The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

SummaryPseudomonas entomophila is an entomopathogenic bacterium that is lethal to Drosophila melanogaster within 1-2 days of ingestion of high doses. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses. Recent studies suggest that its virulence relies on its ability to cause irreversible damages to the intestinal epithelium, in contrast to what is observed with milder pathogenic bacteria such as Erwinia carotovora carotovora Ecc15 or Pseudomonas aeruginosa PA14. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity. Here, we report the identification of the pvf genes, whose products are involved in production of a secondary metabolite involved in P. entomophila virulence. A pvf mutant is impaired in its ability to persist within the gut, to trigger the fly immune responses and to inflict gut damages. The expression of several genes is affected in a pvf mutant, independently of the Gac system. Moreover, growing a pvf mutant in medium supplemented with supernatant extracts from either the wild-type strain or a gacA mutant restore its pathogenicity. Collectively, our results indicate that we identified genes involved in the synthesis of a signalling molecule that controls P. entomophila virulence independently from the Gac system.

The UDP-N-acetylmuramate:L-alanyl-␥-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-␥-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra-and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.The biosynthesis of bacterial cell wall peptidoglycan (murein) is a complex process involving many cytoplasmic and membrane steps (for a review, see reference 54). The main cytoplasmic precursors are a series of seven nucleotide compounds, ranging from UDP-N-acetylglucosaminewhose sequential formation is catalyzed by a set of highly specific enzymes designated the Mur synthetases MurA to MurF (54). Subsequent steps, which occur in the membrane, consist of the transfer of the MurNAc-pentapeptide and GlcNAc motifs to the undecaprenyl phosphate carrier lipid, generating lipid II, which is then translocated to the outer side of the membrane and used for polymerization reactions catalyzed by the penicillin-binding proteins.The cell wall should not be considered a static structure since permanent remodeling necessarily occurs during cell growth and division. This is believed to a result of balanced functioning of murein-hydrolyzing and murein-synthesizing activities, with the former degrading the old peptidoglycan structure to allow insertion of new material. In E. coli, as many as 18 murein hydrolases have been identified, and these enzymes belong to six different families and include lytic transglycosylases, amidases, and endopeptidases; there is a specific hydrolase for almost every covalent bond in the murein (24). The remodeling of the murein by these different "autolysins" results in dramatic turnover, estimated at 40 to 50% per generation time. Some cell wall peptides are rel...

The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm Peptidoglycan is a giant macromolecule composed of alternating N-acetylglucosamine and N-acetylmuramyl residues cross-linked by short peptides. Its biosynthesis is a complex two-stage process. The first stage consists of the formation of the disaccharide-pentapeptide monomer unit, whereas the second stage concerns the polymerization reactions (1). The assembly of the peptide part of the monomer unit is ensured by a series of enzymes (MurC, MurD, MurE, and MurF) called the Mur synthetases. It has been shown that the Mur synthetases constitute a family of enzymes with common mechanistic and structural features (2, 3).Synthetase MurE catalyzes the addition of the third amino acid residue of the peptide chain. This residue, generally a diamino acid, varies among the bacterial species: meso-diaminopimelic acid (meso-A 2 pm) 3 for most Gram-negative bacteria and bacilli, L-lysine for most Grampositive bacteria, L-ornithine, meso-lanthionine, LL-A 2 pm, L-diaminobutyric acid, L-homoserine, etc. in particular species (4). In many organisms, the third residue is involved in the cross-linking of the macromolecule; in those cases, the incorporation by MurE of a "wrong" amino acid results in cell lysis (5). Therefore, MurE must be endowed with a high specificity to select the "right" amino acid among closely related amino acids that coexist within the cell. Recently, the crystallization of MurE from Escherichia coli allowed Gordon et al. (6) to decipher the structural bases for this high specificity.Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors (7). The analysis of its peptidoglycan revealed the absence of A 2 pm and the presence of both enantiomers of lysine. In this paper, we describe the properties of purified MurE from this bacterial species; in particular, we demonstrate that it is capable of adding L-and D-lysine to UDP-N-acetylmuramoyl (MurNAc)-L-Ala-D-Glu with comparable efficiencies but in different ways. The explanation of this finding in terms of known consensus sequences in MurE proteins is discussed. Moreover, we bring evidences of its physiological relevance by showing that: (i) the novel D-lysinecontaining nucleotide is a substrate for MraY from T. maritima in vitro and (ii) the overexpression of T. maritima murE in E. coli results in important lysine incorporation into the peptidoglycan of the host.