title: Membrane localization of the ToxR winged-helix domain is required for TcpP-mediated virulence gene activation in Vibrio cholerae.
abstract: ToxR is a bitopic membrane protein that controls virulence gene expression in Vibrio cholerae. Its cytoplasmic domain is homologous to the winged helix-turn-helix ('winged helix') DNA-binding/transcription activation domain found in a variety of prokaryotic and eukaryotic regulators, whereas its periplasmic domain is of ill-defined function. Several genes in V. cholerae are regulated by ToxR, but by apparently different mechanisms. Whereas ToxR directly controls the transcription of genes encoding two outer membrane proteins, OmpU and OmpT, it co-operates with a second membrane-localized transcription factor called TcpP to activate transcription of the gene encoding ToxT, which regulates transcription of cholera toxin (ctxAB) and the toxin-co-regulated pilus (tcp). To determine the requirements for gene activation by ToxR, different domains of the protein were analysed for their ability to control expression of toxT, ompU and ompT. Soluble forms of the cytoplasmic winged-helix domain regulated ompU and ompT gene expression properly but did not activate toxT transcription. Membrane localization of the winged helix was sufficient for both omp gene regulation and TcpP-dependent toxT transcription, irrespective of the type of periplasmic domain or even the presence of a periplasmic domain. These results suggest that (i) the major function for membrane localization of ToxR is for its winged-helix domain to co-operate with TcpP to activate transcription; (ii) the periplasmic domain of ToxR is not required for TcpP-dependent activation of toxT transcription; and (iii) membrane localization is not a strict requirement for DNA binding and transcription activation by ToxR.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: J Adam
authors.initials: JA
authors.lastname: Crawford
authors.forename: Eric S
authors.initials: ES
authors.lastname: Krukonis
authors.forename: Victor J
authors.initials: VJ
authors.lastname: DiRita
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis / Q000235:genetics; D000483:Alleles; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins; D008958:Models, Molecular; D016296:Mutagenesis; D010447:Peptide Hydrolases; D019897:Periplasm; D018272:Porins / Q000096:biosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C096349:OmpU protein, Vibrio cholerae; D018272:Porins; D011993:Recombinant Fusion Proteins; C097244:TCPP protein, Vibrio cholerae; D014157:Transcription Factors; C499588:ompT protein, E coli; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; C075626:tcpN protein, Vibrio cholerae; D010447:Peptide Hydrolases