Co-ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR-regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR-dependent toxT expression are discussed.

Virulence gene expression in Vibrio cholerae is postulated to involve ToxR-dependent activation of the toxT gene followed by ToxT activation of virulence genes, including several of those involved in biogenesis of the toxin-coregulated pilus. ToxR is a transmembrane, DNA-binding protein which is a member of the OmpR subclass of two-component activator systems in bacteria. Data presented in this report demonstrate that ToxT is similar to the AraC family of transcriptional activators identified in a variety of gram-negative bacteria. The toxT open reading frame begins approximately 200 nucleotides from the end of the tcpF gene, which is part of a cluster of genes responsible for production of the toxin-coregulated pilus. Accumulation of toxT specific mRNA is ToxR dependent and is modulated by environmental conditions that modulate expression of the regulon. Within the intergenic region between tcpF and toxT is a potential stem-loop structure of an unusual nature which may play a role in regulating expression of toxT mRNA. Experiments with tcpF and toxT cloned behind a strong, constitutive promoter suggest that the two genes can be cotranscribed, but Northern (RNA) blot analysis of V. cholerae suggests that if they are, steady-state levels of their messages may be controlled by a posttranscriptional mechanism. Possible mechanisms for ToxR-dependent expression of toxT are discussed.

Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein. These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin. ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade. ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB). The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites. Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V. cholerae. In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter. These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters.Expression of many virulence genes in the human diarrheal pathogen Vibrio cholerae is under direct control of the ToxT protein (5, 7, 13). ToxT is a member of the AraC family of transcriptional activators (13) and activates expression of genes required for production of the toxin-coregulated pilus (tcp) (7, 33), accessory colonization factor (acf) (24, 25), and cholera toxin (ctxAB) (7). Expression of toxT is mediated by the ToxR protein (7, 12, 13), a transcription activator protein in the inner membrane that shares homology with members of the two-component family of response regulators found in a variety of bacterial species (22,30). A third regulatory protein required for wild-type levels of virulence gene expression is the ToxS protein, a membrane protein postulated to stabilize ToxR for DNA binding (6,20).Expression of virulence genes in V. cholerae is proposed to occur via a regulatory cascade in which ToxR, in conjunction with ToxS, activates expression of toxT and the ToxT protein directly activates expression of virulence genes (5, 7). In this model, ToxR-dependent expression of toxT is a critical step in virulence regulation in V. cholerae. The precise requirements for ToxR-dependent expression of toxT are unknown, and, since ToxR does not directly activate toxT expression when assayed in Escherichia coli, we speculate that additional V. cholerae factors may play a role (12, 31).The molecular aspects of ToxR-dependent transcription activation are not clear. The ToxR-binding sites in the ctxAB and toxT promoters have little sequence similarity. In the ctxAB promoter, ToxR b...