The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis

Abstract

Physiological properties of the murG gene product of Escherichia coli were investigated. The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDPMurNAc-pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors. The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphorylMurNAc-(pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I. In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway. Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed. In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different cytoplasmic and membrane steps (10,49). Conditional-lethal mutants altered at different levels of this metabolic pathway have been described previously, and most of the mutations have been mapped (5,43,49,50,52,56). In particular, the 2-min region of the Escherichia coli chromosome contains a large cluster of genes from pbpB to envA that code for proteins involved in different aspects of peptidoglycan synthesis and cell division. The organization of the genes in this cluster is now completely elucidated, and the nucleotide sequence of the whole 17-kbp region has been determined (11-13, 16, 37, 39, 41; see reference 35 for references before 1990). The genes, tightly packed and overlapping in many cases, appear in the following order: pbpB-murE-murF-ORF-Y-murD-ftsW-murGmurC-ddl-ftsQ-ftsA-ftsZ-envA. The murC, murD, murE, murF, and ddl gene products were identified previously as L-alanine, D-glutamic acid, meso-2,6-diaminopimelic acid (DAP), D-alanyl-D-alanine-adding enzymes, and D-alanine: D-alanine ligase, respectively (10, 26-28, 32, 35, 43), which are involved in the synthesis of the soluble nucleotide precursors from UDP-N-acetylmuramic acid (UDP-MurNAc) to UDP-MurNAc-L-Ala-y-D-Glu-meso-DAP-D-Ala-DAla (UDP-MurNAc-pentapeptide). These genes are flanked on the left by a cell division gene named pbpB (1, 52) or ...

Two Proteins Homologous to the N- and C-terminal Domains of the Bacterial Glycosyltransferase Murg Are Required for the Second Step of Dolichyl-linked Oligosaccharide Synthesis in Saccharomyces cerevisiae

“…second step of LLO biosynthesis in S. cerevisiae correspond to the two protein domains that have been shown to be required for UDP-sugar:undecaprenyl pyrophosphoryl monosaccharide glycosyltransferases involved in bacterial cell wall glycoconjugate biosynthesis pathways (9,26,29). These two protein domains are usually formed by a single polypeptide chain, but in a few instances the domains correspond to separate polypeptide chains encoded by different genes (26).…”

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

“…S4). MurG, the glycosyltransferase required for conversion of lipid I into lipid II (20). Both depletion strains were inducer-dependent for growth and exhibited a reduction in optical density upon removal of IPTG (Fig.…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…Once in the cytoplasm, the muropeptides are processed by various enzymes to form lipid II composed of UDPGlcNAcMurNac pentapeptide attached to a hydrophobic undecaprenol-pyrophosphate group [33,36]. Lipid II is flipped across the cytoplasmic membrane into the periplasmic space, where GlcNAcMurNac pentapeptide is reincorporated into the growing cell-wall (Figs 1 and 2).…”

“…MurG catalyses the addition of GlcNAc to the MurNAc moiety of lipid I to yield lipid II [32,36]. Lipid II is flipped over into the periplasmic space using a 'flippase' enzyme [37].…”