Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons required for SNF, a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis revealed that only four gene regions, accounting for <12% of the total sequences of pSymA and pSymB that, were essential for SNF. In the first part of the thesis, I report the cloning of these two pSymA SNF-essential regions on a plasmid (pTH3255) in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain (the strain was named as RmP4291 after integration). Plant root dry weight and nitrogenase-catalyzed acetylene reduction assays were carried out on RmP4291 with four host plants, including Medicago sativa, Medicago truncatula, Melilotus alba and Melilotus officinalis. Nodule kinetic assays were also performed on RmP4291 and RmP110(wt). The results showed that the SNF-essential regions from pSymA were sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with all the host plants tested, except a significant reduction (~40%) in SNF by RmP4291 was noticed on M. officinalis compared to that by wildtype S. meliloti. A higher alfalfa nodulation efficiency of RmP4291 compared to that of wildtype RmP110 was also discovered. In the second part of the thesis, a histochemical staining method for S. meliloti nodules was developed by integrating the marker genes gusA (β-glucuronidase) and celB (β-glucosidase) into the S. meliloti genome. This staining method was found to be useful in the study of nodule competitiveness. A nodule competition assay was carried out between RmP4291 and RmP110 using the new staining method. RmP4291 was found to be significantly reduced in nodulation competitiveness compared to wildtype S. meliloti. The development of the histochemical staining method for S. meliloti nodules will accelerate the identification of genes required for nodule competitiveness in the organism, which will be of crucial importance to the construction of the minimal genome strains with high SNF efficiency. / Thesis / Master of Science (MSc) / Nitrogen is one of the critical elements for life. Biological nitrogen fixation plays a crucial role in providing fixed nitrogen for the ecosystem on Earth. Our Laboratory has endeavored to establish a minimal symbiotic genome in Sinorhizobium meliloti, a model nitrogen fixing bacterium which forms symbiosis with certain kinds of legumes. Building this minimal symbiotic genome will improve our understanding of the symbiotic nitrogen fixation process in S. meliloti at gene level. It may also help in eventually introducing a nitrogen fixation system into other organisms. In this study, the minimal symbiotic genome of the pSymA replicon in S. meliloti was constructed. In addition, a staining method to detect specific S. meliloti strains in nodules was established. This method is potentially useful in finding genes related to nodule competitiveness, and these are potentially important for augmenting the genes that constitute the minimal symbiotic genome.

Computational evolutionary analyses, particularly phylogenetics and ancestral reconstruction, have been extensively exploited under different algorithms and evolutionary models to better understand genome evolution from both small- and large-scale perspectives in order to assign genotypes based on assortment, resolve species relationships and gene annotation issues, further understand gene gain/loss within individual gene families, measure functional divergence among homologs, and infer ancestral character states. These evolutionary studies provide us with insights into biologically relevant issues including paleoenvironments inferred from resurrected proteins, developmental physiology associated with functional divergence of duplicated genes, viral epidemics and modes of transmission in attempt to better prepare, prevent and control diseases, evolution of lineage-specific pathogenicity, and attempts to create a synthetic ancient organism that would benefit the field of synthetic biology. Our work also provides us with greater insights into the accuracies and limitations of ancestral sequence reconstruction methods. In total, our work highlights the diverse questions that evolutionary studies attempt to address and the different biological levels that can be studied to answer these questions.