FIG 6.

Motility regulation. (A) Soft-agar motility assay. Bacterial cultures were stabbed in the middle of each soft-agar plate and incubated at 37°C for 16 h. The experiments were performed four times in quadruplicate. (B to D) β-Galactosidase activity assay for expression of fliA (B), fliC (C), and flhDC (D). fliA-lacZ, fliC-lacZ, and flhDC-lacZ transcriptional fusion strains were grown at 37°C in LB medium with shaking until the OD reached 0.5. β-Galactosidase activity was measured. The values shown are means plus standard deviations for triplicate samples from three independent experiments. Significant differences are indicated by asterisks (***, P < 0.0001 compared to the WT and mutant). (E) Nonradioactive EMSA of binding of (FnrD154A)₂-His₆ to the promoter regions. PCR products of the fliC promoter region were used as probes at 300 ng per reaction mixture. Purified (FnrD154A)₂-His₆ fusion protein was added to each reaction mixture at different concentrations, as indicated; ydfZ promoter region DNA probes with and without the FNR protein were used as positive controls, and fliC coding region DNA probes with and without the FNR protein were used as negative controls. DNA fragments were stained with SYBR green.