Insertional mutagenesis, cloning and expression of gentisate pathway genes from Pseudomonas alcaligenes NCIB 9867

Abstract

An artificial transposon, Omegon‐km, was used for insertional mutagenesis of Pseudomonas alcaligenes NCIB 9867. Insertion mutants were recovered at approximately 6 times 10‐6 per recipient cell; 12.6% of the Omegon‐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‐hydroxy‐4‐methylbenzoate specific 6‐hydroxylase, but expressed low levels of gentisate 1,2‐dioxygenase. Chromosomal DNA encoding gentisate pathway genes adjacent to Omegon‐km insertions were subcloned into a pRK415 vector and expressed in a cured derivative of Ps. putida NCIB 9869. Removal of both transcriptional and translational stop signals by subcloning was found to relieve suppression of all three enzymes, thus confirming the polar nature of the insertion. It was postulated that both xylenol methylhydroxylase and 6‐hydroxylase were present together in a single transcriptional and translational unit, separate from gentisate 1,2‐dioxygenase. The use of Omegon‐km, a transposable element for in vivo insertional mutagenesis, has enabled the rapid cloning of gentisate pathway genes and the construction of a restriction map comprising of a gene cluster involving at least three enzymes for 2,5‐xylenol degradation.