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Mol Microbiol. 2011 Mar;79(5):1248-59. doi: 10.1111/j.1365-2958.2010.07518.x. Epub 2011 Jan 10.

Cooperative amino acid changes shift the response of the σ⁵⁴-dependent regulator XylR from natural m-xylene towards xenobiotic 2,4-dinitrotoluene.

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  • 1Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.

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.

© 2011 Blackwell Publishing Ltd.

PMID:
21205010
[PubMed - indexed for MEDLINE]
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