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

Previously, we reported a novel regulator of biofilm () with a nonsense mutation in the superbiofilm-elaborating strain JP080. Intriguingly, the complementation of JP080 with wild-type 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 , which altered its 35th amino acid (Ala35Thr). To further study the mechanism of superbiofilm elaboration in JP080, we reconstructed the same mutations of and in the strain FK300 and analyzed the phenotypes. The mutation of (A331T) increased biofilm elaboration, as previously demonstrated; similarly, an mutation increased poly--acetylglucosamine and biofilm production in strain FK300. Furthermore, our analyses indicated that the double mutant of and produced significantly more biofilms than the single mutants. Additionally, gel shift analysis revealed that the from JP080 lost its ability to bind to the promoter region. These findings suggest that the mutation in JP080 may result in a nonfunctional protein. We compared operon expression in an single mutant, single mutant, and and double mutant to the wild type. The and mutants showed increased operon transcription by approximately 19- and 79-fold, respectively. However, the and double mutant showed an approximately 350-fold increase, indicating the synergistic effects of and on JP080 biofilm elaboration. Consequently, we concluded that the double mutations and synergistically increased operon transcription, resulting in a superbiofilm phenotype in Staphylococcus aureus. Poly--acetylglucosamine (PNAG) is a major component of S. aureus biofilm. PNAG production is mediated by the products of four genes, encoded in the operon, and the major negative regulator of this operon is IcaR encoded just upstream of . Previously, we reported another negative regulator, Rob, through gene expression analysis of clinically isolated superbiofilm-elaborating strain JP080. The gene is encoded at different loci distant from the operon. Here, we report that JP080 also carried a mutation in 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.