title: Identification of residues critical for the function of the Vibrio cholerae virulence regulator ToxT by scanning alanine mutagenesis.
abstract: Virulence factor expression in Vibrio cholerae is controlled by the transcriptional regulatory protein ToxT. ToxT activates transcription of the genes encoding cholera toxin (ctx) and the toxin co-regulated pilus (tcp), as well as accessory colonization factor (acf) genes. Previous studies of ToxT, a member of the AraC family of proteins, have revealed that it consists of two domains, an N-terminal dimerization and environmental sensing domain, and a C-terminal DNA binding domain. In this study, comprehensive scanning alanine mutagenesis was utilized to identify amino acids critical for the function of ToxT. Forty-eight proteins with Ala substitutions (of 267 total) exhibited defects in ToxT-dependent activation (>90% reduction) in both a V. cholerae acfA-phoA reporter strain and a Salmonella typhimurium ctxAp-lacZ reporter strain. Most of these mutant proteins also caused reductions in cholera toxin (CT) and toxin coregulated pilus (TCP) expression in a DeltatoxT V cholerae strain under in vitro virulence factor inducing conditions. Further analysis with a LexA-based reporter system revealed that one of the 20 Ala substitutions in the N terminus (F151A) diminishes dimerization, and this residue is located in a region of predicted alpha-helical structure, thus identifying a putative dimer interface. Ala substitutions in two putative helix-turn-helix (HTH) recognition helices that caused differential promoter activation (K203A and S249A) did not appear to alter specific DNA binding, suggesting these residues contribute to other aspects of transcriptional activation. A number of Ala substitutions were also found that result in a higher level of ToxT transcriptional activity, and these mutations were almost exclusively found within the N terminus, consistent with this domain being involved in modulation of ToxT activity. This study illuminates the contribution of specific amino acids to the dimerization, DNA binding, and transcriptional activity of ToxT.
authors.affiliation: South Texas Center for Emerging Infectious Diseases, University of Texas San Antonio, San Antonio, TX 78249, USA.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Childers
authors.forename: Gregor G
authors.initials: GG
authors.lastname: Weber
authors.forename: Michael G
authors.initials: MG
authors.lastname: Prouty
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Castaneda
authors.forename: Fen
authors.initials: F
authors.lastname: Peng
authors.forename: Karl E
authors.initials: KE
authors.lastname: Klose
mesh_terms: D000409:Alanine / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000235:genetics* / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics
chemical_list: C093353:AcfA protein, Vibrio cholerae; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D037521:Virulence Factors; C075626:tcpN protein, Vibrio cholerae; D000469:Alkaline Phosphatase; D000409:Alanine