Characterization of toxT translation and virulence gene activation in Vibrio cholerae

Vibrio cholerae is a gram-negative aquatic bacterium responsible for the disease cholera. Virulence factor expression in this organism is highly controlled. ToxT is the major transcription factor activating expression of ctx and tcp genes, resulting in the synthesis of cholera toxin (CT) and toxin-coregulated pilus (TCP). ToxT is a member of the AraC/XylS family of transcriptional activators and consists of an N-terminal dimerization/environmental sensing domain and a C-terminal DNA binding domain. In this study we focused our attention to characterizing the ToxT DNA binding domain and temperature dependent toxT translation.

Using the P22 challenge phage system we identified all the critical ToxT residues needed for ToxT-DNA binding at the ctxA promoter. We concluded that ToxT helix-turn-helix 2 (HTH2) establishes stronger and more specific binding than the residues within HTH1. Using promoter- lacZ constructs, we were able to identify nucleotides important for ToxT-DNA binding.

We have identified a motif in the 5'untranslated region (UTR) of toxT that resembles an RNA thermometer, predicted to provide temperature control over translation. This FourU motif is characterized by four consecutive uridine residues that are situated as an "Anti-Shine Dalgarno (anti-SD)" in the secondary RNA structure. The FourU element basepairs with the Shine Dalgarno (SD) to regulate ribosome access to the mRNA. We showed that toxT translation is enhanced at body temperature (37°C). The introduction of two mutations (U5C and U7C) predicted to strengthen the SD-anti-SD interaction were able to prevent toxT translation.