title: Vibrio cholerae AphA uses a novel mechanism for virulence gene activation that involves interaction with the LysR-type regulator AphB at the tcpPH promoter.
abstract: AphA is required for expression of the Vibrio cholerae virulence cascade and for its regulation by quorum sensing. In order to activate transcription, AphA functions together with a second protein, the LysR-type regulator AphB, at the tcpPH promoter. As AphA is a member of a new and largely uncharacterized regulator family, random mutagenesis was used to gain insights into how this protein activates transcription. As shown here, 17 amino acid substitutions were identified in AphA that reduced expression of the tcpPH promoter and prevented the protein from binding DNA. The amino acids involved in DNA recognition inferred from a dominant-negative analysis were located throughout the N-terminal domain from amino acids 18 to 67. This region of AphA has a conserved domain architecture similar to that of MarR, a multiple antibiotic resistance repressor. The analogous positions of the dominant-negative mutations in AphA and MarR confirm that the DNA-binding domains of these proteins are similar and indicate that AphA is a new member of the winged helix family of transcription factors. We also show that AphB is capable of rescuing two of the DNA binding-defective AphA mutants, suggesting that the proteins interact directly on the DNA. Disruption of this interaction by insertion of half a helical turn between the two binding sites prevented AphB from rescuing the mutants and prevented the expression of the virulence cascade in a wild-type background. These results provide a novel mechanism for the initiation of virulence gene expression at tcpPH.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755, USA.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Kovacikova
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.forename: Karen
authors.initials: K
authors.lastname: Skorupski
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D014774:Virulence / Q000235:genetics
chemical_list: C120327:AphB protein, Vibrio cholerae; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C099204:TcpH protein, Vibrio cholerae; D015534:Trans-Activators; D014157:Transcription Factors; D036781:Fimbriae Proteins; C073470:LysR protein, Bacteria