Characterization of the SarA virulence gene regulator of Staphylococcus aureus

Staphylococcus aureus is a potent human pathogen that expresses a large num ber of virulence factors in a temporally regulated fashion. Two pleiotropic ally acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system f rom the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of b oth agr operons. We have cloned the sarA gene, expressed SarA in Escherichi a coil and purified the recombinant protein to apparent homogeneity. The pu rified protein was found to be dimeric in the presence and absence of DNA a nd to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-aff inity binding sites composed of two half-sites each. Quantitative electroph oretic mobility shift assays (EMSAs) were used to derive equilibrium bindin g constants (K-D) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragme nt including all three binding sites. Our data indicate that SarA regulatio n of the agr operons involves binding to multiple half-sites and may involv e other sites located downstream of the promoters.