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

Abstract

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 ⌬YvqF mutants, which were similar to those of ⌬VraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ⌬VraS mutants differed phenotypically from those of ⌬YvqF and ⌬VraR mutants on many non-ß-lactam antibiotics. ⌬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 ⌬YvqF and ⌬VraR mutants. Subpopulations of ⌬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 ⌬VraS than a ⌬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.

Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation

“…At a 95% confidence interval, we estimate at least a four order of magnitude higher dimerization propensity when BeF 3 − is present compared with its absence (Table 1). A shift in the protein distribution from a sedimentation coefficient of 2.2 Svedberg (S) units when BeF 3 − is absent, to 3.2 S when BeF 3 − is present, was observed using sedimentation velocity AUC analysis (Fig. 1B).…”

“…Purified VraR protein was crystallized in the absence and presence of beryllofluoride. Diffraction data for crystals of apo VraR and VraR in complex with Mg 2+ and BeF 3 − were collected at Cornell High Energy Synchrotron Source beamline F1 and Brookhaven National Synchrotron Light Source beamline X29, respectively, and the structure determined by molecular replacement in both cases. Detailed methods for protein crystallization, diffraction data collection, and structure determination are provided in SI Materials and Methods.…”

“…Mutations or deletions within genes encoding either the sensor histidine kinase VraS or the response regulator VraR restrict antibiotic resistance to both β-lactam and glycopeptide antibiotics across a range of S. aureus strains (2)(3)(4). Furthermore, deletion of the vraSR operon in a clinical USA300 MRSA strain generated an isogenic mutant, that unlike the parent strain, could be treated effectively with the β-lactam oxacillin in mouse models of both skin and lung infections (5).…”

Lantibiotic Resistance

“…In the absence of VraSR, S. aureus shows a significant increase in susceptibility to various cell wall synthesis inhibitors and antimicrobials, including nukacin ISK-1, oxacillin, cefoxitin, tunicamycin, cycloserine, daptomycin, vancomycin, and bacitracin (124,153,157). The vraSR genes have been found to be more highly expressed in some vancomycin-intermediate S. aureus (VISA) strains than in vancomycin-susceptible S. aureus (VSSA).…”

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

“…Inactivation of vraSR both decreases the resistance of different S. aureus strains to ␤-lactams and vancomycin and converts the homogeneous oxacillin resistance phenotype into a highly heterogeneous resistance phenotype (15,25). In contrast, a constitutively active vraSR operon, caused by a single-point mutation in vraS, leads to increased teicoplanin resistance of clinical glycopeptideintermediate S. aureus strains (19).…”