Cooperative amino acid changes shift the response of the σ54-dependent regulator XylR from natural m-xylene towards xenobiotic 2,4-dinitrotoluene.
Mol Microbiol
; 79(5): 1248-59, 2011 Mar.
Article
em En
| MEDLINE
| ID: mdl-21205010
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.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Fatores de Transcrição
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Xilenos
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Xenobióticos
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Regulação Bacteriana da Expressão Gênica
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Pseudomonas putida
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Substituição de Aminoácidos
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Dinitrobenzenos
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Proteínas de Ligação a DNA
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RNA Polimerase Sigma 54
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article