GENE-TRANSFER IN THE AQUATIC ENVIRONMENT - PERSISTENCE AND MOBILIZATION OF THE CATABOLIC RECOMBINANT PLASMID PD10 IN THE EPILITHON

This study investigated mobilization and persistence of the recombinant, catabolic plasmid pD10 in laboratory microcosms by natural mobilizing plasmids, recently isolated from epilithic bacteria by exogenous isolation in Pseudomonas putida, as opposed to their isolation in the strains in which they occur in situ. Initial experiments in simple, beaker microcosms were used to optimize conditions for selection of the donor, recipient and transconjugant populations. Studies in a recirculating stream microcosm showed that donor, P. putida KT2440(pD10, pQKH6), and recipient, P. putida UWC6, populations, although declining with time, were able to survive for three weeks and that colonization and survival was predominantly in the epilithon of the microcosm rather than in the liquid phase. Transconjugants UWC6(pD10, pQKH6) were also isolated from epilithon, showing that plasmid pD10 had been mobilized by plasmid pQKH6. The donor strain was able to survive at 100-fold greater numbers than the recipient, but transconjugants were not isolated after day 15. Separate inoculation of the donor and recipient strains into the microcosm showed that they were able to colonize other stones, where transconjugants then arose as a direct result of plasmid transfer within the epilithon. A catechol 2,3-dioxygenase gene, tdnC, was used to facilitate identification of donors and transconjugants. Preliminary laboratory matings showed that pD10 transferred to indigenous epilithic strains identified as Pseudomonas fluorescens, P. chlororaphis and P. aureofaciens. These results suggest that in the absence of selection, mobilization of introduced recombinant genes encoded by pD10 occurs at easily detectable frequencies, even in an oligotrophic environment.