Abstract
ToxR facilitates TcpP-mediated activation of the toxT promoter in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin secretion and toxin coregulated pilus expression. ToxR binds a region from -104 to -68 of the toxT promoter, from which ToxR recruits TcpP to the TcpP-binding site from -53 to -38. To precisely define the ToxR-binding site within the toxT promoter, promoter derivatives with single-base-pair transversions spanning the ToxR-footprinted region were tested for transcription activation and DNA binding. Nine transversions between -96 to -83 reduced toxT promoter activity 3-fold or greater, and all nine reduced the relative affinity of the toxT promoter for ToxR at least 2-fold, indicating that activation defects were due largely to reduced binding of ToxR to the toxT promoter. Nucleotides important for ToxR-dependent toxT activation revealed a consensus sequence of TNAAA-N(5)-TNAAA extending from -96 to -83, also present in other ToxR-regulated promoters. When these consensus nucleotides were mutated in the ompU, ompT, or ctxA promoters, ToxR-mediated regulation was disrupted. Thus, we have defined the core ToxR-binding site present in numerous ToxR-dependent promoters and we have precisely mapped the binding site for ToxR to a position three helical turns upstream of TcpP in the toxT promoter.