The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter

ToxR is required in Vibrio cholerae for transcriptional activation of the t oxT gene, the protein product of which activates numerous genes involved in virulence. Although ToxR cannot activate the toxT promoter in Escherichia coli, the products of the tcpPH operon are shown here to activate the toxT promoter, and co-expression with ToxRS enhances activation. An identical pa ttern was seen in a Delta fcpP Delta toxR strain of V. cholerae when TcpPH or ToxRS was expressed from plasmids. Although overexpression of the TcpP/H proteins in V. cholerae partially complemented both a Delta toxR strain an d a Delta tcpP Delta toxR double mutant for toxin production and toxT-lacZ activation, the presence of ToxR greatly increased their expression. Analys is of a toxT-lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR bind ing site. This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, whic h showed that TcpP interacts with the promoter region from -51 to -32, wher eas ToxR protected a region from -100 to -69. In addition, membranes contai ning endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP. Characterization of a number of tcpP s ubstitution mutants revealed one derivative (TcpP-H93L) that, when overexpr essed, was markedly defective for toxT activation, cholera toxin and TcpA ( toxin co-regulated pilus) production and DNA binding; however, toxT activat ion by TcpP-H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation. Two add itional mutant derivatives, TcpP-W68L and TcpP-R86A, failed to activate tox T or direct toxin and TcpA production in the presence or absence of ToxR. B oth TcpP-W68L and TcpP-R86A, like TcpP-H93L, were defective for DNA binding . Finally, a ToxR mutant derivative, ToxR-G80S, served to separate the diff erent roles of ToxR on different promoters. Although ToxR-G80S was ineffici ent at activating the ompU promoter in V. cholerae (ompU encodes an outer m embrane porin regulated by ToxR), it was fully capable of activating the to xT promoter. These data suggest that ToxR is not a direct activator in the toxT expression system but, instead, enhances the activity of TcpP, perhaps by recruiting it to the toxT promoter under conditions in which expression levels of TcpP are too row for it to activate toxT efficiently on its own.