Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.

BackgroundStaphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents.ResultsWe have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes.ConclusionCWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.

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.

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.

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