FIG 4.

Indole is an agonist of ToxR activity. (A) ToxR and OmpU Western blots. WT strain JB58 was cultured in T medium supplemented with amino acids (NRES) or increasing concentrations of indole for 4 h, and culture aliquots were collected, normalized by determining the OD₆₀₀, and used for Western blotting with an anti-ToxR (bottom) or an anti-OmpU (top and middle) polyclonal antibody. A ΔtoxRS mutant grown in T medium was included as a marker for ToxR and OmpU. The asterisk indicates a nonspecific band that was used as a loading control. (B) Indole does not affect toxR expression. WT strain JB58 carrying a toxRS-lacZ reporter was cultured in T medium with the indicated indole concentrations for 4 h when β-galactosidase was quantified. The data are means ± the SD for three independent experiments. (C and D) Indole increases the expression of ToxR-dependent genes. The indicated V. cholerae strains harboring leuO-lacZ or ompU-lacZ reporters were cultured under the conditions described above, and gene expression was quantified by determining the β-galactosidase activity. The data are means ± the SD for at least three independent experiments. Statistical significance was determined by two-way ANOVA (*, P ≤ 0.05 relative to the methanol control).