Figure 7
bsmT is epistatic to bsmR and regulates bsmR transcription through a positive feedback loop. (a) Biofilm levels of the bacterial strains quantified by crystal violet staining. (b) Relative quantification of biofilm formation. (c) Swimming motility analyses of bacterial strains grown on rich NYG plates containing 0.15% agar. (d) Relative quantification of the bacterial swimming zone diameters of the strains in (c). (e) Growth curves of bacterial strains grown in rich NYG medium at 28 °C. (f,g) bsmT positively regulates transcription of the bsmR operon. mRNA amounts were determined by semiquantitative RT-PCR (f) and quantitative RT-PCR (g), respectively. In (f), the bands represent PCR amplification products obtained using the cDNA generated from the total RNAs of the indicated strains. The tmRNA amplification product served as the loading control. −RT represents the negative control to detect the presence of contaminating DNA. (h) Electrophoretic mobility shift assay shows the direct binding of BsmT to the promoter region of the bsmR operon. 1 fmol of 32P-labeled double-stranded DNA corresponding to the promoter region of the bsmR operon was incubated at 28 °C in reaction buffer together with BsmT protein. Increasing amounts of unlabeled DNA served as the competitor. The black arrow indicates the position of the protein-DNA complex. (b,d,e and g) Each value is the average of three replicates. Error bars represent the standard deviations (n = 3). *p < 0.05, as determined by ANOVA. The qRT-PCR results (g) are representatives of triplicate repeatable experiments.
