On for maximal induction of vanA and vanR by vancomyin. Furthermore, we show that the increase in vanA operon gene expression from midlog to stationary growth phase is dependent upon an intact VraTSR cell wall stress sensing system. This suggests that vanA expression is induced as cells sense that growth is slowing. This phenomenon could be linked to a signal generated by increased autolysis and slowing of peptidoglycan precursor incorporation into 1676428 the cell wall. It is worth noting that although prior studies have examined the inducibility of growth and D-Ala-D-Lac peptidoglycan precursor production in VRSA strains, this is the first study to examine vanA gene expression in S. aureus. Previously, vraTSR has only been shown to influence expression of native staphylococcal genes. This study now shows that despite being a native gene encoded on the staphylococcal chromosome, vraTSR can be utilized by S. aureus to control the expression of heterologous cell wall biosynthesis operon that is acquired horizontally with the advantage of conferring antibiotic resistance. This represents a particularly Salmon calcitonin custom synthesis clever strategy since both van operon expression and vraTSR are induced by vancomycin. This study confirms and extends a prior study in which the effect of vraTSR on vancomycin resistance had been tested in a VRSA Acid Yellow 23 strain containing the vanA operon. Gardete et al. produced a strain COLVA200DvraS by introducing the plasmid from strain VRS1 into strain COL and deleting vraTSR. In contrast, the approach taken in this study was to delete the vraTSR operon from the native clinical VRS1 strain which is the source of the plasmid used to construct COLVA200DvraS. Moreover, the strain used in our study belongs to the same clonal cluster as all other clinical VRSA isolates reported, clonal cluster 5. In contrast COLVA200 DvraS belongs to ST250 from clonal cluster 8. It was interesting that the vraTSR deletion in the clinical VRSA isolate decreased the vancomycin MIC to a greater extent than seen in the lab derivedstrain. Since both strains harbor the same vanA containing plasmid from VRS1, this provides evidence that factors in addition to vraTSR can account for differences in the level of vanA mediated vancomycin resistance among naturally occurring clinical isolates. This is consistent with historical data for oxacillin resistance. Although vraTSR affects resistance to oxacillin, strain specific factors other than vraTSR also influence the level of oxacillin resistance. We observed a slight paradox on the effect of vraTSR during steady state vanA induction in midlog phase expression of vanA and vanX genes relative to vanR expression. Whereas midlog phase cultures of VRS1Dvra and VRS1 expressed similar levels of vanA and vanX, vanR expression was drastically higher in VRS1Dvra compared with VRS1. Nevertheless, at stationary phase vanR gene expression was drastically lower in the the vraTSR mutant compared with the wildtype, as it was for vanA and vanX. vraTSR Control of vanA Operon Expression in VRSA Although vancomycin resistance decreases by 16 fold with a vraTSR operon deletion and is statistically significant, it may not be clinically important, as the vancomycin MIC still remains in the resistant range. It is possible however, that chemical inhibitors of VraTSR might be able to synergize with vancomycin to improve therapy of VRSA and VISA infections. This proof of principle remains to be tested in animal models of vancomycin therapy of vraTSR mutants, as.On for maximal induction of vanA and vanR by vancomyin. Furthermore, we show that the increase in vanA operon gene expression from midlog to stationary growth phase is dependent upon an intact VraTSR cell wall stress sensing system. This suggests that vanA expression is induced as cells sense that growth is slowing. This phenomenon could be linked to a signal generated by increased autolysis and slowing of peptidoglycan precursor incorporation into 1676428 the cell wall. It is worth noting that although prior studies have examined the inducibility of growth and D-Ala-D-Lac peptidoglycan precursor production in VRSA strains, this is the first study to examine vanA gene expression in S. aureus. Previously, vraTSR has only been shown to influence expression of native staphylococcal genes. This study now shows that despite being a native gene encoded on the staphylococcal chromosome, vraTSR can be utilized by S. aureus to control the expression of heterologous cell wall biosynthesis operon that is acquired horizontally with the advantage of conferring antibiotic resistance. This represents a particularly clever strategy since both van operon expression and vraTSR are induced by vancomycin. This study confirms and extends a prior study in which the effect of vraTSR on vancomycin resistance had been tested in a VRSA strain containing the vanA operon. Gardete et al. produced a strain COLVA200DvraS by introducing the plasmid from strain VRS1 into strain COL and deleting vraTSR. In contrast, the approach taken in this study was to delete the vraTSR operon from the native clinical VRS1 strain which is the source of the plasmid used to construct COLVA200DvraS. Moreover, the strain used in our study belongs to the same clonal cluster as all other clinical VRSA isolates reported, clonal cluster 5. In contrast COLVA200 DvraS belongs to ST250 from clonal cluster 8. It was interesting that the vraTSR deletion in the clinical VRSA isolate decreased the vancomycin MIC to a greater extent than seen in the lab derivedstrain. Since both strains harbor the same vanA containing plasmid from VRS1, this provides evidence that factors in addition to vraTSR can account for differences in the level of vanA mediated vancomycin resistance among naturally occurring clinical isolates. This is consistent with historical data for oxacillin resistance. Although vraTSR affects resistance to oxacillin, strain specific factors other than vraTSR also influence the level of oxacillin resistance. We observed a slight paradox on the effect of vraTSR during steady state vanA induction in midlog phase expression of vanA and vanX genes relative to vanR expression. Whereas midlog phase cultures of VRS1Dvra and VRS1 expressed similar levels of vanA and vanX, vanR expression was drastically higher in VRS1Dvra compared with VRS1. Nevertheless, at stationary phase vanR gene expression was drastically lower in the the vraTSR mutant compared with the wildtype, as it was for vanA and vanX. vraTSR Control of vanA Operon Expression in VRSA Although vancomycin resistance decreases by 16 fold with a vraTSR operon deletion and is statistically significant, it may not be clinically important, as the vancomycin MIC still remains in the resistant range. It is possible however, that chemical inhibitors of VraTSR might be able to synergize with vancomycin to improve therapy of VRSA and VISA infections. This proof of principle remains to be tested in animal models of vancomycin therapy of vraTSR mutants, as.