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.

The transmembrane DNA-binding protein, ToxR, of Vibrio cholerae is a global transcriptional regulator of virulence gene expression. ToxR has been shown to interact with promoter regions upstream of both the ctxAB operon encoding cholera toxin, and the regulatory gene toxT. Deletion analysis has shown that a repeated sequence, TTTTGAT, is required for ToxR binding and activation of the ctxAB promoter. However, this sequence is not found upstream of the toxT promoter. Genetic selections using P22 challenge phages were used to define sites within the promoter for ctxAB which are critical for ToxR-DNA interactions. Single-base-pair changes and deletion mutations that impair ToxR binding cluster within two regions: -57 to -69 within two of three tandem TTTTGAT sequences; and from -39 to -47, between the repeat sequences; and the -35 region of the promoter. ToxR does not bind to a synthetic target that has three tandem repeats which lack a flanking upstream and downstream sequence. These results suggest that the ToxR-binding site lies immediately upstream of the - 35 position of the ctx promoter, and that the affinity of ToxR binding to this site is influenced by the repeat sequences.

ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in Vibrio cholerae. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12 toxR andtoxS positive control mutants producing variant ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in toxR specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven toxR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion oftoxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.

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