title: 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 DeltatcpPDeltatoxR 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 DeltatoxR strain and a DeltatcpPDeltatoxR 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.
authors.affiliation: Unit for Laboratory Animal Medicine, and Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0614, USA.
authors.forename: E S
authors.initials: ES
authors.lastname: Krukonis
authors.forename: R R
authors.initials: RR
authors.lastname: Yu
authors.forename: V J
authors.initials: VJ
authors.lastname: Dirita
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014734:Vibrio cholerae / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C097244:TCPP protein, Vibrio cholerae; D015534:Trans-Activators; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; C075626:tcpN protein, Vibrio cholerae