Figure 3

RovM represses hmsHMSF expression directly. (a,b) RovM repress the expression of the hms operon. The β-galactosidase activity (a) or relative expression measured by quantitative RT-PCR (b) in the indicated bacterial strains was determined. (c) RovM binds the hmsHFRS promoter. Biotin-labelled probe, unlabelled probe or an unrelated fragment was incubated with RovM [0, 0.13, 0.27, 0.54 and 0.108 µM] or BSA [5 µM]. The protein-DNA complexes were detected by streptavidin- conjugated HRP and chemiluminescent substrate. Unlabelled promoter was added to determine the binding specificity of RovM. Bio-P _hmsHFRS : biotin-labelled hmsHFRS promoter; P _hmsHFRS : unlabelled hmsHFRS promoter; URD: unrelated fragment (uncropped version was shown in Fig. S4b). (d) Identification of the RovM-binding site within the hmsHFRS promoter using a DNase I footprinting assay. (e) Nucleotide sequences of the hmsHFRS promoter region. Putative −35 and −10 elements of the hmsHFRS promoter are boxed. +1 denotes the transcription start point. The RovM-binding sites identified using the DNase I footprinting assays are indicated by shading. (f) Aggregates formed in WT, ΔrovM, ΔrovMΔhmsHFR and ΔrovM(rovM) grown in M9 to the late exponential phase. Data are presented as the mean values ± SD calculated from three sets of independent experiments. *P < 0.05; n.s., not significant.