The wing of the ToxR winged helix-turn-helix domain is required for DNA binding and activation of toxT and ompU

Abstract

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT.

Host stimuli and operator binding sites controlling protein interactions between virulence master regulator ToxR and ToxS in Vibrio cholerae

“…Despite its important role for virulence and environmental adaption, the exact mechanism of ToxR signal transduction and transcription factor activation remains to be characterized. Many studies, summarized above and recently published by Morgan et al (2019), showed evidence for ToxR dimerization. However, no detailed information about the interaction interface and orientation is available.…”

“…As is known in w-HTH protein family members, dimerization via such motifs occurs due to the close localization of the monomers after their binding to the DNA operator sequences and the subsequent interaction of the N-terminal winged helix (Littlefield and Nelson, 1999). A recent study sheds light on such mechanisms for ToxR (Morgan et al, 2019). Therein, alanine-scanning mutagenesis was performed to characterize the w-HTH domain.…”

σE controlled regulation of porin OmpU in Vibrio cholerae

“…Additional data show, as discussed below, that suppressor mutations in the ompU expression control region were selected for rescue of cell growth, as the levels of OmpU in Δ rpoE strains play a critical role. As established earlier, the ompU promoter control region consists of three ToxR binding sites, O1‐O3, each separated by a spacer region (Goss et al, 2013 ; Morgan et al, 2019 ). O3 is located immediately upstream of the core promoter, followed by O2 and O1, respectively.…”

“…This inversion of OMP production is ensured by activation of the main virulence regulator ToxR upon host entry, for example by bile salts (Lembke et al, 2018 ). ToxR recognizes AT‐rich DNA elements located upstream of a particular CDS with its winged helix‐turn‐helix motif (Goss et al, 2013 ; Morgan et al, 2019 ). These specific ToxR boxes are each separated by five base pairs.…”

Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae

“…ToxR directly repress the ompT promoter and activate the ompU promoter, but involves a second activator, TcpP, to initiate the toxT promoter that encodes the transcription factor. Modulation of OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization (Provenzano and Klose, 2000 ; Morgan et al, 2019 ). Strains expressing only the OmpT show considerably reduced in vitro expression of virulence factors and intestinal colonization (Provenzano and Klose, 2000 ).…”