Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of nitrate, peptide, dicarboxylate and phytohprmone transporters. Experiments with Mtnip/latd mutants demonstrats a defective nitrate response associated with low (250 μM) external nitrate concentration rather than high (5 mM) nitrate concentration. This suggests that the mutants have defective nitrate transport. To test if MtNIP/LATD was a nitrate transporter, Xenopus laevis oocytes and Arabidopsis thaliana mutant plants Atchl1-5, defective in a major nitrate transporter AtNRT1.1(CHL1), were used as surrogate expression systems. Heterologous expression of MtNIP/LATD in X. laevis oocytes and Atchl1-5 mutant plants conferred on them the ability to take up nitrate from external media with high affinity, thus demonstrating that MtNIP/LATD was a high affinity nitrate transporter. Km for MtNIP/LATD was determined to be approximately160 μM in the X. laevis system and 113 μM in the Arabidopsis Atchl1-5 mutant lines thus supporting the previous observation of MtNIP/LATD being a high affinity nitrate transporter. X. laevis expressing the mutant Mtnip-1 and Mtlatd, were unable to transport nitrate. However X. laevis oocytes, expressing the less severe mutant allele Mtnip-3 were able to transport nitrate suggesting another role of the Mtnip/latd besides high affinity nitrate transport. Experimental evidence suggested that MtNIP/LATD might transport another substrate beside nitrate. MtNIP/LATD levels are regulated by phytohormones. Experiments performed with ABA (abscisic acid), IAA (indole acetic acid) and histidine as substrates in X. laevis system show that the MtNIP/LATD mRNA injected oocytes efflux IAA but do not transport histidine or ABA. When wild type A17 and mutant Mtnip-1 and Mtnip-3 plants, grown in the presence of different sources of nitrogen were screened in herbicide chlorate, a structural analog of nitrate, the A17 and Mtnip-3 mutant showed levels of susceptibility that was different from mutant Mtnip-1 lines. Evidence suggested that the amount of chlorate transported into the plants were regulated by the C:N status of the A17 and Mtnip-3 plants. This regulation was missing in the Mtnip-1 lines thus suggesting a sensor function of MtNIP/LATD gene.

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No description available.

Investigations of the nitrogen fixing (nif) genes in the cyanobacterium Nostoc commune UTEX 584 revealed a gene encoding a hemoprotein, named cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. Cyanoglobin possesses a high oxygen affinity. The study presented here investigated the functional role of cyanoglobin, and encompassed the determination of the kinetic basis for the high oxygen affinity of cyanoglobin through kinetic studies utilizing stopped-flow spectrophotometry and flash photolysis. In addition, studies of cyanoglobin, in the presence of a variety of ligands, employed as structural probes of the distal pocket architecture, are presented. These data are interpreted in terms of structural models of cyanoglobin produced by homology modelling and hemoglobins with known crystal structures. Cyanoglobin coordinated oxygen and a variety of ligands with high rates of association, which explained the high oxygen affinity of cyanoglobin. Cyanoglobin possessed high rates of autoxidation and hemin loss. The ligand binding behavior of cyanoglobin was more similar to leghemoglobin than to sperm whale myoglobin. The ligand binding behavior of cyanoglobin is explained in terms of a highly reactive, and solvent exposed, heme-iron. The 5' region of glbN interacted with NtcA, the global regulator of nitrogen metabolism in cyanobacteria, which may provide an indication of the nitrogen deprivation signal required for cyanoglobin expression in vivo. Finally, the isolation and N-terminal sequencing of a potential cyanoglobin homolog in Anabaena sp. strain PCC 7120 is presented. Collectively, the data obtained in this study may support the model of cyanoglobin function described by Hill, et al., that cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal. / Ph. D.

This investigation has determined the presence of biological nitrogen fixation in two reservoirs in the southwestern United States: Lake Arlington and Lake Ray Hubbard. Subsequent tests have gathered baseline data on the effects of various biological, chemical, and physical parameters on in situ nitrogen fixation in these reservoirs. Of specific importance is the relationship between nitrogen fixation arid occasional blooms of blue-green algae which produce such problems as testes and odors in these water-supply impoundments.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [90]-114).

Thesis (M.S.)--University of Missouri-Columbia, 2006. / "August 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Les premières formes de vie sur Terre seraient apparues durant l’Archéen, il y a 4 à 2,5 milliards d’années. Durant cette période, les océans et l’atmosphère étaient anoxiques. Vers la fin de cet éon, la concentration en dioxygène a brusquement augmenté grâce à la photosynthèse, contribuant à la Grande Oxygénation de la Terre. Toutefois, en raison de la rareté des microorganismes fossiles connus, les métabolismes actifs à cette époque restent mal compris. Le fractionnement des isotopes stables du carbone est souvent utilisé comme un critère de biogénicité et pour l’appréciation des voies métaboliques présentes. Ces fractionnements peuvent être le résultat d’au moins six à huit voies de fixation du carbone. Pour étudier l’histoire évolutive des voies de fixation du carbone et de déterminer leur ordre d’émergence, j’ai appliqué une approche phylogénomique sur l’importante diversité microbienne récemment découverte. Le but était d’identifier les voies responsables des signatures isotopiques du carbone datant de l’éon Archéen inférieur (>3,2 milliards d’années). Le premier chapitre constitue une revue récente sur la diversité, l’écologie et l’évolution des Archaea. J’ai construit une phylogénie de référence des Archaea, robuste et incluant un nombre important de nouveaux génomes. Cette phylogénie m’a permis de mettre en évidence de nouveaux clades d’Archaea pour lesquels j’ai proposé des nouveaux noms. De plus, j’ai examiné la distribution des gènes marqueurs classiquement utilisés dans la taxonomie des Archaea. Dans le chapitre 2, j’ai assemblé différents jeux de données pour construire des phylogénies de référence pour les bactéries. Ceci m’a permis de discuter la classification au sein de ce domaine et la position de quelques groupes proches de la racine. Ces phylogénies des Archaea et Bacteria m’ont servi de cadre pour retracer l’évolution des voies de fixation du carbone. J’ai ensuite étudié la voie de Wood-Ljungdahl (WL) qui est considérée comme la forme la plus ancienne de fixation du carbone mais dont les origines restent encore controversées. J’ai assemblé des banques de données locales englobant 6400 génomes et couvrant toute la diversité connue des archées et des bactéries. Ces banques ont été utilisées pour des recherches exhaustives des homologues des enzymes de la branche carbonyle (chapitre 3) et méthyle basée sur la tétrahydrométhanoptérine (H4MPT; chapitre 4) de la voie de WL. Ces analyses m’ont permis d’inférer la présence d’une forme fonctionnelle de la branche carbonyle chez LUCA (Last Universal Common Ancestor). Cette voie a ensuite été héritée verticalement chez les archées et bactéries en gardant la co-localisation de ses gènes, à l’exception de quelques rares transferts intra et inter-domaines. La branche méthyle-H4MPT semble être apparue chez les archées puis transférée aux bactéries chez lesquelles elle serait impliquée dans la syntrophie ou l’assimilation du carbone. A la suite de gains et de pertes de gènes au sein de cette branche, elle a ensuite été successivement adaptée pour la méthylotrophie anaérobie, la détoxification du formaldéhyde, et la méthylotrophie aérobie. Ces résultats indiquant l’origine de la voie de WL à l’Archéen m’ont permis d’interpréter les signatures isotopiques du carbone et d’apporter des éléments sur la composition de l’atmosphère à la fin de cet éon. Enfin, dans le chapitre 5, j’ai étudié l’histoire évolutive des autres voies de fixation du carbone (Calvin-Benson-Bassham, Reductive Hexulose Phosphate, reverse Krebs, 3-hydroxypropionate bicycle, 3-hydroxypropionate/4-hydroxybutyrate, dicarboxylate/4-hydroxybutyrate). Mes résultats préliminaires m’ont permis de discuter la présence possible de ces voies pendant l’Archéen. / Life on Earth emerged during the Archean Eon (4-2.5 billion years ago). At the time the oceans and atmosphere were anoxic, and oxygen rose at the end of the Eon as a result of oxygenic photosynthesis, in what is known as the Great Oxygenation Event. Anaerobic microorganisms and metabolisms are expected to have operated at the time. However, the specifics are poorly understood, since the fossil record is scarce. The fractionation of stable carbon isotopes is often used as a criterion of biogenicity but also to interpret possible metabolic processes. Such fractionations can arise from at least six to eight different carbon fixation pathways. I took advantage of the newly available microbial diversity, and applied a phylogenomic approach to elucidate the evolutionary history of carbon fixation pathways, and determine their relative order of emergence. The aim was to deduce which ones would have been responsible for the isotopic signatures in the lower Archean (before 3.2 billion years). In the first Chapter, I reviewed the recent literature on the diversity, ecology, and evolution of Archaea. I constructed a well-resolved reference phylogeny taking into account all the novel lineages, for which genomic information has recently become available. I assigned names to some of them, as well as to some of the taxonomic units that were recovered from the phylogeny. Then I examined the distribution of genes that have been used in the past as taxonomic markers for the Archaea. Similarly, in Chapter 2, I constructed well-resolved bacterial phylogenies using different datasets, and used them to map the distribution of potential marker genes. I then discussed the taxonomic classification of Bacteria above phylum level, and the position of some possibly deep-branching phyla. From these endeavors, I gleaned highly resolved phylogenies of Bacteria and Archaea which were then used to map the evolution of carbon fixation pathways. Next, I analyzed the evolution of the Wood-Ljungdahl pathway. It is believed to be the most ancient form of carbon fixation but its origins have been controversial. I assembled local databanks of over 6400 genomes of Bacteria and Archaea encompassing all their known diversity. These were used to perform exhaustive homology searches for the components of the carbonyl (Chapter 3) and tetrahydromethanoperin (H4MPT; Chapter 4) methyl branches. A functional form of the carbonyl branch was found to date back to the Last Universal Common Ancestor. It was then inherited mostly vertically across Bacteria and Archaea with its genes remaining co-localized, except for a few rare intra and interdomain transfers. The H4MPT branch seems to have originated in Archaea and was subsequently transferred to Bacteria where its original role was probably related with hydrogen syntrophy or as a carbon assimilation electron sink. Afterward, through gene gains and losses linking the branch with other pathways, it came to be used in anaerobic methylotrophy and formaldehyde detoxification, and finally in aerobic methylotrophy. These results highlight a presence of the Wood-Ljungdahl pathway throughout the Archean, and also allow me to discuss possible inferences on the composition of the atmosphere and the interpretation of some late Archean carbon isotopic signatures.Finally, in Chapter 5, I attempt to determine the earliest possible origin for the remaining carbon fixation pathways (Calvin-Benson-Bassham, Reductive Hexulose Phosphate, reverse Krebs, 3-hydroxypropionate bicycle, 3-hydroxypropionate/4-hydroxybutyrate, dicarboxylate/4-hydroxybutyrate), by studying the evolution of their marker genes. I managed to deduce some possible constraints about the presence of these pathways in the Archean. My results contribute to expanding our knowledge on early life, the Last Universal Common Ancestor, and the evolution of carbon fixation. They also shed light on the processes on the Archean Earth from the perspective of microbial evolution.

The thermophilic bacterium designated NT-7 was shown to reduce acetylene to ethylene at 35 C. It was found that the organism does not reduce acetylene when it is grown in Burk's medium with 0.3 per cent (w/v) NH4 N0 3 . Reduction of acetylene at 55 C could not be demonstrated due to insolubility of acetylene in Burk's medium at this temperature. It was shown that the bacterium NT-7 can not grow in the absence of atmospheric nitrogen at 55 C when combined nitrogen is not supplied with the nutrient medium. All these characteristics were used to prove that NT-7 is a nitrogen fixing bacterium. Identification procedures confirmed a previous finding that the organisms are rod shaped cells possessing endoepores. Further tests showed that NT-7 is obligately aerobic and motile.