H-NS and ToxT Inversely Control Cholera Toxin Production by Binding to Overlapping DNA Sequences

Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by the production of cholera toxin (CT). A complex regulatory cascade controls the production of CT and other virulence factors. However, ultimately, a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (PctxAB). The toxboxes overlap the two promoter-proximal GATTTTT heptad repeats found within PctxAB in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be located within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for the control of PctxAB transcription proposes complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNA polymerase (RNAP) contacts. The goal of this study was to determine more precisely where H-NS binds to PctxAB and test the hypothesis that ToxT completely displaces H-NS from the PctxAB promoter before activating transcription. The results suggest that H-NS binds only to the region of PctxAB encompassing the heptad repeats and that ToxT displaces H-NS only from its most promoter-proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at PctxAB. IMPORTANCE H-NS is a global negative regulator of transcription in Gram-negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses the transcription of cholera toxin genes by binding to a large region upstream of its promoter and that the virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT displaces only H-NS bound to the most promoter-proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher-resolution mechanism for the antirepression of H-NS in the control of cholera toxin production.