Determination of VraR Binding Activity on Promoters of fmtA, murZ, sgtB and pbp2

VraSR two-component system rapidly senses the cell wall damage by antibiotics and positively modulates a set of genes (VraSR Regulon) to enhance the resistance phenotype in S. aureus. fmtA, murZ, sgtB and pbp2 are members of VraSR Regulon, and they are involved in cell wall peptidoglycan synthesis in response to cell wall inhibitors such as β-lactams. We investigated VraR binding activity on fmtA promoter/its mutants by in vitro and in vivo experiments. We found VraR can bind to two conserved motifs on fmtA promoter in the formation of a dimer and up-regulate the transcription of fmtA under oxacillin conditions. We also found fmtA had two transcription start sites: -157G was responsible for maintenance of a basal level expression, and -195G was induced by oxacillin treatment. We reported VraR/phosphorylated-VraR binding sequences on promoters of murZ, sgtB and pbp2. Putative transcription start sites of murZ, sgtB and pbp2 were also identified.