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

Abstract

ToxR is required in Vibrio cholerae for transcriptional activation of the toxT 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 pattern was seen in a ΔtcpPΔtoxR 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 ΔtoxR strain and a ΔtcpPΔtoxR double mutant for toxin production and toxT–lacZ activation, the presence of ToxR greatly increased their expression. Analysis of a toxT–lacZ promoter deletion series demonstrated that TcpP was able to interact functionally with the toxT promoter downstream of the ToxR binding site. This was confirmed using electrophoretic mobility shift assays of this toxT promoter deletion series and DNase I footprinting analysis, which showed that TcpP interacts with the promoter region from −51 to −32, whereas ToxR protected a region from −100 to −69. In addition, membranes containing endogenous levels of ToxR bound more readily to the toxT promoter than did membranes containing only TcpP. Characterization of a number of tcpP substitution mutants revealed one derivative (TcpP‐H93L) that, when overexpressed, was markedly defective for toxT activation, cholera toxin and TcpA (toxin co‐regulated pilus) production and DNA binding; however, toxT activation by TcpP‐H93L was restored in the presence of ToxR, suggesting that ToxR can provide the promoter recognition function for toxT activation. Two additional mutant derivatives, TcpP‐W68L and TcpP‐R86A, failed to activate toxT or direct toxin and TcpA production in the presence or absence of ToxR. Both TcpP‐W68L and TcpP‐R86A, like TcpP‐H93L, were defective for DNA binding. Finally, a ToxR mutant derivative, ToxR‐G80S, served to separate the different roles of ToxR on different promoters. Although ToxR‐G80S was inefficient at activating the ompU promoter in V. cholerae (ompU encodes an outer membrane porin regulated by ToxR), it was fully capable of activating the toxT 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 low for it to activate toxT efficiently on its own.

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

“…CRP bound to -310 et al, 1998;Li et al, 2000). Krukonis et al (2000) recently showed that, at the toxT promoter, ToxR acts as a coactivator of TcpP, demonstrating for the first time the possibility that ToxR-dependent transcription regulation involves other transcription factors in addition to RNAP. Our finding that ToxR represses ompT transcription through an antiactivation mechanism further illuminates the important aspect of ToxR as a global transcription regulator.…”

“…Using phage CP-T1ts-mediated generalized transduction (Hava and Camilli, 2001), crp was inactivated in a toxR mutant of 395, EK307 (Krukonis et al, 2000), generating the double mutant SCL100 (toxR -crp -). The growth phasedependent regulation of OmpT expression in wild-type 395 was then compared with that in EK307 (toxR -), KSK377 (crp -) and SCL100 (toxR -crp -).…”

“…The ToxR regulon is organized into two branches in a cascade fashion (DiRita et al, 1991;Champion et al, 1997). In one branch, ToxR works as a co-activator of another transmembrane DNA-binding protein TcpP to activate transcription of ToxT, an AraC family transcription activator (Higgins et al, 1992;Häse and Mekalanos, 1998;Krukonis et al, 2000). ToxT then directly activates transcription of the genes encoding CT and TCP and autoregulates its own gene (Yu and DiRita, 1999).…”

“…It was proposed that the binding of ToxR to a region extending from -238 to -149 of the promoter is followed by co-operative binding to the downstream regions extending from -116 to -58 and from -53 to -24, where ToxR interacts directly with RNA polymerase (RNAP) and activates transcription (Crawford et al, 1998). For toxT, ToxR binding to the -108 to -65 region of the promoter recruits TcpP to bind downstream at -58 to -44, where TcpP can activate transcription directly (Krukonis et al, 2000;Li et al, 2000). ToxR was proposed as a repressor of OmpT because the expression of this protein is dramatically increased in a toxR mutant .…”

The H-NS protein represses transcription of the eltAB operon, which encodes heat-labile enterotoxin in enterotoxigenic Escherichia coli, by binding to regions downstream of the promoter

“…Transcriptional regulation of CT has been extensively studied. Expression of the ctx gene encoding CT is controlled by a regulatory cascade involving two cytoplasmic membrane pairs, ToxS/ToxR and TcpP/ TcpH, and an AraC-like activator, ToxT (DiRita et al, 1991;Higgins et al, 1992;Skorupski & Taylor, 1997;Hase & Mekalanos, 1998;Krukonis et al, 2000). In contrast, little is known about the regulation of the eltAB operon.…”