Strain-dependent toxT expression, rather than ToxT activity, governs virulence gene regulation in Vibrio cholerae

The AraC-type transcriptional activator ToxT is a central regulator of <i>Vibrio cholerae</i> virulence, directly controlling expression of the major virulence genes <i>ctxAB</i> and <i>tcpA</i>. Although biotype-specific culture conditions have been widely used to study virulence gene regulation, virulence gene expression patterns are not conserved across <i>V. cholerae</i> strains, particularly within the El Tor biotype. In this study, we investigated strain- and allele-dependent control of <i>toxT</i> expression using isogenic derivatives harboring chromosomally encoded His-tagged <i>toxT</i> alleles, enabling direct comparison of <i>toxT</i> transcription, ToxT protein production, and downstream virulence gene activation. All <i>toxT</i> alleles retained transcriptional activator function once expressed; in the classical biotype strain O395, four different <i>toxT</i> alleles activated virulence gene expression, although the expression levels of <i>tcpA</i> and <i>ctxAB</i> varied among alleles. In contrast, in the El Tor prototype strain N16961, <i>toxT</i>-AY and <i>toxT</i>-AF supported virulence gene expression at 30°C, whereas <i>toxT</i>-SY and <i>toxT</i>-SF were transcriptionally repressed. By comparison, the atypical El Tor strain IB5230 exhibited robust expression of <i>toxT</i>-SY and <i>toxT</i>-SF, with activation of downstream virulence genes even at 37°C, a temperature relevant to the intestinal environment. Together, these results demonstrate that <i>toxT</i> expression functions as a regulatory gate within the canonical ToxR regulon and represents a critical control point governing strain-specific virulence gene regulation and pathogenic potential in <i>V. cholerae</i>.