INSERTIONAL MUTAGENESIS, CLONING AND EXPRESSION OF GENTISATE PATHWAY GENES FROM PSEUDOMONAS-ALCALIGENES NCIB-9867

An artificial transposon, Omegon-km, was used for insertional mutagene sis of Pseudomonas alcaligenes NCIB 9867. Insertion mutants were recov ered at approximately 6 x 10(-6) per recipient cell; 12.6% of the Omeg on-km insertion mutants were catabolic mutants deficient in gentisate pathway enzymes. A specific insertion mutant, p44S, was found to lack two gentisate pathway enzymes, viz. xylenol methylhydroxylase and 3-hy droxy-4-methylbenzoate specific 6-hydroxylase, but expressed low level s of gentisate 1,2-dioxygenase. Chromosomal DNA encoding gentisate pat hway genes adjacent to Omegon-km insertions were subcloned into a pRK4 15 vector and expressed in a cured derivative of Ps. putida NCIB 9869. Removal of both transcriptional and translational stop signals by sub cloning was found to relieve suppression of all three enzymes, thus co nfirming the polar nature of the insertion. It was postulated that bot h xylenol methylhydroxylase and 6-hydroxylase were present together in a single transcriptional and translational unit, separate from gentis ate 1,2-dioxygenase. The use of Omegon-km, a transposable element for in vivo insertional mutagenesis, has enabled the rapid cloning of gent isate pathway genes and the construction of a restriction map comprisi ng of a gene cluster involving at least three enzymes for 2,5-xylenol degradation.