Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWW0) from Pseudomonas putida mt-2: Evidence for the positive nature of the regulation by the xylR gene

Summary The hypothesis that the early enzymes of the degradative pathway determined by the TOL plasmid pWW0 are positively regulated by the product of the xylR gene has been tested by constructing a strain which is a partial diploid for the TOL genes. Two parental plasmids were first constructed by in vivo methods, neither of which could determine the ability to grow on m-xylene, one of the primary substrates of the plasmid degradative pathway, because of mutations. One of these, pWW0-216, was a derivative of pWW0 but carried a xylR ^- allele and a copy of the Tn401 transposon, encoding carbenicillin resistance. The other plasmid, pWW0-152, was a derivative of the promiscuous R plasmid RP4 into which had been translocated part of a pWW0 plasmid carrying a wild type xylR ⁺ allele but with a defective xylA, the structural gene for xylene oxidase. When these two plasmids were mated into the same strain, all the transconjugants examined grew on m-xylene and one representative of these, PaW 219, was shown to contain induced levels of xylene oxidase when grown under inducing conditions. The possibility that ability to utilise m-xylene was due to recombination between or reversion of the coexisting plasmids was eliminated by demonstrating that the two parental plasmids segregated on mating out from PaW 219. It is concluded therefore that xylR ⁺ is transdominant to xylR ^-, and that its gene product is a positive regulator.