<p> Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate with the simultaneous reduction of a nicotinamide cofactor. It was previously reported that the nitrogen-fixing bacterium, Sinorhizobium meliloti, has two malic enzymes, a diphosphopyridine-dependent malic enzyme (DME) and a triphosphopyridine-dependent malic enzyme (TME). The dme gene is essential for symbiotic nitrogen-fixation in alfalfa root nodules and this symbiotic requirement cannot be met through increased expression of tme. In order to determine if a metabolic difference exists between the dme and tme mutants which might explain the symbiotic phenotypes, we conducted an analysis of intracellular and extracellular polar metabolomes. Differences noted between the intracellular profiles of the dme and tme mutant strains hinted at osmotic stress or a disturbance in central carbon metabolism. Extracellular studies indicated that dme mutant cells excreted at least 10-fold greater concentrations of both malate and fumarate. When considered together, the metabolic data implies that the DME enzyme is primarily responsible for the conversion of malate to pyruvate to generate acetyl-CoA whereas the TME enzyme must serve a secondary function within the cell.</p> <p> While the C-terminal 320 amino acid regions from both DME and TME are similar in sequence to phosphotransacetylase enzymes, enzyme assays with DME and TME proteins have failed to detect PTA activity. Here we report that the chimeric malic enzyme structure is conserved among various gram negative bacteria including Agrobacterium tumefaciens, Escherichia coli, Bradyrhizobium japonicum and Porphyromonas gingivalis. Moreover these chimeric proteins are also present in the archaebacteria. Halobacterium salinarum and Haloarcula marismortui. To further our understanding of the functions of DME and TME in S. meliloti, we have fused protein domains from DME to an affinity tag consisting of strepII and a calmodulin binding peptide. To identify proteins interacting with this fusion, we expressed these protein fusion constructs in S. meliloti, prepared extracts containing the soluble proteins and passed these through tandem affinity chromatography columns. All proteins that coeluted with the fusion proteins appeared to be interacting with antibodies specific for the DME protein and so may have been aggregates or break-down products of DME.</p> / Thesis / Master of Science (MSc)

In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)

No description available.

Des génomes entiers de bactéries sont séquencés en nombre croissant. Parallèlement sont mis en place des programmes d'analyse systématique de l'expression des gènes et des protéines dans différentes conditions. La compréhension du fonctionnement d'un organisme nécessite une annotation des fonctions des gènes et l'intégration de ces données dans des schémas fonctionnels. Les voies métaboliques constituent une classe de fonctions permettant d'aborder ce problème d'intégration, elles sont bien répertoriées chez de nombreux organismes et sont accessibles à l'expérimentation.<br />Dans un premier temps, nous avons développé une méthode automatique de prédiction de fonction spécifique des enzymes. Cette méthode nommée PRIAM (PRofils pour l'Identification Automatique du Métabolisme) repose sur la nomenclature des enzymes et sur la construction automatique d'un jeu de profils spécifiques des fonctions enzymatiques. Puis, cette méthode permet d'identifier les enzymes dans un génome complet et de visualiser les résultats obtenus sur les graphes des voies métaboliques de la base de données KEGG. <br />Dans un second temps, cette méthode a été appliquée sur le génome de la bactérie fixatrice d'azote Sinorhizobium meliloti et nous a permis l'analyse des voies métaboliques spécifiques de cet organisme symbiote.

Contrary to the predictions of numerous theoretical models, variation in partner quality continues to persist in mutualisms, including in the symbiosis between legumes and rhizobia. One potential explanation for the maintenance of this genetic diversity is genotype × genotype interactions, however it is unknown which genetic regions might underlie these interactions. To investigate this question, I performed a quantitative trait loci mapping experiment with two different rhizobium strains to locate potential regions of the genome influencing genotype × genotype interactions between the legume Medicago truncatula and its symbiont Sinorhizobium meliloti. I found no evidence for genotype × genotype or QTL × rhizobium interactions, however some of the QTLs colocalized with genes involved in the symbiosis signaling pathway, suggesting variation in these genes could potentially affect plant performance and fitness traits. These findings have important implications for the evolutionary interactions between legumes and rhizobia, and the genetic architecture of Medicago truncatula.

Contrary to the predictions of numerous theoretical models, variation in partner quality continues to persist in mutualisms, including in the symbiosis between legumes and rhizobia. One potential explanation for the maintenance of this genetic diversity is genotype × genotype interactions, however it is unknown which genetic regions might underlie these interactions. To investigate this question, I performed a quantitative trait loci mapping experiment with two different rhizobium strains to locate potential regions of the genome influencing genotype × genotype interactions between the legume Medicago truncatula and its symbiont Sinorhizobium meliloti. I found no evidence for genotype × genotype or QTL × rhizobium interactions, however some of the QTLs colocalized with genes involved in the symbiosis signaling pathway, suggesting variation in these genes could potentially affect plant performance and fitness traits. These findings have important implications for the evolutionary interactions between legumes and rhizobia, and the genetic architecture of Medicago truncatula.

Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant. / One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate. / All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis.

No description available.

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.