Molecular cloning and transcriptional analysis of the alkyl hydroperoxide reductase genes from Pseudomonas putida KT2442

Abstract

Pseudomonas putida KT2442TOL (formerly designated TOL), a toluene-resistant variant of strain KT2442 constitutively overexpressed several proteins. The most abundantly produced 24-kDa soluble protein was found to be similar to AhpC, the small subunit of alkyl hydroperoxide reductase. Molecular cloning of the P. putida ahpC based on the N-terminal sequence allowed cloning of closely located ahpF, the large subunit of alkyl hydroperoxide reductase. The deduced amino acid sequences of these genes showed high similarity with corresponding bacterial homologues. Results of RNA transcriptional analyses suggested that P. putida ahpC and ahpF were co-transcribed. A lower level expression of the ahpF may result from an attenuation of transcription by stem-and-loop structures located between two genes. oxyR, the known expression regulatory gene of ahpC-ahpF, was separately cloned and a point mutation that rendered an amino acid change (Phe¹⁰⁶ to Ile) in OxyR was observed. Reverse mutation of the oxyR gene by allelic exchange in P. putida KT2442TOL revealed that this mutation was the cause of the overexpression. About 50% of the reverse mutated cells lost colony-forming ability under toluene, indicating the mutation of oxyR that contributes to overexpression of the oxyR-regulated genes has some relationship with the solvent resistance, but their contribution was not significant.