thesis published Hande Acar author 2DDF136A-F248-11E8-B48F-1D18A9856A870000-0003-1986-9753 Jonathan P Bollback supervisor JoBo department GradSch department Selective Barriers to Horizontal Gene Transfer project Horizontal gene transfer (HGT), the lateral acquisition of genes across existing species boundaries, is a major evolutionary force shaping microbial genomes that facilitates adaptation to new environments as well as resistance to antimicrobial drugs. As such, understanding the mechanisms and constraints that determine the outcomes of HGT events is crucial to understand the dynamics of HGT and to design better strategies to overcome the challenges that originate from it. Following the insertion and expression of a newly transferred gene, the success of an HGT event will depend on the fitness effect it has on the recipient (host) cell. Therefore, predicting the impact of HGT on the genetic composition of a population critically depends on the distribution of fitness effects (DFE) of horizontally transferred genes. However, to date, we have little knowledge of the DFE of newly transferred genes, and hence little is known about the shape and scale of this distribution. It is particularly important to better understand the selective barriers that determine the fitness effects of newly transferred genes. In spite of substantial bioinformatics efforts to identify horizontally transferred genes and selective barriers, a systematic experimental approach to elucidate the roles of different selective barriers in defining the fate of a transfer event has largely been absent. Similarly, although the fact that environment might alter the fitness effect of a horizontally transferred gene may seem obvious, little attention has been given to it in a systematic experimental manner. In this study, we developed a systematic experimental approach that consists of transferring 44 arbitrarily selected Salmonella typhimurium orthologous genes into an Escherichia coli host, and estimating the fitness effects of these transferred genes at a constant expression level by performing competition assays against the wild type. In chapter 2, we performed one-to-one competition assays between a mutant strain carrying a transferred gene and the wild type strain. By using flow cytometry we estimated selection coefficients for the transferred genes with a precision level of 10-3,and obtained the DFE of horizontally transferred genes. We then investigated if these fitness effects could be predicted by any of the intrinsic properties of the genes, namely, functional category, degree of complexity (protein-protein interactions), GC content, codon usage and length. Our analyses revealed that the functional category and length of the genes act as potential selective barriers. Finally, using the same procedure with the endogenous E. coli orthologs of these 44 genes, we demonstrated that gene dosage is the most prominent selective barrier to HGT. In chapter 3, using the same set of genes we investigated the role of environment on the success of HGT events. Under six different environments with different levels of stress we performed more complex competition assays, where we mixed all 44 mutant strains carrying transferred genes with the wild type strain. To estimate the fitness effects of genes relative to wild type we used next generation sequencing. We found that the DFEs of horizontally transferred genes are highly dependent on the environment, with abundant gene–by-environment interactions. Furthermore, we demonstrated a relationship between average fitness effect of a gene across all environments and its environmental variance, and thus its predictability. Finally, in spite of the fitness effects of genes being highly environment-dependent, we still observed a common shape of DFEs across all tested environments. https://research-explorer.ista.ac.at/download/1121/6814/PhDThesis_HandeAcar_1230.pdf application/pdf https://research-explorer.ista.ac.at/download/1121/9184/2016_Thesis_HandeAcar.pdf application/pdfno Institute of Science and Technology Austria2016 eng 2663-337X 75 Acar H. Selective barriers to horizontal gene transfer. 2016. H. Acar, Selective Barriers to Horizontal Gene Transfer, Institute of Science and Technology Austria, 2016. Acar, Hande. “Selective Barriers to Horizontal Gene Transfer.” Institute of Science and Technology Austria, 2016. Acar, Hande. <i>Selective Barriers to Horizontal Gene Transfer</i>. Institute of Science and Technology Austria, 2016. Acar H. 2016. Selective barriers to horizontal gene transfer. Institute of Science and Technology Austria. Acar, H. (2016). <i>Selective barriers to horizontal gene transfer</i>. Institute of Science and Technology Austria. H. Acar, “Selective barriers to horizontal gene transfer,” Institute of Science and Technology Austria, 2016. 11212018-12-11T11:50:16Z2026-07-29T11:17:47Z