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The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR B S B gene encoding a two-component regulatory system and the vanY B WH B BX B resistance genes that are transcribed from promoters P RB and P YB respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR B and vanY B, respectively, that allowed identification of −10 and −35 promoter motifs. The VanRB protein was overproduced in Escherichia coli, purified and phosphorylated (VanRB-P) non-enzymically with acetylphosphate. VanRB-P and VanRB specifically bound to P RB and P YB promoters. VanRB bound at a single site at position −32.5 upstream from the P RB transcriptional start site and at two sites at positions −33.5 and −55.5 upstream from that of P YB. The proximal VanRB binding site overlapped the −35 region of both promoters. VanRB was converted from a monomer to a dimer upon acetylphosphate treatment. VanRB-P had higher affinity than VanRB for its targets and appeared more efficient than VanRB in promoting open complex formation with P RB and P YB. In the absence of regulator, E. coli RNA polymerase was able to interact with P RB but not with P YB. Phosphorylation of VanRB significantly increased promoter interaction with RNA polymerase and led to an extended and modified footprint. In vitro transcription assays showed that VanRB-P activates P YB more strongly than P RB. Analysis of the protected regions revealed one copy of a 21 bp sequence in the P RB promoter and two copies in the P YB promoter which may serve as recognition sites for VanRB and VanRB-P binding that are required for transcriptional activation and expression of vancomycin resistance. [ABSTRACT FROM AUTHOR]

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d -alanine: d -alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanS B gene designated vanS BΔ . The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBΔ histidine 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 , VanSBΔ and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBΔ histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBΔ was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB . Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d -alanyl- d -alanine ending pentapeptide and to constitutive synthesis of d -alanyl- d -lactate terminating peptidoglycan precursors, following loss of d -alanine: d -alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanS B may favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB-type strains. [ABSTRACT FROM AUTHOR]

The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate. VanR(B)-P and VanR(B) specifically bound to P(RB) and P(YB) promoters. VanR(B) bound at a single site at position -32.5 upstream from the P(RB) transcriptional start site and at two sites at positions -33.5 and -55.5 upstream from that of P(YB). The proximal VanR(B) binding site overlapped the -35 region of both promoters. VanR(B) was converted from a monomer to a dimer upon acetylphosphate treatment. VanR(B)-P had higher affinity than VanR(B) for its targets and appeared more efficient than VanR(B) in promoting open complex formation with P(RB) and P(YB). In the absence of regulator, E. coli RNA polymerase was able to interact with P(RB) but not with P(YB). Phosphorylation of VanR(B) significantly increased promoter interaction with RNA polymerase and led to an extended and modified footprint. In vitro transcription assays showed that VanR(B)-P activates P(YB) more strongly than P(RB). Analysis of the protected regions revealed one copy of a 21 bp sequence in the P(RB) promoter and two copies in the P(YB) promoter which may serve as recognition sites for VanR(B) and VanR(B)-P binding that are required for transcriptional activation and expression of vancomycin resistance.