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H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin-and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

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Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the ؊35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the ␣-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.Vibrio cholerae is the etiological agent of the human diarrheal disease cholera. Two virulence factors that are produced by V. cholerae and are essential for disease are toxin-coregulated pilus (TCP) (62) and cholera toxin (CT) (28). TCP is a type IV pilus that is assembled by polymerization of the pilin subunit, TcpA, and forms long filaments that laterally associate into bundles. Expression of TCP in vitro results in autoagglutination of the bacterium, and TCP-mediated bacterium-bacterium interactions in vivo facilitate microcolony formation on the intestinal epithelium. Pilus biogenesis requires at least 9 other proteins in addition to TcpA, which are encoded in an operon located on the Vibrio pathogenicity island (VPI). The pilus biogenesis apparatus plays a second role in colonization by secreting the cotranscribed, soluble colonization factor TcpF, which is also essential for colonization but for which a mechanism remains unknown (29, 30). The second main virulence factor, CT, is a potent A 1 B 5 subunit, ADP-ribosylating toxin that is responsible for the severe watery diarrhea that is associated with cholera. CT is encoded by the ctxAB operon, which is located on the lysogenic bacteriophage CTX. The VPI and CTX were both acquired by horizontal gene transfer. TCP serves as the high-affinity receptor for CTX that links TCP production to ctx acq...

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