Spherical E. coli due to elevated levels of D-alanine carboxypeptidase

Markiewicz, Z; Broome-Smith, Jenny K.; Schwarz, Uli; Spratt, Brian G.

doi:10.1038/297702a0

Cited by 97 publications

(82 citation statements)

References 14 publications

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“…dacA is the last gene in a 7.2-kbp sequenced cluster of genes involved in cell wall biosynthesis (33). We sought to avoid cloning the intact dacA structural gene because cells containing multicopy plasmids carrying this gene are unusually detergent sensitive and have an aberrant cell shape (31). The orientation of the cloned PvuII chromosomal fragment in plasmid pCTV616 was verified by DNA sequencing (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

1991

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We report the isolation and genetic characterization of novel TnlOdTc and TnlOOOdKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The TnlOdTc and TnlOOOdKn mutations define two genes, lpA and lipB, involved in lipoic acid biosynthesis. Two representative alleles (lip-2 and lip-9) from the previously reported genetic class of lipoic acid auxotrophic mutants (A. A. Herbert and J. R. Guest, J. Gen. Microbiol. 53:363-381, 1968) were assigned to the lipA complementation group. We have cloned the E. coli lip locus and developed a recombinant plasmid-based genetic system for fine-structure physical-genetic mapping of mutations in this region of the E. coli chromosome. We also report that a recombinant plasmid containing a 5.2-kbp PvuH restriction fragment from the E. coli lip locus produced three proteins of approximately 8, 12, and 36 kDa by using either a maxiceli or in vitro transcription translation expression system. The 36-kDa protein was identified as the gene product encoded by the lipA locus. Finally, we have identified a previously unreported lipoylated protein that functions in the glycine cleavage system of E. coli.

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Section: Resultsmentioning

confidence: 99%

Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

1991

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“…A regulatory role is indicated by their interference with the levels of pentapeptidyl residues, which represent the donors for the crucial murein cross-linking reaction catalyzed by DD-transpeptidases (14). Experimental evidence seems to support this proposal (21,25), and interestingly mutants lacking all three DD-carboxypeptidases have not been obtained yet. Penicillin-insensitive LD-carboxypeptidases that remove the terminal D-Ala from the tetrapeptidyl moiety -L-Ala-DGlu-m-A2pm-D-Ala have also been identified in E. coli (1,15,23,24).…”

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confidence: 90%

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography

1992

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An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10-4 M, and the Vm.x was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10-5 M.Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.A wide range of bacterial carboxypeptidases cleaving off terminal alanine residues from the peptide substituents of murein have been described (28). Three DD-carboxypeptidase activities in Escherichia coli, are known to specifically hydrolyze the D-alanyl-D-alanine terminus of the pentapeptidyl sidechain -L-Ala-D-Glu-m-A2pm-D-Ala-D-Ala (13). Being penicillin-sensitive enzymes, they belong to the family of penicillin-binding proteins (PBPs). Whereas PBP 5 and PBP 6 are specific carboxypeptidases (29), PBP 4 has been found, in addition to its DD-carboxypeptidase activity, to hydrolyze the murein-cross-linking DD-peptide bond between D-Ala and m-A2pm, thus also showing endopeptidase activity (19).The DD-carboxypeptidases have been studied in considerable detail as a model system for the interaction of P-lactams with the active sites of penicillin-sensitive enzymes (9, 16). However, the specific function of these carboxypeptidases in the process of growth and division of the murein sacculus is still not understood. A regulatory role is indicated by their interference with the levels of pentapeptidyl residues, which represent the donors for the crucial murein cross-linking reaction catalyzed by DD-transpeptidases (14). Experimental evidence seems to support this proposal (21, 25), and interestingly mutants lacking all three DD-carboxypeptidases have not been obtained yet. Penicillin-insensitive LD-carboxypeptidases that remove the terminal D-Ala from the tetrapeptidyl moiety -L-Ala-DGlu-m-A2pm-D-Ala have also been identified in E. coli (1,15,23,24). One LD-carboxypeptidase present in E. coli has been purified and determined to be a monomeric protein with a molecular mass of 12 kDa (24). Another LD-carboxypeptidase has been partially purified and reported to be an 86-kDa protein consisting of two 43-kDa polypeptides (2). This enzyme can easily be extracted from cells by Tris-EDTA, suggesting a localization in the periplasm (1). In synchronized cultures, the enzyme was found to be most active at the time of cell division. The activity of the enzyme has been proposed to be controlled by a still unidentified inhibitor (2).Re...

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“…Morphological defects accompanying the loss of PBP5 are visible by microscopic inspection (185), and shape changes with slight abnormalities at the poles are detectable by fluorescence-activated cell sorting (170). The overproduction of PBP5 is lethal, causing E. coli to grow as spherical cells before lysing (162,247). The proposed function of PBP5 is to regulate the availability of pentapeptide subunits for the formation of the cross-linkages by transpeptidation (48,205).…”

Section: Penicillin-binding Peptidasesmentioning

confidence: 99%

Peptidoglycan Hydrolases of Escherichia coli

Heijenoort

2011

Microbiol Mol Biol Rev

190

233

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SUMMARY The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.

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Spherical E. coli due to elevated levels of D-alanine carboxypeptidase

Cited by 97 publications

References 14 publications

Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography

Peptidoglycan Hydrolases of Escherichia coli

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