Table 1.
Strains and Plasmids used in this study.
Table 2.
Oligonucleotides and qRT-PCR probes used in this study.
Figure 1.
Comparison of VRS1, VRS1Δvra and VRS1 CΔvra growth curves in absence (Van0) and presence of vancomycin at concentrations of 32 µg/ml and 512 µg/ml (Van 32 and Van 512).
Colonies were picked from TSA, diluted to a density equivalent to a 0.5 McFarland standard in 0.9% saline. This inoculum was diluted to 5×105 CFU/ml and dispensed in a volume of 0.6 ml in each well.
Figure 2.
Comparison of MICs of the wild type VRS1, isogenic vraTSR deletion strain VRS1Δvra and complemented strain, VRS1 CΔvra.
(A) MIC for Vancomycin (B) MIC for Oxacillin. The bars represent the SD of the mean MIC from at least 4 independent determinations. The lack of error bars indicates a SD of 0. Vancomycin was tested in BHI at 0, 8, 16, 32, 64, 128, 256, and 512 µg/ml. Oxacillin was tested in TSB containing 2% NaCl at 0, 8, 16, 32, 64 and 128 µg/ml; Tetracycline (5 µg/ml) was used to test strain VRS1cΔvra to maintain the complementation plasmid pVRASR2.
Figure 3.
Effect of deletion of the vraTSR operon on steady state expression of the vanA operon genes by qRT-PCR.
Subinhibitroy concentration of vancomycin (2 µg/ml) was present in the growth media in two successive passages to maintain constitutive expression of vanA. The cultures were harvested at midlog (OD600 0.5) and stationary (OD600 1.0) growth phases as described in Materials and Methods. Expression was quantified by relative quantification (RQ) using the comparative ΔΔCt method and the wild type strain VRS1 at midlog growth phase (OD600 0.5) as the reference (after correction of each probe with an endogenous control). The gyrB gene probe labeled with the fluorophore Cy5 was used as the endogenous housekeeping gene and FAM-labeled probes for target genes vanA, vanX and vanR were used. The reactions were performed with three biological replicates with three technical replicates. * indicates p<0.05 compared with strain VRS1 at same time point. Error bars reflect propagated error calculated starting with the SD from the average CT in each replicate.
Figure 4.
Effect of deletion of vraTSR on vanA and vanR gene induction by vancomycin.
Expression of vanA operon genes in wildtype VRSA clinical strain (VRS1), the vraTSR deletion mutant (VRS1Δvra) and the vraTSR-complemented mutant (VRS1cΔvra), as measured by qRT-PCR. Vancomycin was added to early log cultures (when cultures reached an OD600 of 0.2) to induce vanA expression and RNA was isolated 1 hour later. Expression of each gene target was evaluated using relative quantification (RQ) with the comparative ΔΔCt method using strain VRS1 treated with vancomycin as the reference (after each probe was normalized to an endogenous control). Error bars reflect the range of RQ values from 3 experimental triplicates. The vanA gene probe was labeled with FAM and used in duplex reactions with the 16S rRNA gene probe labeled with fluorophore Cy5 as the endogenous control. A FAM-labled vanR probe was used in a duplex reaction with a gyrB probe labeled with fluorophore Cy5 as the endogenous control. The choice of the endogenous controls were based on compatibility with the target in the duplex reaction.