The Impact of Selected Gene Mutations to the Activation and DNA Binding Activity of the Transcription Factor VraR

VraSR two-component system in S. aureus senses bacterial cell wall damage caused by antibiotics (vancomycin or -lactams) and regulates a set of genes. We have undertaken the study of the role of certain amino acids in the function of VraR, which undergo mutation in clinical S. aureus resistant strains and analyzed their effects on the VraSR signal transduction mechanism. In particular, we have looked at the role of Met-13 in VraR phosphorylation induced activation. M13A failure to be activated by phosphorylation resulted in a weaker binding to DNA. Moreover, we investigated VraR E59D, S164P, A113V, and S164A binding activity to the PvraSR by DNase-I footprinting. Finally, in vivo studies on the effects of substitutions of the above residues on the mRNA level of a number of VraSR regulon genes (fmtA, pbp2, and sgtb) provide insights as to how substitutions at certain positions may be beneficial to the S. aureus resistance to antibiotics.