DNA-Binding Activity of the Vancomycin Resistance Associated Regulator Protein VraR and the Role of Phosphorylation in Transcriptional Regulation of the vraSR Operon

Abstract

In Staphylococcus aureus the VraSR two-component system acts as a sentinel that can rapidly sense cell wall peptidoglycan damage and coordinate a response to enhance the resistance phenotype. VraR is a transcription factor and its cognate kinase, VraS, modulates the DNA-binding activity of VraR by regulating its phosphorylation state and hence its dimerization state. Here we provide the first report on the VraR transcriptional activity by investigating the interaction with the vraSR operon control region. We found that this region contains three VraR-binding sites, each with unique VraR-binding features. VraR binding to the most conserved site is phosphorylation independent, and dimerization is proposed to be induced through binding to DNA. By contrast, binding to the less conserved site requires phosphorylation of VraR. This site overlaps with the binding site of the sigma subunit of the RNA polymerase complex, suggesting that VraR could be interacting with the transcription machinery in the presence of the cell wall stress signal. Mutagenesis studies on the VraR binding sites suggest that there is directionality in the binding of VraR to the target DNA, probably dictated by VraR dimerization. We also constructed a P(vraSR)-fused lux operon reporter vector to investigate in vivo the significance of our in vitro studies. These studies show that upon cell wall stress, induced by oxacillin, the expression level of the lux operon goes up and it is affected by the integrity of the two identified VraR-binding sites in agreement with the in vitro studies. Further, they demonstrate that the VraR most conserved binding site is essential to the vraSR operon expression. On the other hand, they suggest that the role of the VraR less conserved site could be that of mediating high levels of vraSR operon expression during cell wall stress conditions.

Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation

“…Domain rearrangements in inactive and active VraR provide an explanation for the greater affinity of phosphorylated VraR for its target DNA sequences relative to the unphosphorylated protein (9). The DNA-binding domain consists of four α helices (Leu148 to Gln209) with a classical helix-turn-helix (HTH) DNA-binding motif that defines members of the NarL/FixJ response regulator subfamily, as previously reported for the NMR structure of the isolated domain (25).…”

“…A fluorescence anisotropy assay was developed to measure the affinity of VraR and VraR M13D with the putative R1 VraR binding site DNA (9). See SI Materials and Methods for experimental conditions and procedure.…”

“…VraS can act as both a kinase and a phosphatase to control the phosphorylation state of the cytoplasmic transcription factor VraR (8). Phosphorylation increases the DNA-binding activity of VraR (9), leading to increased expression of the vraSR operon and a regulon of at least 42 additional genes, many of which are involved in cell wall synthesis (10).…”

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

“…The DNA sequence derived from the vraSR operon control region, which encompasses nucleotides Ϫ121 to ϩ26, was amplified by PCR using the genomic DNA of S. aureus RN4220 as a template and was cloned in the blunt-end vector pSTBlue-1 as previously described (2). The resulting plasmid was named pSTblue-1:: P vraSR .…”

“…1), referred to as the R1, R2, and R3 sites (2). VraR binding sites centered at positions Ϫ60 and Ϫ35 were analyzed independently, and in vitro studies showed binding of VraR to R1 was not affected by phosphorylation of VraR, while unphosphorylated VraR did not bind to R2.…”