FIG 5 .

ToxR regulation of leuO expression. (A) Expression of leuO-lacZ in the WT, ΔtoxT, ΔtoxRS, and ΔtcpPH strains. The strains were grown under AKI conditions and assayed for β-galactosidase activity at the indicated times. The results are representative of three experiments ± SD. (B) Effect of 1 mM cFP on aphA, aphB, and leuO expression of the WT and ΔtoxRS strains following growth under AKI conditions. RNA was isolated at 3 h and used for qRT-PCR. The expression ratios are the cFP-treated versus DMSO control culture and represent the means ± SEM for three biological replicates. *, P < 0.01 from a hypothetical value of 1.0. (C) Expression of leuO in the ΔtoxRS strain following growth in the presence and absence of cFP. The ΔtoxRS strain containing pBAD18 or pBAD18-toxRS was grown under AKI conditions in broth containing 0.02% arabinose and 1 mM cFP or an equivalent volume of DMSO. RNA was isolated at 3 h and used for qRT-PCR. The leuO expression ratios in the ΔtoxRS(pBAD18-toxRS) versus ΔtoxRS(pBAD18) strains are presented. The results are the mean for three biological replicates ± SEM. “**” indicates significance at P < 0.001. (D) ToxR Western blot of the toxR mutant or the WT grown under AKI conditions in the absence or presence of 1 mM cFP. Culture aliquots were collected at the indicated time points, normalized by OD, and processed for ToxR Western blotting. The band above ToxR is a nonspecific immunoreactive protein that serves as a loading control.