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A major constituent of manyStaphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or polyN -acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of theicaADBC operon, which is negatively regulated by the divergently transcribedicaR gene. We previously reported the identification of a gene,rbf , involved in the positive transcriptional regulation oficaADBC transcription by repressingicaR inS. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream oficaR oricaA , suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulatesica 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 activatingicaADBC . Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream oficaR within theicaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to thesarX promoter inS. aureus . These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation inS. aureus .

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