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 (OPl)-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 pWWO. 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.In soil isolates of Pseudomonas spp. the ability to catabolize toluene and substituted toluenes via benzoate and substituted benzoates (20, 29) appears to be almost always plasmid encoded (27), but the plasmids themselves differ considerably in properties and structure (5,28).Horizontal transfer on plasmid vectors through different members of the soil microbial population undoubtedly imposes selection pressures on catabolic genes different from those which are chromosomally located. One way of monitoring and assessing these differences is to compare the structure and organization of catabolic genes on isofunctional plasmids isolated from different geographical locations.The archetypal TOL plasmid pWWO (117 kilobase pairs [kbp]) was found in Pseudomonas putida mt-2 (23, 27), a strain originally isolated in Japan in the 1950s. The majority of molecular biological studies on the catabolic structural genes of the toluene-xylene pathway have been performed on pWW0 or its derivatives (7,10,11,15,16,21). More recently, we isolated P. putida MT53 in North Wales and initiated studies on its TOL plasmid, the 105-kbp plasmid pWW53, and its RP4 cointegrate, pWW53-4 (18). The DNA of its second operon (xylDLEGF. . ,), coding for the enzymes converting benzoic acids to central metabolites, has the same gene order as the corresponding operon on pWWO, and its restriction map shows a number of common sites. This paper reports parallel studies on the first operon of the pathway (xylCAB) responsible for the conversion of toluenes to benzoates. MATERIALS AND METHODSBacterial strains and media. The Escherichia coli and P. putida strains used or constructed in this study are listed in been described elsewhere (19,29). To maintain recombinant plasmids in E. coli, streptomycin sulfate, kanamycin, ampicillin, and tetracycline were added to final concentrations of 15, 15, 25, and 7.5 p.g/ml, respectively, when appropriate. In Pseudomonas hosts, streptomycin and kanamycin were added at 150 and 50 ,ug/ml, respectively.Enzyme assays...
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