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Chapter 11 : Structure and Biosynthesis of the Murein (Peptidoglycan) Sacculus
Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology
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This chapter reviews the murein structure and biosynthesis and the possible mechanism(s) of enlargement during growth of gram-negative bacteria. Unless otherwise stated, the presented data were obtained in studies with Escherichia coli, which is the most extensively studied gram-negative bacterium with respect to murein structure and biosynthesis. The major subunits in the murein of E. coli are the disaccharide tetrapeptide monomer and the DD-cross-linked bisdisaccharide tetratetrapeptide dimer. The layered murein cannot be perfect for two reasons. First, compared with the dimensions of the cell, the glycan strands are rather short. Second, the percentage of cross-linked peptides is slightly lower than the theoretical value of 50%. The final steps in murein synthesis take place at the periplasmic side of the cytoplasmic membrane and involve two reactions. First, the murein glycan strands are oligomerized by transglycosylation, and second, the peptide cross-links are formed by transpeptidation. Of the six known lytic transglycosylases of E. coli, only one is soluble (Slt70), whereas five are lipoproteins anchored to the outer membrane (MltA, MltB, MltC, MltD, and EmtA) and face into the periplasm. Murein hydrolases are enzymes that cleave covalent bonds in the murein sacculus or in murein fragments. Morphogenesis of E. coli seems rather simple with two phases in the cell cycle. Most models have in common that the enlargement of the sacculus is achieved by the insertion of new glycan strands and that both synthesis of new murein and hydrolysis of bonds within the existing sacculus are combined.
(A) A cryo-transmission electron microscopy picture of a frozen-hydrated section of an E. coli cell. The murein layer (PG) is embedded in the envelope between the cytoplasmic membrane (PM) and the outer membrane (OM); bar, 200 nm. ( Taken from Matias et al. [2003 ] with permission.) The other transmission electron microscopy pictures show isolated murein sacculi from E. coli (B), Caulobacter crescentus (C), and Pseudomonas aeruginosa (D). B to D, bar, 500 nm.
Structure of murein from gram-negative bacteria. (A) The murein glycan strands consist of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues. A 1,6-an-hydroMurNAc residue is present at one chain end. R, peptide. (B) Structures of monomeric, dimeric, and trimeric peptides. There are two types of cross-links (DD and LD). In the murein, the L-Ala residue of the peptide is attached to the lactyl group of the MurNAc residue of the glycan strands. iGlu (iso-glutamate), the γ-carboxyl group of Glu, is linked to the m-A2pm residue. (C) The murein of some bacteria contains an O-acetyl group (in bold) at C-6 of a fraction of the MurNAc residues. (D) Structure of the covalent linkage between Braun’s lipoprotein (Lpp) and the tripeptide in the murein.
Architecture of murein. (A) The peptides (arrows) protrude helically from the glycan strands. Light grey bar, GlcNAc; dark grey bar, MurNAc. (B) Model for the architecture of a murein layer. The glycan strands are indicated as bold zigzag lines. Arrows indicate the cross-linked peptides in the murein layer (xy plane). The non-cross-linked peptides pointing up and down are shown as dotted lines. A tessera is the smallest unit (pore) formed by two glycan strands and two peptide cross-links. (C) Model of the murein sacculus. The glycan strands (lines) run in the direction perpendicular to the long axis of the cell (x direction in B), whereas the peptide cross-links (arrows) are in the direction of the long axis (y direction in B). About 70 to 120 glycan strands of average length are required for one circumference, and about 500 to 1,000 glycan strands are arranged in parallel to cover the length of the cell. Most of the surface of the sacculus is made of a single layer. (Reproduced from Holtje  with permission.)
Murein synthesis reaction with lipid II as substrate.(A) The glycan strands are oligomerized by transglycosylation. PP, pyrophosphate; upr, undecaprenyl. (B) The dd-cross-links are formed by transpeptidation by a penicillin-binding protein (PBP). The intermediate peptidyl-enzyme complex is shown. (left) Donor peptide; (right) acceptor peptide; G, GlcNAc; M, MurNAc.
Murein hydrolysis. (A) Cleavage sites of different classes of murein hydrolases in high-molecularweight murein.A, N-acetylmuramyl-l-alanine amidase; LT, lytic transglycosylase;dd-EP, dd-endopeptidase;LD-EP, LD-endopeptidase; DD-CP, DD-carboxypeptidase; LD-CP, LD-carboxypeptidase. (B) Cleavage of a murein glycan strand by an exo-specific lytic transglycosylase (LT) with concomitant formation of a 1,6-anhydro bond at the MurNAc residue.
Model of the enlargement of the murein layer proposed by Park (1996) . (A) Hydrolysis of cross-links precedes the insertion of a new glycan strand, connecting new and old material. (B) After some time, connections between two new strands are also made. (Reproduced from Park  with permission.)
Three-for-one growth model proposed by Höltje. A triplet of three new strands is attached to the sacculus and is inserted by the concomitant removal of one old strand (docking strand). During elongation, one strand of the triplet (the primer strand) is preformed (left side). During cell division, all three strands are newly synthesized (right side). Below are shown the hypothetical murein synthesis multienzyme complexes for the enlargement of the sacculus. Complexes active during elongation contain PBP2, and complexes active during cell division contain PBP3. TG, transglycosylase;TP, transpeptidase; EP, endopeptidase; LT, lytic transglycosylase;A, Nacetylmuramyl-L-alanine amidase. (Reproduced from Höltje  with permission.)
The periplasmic murein synthases and hydrolases in E. coli