Figure 4. Effects of putative CRP binding sites on the tfoX^VC expression.

(A) The structural organizations of the tfoX^VC promoter and transcriptional fusions. Two putative CRP binding sites were found in the upstream region of tfoX^VC centered at positions −84.5 and −41.5. (B) A deletion analysis of the tfoX^VC promoter. V. cholerae strains C7258∆lacZ (WT) and WL7258∆lacZ (∆crp) containing the p2tfoXVC–lacZ and p3tfoXVC–lacZ fusion, respectively, were grown at 37 °C to mid-log phase. (C) The promoter activities of wild-type and CBS mutated fusions. C7258∆lacZ containing ptfoXVC–lacZ, ptfoXVC-lacZ-CBS1M, ptfoXVC-lacZ-CBS2M, or ptfoXVC-lacZ-CBS1M+2M were grown at 37 °C to the mid-log phase. The β-galactosidase activity was measured as described in the Methods. The mutated bases in fusions were constructed by site-directed mutagenesis and underlined. (D) V. cholerae strains C7258 (WT), C7258∆ptfoX-CBS1M, C7258∆ptfoX-CBS2M and C7258∆ptfoX-CBS1M+2M were grown in LB medium to late-log phase. The tfoX^VC and pilB, chiA-1 and chiA-2 mRNA abundances were measured by qRT-PCR. Error bars indicate the standard deviations of three independent cultures. The “WT” bar was set to 1 and used as a reference to calculate subsequent expression values. ***Significantly different from the wild-type strain (t-test, P < 0.0003). *Significantly different from the wild-type strain (t-test, P < 0.05).