Pseudomonas putida Alkylhydroperoxide Reductase: Molecular Cloning of the ahpC and ahpF Genes
Fumiyasu FUKUMORI* and Koki HORIKOSHI
The DEEPSTAR group, Japan Marine Science and Technology Center, 2-15 Natsushima, Kanagawa 237, Japan
After Inoue and Horikoshi first described that a Pseudomonas putida strain that could grow under toluene and introduced logPow values as a toxicity index of organic solvents (1), several reports documenting the isolation of organic solvents-resistant bacteria have appeared (Reviewed in Ref.2). Recent studies on the mechanisms of organic solvents tolerance have mainly focused on changes in fatty acid composition of membrane lipids and solvent efflux systems. Removal of solvents by transformation has also been considered as a possible tolerance mechanisms, yet, little is known. P putida KT2442 is intrinsically toluene-sensitive, however, a long term liquid cultivation under xylene followed by toluene allowed us to obtain the toluene- resistant variant strain TOL which constitutively overexpressed a 24 kDa soluble protein. Its N-terminal amino acid (aa) sequence resembled that of the Escherichia coli ahpC gene product which is the catalytic component of an alkylhydroperoxide reductase (AHPR) (3). This result may suggest a general role of AHPR in the organic solvents resistance because ahpC has been reported as an organic solvents-resistant gene (4). Here we report the molecular cloning of the P putida ahpCF genes.
The ahpC gene has been cloned by using an oligonucleotide probe specifically designed for its N-terminal aa sequence. The deduced aa sequence of an open reading frame (ORF) completely matched the AhpC sequence up to the 30th residue except for the N-terminal methionine. The mature AhpC consists of 186 aa residues with a calculated molecular mass of 20,376 Da, which is fairly close to the Mr of 24,000 estimated by SDS-PAGE. Downstream of ahpC another ORF was found, designated as ahpF, which possibly encodes for a protein of 520 aa residues with a calculated molecular mass of 55,349 Da. The aa sequences of the ahpC and ahpF gene products showed 69 and 64% overall identity, respectively, to those of the small and large subunit of E. coli AHPR. oxyR, a probable regulatory gene of ahpC was not found adjacent to these genes. The ahpF gene was not overexpressed in the strain TOL, although it is located downstream of ahpC. We have noticed two stable stem and loop structures which may act as transcriptional terminators in the ahpC-ahpF intergenic region. These two genes may be divergently transcribed as was observed in a Xanthomonas strain (5).
