Fig. 4

Both c-di-GMP binding sites of BrlR affect BrlR-DNA binding. a Electrophoretic mobility shift assays (EMSAs) demonstrating specificity of BrlR binding. A 36-bp FAM-labeled PbrlR or PpscE-F DNA (0.375 pmol) was incubated with increasing amounts of BrlR protein (the concentrations are noted in the panel). The results were quantified by band densitometry (right). Error bars are s.d. for triplicate experiments. b BrlR-DNA gel mobility shift assays using FAM-labeled PbrlR DNA (0.375 pmol) in the absence or presence of increasing concentrations of c-di-GMP and c-di-AMP. A total of 225 pmol of purified BrlR was used. The BrlR-DNA complexes were quantified by band densitometry (right). c–e BrlR-DNA gel mobility shift assays using BrlR mutants and PbrlR DNA in the absence and presence c-di-GMP. The protein-DNA complexes within the top red rectangle are overexposed. c The mutants for two c-di-GMP binding sites. d The single point mutants of the first c-di-GMP binding site. e The single point mutants of the second c-di-GMP binding site. The BrlR-DNA complex was quantified by band densitometry on the right side of the gel. f Far-UV CD spectra (200–250 nm) were obtained for the wild-type BrlR and its related mutants, which were collected at ∼20 µM at 25 °C on a Jasco J-810 spectropolarimeter. g Use of plasmid-borne lacZ transcriptional fusions to investigate the effect of BrlR protein on its own promoter at low and high c-di-GMP levels in P. aeruginosa. The promoter activities of brlR were tested in ΔbrlR strains, and plasmid pHERD20T-PA4843 and pHERD20T were used to regulate c-di-GMP concentrations. An empty lacZ vector was used as a negative control. Error bars, s.d., obtained from triplicate experiments, n = 3. h The relative transcriptional changes in brlR are presented for P. aeruginosa PAO1 harboring pHERD20T-PA4843 and pHERD20T. Transcription levels of 16 S RNA were used as controls. Error bars, s.d., obtained from triplicate experiments, n = 3