Characterization of Functional Domains of the Vibrio Cholerae Virulence Regulator ToxT

Mol Microbiol. 2005 Nov;58(4):1143-56. doi: 10.1111/j.1365-2958.2005.04897.x.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / analysis
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / physiology
  • Bile / metabolism
  • Binding Sites
  • DNA Mutational Analysis
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Electrophoretic Mobility Shift Assay
  • Gene Fusion
  • Genes, Reporter
  • Luciferases / analysis
  • Luciferases / genetics
  • Protein Binding
  • Protein Structure, Tertiary
  • Serine Endopeptidases / analysis
  • Serine Endopeptidases / genetics
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics*
  • Transcription Factors / physiology
  • Transcription, Genetic
  • Vibrio cholerae / genetics*
  • beta-Galactosidase / analysis
  • beta-Galactosidase / genetics

Substances

  • Bacterial Proteins
  • DNA-Binding Proteins
  • LexA protein, Bacteria
  • Transcription Factors
  • tcpN protein, Vibrio cholerae
  • Luciferases
  • beta-Galactosidase
  • Serine Endopeptidases