SUMMARY: pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from MT53, which contains 36 kbp of pWW53 DNA inserted close to the 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 operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase () and 4-oxalocrotonate tautomerase () were shown by subcloning and enzyme assay to lie at the distal end of the pathway operon. Although 2-oxopent-4-enoate hydratase () and 4-hydroxy-2-oxovalerate aldolase () could be detected on a large cloned dIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between and . The gene order in the pathway operon is therefore . The regulatory genes and were located close to and downstream of the 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 pathway operon which support low constitutive expression of the structural genes downstream in hosts but not in .


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