| Biofilms provide an environment conducive for horizontal gene transfer; the dissemination, uptake and maintenance of genes that potentially provide beneficial genetic traits to the recipient (e.g. metabolic enhancement or antibiotic resistance). Natural genetic transformation, the active cellular uptake of free DNA, is one of three horizontal gene transfer mechanisms and this dissertation's focus. Confocal laser scanning microscopy (CLSM), an integral tool used in this study, allowed for nondestructive, in situ, three-dimensional quantification of biofilm architecture and natural genetic transformation.;The goal of this dissertation was to (i) determine the interrelationship between Acinetobacter baylyi biofilm architecture and natural genetic transformation to discern natural genetic transformation as a means for facilitating recalcitrant contaminant biodegradation in engineered systems, (ii) address difficulties in continuous online CLSM monitoring of A. baylyi biofilm architecture, and (iii) develop improvements to the user-friendliness and reliability of CLSM image analysis.;A. baylyi biofilm architecture was found to directly influence transformation frequency (the ratio of genetically transformed cells to total recipients) and transformant abundance and location. Biofilm extracellular polymeric substance (EPS) architecture had a greater relationship to natural genetic transformation than cellular architecture or the combined architecture of both cells and EPS. The major role of EPS may facilitate plasmid DNA binding and stabilization for cellular uptake.;Initial studies investigated the potential of continuously monitoring A. baylyi biofilm architecture using CLSM, which requires fluorescence excitation. Because A. baylyi is not naturally florescent and could not be labeled with a gene encoding fluorescence, fluorescent nucleic acid staining was considered. Unfortunately, continuous monitoring required repetitive staining that detrimentally affected biofilm development. Subsequently, studies requiring continuous biofilm monitoring were limited to visualization at specified time points by sacrificing replicate biofilms to nucleic acid staining. This initial study also raised concerns about current biofilm image analysis methods found to be biased by images lacking biofilm visualization. The novel program, Auto PHLIP-ML, was developed to resolve this bias by implementing a method to iteratively exclude these extraneous images.;Presented findings will help improve the utilization of natural genetic transformation as a means to engineer specialized biodegradative systems and contribute to more advanced image analysis methods. |
