Fig. 3: VraR is a negative regulator of agr and spa in USA300.
From: Modulation of MRSA virulence gene expression by the wall teichoic acid enzyme TarO
a Disk diffusion assay for induction of vraX-lacZ by tunicamycin. b Expression of vraX-lux in S. aureus strains grown in TSB medium supplemented with or without tunicamycin (Tuni). Data from n = 4 biological replicates are reported as the mean ± SD. c Expression of psmα-lux (left panel) and the corresponding growth curve (right panel) in S. aureus strains grown in TSB medium supplemented with or without tunicamycin (Tuni). Data from n = 6 biological replicates are reported as the mean ± SD. d Effect of tunicamycin treatment on the expression levels (log2 fold changes) of psmα-lux in S. aureus strains cultured in TSB medium for 25 h (in c). Data from n = 6 biological replicates are reported as the mean ± SD. Statistical analysis was performed using Student’s two-tailed unpaired t-test. e, f Representative images (e) and quantitative analysis (f) of Western blotting for SpA in S. aureus strains grown in TSB medium supplemented with (+) or without (−) tunicamycin (0.5 μg/ml) for 3 h. SrtA is loading controls; the results are reported as relative expression levels (log2 fold changes) compared with the tunicamycin-untreated WT USA300. Data represent mean ± SD from n = 3 independent experiments. Statistical analysis was performed using two-tailed one-sample t-test (when compared with the tunicamycin-untreated WT USA300 control, which was set to a fold change of 1) or otherwise with Student’s two-tailed unpaired t-test. USA300 and ΔvraR harbor an empty pYJ335-1 vector as control; ΔvraR/c denotes the complemented strain of ΔvraR (ΔvraR/p-vraR). g Electrophoretic mobility shift assay (EMSA) showing the DNA-binding ability of purified His6-VraR to the promoter DNA fragments of spa (spa-p), vraX (vraX-p), and vraRS (vraRS-p). Control DNA, a 228-bp DNA fragment (coa-p) covering the promoter region of coa gene. h, i Electropherograms show the protection pattern of vraX (h) and spa (i) promoters after digestion with DNase I following incubation in the absence (−) and presence (+) of His6-VraR (6 μmol/L). The protected regions (relative to the start codon) are underlined.
