Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus

Fibronectin-binding proteins (FnBPs) are thought to be important for the at tachment of Staphylococcus aureus during infection. The regulation of the g enes fnbA and fnbB by the global regulatory loci sar and agr was examined u sing site-specific regulatory mutants of S. aureus strain Newman. The resul ts from binding assays using both aqueous and solid-phase fibronectin as we ll as ligand blotting with biotinylated fibronectin showed that the express ion of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar-agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA-specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB p romoter activity in promoter fusion assays was not affected by either sar o r agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post-tra nscriptional level. Gel shift assays demonstrated that SarA binds to the fn bA promoter fragments, probably as a dimer. DNase I footprinting assays wit h SarA revealed a protected area of 102 bp upstream of fnbA.