Conjugative transfer of plasmid pJP4 in defined biofilms and catabolic activity of transconjugant cells

Horizontal gene transfer facilitates the successful exploitation of plasmids and other mobile genetic elements in the bioaugmenation of polluted environments. Biofilms promote plasmid stability and may enhance transfer frequency as well as the host range of mobile genetic elements that are transferred horizontally. In many cases, the conjugative transfer of catabolic genes to recipient cells does not result in a functional phenotype of the plasmid in recipient cells. The functionality of degrading phenotype might depend on the genetic characteristics of transconjugants and environmental conditions. The first objective of this project was to study the conjugative transfer of plasmid pJP4 (which carries genes that encode catabolic functions for the degradation of a herbicide, 2,4-dichlorophenoxyacetic acid-2,4-D) in Pseudomonas putida biofilms in the absence of selective pressure. Transconjugant cells were detected microscopically based on their plasmid-conferred red fluorescent protein (DsRed) fluorescence. The results indicated that plasmid pJP4 was transferred from the donor P. putida strain to recipient P. putida biofilms under non selective pressure condition. The second objective of this study was to investigate 2,4-D degradation capability of a plasmid pJP4 transconjugant strain (isolated from a soil bacterial community) in a batch culture system as well as plasmid stability in the planktonic cultures in the absence of selective pressure. The results showed that the reactor with plasmid pJP4 transconjugant strain contained less than 5% of the initial concentration (221mg/L) of 2,4-D (concentration of 2,4-D was measured spectrophotometrically at 230 nm) after 12 days whereas, the reactors with donor P. putida SM1443 carrying plasmid pJP4, P. putida ATCC12633, transconjugant strain without a plasmid and a soil microbial community contained 30%, 60%,80% and 37% of the initial concentration of 2,4-D respectively. The capability of plasmid pJP4 transconjugant strain to maintain its plasmid (plasmid loss rate) in planktonic cultures under non-selective pressure condition was also observed. Plasmid pJP4 was lost from almost 70% of the transconjugant population within 24 generations (the presence of plasmid in planktonic cultures was detected over time by confocal laser scanning microscopy and spread plate techniques). These observations may be useful for future plasmid pJP4 mediated genetic bioaugmentation studies of polluted environmental samples.