~~~Pseudomonasputida MT15 contains a large plasmid, pWW15, of about 250 kbp, which encodes the genes for toluene and xylene catabolism. Growth on benzoate selects strongly against the wild-type and results in the segregation of three phenotypically distinguishable mutant types. (1) B1 mutants, which have lost the complete plasmid.(2) B3 mutants, in which the plasmid has undergone a large deletion of about 90 kbp which appears to affect the regulation of the catabolic enzymes; these mutants retain the ability to grow on m-xylene and toluene (Mxy+ Tln+) but no longer grow on the metabolite of m-xylene, m-toluate (Mtol-). (3) A novel class not previously described, the B5 mutants, which still grow well on toluene but grow very poorly on m-xylene and do not grow on m-toluate (Mxy-Tln+ Mtol-). The B5 mutants appear to share the regulatory lesion of the B3 mutants but in addition do not express the xylF and xylG gene products, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semialdehyde dehydrogenase. The plasmids in the B5 mutants have also undergone a deletion of about 90 kbp similar to, but distinguishable from, that in the B3 mutants.Both B3 and B5 mutants can revert to growth on m-toluate. The revertants all show elevated constitutive levels of catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase which are not further induced by m-toluate. The reversion is accompanied by the tandem amplification of a region of 23-28 kbp on either side of the original deletion. As a result of Southern hybridizations, it was shown that the amplified region contains the structural genes of some of the enzymes which metabolize m-toluate but not the enzymes which convert m-xylene to m-toluate.

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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pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4. A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon. Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI. The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH. The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0. Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E. coli.

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 (C230) were cloned from pWW15. The gene for C2301 was located on the 2.1-kbp XhoI fragment Xh, whereas that for C2301I 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 C2301 from its 3' end. Thus, only C230II was expressed in the host MT15-510. Conversely, in RP4::pWW15 cointegrate plasmid pWW15-1003, only the C2301 gene was present. The expression of C230 activity from these two derivative plasmids and from the wild-type pWW15 showed that only C230I 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.