Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine.
Cloning and nucleotide sequencing indicated that transposon Tn1546 from Enterococcus faecium BM4147 encodes a 23,365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.
Department of Biochemistry, University of Cambridge, UK.
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Reynolds
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Depardieu
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Dutka-Malen
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Arthur
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Courvalin
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D019729:Carbon-Oxygen Ligases; D004251:DNA Transposable Elements / Q000235:genetics*; D004151:Dipeptides / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006868:Hydrolysis; D007773:Lactates / Q000378:metabolism; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000378:metabolism; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D017334:Teicoplanin / Q000494:pharmacology*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014640:Vancomycin / Q000494:pharmacology*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004151:Dipeptides; D007773:Lactates; D010457:Peptidoglycan; D011773:Pyruvates; D014157:Transcription Factors; C071437:VanA ligase, Bacteria; C078877:alanyllactate; C073902:VanR protein, bacteria; C073901:VanS protein, Enterococcus; C070700:VanH protein, Enterococcus faecium; C002956:alanylalanine; D017334:Teicoplanin; D014640:Vancomycin; D019289:Pyruvic Acid; D011494:Protein Kinases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase; D008025:Ligases; D019729:Carbon-Oxygen Ligases; D000409:Alanine