Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8
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TfdT is a LysR-type transcriptional regulator that activates the transcription of the chlorocatechol degradative gene operon tfdCDEF of the chlorobenzoate-degrading bacterium Burkholderia sp. NK8. To identify the amino acids involved in the effector recognition by TfdT, a polymerase-chain-reaction-based random mutagenesis protocol was applied to introduce mutations into the tfdT gene. Nine types of TfdT mutant bearing a single-amino-acid substitution at positions, Lys-129, Arg-199, Val-226, Val-246, and Pro-267 were obtained on the basis of their altered effector profiles and enhanced responses particularly to 2-chlorobenzoate, 2-aminobenzoate, and 2,6-dichlorobenzoate. All the TfdT mutants showed enhanced response to the effectors with a chloro-group in C-2 of benzoic acid. A homology model of wild-type TfdT was built on the basis of the crystal structure of CbnR with SwissModel. In this model, residues corresponding to the mutation sites of isolated TfdT mutants were located at the interface between the domains RD-I and RD-II. The findings that these TfdT mutants expressed altered effector specificities and enhanced responses to specific effectors suggest that these five residues are involved in effector binding by TfdT.
KeywordsLysR-type transcriptional regulator Chlorocatechol ortho-cleavage pathway TfdT Random mutagenesis Burkholderia sp. NK8 Chlorobenzoate
The authors thank Drs. Yoshiyuki Ohtsubo and Masataka Tsuda for helpful advice on the experiments. This work was supported by a Grant-in-aid (Hazardous Chemicals) from the Ministry of Agriculture, Forestry, and Fisheries of Japan (HC-07-2324-1).
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