1887

Abstract

Group A (GAS) possesses a complex regulatory system enabling the organism to colonize a range of physiologically distinct host sites. Within this network of regulators is the streptococcal regulator of virulence (Srv). Srv is a member of the CRP/FNR family of transcriptional regulators and is most similar to pleiotropic regulatory factor A (PrfA), a positive regulator of virulence in . Members of this family possess a characteristic C-terminal helix–turn–helix motif (HTH) that facilitates binding to DNA targets. Genome scanning identified four targets in GAS that were similar to the consensus DNA target recognized by PrfA. Furthermore, previous amino acid sequence alignments identified conserved residues within the Srv HTH which are necessary for function in PrfA and CRP. Here we investigated the ability of Srv to interact with DNA and evaluated the role of the HTH in this interaction. Purified recombinant Srv (rSrv) was found to co-purify with an untagged form of Srv. Glutaraldehyde cross-linking and gel-filtration chromatography indicated that this co-purification is likely due to the ability of Srv to oligomerize. Electrophoretic mobility shift assays (EMSAs) demonstrated that rSrv retarded the mobility of DNA targets and a supershift analysis confirmed the observation was rSrv-dependent. Competition EMSA indicated that rSrv had a higher relative affinity for the DNA targets studied than non-specific DNA. Site-directed mutagenesis of residues predicted to be in or near the HTH resulted in a decrease or abrogation of DNA binding. Complementation of MGAS5005Δ with one of these site-directed mutants failed to restore wild-type SpeB activity. Taken together, these data suggest that the Srv HTH is necessary for DNA binding and Srv function.

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2008-07-01
2019-11-12
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