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.

Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAcpentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.Colicins are plasmid-encoded toxins, synthesized and released in the growth medium by Escherichia coli, which kill susceptible E. coli strains and closely related bacterial species (1-3). Strains are protected against the colicin they produce by concomitant expression of a highly specific immunity protein.The lethal action of colicins can be divided into three steps:binding to a specific outer membrane receptor protein, translocation through the cell envelope, and finally interaction with the target and killing effect. To each of these steps corresponds a specific protein domain, the different colicins showing a similar three-domain structural organization. Depending on the import pathway they parasitize to enter the cells, the Tol system or the TonB system, colicins are classified into two groups A and B, respectively (3). The mode of action of colicins is variable: formation of voltage-gated pores in the cytoplasmic membrane (e.g. colicins A, B, E1, Ia, N), inhibition of protein synthesis (e.g. colicins D and E3), enzymatic degradation of cellular DNA or 16S rRNA (e.g. colicin E2) and inhibition of cell envelope biosynthesis (colicin M, see below).Various bacterial cell envelope polysaccharides (peptidoglycan, O-antigen, teichoic acid, capsular polysaccharide) in both Gram-negative and Gram-positive bacteria have a lipid-linked intermediary stage in their biosynthesis that is dependent on the essential carrier lipid undecaprenyl phosphate (C 55 -P) 4 (4 -15) (Scheme 1). In the peptidoglycan pathway, this lipid is needed for the synthesis and trans...

During the biogenesis of bacterial cell-wall polysaccharides, such as peptidoglycan, cytoplasmic synthesized precursors should be trafficked across the plasma membrane. This essential process requires a dedicated lipid, undecaprenyl-phosphate that is used as a glycan lipid carrier. The sugar is linked to the lipid carrier at the inner face of the membrane and is translocated toward the periplasm, where the glycan moiety is transferred to the growing polymer. Undecaprenyl-phosphate originates from the dephosphorylation of its precursor undecaprenyl-diphosphate, with itself generated by de novo synthesis or by recycling after the final glycan transfer. Undecaprenyl-diphosphate is de novo synthesized by the cytosolic cis-prenyltransferase undecaprenyldiphosphate synthase, which has been structurally and mechanistically characterized in great detail highlighting the condensation process. In contrast, the next step toward the formation of the lipid carrier, the dephosphorylation step, which has been overlooked for many years, has only started revealing surprising features. In contrast to the previous step, two unrelated families of integral membrane proteins exhibit undecaprenyldiphosphate phosphatase activity: BacA and members of the phosphatidic acid phosphatase type 2 super-family, raising the question of the significance of this multiplicity. Moreover, these enzymes establish an unexpected link between the synthesis of bacterial cell-wall polymers and other biological processes. In the present review, the current knowledge in the field of the bacterial lipid carrier, its mechanism of action, biogenesis, recycling, regulation, and future perspective works are presented.

Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg 2؉ -dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.Certain Escherichia coli strains produce and release in the growth medium toxins designated colicins in order to kill competitors belonging to the same species or to related species (8,28,29). The various modes of action of colicins include formation of pores in the cytoplasmic membrane, inhibition of protein synthesis, enzymatic degradation of cellular DNA or 16S rRNA, and interference with cell envelope biosynthesis. Colicins and proteins conferring immunity to the producer are generally encoded by plasmids. Depending on the import pathway they use to enter the cells, the Tol or TonB system, the colicins have been classified in group A or B, respectively. Their lethal action occurs in three steps: binding to a specific outer membrane receptor protein, translocation through the cell envelope, and interaction with the target leading to the bactericidal effect. A specific protein domain corresponds to each of the three steps, and the different colicins display a similar three-domain structural organization (8).Colicin M (ColM) exhibits a unique mode of action, as it is the only colicin known to interfere with peptidoglycan biosynthesis and to cause cell lysis (35). This class B colicin is internalized via the TonB translocation machinery and uses the FhuA outer membrane protein as the receptor. ColM lacks peptidoglycan-degrading activity and acts synergistically with ␤-lactam antibiotics, which inhibit the last polymerization step of peptidoglycan synthesis performed by penicillin-binding proteins (34,35). Since ColM inhibited both peptidoglycan synthesis and lipopolysaccharide O-antigen sy...

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124 -271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the fulllength colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surfaceexposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

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Background: Pathogenic Pseudomonas aeruginosa strains produce a colicin M-like bacteriocin exhibiting peptidoglycan lipid II-degrading activity. Results: We have determined the crystal structure of the Pseudomonas aeruginosa PaeM bacteriocin and functionally characterized its C-terminal activity domain. Conclusion:This study highlights structural plasticity of the active site of this enzyme family. Significance: The PaeM pyocin could potentially be exploited as antibacterial agent.