The organization of the Pm promoter of the TOL plasmid reflects the structure of its cognate activator protein XylS.
The toluate catabolic operon carried by the TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the benzoate-responsive XylS protein which, when activated, stimulates transcription from the operon promoter Pm. In this study, the mode in which XylS effects the activity of the Pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. Substitution of His31Asp/Ser32Pro,Leu113Pro,Phe214Leu/Glu215A sp/Arg216Pro or Thr312Pro, all predicted to disrupt the secondary structure, renders XylS inactive and unable to compete with the native protein for activation of Pm. In contrast, Pro substitutions at Ser64 or Gly160 had no or only minor effects on XylS activity, and a protein with residual capacity to activate Pm resulted when Glu11 was altered to Pro. None of a collection of truncated variants, of XylS protein deleted for more than 10 terminal amino acids retained activity or were able to negatively interact the wild-type XylS protein. These data indicate that amino- and carboxy-terminal portions of the protein sequence depend on each other for the maintenance of their functional structure, rather than being arranged as independent domains. Pm promoter derivatives, in which the two half-sites of the direct repeat sequence that confers responsiveness to XylS were separated and/or inverted by one-half or one complete DNA helix turn, lost responsiveness in vivo. These results favour a model for XylS-mediated activation of Pm in which a tandem dimer is the protein form that binds the Pm promoter.
Gesellschaft für Biotechnologische Forschung, Bereich Mikrobiologie, Braunschweig, Germany.
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Kessler
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Timmis
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de Lorenzo
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004268:DNA-Binding Proteins; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics*; D017384:Sequence Deletion; D014050:Toluene / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; D014050:Toluene