Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA

Abstract

Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein. These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin. ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade. ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB). The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites. Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V. cholerae. In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter. These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters.Expression of many virulence genes in the human diarrheal pathogen Vibrio cholerae is under direct control of the ToxT protein (5, 7, 13). ToxT is a member of the AraC family of transcriptional activators (13) and activates expression of genes required for production of the toxin-coregulated pilus (tcp) (7, 33), accessory colonization factor (acf) (24, 25), and cholera toxin (ctxAB) (7). Expression of toxT is mediated by the ToxR protein (7, 12, 13), a transcription activator protein in the inner membrane that shares homology with members of the two-component family of response regulators found in a variety of bacterial species (22,30). A third regulatory protein required for wild-type levels of virulence gene expression is the ToxS protein, a membrane protein postulated to stabilize ToxR for DNA binding (6,20).Expression of virulence genes in V. cholerae is proposed to occur via a regulatory cascade in which ToxR, in conjunction with ToxS, activates expression of toxT and the ToxT protein directly activates expression of virulence genes (5, 7). In this model, ToxR-dependent expression of toxT is a critical step in virulence regulation in V. cholerae. The precise requirements for ToxR-dependent expression of toxT are unknown, and, since ToxR does not directly activate toxT expression when assayed in Escherichia coli, we speculate that additional V. cholerae factors may play a role (12, 31).The molecular aspects of ToxR-dependent transcription activation are not clear. The ToxR-binding sites in the ctxAB and toxT promoters have little sequence similarity. In the ctxAB promoter, ToxR b...

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

“…Expression of BcrR in E. faecalis and E. coli-To study membrane-bound DNA-binding proteins, DNA binding assays have been either performed with inverted membrane vesicles that may contain other potentially contaminating proteins (10,35), soluble variants of the protein (36,37), in the presence of detergent (38), or by reconstitution of purified protein into liposomes (39). The level of BcrR expression in native membranes of E. faecalis was too low for EMSAs with bcrABD promoter DNA (data not shown).…”

Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli

“…For example, the amino-terminal portion of ToxR shares homology with transcriptional activators of the two-component family of bacterial proteins (see below) that are involved in regulating gene expression in response to environmental stimuli Ottemann et al, 1992). A number of these conserved amino acid residues are important for ToxR to bind DNA and activate transcription (Ottemann et al, 1992;Higgins and DiRita, 1996). When the periplasmic domain of ToxR was fused to alkaline phosphatase, a protein that is active as a dimer, the resulting ToxR-PhoA fusion permitted expression of the ToxR regulon under conditions of high osmolarity that are normally repressive for its expression .…”

A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains

“…Sequences within this promoter are bound by ToxR in gel-shift experiments (Higgins and DiRita, 1994). Interaction between ToxR and the toxT promoter requires the upstream half-site of an inverted repeat element for activation and DNA binding by ToxR (Higgins et al, 1992;Higgins and DiRita, 1996). The ToxR-binding site in the toxT promoter differs in primary sequence from its binding site in the ctxAB promoter, which is characterized by a heptad repeat, TTTTGAT, as well as sequences downstream of the heptad repeat Pfau and Taylor, 1996).…”

“…ToxR shares extensive similarity in its amino terminus with the DNA-binding domain of the OmpR subclass of response regulators in bacteria Ottemann et al, 1992), and mutations in the OmpRhomologous residues abolish the ability of ToxR to bind promoter DNA (Ottemann et al, 1992;Higgins and DiRita, 1996). The carboxyl terminus of ToxR is in the periplasm , where it interacts with another regulatory protein, called ToxS (Miller et al, 1989;DiRita and Mekalanos, 1991).…”