Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

Abstract

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

FIG. 1.

Detection of megaplasmids in different Sphingomonas strains by PFGE. Lane 1, S. wittichii RW1; lane 2, Sphingomonas sp. A175; lane 3, Sphingomonas sp. SS3; lane 4, S. yanoikuyae B1; lane 5, S. paucimobilis EPA505; lane 6, S. chlorophenolica; lane 7, S. subterranea; lane 8, S. aromaticivorans F199; lane 9, S. stygia; lane 10, λ-DNA standard.

FIG. 2.

PFGE analysis of the plasmids from different Sphingomonas strains with (a) or without (b) S1 nuclease treatment. Lane 1, S. aromaticivorans B0695; lane 2, S. xenophaga BN6; lane 3, Sphingomonas sp. K39; lane 4, S. herbicidovorans; lane 5, S. subterranea; lane 6, Sphingomonas sp. A175; lane 7, S. paucimobilis Q1; λ, λ-DNA standard.

FIG. 3.

PFGE analysis of the plasmid profile of S. wittichii RW1 and Sphingomonas sp. HH69 (right) and hybridization of the genomic DNAs with a labeled dxnA1A2 probe (left).

FIG. 4.

PFGE of the total DNA of different transconjugants, which were obtained after the conjugation of S. xenophaga BN6 AKE2/5 Km^r with Sphingomonas sp. SS3 (right), and Southern hybridization of the gel using a neo probe (left). Lanes: 1, Sphingomonas sp. SS3; 2, S. xenophaga AKE2/5; 3 to 7. transconjugants; 8, λ DNA standard.

FIG. 5.

PFGE of the total DNA of different transconjugants, which were obtained after the conjugation of S. aromaticivorans F199 Km^r with Sphingomonas sp. HH69 (left) and Southern hybridization of DNA from the gel using a dxnA1A2 probe (right). Lane 1, λ-DNA standard; lane 2, S. aromaticivorans F199 Km^r; lane 3, Sphingomonas sp. HH69; lane 4, Sphingomonas sp. HH69-1 transconjugant; lane 5, Sphingomonas sp. HH69-2 transconjugant; lane 6, Sphingomonas sp. HH69-3 transconjugant.