φ×174). The phage

produces a protein E, that inhibits a cellular protein involved in synthesis of the cell wall

membrane protein (MraY).

Fig.14: One gene lysis of phage enzyme

Two gene system: In this system, the phage produces a holin protein and a lysozyme. The holin

integrates into the inner membrane and, when its density reaches a threshold, it creates gaps,

letting the lysozyme through to degrade the cell wall.

Fig.15: Two gene lysis of phage enzyme

The outer membrane is present in Gram-negative cells while in Gram-positive bacteria cell wall

polymers (all of them termed as ‘secondary cell wall polymers’, SCWP) such as Teichoic,

teichuronic acids, or other neutral or acidic polysaccharides are linked to the peptidoglycan

chains. After infection by bacteriophages, murein hydrolases encoded by phage genomes are

produced during the late phase of the lytic cycle. Endolysins gain access to their substrate, the

bacterial cell wall, when at a genetically programmed moment holin, a small phage-encoded

membrane protein, disrupts the membrane. These hydrolases act at different points like the

glycoside linkage and also at the inter-peptide region cleaving the peptide cross bridge at

different points.

Fig.16: Different parts of bacterial cell wall and mechanism of action of bacterial

hydrolase.

3. Antibiotics degrading Bacterial Cell Wall:

Fig.17: Different antibiotics inhibiting bacterial cell wall

a) Penicillins: They interfere with the last step of bacterial cell wall synthesis

(transpeptidation or cross-linkage1), resulting in exposure of the osmotically less stable

membrane. Cell lysis can then occur, either through osmotic pressure or through the

activation of autolysins. These drugs are thus bactericidal. The success of a penicillin

antibiotic in causing cell death is related to the antibiotic's size, charge, and

hydrophobicity. Penicillins are only effective against rapidly growing organisms that

synthesize a peptidoglycan cell wall.

Penicillins inactivate numerous proteins on the bacterial cell membrane. These penicillin-

binding proteins (PBPs) are bacterial enzymes involved in the synthesis of the cell wall

and in the maintenance of the morphologic features of the bacterium. Exposure to these

antibiotics can therefore not only prevent cell wall synthesis but also lead to morphologic

changes or lysis of susceptible bacteria. The number of PBPs varies with the type of

organism.

Some PBPs catalyze formation of the cross-linkages between peptidoglycan chains.

Penicillins inhibit this Trans peptidase-catalyzed reaction, thus hindering the formation of

cross-links essential for cell wall integrity. As a result of this blockade of cell wall

synthesis.

b) Cephalosporins: It has a 6 membered sulfur containing dihidrothiaizine ring. Changes in

side chain R groups gives changes in spectrum of activity, pharmacokinetics, etc.

Mechanism of action: binds to penicillin binding proteins and inhibition of formation of

cell wall

Fig.18: Structure of cephalosporin

c) Carbapenem: Like all other β-lactams, they inhibit bacterial cell wall synthesis by

binding to and inactivating relevant transpeptidases, known as penicillin binding proteins

(PBPs). In Escherichia coli, imipenem inhibits the Trans peptidase activities of PBPs-1A,

-1B and -2, and the D-alanine carboxypeptidase activities of PBP-4 and PBP-5. It also

causes strong inhibition of the transglycosylase activity of PBP-1A while it inhibits the

Trans peptidase activity of PBP-3 only weakly, which is consistent with the finding that it

has low binding affinity to PBP-3 and does not inhibit septum formation by the cells.3

Fig.19: Effectiveness of Imipenem over Meropenem

d) Monobactums:

The Monobactums, having a unique monocyclic beta-lactam nucleus, are structurally different

from other beta-lactam antibiotics (e.g., penicillins, cephalosporins, and cephamycins). The

sulfonic acid substituent in the 1-position of the ring activates the beta-lactam moiety; an

aminothiazolyl oxime side chain in the 3-position and a methyl group in the 4-position confer the

specific antibacterial spectrum and beta-lactamase stability. They are useful in the treatment of

Pseudomonas and other Gram negative bacteria but against Gram positive bacteria.

Fig.20: Structure of Monobactum (Aztreonam)

e) Vancomycin: It is a stage II cell wall biosynthesis inhibitor. It is effective again Gram

positive bacteria, but cannot penetrate the Gram negative bacteria cell wall. Its

mechanism of action is to bind to the D-Alanyl-D-Alanine portion of the dipeptide at the

end of the chain. Vancomycin forms three hydrogen bonds with the D-Ala-D-Ala portion

of the cell wall. This inhibits it from being transported to the cell wall for cross linking.

Vancomycin is a very large, lipophilic molecule that does a lot of protein binding. This

lipophilicity is what allows it to get into the cell wall of the Gram positive bacteria, but

prevents it from getting into the porins of Gram negative bacteria.

Fig.21: Action of Vancomycin on bacterial cell wall

f) Clavulinic acid: It is a beta lactamase inhibitor binding to the serine residue of the

enzyme, rendering it ineffective. It does not have any anti bacterial action of its own but

when given along with an antibiotic it becomes extremely useful in controlling bacteria.

Fig.22: Action of Clavulinic acid

Fig.23: Different sites of antibiotic activity in cell wall biosynthesis of bacteria

4. Auto degradation of Cell wall by bacterial enzymes:

a) N -Acetylmuramyl-L-alanine amidases (MurNAc-LAAs) : They cleave the amide

bond between MurNAc and the N-terminal L-alanine residue of the stem peptide. These

Enzymes, also referred to in the literature as peptidoglycan amidases or amidases, are

found in bacterial and bacteriophage or prophage genomes. In most cases, bacterial ¨

MurNAc-LAAs are members of the bacterial autolytic system and carry a signal peptide

in their N-termini that allow their transport across the membrane. Five MurNAc-LAAs

are present in E. coli: AmiA, AmiB, and AmiC.

Fig.24: Amidase action

b) N -Acetyl-b-D-glucosaminidases (N-acetylglucosaminidases): They are widespread in

bacteria; they hydrolyse the glycosidic bond between N-acetyl-b-D glucosamine residues

and adjacent monosaccharides in different oligosaccharide substrates including

peptidoglycan, chitin and N-glycans. Most of these enzymes have a catalytic domain

belonging to the protein family (Pfam) 01832.

Fig.25: Action of N-acetylglucosaminidase