The investigator is interested in how bacteria adapt to contaminated soils. The type of adaptation that is the focus of this proposal is acquisition of plasmids encoding naphthalene degrading enzymes. Dr. Duncan's hypothesis is that rearrangements and other modifications of plasmids encoding naphthalene degrading enzymes or enzymes for degrading other environmental pollutants occur as the plasmids are transferred by conjugation to different hosts and these mutations may contribute to adaptation of bacteria to environments containing naphthalene or other toxic compounds. In a previous study, the investigator and others found that in a sterile soil microcosm inoculated with strains of Pseudomonas putida, transfer of a plasmid encoding naphthalene genes occurred. As expected, acquisition of a naphthalene plasmid by an isogeneic strain increased the fitness of the strain somewhat in soil containing naphthalene. An even greater increase in fitness was seen when a nonisogeneic strain, R, acquired the plasmid. This increase was seen when the donor, N, and R were grown together either in the presence or in the absence of naphthalene. A number of independent transconjugants were isolated from different microcosms. Specifically, Dr. Duncan proposes to characterize the plasmids and chromosomes of ten transconjugants that arose from preliminary microcosm experiments involving an ATCC Pseudomonas strain carrying the NAH7 plasmid and an unrelated strain which does not degrade naphthalene. The plasmids will be screened initially for plasmid size (on gels), presence of nahAB (by Southern hybridization to probes) and for the size of the two PCR generated fragments from NAH7 (to detect rearrangement). Second, a more extensive characterization will be performed on these plasmids by restriction endonuclease mapping and Southern hybridization to further identify molecular changes from the structure of NAH7. The chromosomes of transconjugants and parental bacteria will be compared by examining PCR products using REP sequence primers or random primers to produce fingerprints. These experiments are designed to show whether chromosomal DNA was exchanged resulting in variation. Finally, the relative fitness of transconjugants will be compared and the plasmid DNA will be transferred from the transconjugants to a plasmid free strain of the original Pseudomonas species to determine whether the increased fitness was due to association of the plasmid with a new host or in sequence changes in the plasmid.
