DNA binding and ToxR responsiveness by the wing domain of TcpP, an activator of virulence gene expression in Vibrio cholerae.
Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR. We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR. Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators. Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR. One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction. We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association.
Krukonis
Eric S
ES
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
DiRita
Victor J
VJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C097244:TCPP protein, Vibrio cholerae; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; C075626:tcpN protein, Vibrio cholerae; D004247:DNA; D012321:DNA-Directed RNA Polymerases