Identification of the TcpP-Binding Site in the <i>toxT</i> Promoter of <i>Vibrio cholerae</i> and the Role of ToxR in TcpP-Mediated Activation

<jats:title>ABSTRACT</jats:title> <jats:p> ToxR-dependent recruitment of TcpP to the <jats:italic>toxT</jats:italic> promoter facilitates <jats:italic>toxT</jats:italic> transcription in <jats:italic>Vibrio cholerae</jats:italic> , initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the <jats:italic>toxT</jats:italic> promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of <jats:italic>toxT</jats:italic> promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne <jats:italic>toxT-lacZ</jats:italic> fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal <jats:italic>toxT</jats:italic> promoter. When present in Δ <jats:italic>toxR V. cholerae</jats:italic> overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N <jats:sub>6</jats:sub> -TGTAA) caused defects in TcpP-dependent <jats:italic>toxT-lacZ</jats:italic> fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the <jats:italic>toxT</jats:italic> promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient <jats:italic>toxT</jats:italic> activation, even in the presence of ToxR. These results suggest that the <jats:italic>toxT</jats:italic> promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the <jats:italic>toxT</jats:italic> promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for <jats:italic>toxT</jats:italic> promoter activation. </jats:p>