Figure 6. BrlR binds c-di-GMP in vitro.

(A) Pulldowns using biotinylated c-di-GMP immobilized to streptavidin magnetic beads demonstrating that BrlR binds c-di-GMP. Increasing concentrations of V5-tagged BrlR were used (1.25-10 μg). A no-protein sample (0 μg) and cell extracts obtained from P. aeruginosa inactivated in brlR (ΔbrlR) were used as controls. BrlR-c-di-GMP binding was detected by immunoblot analysis using anti-V5 antibodies. V5-tagged FleQ and BdlA, having apparent mass of 58.3 and 49.9 kDa, respectively, were used as controls. (B) Concentration-dependent binding of biotinylated c-di-GMP by BrlR. Data are based on relative intensity of bands detectable following probing with anti-biotin antibodies and subsequent analysis using ImageJ. K_D, dissociation constant for for c-di-GMP binding to BrlR. (C) BrlR-c-di-GMP binding assays in the presence of increasing concentrations of non-biotinylated c-di-GMP (c-di-GMP-NB), GTP (GTP-NB) and cyclic AMP (cAMP-NB). BrlR-c-di-GMP binding was detected by immunoblot analysis using anti-V5 antibodies. (D) Detection of BrlR-bound c-di-GMP by HPLC analysis. Detection was based on absorbance at 253 nm. C-di-GMP was extracted from BrlR-pulldowns in the absence and presence of anti-V5 antibodies by heat and ethanol extraction. A total of 2 mg of cell extract obtained from P. aeruginosa PAO1/pMJT-brlR-His₆V5 was used per pulldown. (E) Immunoblot analysis demonstrating absence/presence of BrlR in pulldown samples prior to (input) and following immunoprecipitation (beads). The respective immunoprecipitation eluates (from beads) were used for c-di-GMP extraction and subsequent HPLC analysis. Immunoblots were probed using anti-V5 antibodies. (F) BrlR oligomerization as determined using immunoblot analysis and crosslinking. Increased dimerization of BrlR is detectable upon multicopy expression of the cyclase PA4843, while only BrlR monomers are detectable upon overexpression of the phosphodiesterase PA2133 as indicated using in vivo crosslinking with dithiobis(succinimidyl propionate) (DSP) and probing with anti-V5 antibodies. Experiments were carried out at least in duplicate and representative data are shown. Error bars represent the standard deviations between replicates