Characterization of functional domains of the Vibrio cholerae virulence regulator ToxT.
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.
Prouty
Michael G
MG
Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Osorio
Carlos R
CR
Klose
Karl E
KE
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
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