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Summary: The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanRBSB gene encoding a two-component regulatory system and the vanYBWHBBXB resistance genes that are transcribed from promoters PRB and PYB respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanRB and vanYB, 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 PRB and PYB promoters. VanRB bound at a single site at position -32.5 upstream from the PRB transcriptional start site and at two sites at positions -33.5 and -55.5 upstream from that of PYB. 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 PRB and PYB. In the absence of regulator, E. coli RNA polymerase was able to interact with PRB but not with PYB. 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 PYB more strongly than PRB. Analysis of the protected regions revealed one copy of a 21 bp sequence in the PRB promoter and two copies in the PYB promoter which may serve as recognition sites for VanRB and VanRB-P binding that are required for transcriptional activation and expression of vancomycin resistance.

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