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.

Made available in DSpace on 2016-04-28T20:18:28Z (GMT). No. of bitstreams: 1 2006-Juliana Solozabal Martins da Rocha.pdf: 3772089 bytes, checksum: 41414ee2cbb97d4f355ca0c9f47ec737 (MD5) Previous issue date: 2006-02-09 / The microfilariaes when found should be identified correctly, for obtaining a correct diagnosis, since Dipetalonema is apathogenic and the infection doesn't request treatment, while Dirofilariosis, known as Heartworm Disease, it can take the dog to the death. This work had as objective to contribute to the diagnosis using histochemical differentiation in twelve sanguine samples of microfilaremics dogs and aid in the recognition of the identified species with that staining, based on the individual acid fosfatase activity and to confirm the use of a commercial kit for this procedure. They were identified Dirofilaria immitis and Dipetalonema reconditum, making possible this technique in the taxonomy of those species. / As microfil?rias quando encontradas devem ser identificadas corretamente, para realiza??o de um diagn?stico preciso, j? que o Dipetalonema ? apatog?nico e a infec??o n?o requer tratamento, enquanto a Dirofilariose, conhecida como Doen?a do Verme do Cora??o, pode levar o c?o ? morte. Este trabalho teve como objetivo contribuir ao diagn?stico pela diferencia??o histoqu?mica em doze amostras sangu?neas de c?es microfilar?micos e auxiliar no reconhecimento das esp?cies identificadas por essa marca??o, baseada na atividade da fosfatase ?cida individual, confirmando a utiliza??o de um kit comercial para este procedimento. Foram identificadas histoquimicamente, Dirofilaria immitis e Dipetalonema reconditum, possibilitando esta t?cnica na taxonomia dessas esp?cies.