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Approximately 10% of sequenced bacterial genomes are multipartite, consisting of two or more large chromosome-sized replicons. This genome organization can be found in many plant, animal, and human pathogens and symbionts. However, the advantage of harbouring multiple replicons remains unclear. One species with a multipartite genome is Sinorhizobium meliloti, a model rhizobium that enters into N2-fixing symbioses with various legume crops. In this work, S. meliloti derivatives lacking one or both of the secondary replicons (termed pSymA and pSymB) were constructed. Phenotypic characterization of these strains, including growth rate, metabolic capacity, and competitive fitness, provided some of the first experimental evidence that secondary replicons evolved to provide a niche specific advantage, improving fitness in a newly colonized environment. These results were further supported by characterizing the symbiotic phenotypes of 36 large-scale pSymA and pSymB deletion mutants. To further this analysis, an in silico S. meliloti genome-scale metabolic network reconstruction was developed and flux balance analysis used to examine the contribution of each replicon to fitness in three niches. These simulations were consistent with the hypothesis that metabolic pathways encoded by pSymB improve fitness specifically during growth in the plant-associated rhizosphere. Phylogenetic analysis of a pSymB region containing two essential genes provided a clean example of how a translocation from the primary chromosome to a secondary replicon can render the secondary replicon essential. Moreover, an experimental analysis of genetic redundancy indicated that 10-15% of chromosomal genes are functionally redundant with a pSymA or pSymB encoded gene, providing an alternative method for how secondary replicons can become essential and influence the evolution of the primary chromosome. Finally, the work presented here provides a novel framework for forward genetic analysis of N2-fixing symbiosis and the identification of the minimal N2-fixing symbiotic genome, which will help facilitate the development of synthetic symbioses. / Thesis / Doctor of Philosophy (PhD) / Many bacteria that enter into symbiotic or pathogenic relationships with plants, animals, and humans contain a genome that is divided into multiple chromosome-like molecules. One example is the N2-fixing legume symbiont Sinorhizobium meliloti, whose genome contains three chromosome-sized molecules. Here, the functions associated with each molecule in the S. meliloti genome were examined through a combination of experimental genetic analyses and computer based simulations. Results from these approaches suggested that adaptation to unique environments selected for the evolution of secondary chromosome-like molecules, with each predominately contributing to growth in a specific environment, including environments associated with an eukaryotic host. The genes on these replicons are therefore prime targets for manipulation of bacterium-host interactions, and represent reservoirs of valuable genes for use in synthetic biology applications. Additionally, the genome reduction approach employed in this study laid out a ground work for identification of the minimal N2-fixing symbiotic genome. This represents a crucial step towards successfully engineering improved nitrogen fixation, and the engineering of synthetic N2-fixing symbioses involving non-legumes and/or non-rhizobia.

Synthetic biology has ushered in a new age of molecular biology with the aim towards practical developments in disciplines ranging from medicine, agriculture, and industry. Presently, it remains difficult to manipulate the genomes of many organisms due to lack of genetic tools. These problems can be circumvented by cloning large fragments of DNA into strains where many genetic tools are in place, such as Saccharomyces cerevisiae. However, this organism is unable to directly transfer cloned DNA to other organisms and is unable to stably maintain DNA with a G+C content >40%. Many organisms relevant in biotechnology often have G+C content DNA >60%, and therefore are difficult to engineer. Here, the soil bacteria Sinorhizobium meliloti was chosen as a host strain to clone and manipulate large fragments of high G+C content DNA. S. meliloti is a Gram-negativeα-proteobacteria that forms symbiotic relationships with legumes to fix nitrogen. It has a multi-partite genome with a G+C content of 62.7% that includes a chromosome (3.65 Mb), the pSymA (1.35 Mb), and pSymB (1.68 Mb) replicons. A restriction endonuclease hsdR mutant strain lacking pSymA and pSymB was created and used in this study. Multi-host shuttle (MHS) vectors were constructed that allow for direct transfer and maintenance of DNA in E. coli, S. cerevisiae, and P. tricornutum. Characterization of strains was conducted to determine transduction, conjugation, and transformation frequencies, as well as stability of MHS plasmids. Furthermore, a proof-of-concept experiment was conducted to clone large plasmids (70-205 kb) with G+C content >58% via site-specific recombination at a landing pad in the MHS vector, which was then verified using colony PCR. This work demonstrates the usefulness of S. meliloti containing a MHS vector for cloning of large fragments with high G+C content DNA, a technology that may be used for several applications in both applied and basic research. / Thesis / Master of Science (MSc) / Synthetic biology is an emerging field that incorporates principles of molecular biology and engineering for the design and construction of biological systems for application in medicine, agriculture, and industry. Presently, it remains difficult to modify genomes of several organisms due to lack of available techniques. Yeast is currently used for the modification of large DNA pieces, however it is unable to transfer and maintain modified DNA with high G+C content. Here, the bacteria Sinorhizobium meliloti was used as a host organism to conduct genetic engineering due to its ability to maintain large DNA pieces with a high G+C content. Characterization experiments were conducted to assess the efficiency of this organism for this task. Using this strain, a proof-of-concept experiment to demonstrate the uptake and maintenance of large, high G+C DNA pieces was completed. This technology may be useful in biotechnology applications for engineering of large DNA pieces from industrially relevant organisms.