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

P>XylR is a sigma 54-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.