An in situ examination of factors determining the spread and maintenance of genes disseminated via horizontal gene transfer in biofilms

Due to their spatial and temporal stability, bacterial biofilms are ideal settings for conjugal plasmid transfer and may have a role as a reservoir for plasmid-encoded genes. The overall objective of the research presented herein was to examine factors that affect the spread, maintenance, and catabolic potential of genes disseminated into biofilms by conjugative transfer of the plasmid pWDL7::rfp.;Preliminary studies were conducted in batch cultures to investigate the plasmid-enhanced transformation of 3-chloroaniline (3-CA) when it was present as sole carbon source, as sole nitrogen source, or with additional carbon present. Results indicated that conjugal plasmid transfer of pWDL7:: rfp can lead to enhanced contaminant removal depending on the recipient. Additionally, radioactive 3-CA uptake in situ was examined using microautoradiography-fluorescence in situ hybridization (MAR-FISH).;Factors that affect the transfer and maintenance of pWDL7::rfp in biofilms were explored in a long-term flow cell experiment, comparing biofilms in the presence and absence of 3-CA as selective pressure to examine the effects of biofilm architecture and selective pressure on conjugation. The plasmid was maintained in biofilms, both in the absence or presence of selective pressure. Conjugation frequency was related to both selective pressure and resulting distinct biofilm architecture, with higher biofilm porosity corresponding to enhanced conjugation efficiency.;The relationship between biofilm architecture and conjugation outcome was further explored in a study where biofilms were grown under different flow rates to produce distinct architectures prior to introducing pWDL7:: rfp. The greatest retention of donor cells, and corresponding higher conjugation frequency, was obtained for a biofilm architecture characterized by high porosity. Biomass distributions were analyzed over biofilm depth, providing novel insight into the spatial relationships of transconjugant, donor, and recipient biomass.;Results from this work demonstrate that conjugative transfer of pWDL7:: rfp can enhance the catabolic potential of recipient strains under certain circumstances, and that conjugative transfer in biofilms can lead to long-term maintenance of plasmids, regardless of whether selective pressure is present. A major conclusion from this work is that the success of conjugation is a function of biofilm architecture---primarily determined by the ability of plasmid donors to penetrate biofilms.