Because of increasing antibiotic resistance, Staphylococcus aureus continues to be a major human pathogen. To develop a novel approach against this pathogen we have tried to understand the genetic control apparatus in an attempt to identify new targets amenable to therapy. Using Tn9l7 mutagenesis , we identified a locus on the S. aureus chromosome, designated sar, that is involved in the regulation of several extracellular and cell wall virulence factors. The sar locus is composed of three overlapping transcripts, each encoding SarA, the major sar regulatory molecule. The SarA protein (14.5 kD) binds to the agr promoter region to modulate transcription of RNAII and RNAIII (the agr regulatory molecule) from the agr P2 and P3 promoters. As agr is a pleiotropic regulator of exoprotein synthesis, our data are consistent with the hypothesis that SarA positively regulates the expression of exoprotein genes via agr. The binding site of SarA on the agr promoter has been mapped to a 29-bp sequence in the P2-P3 interpromoter region. Sequence alignment revealed a conserved """"""""SarA recognition motif"""""""" upstream of the -35 promoter boxes of several sar target genes (e.g. hla, spa and fnbB) that is homologous with the 29-bp sequence. Deletion of the """"""""SarA recognition motif"""""""" in the agr and the spa promoter regions renders the respective genes unresponsive to the effect of the sar locus. To verify the hypothesis that SarA binds to a conserved SarA recognition motif in various target genes to modulate transcription we propose to examine the interactions of SarA with target promoters (hla, fnbB and spa) lacking the SarA recognition motif. These studies will be followed by footprinting and in vitro transcription assays of target promoters in the presence of SarA. These in vitro data will be confirmed by in vivo transcription study of S. aureus cells carrying sar target genes lacking the SarA recognition motif. A corollary to our hypothesis is that the activation of sar target genes may depend on the SarA protein level which, in turn, may be controlled by SarA and genetic elements within the extensive 800-bp sar promoter region. Additionally, a l3 kD protein, designated SarR, may bind to the sar promoter region to modulate sar transcription and ultimately SarA expression. We thus propose to evaluate the contribution of these genetic elements and regulatory proteins in regulating SarA expression and hence target gene transcription. The results of these studies will provide a unifying hypothesis for sar-mediated regulation whereby SarA binds to the conserved SarA recognition motif to control target gene transcription and that activation of these promoters is dependent on the SarA protein levels. This knowledge is indispensable if we are to design synthetic analogs to interfere with the expression of virulence genes controlled by the sar locus in the future.
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