Binding sites of VanRB and σ70 RNA polymerase in the vanB vancomycin resistance operon of Enterococcus faecium BM4524

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.