Transposition of plasmid DNA segments specifying hydrocarbon degradation and their expression in various microorganisms

Abstract

The conjugative TOL plasmid (75 Mdal [megadaltons]), specifying biodegradation of xylenes, toluene and trimethylbenzene derivatives, undergoes dissociation in Pseudomonas aeruginosa PAO to a nonconjugative TOL* plasmid (28 Mdal) and a transfer plasmid termed TOL.DELTA. (48 Mdal). The TOL* plasmid is rendered transmissible through introduction of a number of conjugative plasmids such as factor K, CAM and TOL.DELTA. but not by the FP2 derivative pRO271. Transfer of TOL* via factor K or TOL.DELTA. is mediated by the formation of plasmid cointegrates; no recombination is observed with CAM. A recombinant RP4-TOL plasmid (76 Mdal), which has lost resistance to tetracycline, was isolated. The TOL* segment can be transposed from this RP4-TOL recombinant plasmid to other antibiotic resistance plasmids such as R702. A segment of DNA, specifying salicylate degradation from SAL plasmid, was transposed onto pAC10, the TOL*- derivative of RP4-TOL recombinant plasmid, which has lost resistance to tetracycline but retains the transfer genes of RP4. Transposition of the salicylate degradative genes onto pAC10 results in the loss of kanamycin resistance. It was possible to isolate SAL+ segregants from pAC10::SAL transposition derivatives that lost the pAC10 plasmid. Such segregants harbor the salicylate degradative genes in the form of a nonconjugative plasmid (SAL*). Transfer of RP4::TOL* or pAC10::SAL* transposition derivatives to Escherichia coli, Salmonella typhimurium, Agrobacterium tumefaciens or Azotobacter vinelandii results in the functional expression of the antibiotic resistance genes but not of the hydrocarbon degradative genes. Such genes are fully expressed on being transferred back to Pseudomonas.