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Pseudomonas putida MT15 contains a 250-kilobase-pair (kbp) TOL plasmid pWW15, encoding toluene and xylene catabolism, which undergoes large spontaneous deletions to give two classes of mutants with altered catabolic phenotypes (H. Keil and P. A. Williams, J. Gen. Microbiol, 131:1023-1033, 1985). 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 two 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 two enzymes were also substantially different. The two structural genes were separated on pWW15 by about 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. Conversely, in RP4::pWW15 cointegrate plasmid pWW15-1003, only the C23OI gene was present. The expression of C23O activity from these two 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 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.

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