Mutational Analyses of Open Reading Frames within the vraSR Operon and Their Roles in the Cell Wall Stress Response of Staphylococcus aureus

The exposure of Staphylococcus aureus to a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. The vraSR genes form part of the four-cistron autoregulatory operon orf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed that orf1 played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF in S. aureus appears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of Delta YvqF mutants, which were similar to those of Delta VraR mutants in which CWSS induction also was completely abolished. Resistance profiles of Delta VraS mutants differed phenotypically from those of Delta YvqF and Delta VraR mutants on many non-beta-lactam antibiotics. Delta VraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than Delta YvqF and Delta VraR mutants. Subpopulations of Delta VraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a Delta VraS than a Delta VraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.