Distribution and molecular relatedness of pSS50-homologous plasmids in bacterial environmental isolates

The 51 kilobase plasmid pSS50, a broad host range IncP1 plasmid from Alcaligenes eutrophus A5, a 4-chlorobiphenyl (4-CBP)-degrading organism isolated from lake sediment, has been used as a probe in colony hybridization of lakewater microcosms and soil lysimeters to monitor population levels of organisms with pSS50-homologous sequences. In this study, organisms with pSS50-homologous plasmids from the colony hybridizations were isolated, checked for the ability to degrade 4-CBP and screened for similarity to A5 or among themselves by chromosomal restriction enzyme analysis combined with pulsed field gel electrophoresis. Also, their pSS50-homologous plasmids were screened for variations by restriction enzyme analysis. Results from characterization of isolates from Lake London lakewater microcosms demonstrated that isolates with chromosomal restriction digest patterns identical to A5 and harboring the pSS50 plasmid were present in Lake London lakewater several years after the isolation of A5 from Lake London sediment. Results of this study also suggest conjugal transfer of pSS50 from A5 to a 4-CBP-degrading lakewater isolate tentatively identified as Pseudomonas facilis in a 4-CBP-enriched, A5-inoculated lakewater microcosm. No large unstable pSS50-homologous plasmids were detected from the lakewater microcosms or soil lysimeters, possibly because they had undergone deletions prior to plasmid analysis. However, a 61 kb pSS50-homologous plasmid, pSS60, which did not undergo deletions, was found in two chromosomal restriction digest groups from 4-CBP-enriched lakewater microcosms. LBS1C1 is a unique isolate harboring pSS60 which degrades 4-CBP. Several isolates which harbor pSS60 but do not degrade 4-CBP and have identical chromosomal restriction digest patterns were also found. Results from characterization of soil lysimeter isolates demonstrate that isolates harboring pSS50 with chromosomal restriction digest patterns identical to that of A5 were present a year after inoculation of A5 into the soil lysimeters, indicating maintenance of the inoculated A5 in the lysimeters. It is possible that the pSS50 plasmids found in five different Pseudomonas strains which did not degrade 4-CBP in A5-inoculated soil lysimeters were transferred to them by A5, although there is no available evidence to determine the source of the pSS50 plasmid in these strains.