Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants

Abstract

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.