Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.