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

The conjugative TOL plasmid (75 Mdal), 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 TOLDelta (48 Mdal). The TOL(*) plasmid is rendered transmissible through introduction of a number of conjugative plasmids such as factor K, CAM, and TOLDelta but not by the FP2 derivative pR0271. Transfer of TOL(*) via factor K or TOLDelta 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, has been 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 has been possible to isolate SAL(+) segregants from pAC10[unk]SAL transposition derivatives that have lost the pAC10 plasmid. Such segregants harbor the salicylate degradative genes in the form of a nonconjugative plasmid (SAL(*)). Transfer of RP4[unk]TOL(*) or pAC10[unk]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, however, are fully expressed on being transferred back to Pseudomonas.

XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy.

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.

Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA.

Conditions optimal for the transformation of Pseudomonas putida and E. coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described. The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent. Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants. The frequency of transformation is drastically reduced by treatment of RP 1 DNA with DNase and by denaturation or sonication. Shearing of RP 1 DNA in vitro and their subsequent introduction in P. putida cells, by transformation, produces transformants that exhibit a wide range of drug-resistant phenotypes, including those which are resistant to neomycin but sensitive to kanamycin. Isolation of such neomycin-resistant but kanamycin-sensitive transformants indicates that there might be two separate mechanisms specified by RP 1 for resistance to the two antibiotics.

Dissociation and interaction of individual components of a degradative plasmid aggregate in Pseudomonas.

The transfer of the OCT plasmid from Pseudomonas oleovorans to Pseudomonas putida strain PpGl results in the acquisition of three independent replicons: OCT, factor K, and the MER plasmid. OCT is a nontransmissible plasmid harboring genes that code for the enzymes responsible for the degradation of n-octane. Factor K is a transfer plasmid capable of mobilizing OCT as well as chromosomal genes but incapable of enhancing transfer frequencies of other transmissible plasmids such as CAM, SAL, or RP-1. MER is a self-transmissible plasmid which can confer resistance to high concentrations of mercury salts. While OCT and MER are incompatible with CAM, factor K is compatible with it. Transmissible plasmids such as SAL, CAM, MER, or RP-1 cannot mobilize OCT to any significant extent, and exert strong repression on factor K-mediated transfer of chromosomal genes.