Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae

Abstract

The membrane proteins Toxr and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera. One of the ToxRS-regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin. To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter. Deletions with a 5' end-point at or downstream of -128, relative to the start site for transcription, did not direct expressions of a lacZ reporter gene in wild-type V. cholerae, although the - 128 promoter fragment did direct ToxRS-dependent reporter gene activity under conditions of ToxR overexpression in E. coli. Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end-point is at -211, but not with -128 or -68 fragments. ToxRS membranes did shift the -128 fragment when added at higher concentrations. DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from -238 to -139, and two downstream sites ranging from -116 to -58 and -53 to -24. Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR-dependent genes, ctxA and toxT. We suggest a model for transcription activation that involves sequential ToxR-binding events to distinct regions in the ompU promoter.