TOL plasmid pWW15 contains two nonhomologous, independently regulated catechol 2,3-oxygenase genes

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Abstract
Pseudomonas putida MT15 contains a 250-kilobase-pair (kbp) TOL plasmid pWW15, encoding toluene and xylene catabolism, which undergoes large spontaneous deletions to give 2 classes of mutants with altered catabolic phenotypes. Two structural genes for catechol 2,3-oxygenase (C23O) were cloned from pWW15. The gene for C23OI was located on the 2.1-kbp XhoI fragment Xh, whereas that for C23OII was found on the 11.5-kbp BamHI fragment BJ. The 2 restriction fragments and the subcloned regions of them showed no similarity in the pattern of restriction digestion, nor did they hybridize with each other. The substrate specificities of the 2 enzymes were also substantially different. The 2 structural genes were separated on pWW15 by .apprx. 100 kbp. In plasmid pWW15-510 of a B5 mutant, the 90-kbp deletion in the plasmid removed most of the intervening DNA, but it also deleted 80% of the gene for C23OI from its 3'' end. Thus, only C23OII was expressed in the host MT15-510. In RP4::pWW15 cointegrate plasmid pWW15-1003, only the C23OI gene was present. The expression of C23O activity from these 2 derivative plasmids and from the wild-type pWW15 showed that only C23OI was induced by growth in the presence of m-toluate, whereas both activities were induced in the presence of m-xylene. These findings cast doubt on the earlier hypothesis that the deletions in B3 and B5 mutants remove a regulatory gene by which m-toluate induces the enzymes necessary for its own catabolism.