Molecular cloning and transcriptional regulation of ompT, a ToxR-repressedgene in Vibrio cholerae

In pathogenic Vibrio cholerae, at least 17 genes are co-ordinately regulate d by ToxR. Most of these genes, including those that encode cholera toxin ( CT), toxin co-regulated pilus (TCP), accessory colonization factor (ACF) an d OmpU, are positively regulated. OmpT is the only identified protein under negative regulation of ToxR. To understand the molecular mechanism by whic h ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR. Sequence analysis revealed that om pT encodes a predicted 35.8 kDa outer membrane porin of V. cholerae. Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon. Both primer extension analysis and promo ter fusion studies showed that ToxR represses OmpT expression at the transc riptional level. Promoter fusion studies also suggest that cyclic AMP recep tor protein (CRP) is involved in ompT activation. Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediate s repression of ompT transcription by directly binding to an A/T-rich regio n between -95 and -30 of the ompT promoter. To further understand how the i nteraction of ToxR with different promoters results in its function as an a ctivator or repressor, we have also mapped the regions on the ctxAB and tox T promoters to which ToxR binds. The regions protected by ToxR on each of t hese promoters are all A/T rich and large in size, although they are positi oned differently relative to each transcriptional start site.