Determination of murein precursors during the cell cycle of Escherichia coli.
(73/97)
A convenient and reliable method has been established that allows a quantitative determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography the lipid-linked intermediates were hydrolysed to release the muropeptides. The accuracy for the measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was +/- 1.9% (SD), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) +/- 10% (SD) and for undecaprenyl-P-P-(GlcNAc-beta 1----4)MurNAc-pentapeptide (lipid II) +/- 5% (SD). The ratio of UDP-MurNAc-pentapeptide:lipid I:lipid II was about 300:1:3 for E. coli MC4100. The relative cellular concentrations of all three precursor molecules were found not to vary throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is not controlled by oscillations in the concentrations of these late murein precursors. (+info)
Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032.
(74/97)
LY146032, a cyclic lipopeptide antibiotic, is an inhibitor of cell wall peptidoglycan biosynthesis in gram-positive bacteria. Although LY146032 at relatively high concentrations inhibited the in vitro polymerization of UDP-linked sugar precursors, inhibition of cell wall formation in intact Staphylococcus aureus and Bacillus megaterium cells did not lead to the accumulation of UDP-N-acetyl-muramyl (MurNAc)-peptide(s). Experiments that measured formation of UDP-MurNAc-peptides revealed that LY146032 inhibited the formation of these nucleotide-linked intermediates. This antibiotic had a disruptive effect on membrane permeability as evidenced by the loss of intracellular potassium immediately after exposure to the drug. The lack of any major disruption of the phosphoenolpyruvate:sugar phosphotransferase system indicated that the membrane is not likely a lethal target for this antibiotic. The findings are consistent with a mechanism by which LY146032 inhibits the formation of precursor molecules utilized in peptidoglycan biosynthesis. The observed membrane effects likely result from transit of the inhibitor to its lethal target site. (+info)
Determination of gene products and coding regions from the murE-murF region of Escherichia coli.
(75/97)
We report the cloning of murE and murF genes and the identification of their gene products. The murE and murF genes encode diaminopimelic acid- and D-alanyl-D-alanine-adding enzymes, respectively, and both genes are involved in cell wall peptidoglycan biosynthesis in Escherichia coli. (+info)
Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.
(76/97)
The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation. (+info)
UDP-N-acetylmuramylpentapeptide as acceptor in murein biosynthesis in Escherichia coli membranes and ether-permeabilized cells.
(77/97)
Two widely used in vitro systems of Escherichia coli capable of synthesizing murein were evaluated by using high-pressure liquid chromatography for murein analysis. Comparison of the composition of murein synthesized by either a membrane preparation or ether-treated cells with native murein revealed that both in vitro systems failed to synthesize murein that was identical to murein formed in vivo. Furthermore, neither system attached the lipoprotein to the murein. Ether-treated cells, however, were superior to the membrane preparation in catalyzing the formation of the remarkable A2pm-A2pm cross-linkage. In both systems an atypical transpeptidation reaction was found to take place in which exogenously supplied UDP-N-acetylmuramylpentapeptide was directly linked to the murein without participation of the bactoprenol lipid carrier. The direct transpeptidation yields preferentially trimeric peptide bridges with the UDP-linked muramylpentapeptide serving as the acceptor. (+info)
Specificity of the uridine-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate synthetase from Escherichia coli.
(78/97)
To investigate the specificity of the uridine-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate synthetase, various compounds mimicking more or less different parts of the UDP-MurNAc-L-Ala-D-Glu substrate were prepared. Their size ranged from that of uridine or L-Ala-D-Glu to that of the whole nucleotide substrate. Chemical synthesis led to N alpha-acyl-dipeptides, in which the acyl group mimicked the MurNAc moiety, and to glycopeptides MurNAc(alpha or beta-Me)-L-Ala-D-Glu, in which the anomeric function is blocked. Partial degradation or chemical modification of the substrate UDP-MurNAc-L-Ala-D-Glu afforded: MurOHNAc-L-Ala-D-Glu, P1-MurNAc-L-Ala-D-Glu, and DDP-MurNAc-L-Ala-D-Glu (DDP = dihydrouridine-diphosphate). All these compounds were tested as substrates or (and) inhibitors of the reaction catalyzed by the A2pm-adding enzyme, which, after partial purification, was obtained in two active forms. Among the compounds tested as substrates, only DDP-MurNAc-L-Ala-D-Glu was a good one. The Km for this compound was 97 microM versus 55 microM for the natural substrate. Among the various compounds tested as inhibitors, only P1-MurNAc-L-Ala-D-Glu and MurNAc(alpha or beta-Me)-L-Ala-D-Glu had a significant inhibitory effect at 1mM. Apparently, no particular portion of the molecule is predominantly responsible for its recognition by the enzyme. In other words, multiple sites located over the whole molecule are required for a proper recognition and determine the high specificity of this activity. Therefore, to obtain efficient competitive inhibitors it is necessary to synthesize molecules very similar in size and structure to the natural substrate. (+info)
Kinetic evidence for an acyl-enzyme intermediate in D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus.
(79/97)
The kinetics of hydrolysis and transpeptidation of the synthetic substrate diacetyl-L-lysyl-D-alanyl-D-alanine and of the natural substrate UDP-acetylmuramyl pentapeptide and related compounds catalyzed by the D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus in the presence of the nucleophiles hydroxylamine or glycine have been examined. These kinetic data suggest that an acyl-enzyme intermediate is formed in the first step of the reaction and that the transpeptidation is the consequence of the partitioning of this intermediate between water and the nucleophile in the second step. (+info)
Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis.
(80/97)
A high-pressure liquid chromatography procedure was developed for the isolation and quantitation of UDP-N-acetylglucosamine, UDP-N-acetylglucosamine-enolpyruvate, and UDP-N-acetylmuramic acid, which are the early cytoplasmic precursors of bacterial peptidoglycan. In exponential-phase cells of Escherichia coli K-12, the intracellular concentration of UDP-N-acetylglucosamine was about 100 microM, whereas that of UDP-N-acetylglucosamine-enolpyruvate was only 2 microM. The phosphoenolpyruvate: UDP-N-acetylglucosamine transferase and UDP-N-acetylglucosamine-enolpyruvate reductase activities were investigated in extracts from E. coli. These activities appeared to be present in amounts sufficient for the ongoing rate of peptidoglycan synthesis. Certain uridine nucleotide peptidoglycan precursors were found to inhibit phosphoenolpyruvate: UDP-N-acetylglucosamine transferase activity. (+info)