title: Characterization of functional domains of the Vibrio cholerae virulence regulator ToxT.
abstract: The toxT gene encodes an AraC family transcriptional activator that is responsible for regulating virulence gene expression in Vibrio cholerae. Analysis of ToxT by dominant/negative assays and a LexA-based reporter system demonstrated that the N-terminus of the protein contains dimerization determinants, indicating that ToxT likely functions as a dimer. Additionally, a natural variant of ToxT with only 60% identity in the N-terminus, as well as a mutant form of ToxT with an altered amino acid in the N-terminus (L107F), exhibited altered transcriptional responses to bile, suggesting that the N-terminus is involved in environmental sensing. The C-terminus of ToxT functions to bind DNA and requires dimerization for stable binding in vitro, as demonstrated by gel shift analysis. Interestingly, a dimerized form of the ToxT C-terminus is able to bind DNA in vitro but is transcriptionally inactive in vivo, indicating that the N-terminus contains determinants that are required for transcriptional activation. These results provide a model for a two-domain structure for ToxT, with an N-terminal dimerization and environmental sensing domain and a C-terminal DNA-binding domain; unlike other well-studied AraC family proteins, both domains of ToxT appear to be required for transcriptional activation.
authors.affiliation: Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Prouty
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Osorio
authors.forename: Karl E
authors.initials: KE
authors.lastname: Klose
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D001646:Bile / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D050939:Gene Fusion; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics; D000099160:LexA Repressor Protein
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008156:Luciferases; D012697:Serine Endopeptidases; D014157:Transcription Factors; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein; C075626:tcpN protein, Vibrio cholerae