New Molecular Mechanism of Superbiofilm Elaboration in a Staphylococcus aureus Clinical Strain.

Previously, we reported a novel regulator of biofilm ( rob ) with a nonsense mutation in the superbiofilm-elaborating strain JP080. Intriguingly, the complementation of JP080 with wild-type rob did not completely abolish its superbiofilm-elaborating phenotype. Therefore, we searched for other possible mutation(s) using complete genome sequence data and found a missense mutation in the gene icaR , which altered its 35th amino acid (Ala35Thr). To further study the mechanism of superbiofilm elaboration in JP080, we reconstructed the same mutations of rob and icaR in the strain FK300 and analyzed the phenotypes. The mutation of rob (A331T) increased biofilm elaboration, as previously demonstrated; similarly, an icaR mutation increased poly- N -acetylglucosamine and biofilm production in strain FK300. Furthermore, our analyses indicated that the double mutant of rob and icaR produced significantly more biofilms than the single mutants. Additionally, gel shift analysis revealed that the icaR from JP080 lost its ability to bind to the ica promoter region. These findings suggest that the icaR mutation in JP080 may result in a nonfunctional protein. We compared ica operon expression in an icaR single mutant, rob single mutant, and rob and icaR double mutant to the wild type. The rob and icaR mutants showed increased ica operon transcription by approximately 19- and 79-fold, respectively. However, the rob and icaR double mutant showed an approximately 350-fold increase, indicating the synergistic effects of icaR and rob on JP080 biofilm elaboration. Consequently, we concluded that the double mutations rob and icaR synergistically increased ica operon transcription, resulting in a superbiofilm phenotype in Staphylococcus aureus. IMPORTANCE Poly- N -acetylglucosamine (PNAG) is a major component of S. aureus biofilm. PNAG production is mediated by the products of four genes, icaADBC encoded in the ica operon, and the major negative regulator of this operon is IcaR encoded just upstream of icaADBC . Previously, we reported another negative regulator, Rob, through gene expression analysis of clinically isolated superbiofilm-elaborating strain JP080. The rob gene is encoded at different loci distant from the ica operon. Here, we report that JP080 also carried a mutation in icaR and demonstrated that IcaR and Rob synergistically regulate PNAG production. We successfully reconstructed these mutations in a wild type, and the double mutant resulted in superbiofilm-elaborating phenotype. We clearly show that loss of function of both IcaR and Rob is the very reason that JP080 is showing the superbiofilm-elaborating phenotype. This study clearly demonstrated there are at least two independent regulators synergistically fine-tuning PNAG production and suggested the complex regulatory mechanism of biofilm production.