Activation of sarX by Rbf Is Required for Biofilm Formation and icaADBC Expression in Staphylococcus aureus

A major constituent of many Staphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or poly N-acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of the icaADBC operon, which is negatively regulated by the divergently transcribed icaR gene. We previously reported the identification of a gene, rbf, involved in the positive transcriptional regulation of icaADBC transcription by repressing icaR in S. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream of icaR or icaA, suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulates ica expression. Here we report that the unknown factor is SarX protein. Results from epistasis assays and genetic complementation analyses suggest that Rbf upregulates SarX, which then downregulates IcaR, thereby activating icaADBC. Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream of icaR within the icaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to the sarX promoter in S. aureus. These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation in S. aureus.