title: Cooperative amino acid changes shift the response of the σ⁵⁴-dependent regulator XylR from natural m-xylene towards xenobiotic 2,4-dinitrotoluene.
abstract: XylR is a σ⁵⁴-dependent transcriptional factor of Pseudomonas putida that activates the Pu promoter of the TOL plasmid upon binding its natural effector, m-xylene. The search for mutants of the signal-sensing module of XylR that respond to the xenobiotic compound 2,4-dinitrotoluene recurrently yields protein variants with a broad effector range. These mutants had amino acid changes not only in the effector recognition moiety (A module), but also in the inter-domain B linker of the protein. A random mutagenesis and selection/counterselection setup was adopted to optimize the 2,4-DNT reaction of XylRv17, one of the best 2,4-DNT responders and thus recreate how this regulator can adjust its specificity to novel effectors by individual changes on the evolving protein. Site-specific mutagenesis was then used to decipher the contribution of individual mutations in XylRv17 and in one of the mutants evolved from it (XylR28) to the 2,4-DNT response. This approach allowed us to capture a new XylR version with novel mutations that fixed the protein in an intermediate stage of the progress from an effector-promiscuous, pluri-potent protein type to a more specific form where the natural response to m-xylene was decreased and the non-native acquired response to 2,4-DNT was increased.
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
authors.forename: Aitor
authors.initials: A
authors.lastname: de Las Heras
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004136:Dinitrobenzenes / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D051877:RNA Polymerase Sigma 54 / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015262:Xenobiotics / Q000494:pharmacology*; D014992:Xylenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004136:Dinitrobenzenes; D014157:Transcription Factors; D015262:Xenobiotics; C074322:XylR protein, Pseudomonas; D014992:Xylenes; C016403:2,4-dinitrotoluene; D051877:RNA Polymerase Sigma 54