Dataset Information

D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174.

ABSTRACT: An open reading frame located 230 nucleotides downstream from the stop codon of vanS(c) and in the opposite direction relative to the other genes of the vanC cluster was identified in Enterococcus gallinarum BM4174. This gene (designated ddl2) encoded a protein of 343 amino acids that had significant predicted structural similarity to D-Ala:D-Ala ligases and displayed 33 and 35% amino acid identity to VanC-1 and the previously reported partial sequence of Ddl from E. gallinarum, respectively. Biochemical characterization by thin-layer chromatography confirmed that Ddl2 is a D-Ala:D-Ala ligase with no detectable D-Ala:D-Ser ligase activity. The vancomycin dependence of Enterococcus faecalis BM4320 (ddl mutant) was lost on electroporation of a plasmid construct expressing ddl2 constitutively. The latter strain was able to grow in the absence of vancomycin, and peptidoglycan precursor analysis under the same conditions indicated the synthesis of pentapeptide[D-Ala] as the main precursor, confirming the activity of Ddl2 in vivo. Expression of ddl and ddl2 in BM4174 was tested by reverse transcription-PCR: results suggested that both D-Ala:D-Ala ligases were expressed concomitantly. Our findings indicate that vancomycin-resistant E. gallinarum BM4174 is likely to express one D-Ala:D-Ser and two D-Ala:D-Ala ligase genes.

SUBMITTER: Ambur OH

PROVIDER: S-EPMC126995 | biostudies-literature | 2002 Jan

REPOSITORIES: biostudies-literature

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D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174.

Ambúr Ole-Herman OH Reynolds Peter E PE Arias Cesar A CA

Antimicrobial agents and chemotherapy 20020101 1

An open reading frame located 230 nucleotides downstream from the stop codon of vanS(c) and in the opposite direction relative to the other genes of the vanC cluster was identified in Enterococcus gallinarum BM4174. This gene (designated ddl2) encoded a protein of 343 amino acids that had significant predicted structural similarity to D-Ala:D-Ala ligases and displayed 33 and 35% amino acid identity to VanC-1 and the previously reported partial sequence of Ddl from E. gallinarum, respectively. Bi ...[more]

PMID: 11751117

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Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174.

Publications

D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174.

Similar Datasets

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