Characterization and transcriptional control of the Vibrio cholerae toxT gene.

Abstract

This work addresses the characterization and regulated expression of the Vibrio cholerae toxT gene. Expression of several virulence determinants in V. cholerae, including cholera toxin, toxin coregulated pilus and accessory colonization factor, is controlled by the ToxR protein. ToxR has been shown to directly activate the transcription of the cholera toxin genes. However, ToxR does not directly activate the expression of other genes under its control. Instead, ToxR mediates its control through the ToxT protein. DNA sequence analysis and homology studies identified ToxT to be a member of the AraC family of transcriptional activators. ToxT can directly activate transcription of many ToxR controlled genes and its expression is ToxR-dependent. The mechanism of ToxR-dependent activation of toxT is unknown, but data presented in this work indicates a direct involvement by ToxR. Primer extension, transcriptional fusion and DNA binding studies identified a ToxR-regulated promoter and an inverted repeat element upstream of this promoter that are required for ToxR binding and transcriptional activation of toxT. DNA binding studies with mutant ToxR proteins and site-directed mutagenesis identified residues of ToxR and specific nucleotides within the inverted repeat that are important for DNA binding and transcriptional activation of toxT. Additional RNA transcript analysis suggests that read-through transcription is involved in the expression of toxT and that the level of toxT specific transcripts is growth phase dependent. Moreover, studies with ToxR in Escherichia coli indicate that an additional Vibrio host factor may be required for ToxR-dependent expression of toxT.