MOLECULAR-INTERACTIONS BETWEEN 2 GLOBAL REGULATORS, SAR AND AGR, IN STAPHYLOCOCCUS-AUREUS

The expression of many virulence determinants in Staphylococcus aureus is controlled by regulatory loci such as agr and sar, We have previou sly shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus, To define the specific molecular interaction, we overexpressed SarA as a glutathione S-transferase (GS T) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector, The purified GST-SarA as well as cleaved SarA were able to bi nd specifically to the P2, P3, and the combined P2-P3 promoter fragmen ts of agr in gel shift assays, Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evid enced by the formation of a supershifted band, The SarA binding site o n the agr promoter, mapped by DNase I footprinting assay, covered a 29 -bp region between the P2 and P3 promoters devoid of any direct repeat s, A synthetic 45-bp fragment encompassing the 29-bp sequence also bou nd the SarA protein in band shift assays, Serial in-frame deletion ana lysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16-124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA(16-124)) could activate agr transcription to a le vel approaching that of the full-length counterpart (SarA(1-124)), Tak en together, these data indicated that SarA is a DNA-binding protein w ith binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription.