Gene Transfer In Aquatic Organisms PDF Download

The study presented in this dissertation focuses on characterizing the rates of horizontal gene transfer (HGT) by conjugation in Gram-negative bacteria under nutrient-limiting conditions. The HGT rates and frequencies of different Gram-negative mating pairs were evaluated and quantified experimentally. Escherichia coli S17-1 was used as a donor strain, while recipient bacteria were Acinetobacter calcoaceticus and E. coli DH5[alpha], Cupriavidus necator JMP134, Pseudomonas aeruginosa PAO1, Burkholderia vietnamiensis G4. A broad-host-range green fluorescence protein (GFP) reporter system, using plasmids pMMB190 and pRK-Km (a pRK415 derivative) was constructed for visual detection and quantification of HGT events under different conditions.The primary data generated in the various mating experiments were used as empirical input data to run a batch HGT kinetics model. The developed mathematical model was derived from a system of mass action rate expressions to predict the increase in transconjugant species under surface and planktonic mating conditions. The model is driven largely by unique intrinsic rate coefficients of gene transfer (k(z)) that were determined for different mating pairs. The nutrient-limiting conditions used in the experiments allowed for gene transfer kinetics to be observed and quantified, largely in the absence of growth, so that gene transfer would not be overestimated. The results of this study indicate that gene transfer varies considerably between donor and recipient mating pairs, and is markedly enhanced in surface-associated incubations ranging from 5.56 x 104 to 3.70 x 102 transconjugants per minute, compared to 2.8 x 102 to 1.56 x 101 per minute under planktonic conditions. The kinetics of gene transfer in most mating pairs appeared to lag at earlier time points ( 3 hours), but increased markedly after 8 hours for surface incubations. Isogenic mating pairs (e.g. E. coli) demonstrated much higher rates of gene transfer under both incubation conditions, despite possessing the slowest growth rates compared to non-isogenic mating pairs (e.g. E. coli with Cupriavidus necator JMP134, Pseudomonas aeruginosa PAO1 or Burkholderia vietnamiensis G4).The results of the various modeling simulations suggest that gene transfer kinetics using the developed mass-action-based model is sufficient for predicting the rate of gene transfer of a bulk mating population for different species when accounting for growth. The accuracy is driven by unique intrinsic rate terms (k(z)) that are expressed for individual mating pairs. Variability of k(z) terms was seen in short mating incubations, but these terms converged to uniform values at later times ( 6 hours). The uniform k(z) values provide accurate predictions of transconjugant increases during simulations, as confirmed by primary experimental data. The combination of k(z) terms along with transport equations can be reduced to a simplified two-equation model for evaluating the distribution and transport of transconjugant species in subsurface aquatic environments. Moreover, transport dynamics of mating pair species can be further characterized using confocal microscopy to generate real-time datasets. Based on the comparison between primary confocal data and simulations from the two-equation model, the Happel sphere-in-cell model is not predictive of species transport and distribution at pore-scale levels, because the model is far too idealized. Based on visual data generated with the HGT reporter system, single-cell gene transfer can be detected and quantified in real time under static and dynamic conditions and evaluated under nutrient-limiting conditions to confirm model predictions.