Rbf Promotes Biofilm Formation by Staphylococcus aureus via Repression of icaR , a Negative Regulator of icaADBC

We previously reported the identification of a gene,rbf , involved in the regulation of biofilm formation byStaphylococcus aureus 8325-4. In an effort to study the mechanism of regulation, microarrays were used to compare the transcription profiles of the wild-type strain with anrbf mutant and anrbf overexpression strain of the clinical isolate UAMS-1. Among the genes affected byrbf overexpression are those of the intercellular adhesion (ica ) locus; however, expression of these genes was not affected by anrbf deletion in the chromosome. TheicaADBC genes are responsible for production of poly-N -acetylglucosamine (PNAG), a major constituent of biofilm. TheicaR gene encodes a negative regulator oficaADBC . In UAMS-1 carrying an Rbf-encoding plasmid, Rbf was found to repressicaR transcription with a concomitant increase inicaADBC expression and increased PNAG and biofilm production relative to isogenic strains lacking the plasmid. Sequencing of therbf gene from UAMS-1 showed that there was a 2-bp insertion affecting the 50th codon of therbf open reading frame, suggesting thatrbf is a pseudogene in UAMS-1. This finding explains why deletion ofrbf had no effect on biofilm formation in UAMS-1. To further characterize the Rbf regulation on biofilm we compared biofilm formation,icaA andicaR transcription, and PNAG production in 8325-4 and its isogenicrbf andicaR single mutants and anrbf icaR double mutant. Our results are consistent with a model whereinrbf represses synthesis oficaR , which in turn results in derepression oficaADBC and increased PNAG production. Furthermore, purifiedrbf did not bind to theicaR oricaA promoter region, suggesting thatrbf controls expression of an unknown factor(s) that repressesicaR . The role ofrbf in controlling theS. aureus biofilm phenotype was further demonstrated in a clinical strain, MW2.