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A six amino acid deletion, partially overlapping the VanS B G2 ATP‐binding motif, leads to constitutive glycopeptide resistance in VanB‐type Enterococcus faecium
Author(s) -
Depardieu Florence,
Courvalin Patrice,
Msadek Tarek
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03771.x
Subject(s) - biology , enterococcus faecium , microbiology and biotechnology , autophosphorylation , operon , alanine , phosphatase , escherichia coli , gene , electrophoretic mobility shift assay , biochemistry , amino acid , kinase , phosphorylation , gene expression , protein kinase a , antibiotics
Summary Enterococcus faeciumclinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomalvanBcluster, including thevanSB/vanRBtwo‐component system regulatory genes.Enterococcus faeciumstrain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. Theddlgene of BM4525 had a 2 bp insertion leading to an impairedd‐alanine:d‐alanine ligase. Sequencing of thevanBoperon in BM4525 also revealed an 18 bp deletion in thevanSBgene designatedvanSBΔ. The resulting six amino acid deletion partially overlapped the G2 ATP‐binding domain of the VanSBΔhistidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base‐pairs. The VanSB, VanSBΔand VanRBproteins were overproduced inEscherichia coliand purified.In vitroautophosphorylation of the VanSBand VanSBΔhistidine kinases and phosphotransfer to the VanRBresponse regulator did not differ significantly. However, VanSBΔwas deficient in VanRBphosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance inE. faeciumBM4525 was therefore a result of the lack of production ofd‐alanyl‐d‐alanine ending pentapeptide and to constitutive synthesis ofd‐alanyl‐d‐lactate terminating peptidoglycan precursors, following loss ofd‐alanine:d‐alanine ligase and of VanSBphosphatase activity respectively. We suggest that the heptanucleotide direct repeat invanSBmay favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB‐type strains.