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The entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from TOL plasmid pWW53 and the position of the genes accurately located. The coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (OP1)-a small open reading frame-xylC (1.6 kbp)-xylA (2.9 kbp)-xylB (1.8 kbp). Within the coding region there was considerable homology with the isofunctional region of the archetypal TOL plasmid pWW0. A central region of 2.9 kbp complemented an xylA (for xylene oxygenase) mutant of Pseudomonas putida mt-2 and was also capable of conferring the ability to convert indole to indigo on strains of Escherichia coli and P. putida. This reaction has been reported previously only for dioxygenases involved in aromatic catabolism but not for monooxygenases. It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo.

Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene-xylene catabolism. pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0. An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived. The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment. The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P. putida PaW130. A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P. putida hosts. Good induction of the enzymes by m-toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P. putida, but little or no regulation was found in E. coli. The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids.

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.