Molecular interactions between two 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 previously 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 (GST) 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 bind specifically to the P2, P3, and the combined P2-P3 promoter fragments 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 evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis 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 (SarA16-124) could activate agr transcription to a level approaching that of the full-length counterpart (SarA1-124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription.
Chien
Y
Y
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York 10021, USA.
Cheung
A L
AL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D016372:RNA, Antisense / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
C095698:Agr protein, Staphylococcus aureus; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C034477:RNA primers; D016372:RNA, Antisense; D012329:RNA, Bacterial; C096035:RNAIII, Staphylococcus aureus; D011993:Recombinant Fusion Proteins; C088021:SarA protein, bacterial; D015534:Trans-Activators; D014157:Transcription Factors; D012313:RNA