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