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Phospholipids and lipopolysaccharide were purified from Haemophilus influenzae type b and combined by sonication to form liposomes which were impermeable to sucrose and to high molecular weight polysaccharides. When outer membrane proteins were incorporated into such liposomes, they became permeable to saccharides up to a molecular weight of about 1,400 daltons. The outer membrane proteins were fractionated on DEAE-Sepharose. Three protein-containing peaks were eluted from the column. Two of these peaks contained mixtures of proteins while the third peak contained only a single species of protein with a molecular weight of 40,000 daltons (40 kDa). When tested in reconstituted vesicles, only the material from this third peak rendered liposomes permeable to sucrose. The molecular weight exclusion limit for the 40 kDa protein matched the estimate of 1,400 daltons determined for the outer membrane. / The relative rates of diffusion of small sugars were determined by measuring the rates of swelling of liposomes reconstituted with the 40 kDa protein. From these rates, a pore diameter of 1.8 nm was estimated using the Renkin equation. / The properties of the 40 kDa protein were also studied using black lipid membranes. When added to the aqueous phase which was bathing a lipid bilayer, this protein caused the conductance of the membrane to increase rapidly. At low protein concentrations, the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1.0 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was only slightly cation-selective: the pores were about 1.6 times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit or from its pore diameter estimated by the liposome swelling assay.

Employing successive chromatography on DEAE-Sepharose and on single-strand (ss) DNA-cellulose, a new form of Neurospora endo-exonuclease has been isolated, a Mg('2+)-dependent ss-DNA-binding endo-exonuclease. The enzyme has been purified to homogeneity from extracts of mitochondria, vesicles, and the "25,000 xg pellet" (a mixture of mitochondria and small vesicles) indicated by the appearance of single protein bands on non-denaturing polyacrylamide gels and the same specific activity (14,000 U/mg) for enzyme from all three sources. The apparent native molecular weight for the endo-exonuclease from vesicles and the 25,000 xg pellet was 80K, but for the enzyme from mitochondria it was 116-130K. The mobility of the mitochondrial endo-exonuclease on non-denaturing polyacrylamide gels was also found to be higher, 0.65 as compared to 0.17 for the vesicle endo-exonuclease. This higher mobility was shown to be due to the presence of phosphomonoester groups associated with the protein. The electrophoresis of the enzyme from the three sources on SDS-polyacrylamide gels revealed that the enzyme is a "complex" of polypeptides of which 49K, 33K, and 12 - 14K polypeptides were common to all three preparations. / The enzymatic properties of the ss-DNA-binding endo-exonuclease from the three sources are identical showing, for the first time, the presence of the same enzyme in two different types of subcellular organelles. The enzyme releases initially large 5'-p-terminated oligonucleotides from ss-DNA in a non-processive manner and di-, tri-, and tetranucleotides from double strand (ds) DNA in strictly processive manner. The endo- and exonuclease activities showed differential Mg('2+) requirements, inhibitions by NaCl and effects of pH and ethidium bromide. The inactivation of both activities by ethoxyformic anhydride was inhibited by ss-DNA, but not by ds-DNA. / The presence of the protease inhibitor, PMSF, during extraction prevented the recovery of ss-DNA-binding endo-exonuclease, indicating the presence of a more intact form of enzyme in vivo. Treatment of an inactive precursor protein with a PMSF-sensitive protease of Neurospora, Bz-TYR-Nanase, resulted in the formation of the ss-DNA-binding endo-exonuclease. Proteolytic conversions of ss-DNA-binding endo-exonuclease to other various forms of sugar-non-specific nucleases which have been characterized previously were confirmed. The relatedness of these nucleases to ss-DNA-binding endo-exonuclease and precursor was demonstrated by preparing antiserum in rabbit using ss-DNA-binding enzyme and testing for immunological cross-reactivity. Cross-reactivity was also seen with a commercial preparation of Neurospora endonuclease and a yeast mitochondria nuclease, but none was seen with pancreatic DNase I or Micrococcal nuclease.

During HIV-1 assembly, Gag, GagPol, and Env are assembled into immature viral particles at the plasma membrane, along with viral genomic RNA and other viral and cellular factors. Among the cellular factors are lysyl-tRNA synthetase (LysRS) and tRNALys3, the primer tRNA for reverse transcription. LysRS is a tRNALys-binding protein, and is the enzyme that aminoacylates tRNALys. It is therefore a likely candidate for the signal which targets tRNALys for packaging into HIV-1. Gag alone among the viral proteins is sufficient to package LysRS, but the additional presence of GagPol is required for tRNALys incorporation as well. We propose that the tRNALys packaging complex includes Gag, GagPol, viral genomic RNA, tRNALys, and LysRS. To study the formation of this complex, we have investigated the following: (1) How does RNA facilitate the interaction between Gag and GagPol? (2) What are the kinetics of assembly of Gag/GagPol complexes in HIV-1? (3) How specific is the interaction of Gag with LysRS? (4) Which cellular pools of LysRS are required for the incorporation of LysRS into virions? / Membrane and RNA have been proposed to act as intracellular scaffolds for aligning Gag molecules and facilitating their interaction with each other. In this thesis, we have examined both the RNA requirement for Gag/GagPol interaction and the membrane site at which this interaction can first be seen. Using both RNAse in vitro and mutations within the nucleocapsid region in either Gag or GagPol in vivo, we have concluded that the interaction of GagPol with Gag required an RNA-facilitated Gag multimerization, but that a direct interaction of GagPol with RNA is not required. We have also found that at steady-state, almost all Gag/Gag and Gag/GagPol complexes are found at detergent-resistant membrane (DRM). Analysis of the localization of newly-synthesized Gag/Gag and Gag/GagPol complexes indicates that after a 10 minute pulse with radioactive 35S-Cys/Met, all newly-synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag complexes move to the membrane, of which 38% is found associated with DRM at 0 minute chase. The remaining membrane-bound Gag associates with DRM after a 30 minute chase. The rapid localization of GagPol to DRM probably reflects the interaction of all newly-synthesized GagPol with the first new-synthesized polymeric Gag to associate with DRM.

Nutrient acquisition by Gram-negative bacteria requires strategies for passage across the outer membrane (OM). Because iron is a vital nutrient for bacteria, we aim to elucidate the molecular mechanism of its uptake. / In an aerobic environment, ferric iron is predominant but exhibits low solubility. To overcome this challenge, bacteria import siderophores, ferric iron chelators. Siderophore import involves its binding to an OM receptor then to a periplasmic binding protein (PBP). Cytoplasmic membrane-embedded protein TonB, through its interaction with the periplasmic face of OM receptors, may provide energy to facilitate nutrient uptake. / To explore the molecular mechanism of TonB-dependent transport, we probed for conformational changes in FhuA, OM receptor for hydroxamate siderophores, in response to TonB binding. We defined epitopes for anti-FhuA monoclonal antibodies (mAbs) and monitored binding of these mAbs to FhuA with and without bound TonB. The most striking changes in binding kinetics were observed for mAb binding to extracellular loops of FhuA. We further explored these changes by conjugating a fluorophore, MDCC, to an extracellular loop of FhuA to monitor changes in fluorescence. Binding of TonB to FhuA quenched MDCC fluorescence. / Investigating events at another stage of TonB-dependent nutrient uptake, we previously discovered interactions between TonB and FhuD, PBP for hydroxamate siderophores. To determine whether such interaction occurs in other TonB-dependent uptake systems, we investigated binding of TonB to BtuF, PBP for cyanocobalamin. By phage display, we predicted a TonB-BtuF interaction by identifying potential binding residues on each protein. Further, we confirmed in vitro interaction between these proteins. TonB-BtuF binding stoichiometry was 1:1 and independent of cyanocobalamin. / In summary, binding of TonB to the periplasmic face of FhuA induced conformational changes in extracellular loops of the receptor that may serve to promote vectorial transport of siderophore. In addition, we have identified a novel, cyanocobalamin-independent interaction between TonB and BtuF. This interaction could serve to situate BtuF to efficiently encounter, bind and deliver substrate for uptake into the cytoplasm. / L'acquisition d'éléments nutritifs par les bactéries gram-négatives nécessite des stratégies de transport au travers de la membrane externe. Le fer étant un élément essentiel pour les bactéries, nous travaillons à élucider le mécanisme moléculaire de ce transport. / En milieu aérobique, le fer se trouve principalement sous sa forme ferrique qui est peu soluble. Pour surmonter cet obstacle, les bactéries importent des sidérophores, un type de chélateurs du fer ferrique. L'acquisition des ces sidérophores implique leur liaison par un récepteur de la membrane externe et par une protéine périplasmique de liaison. L'énergie nécessaire au transport de sidérophores à travers la membrane externe pourrait être fournie par TonB, une protéine de la membrane cytoplasmique qui se lie à la face périplasmique du récepteur de la membrane externe. / Pour étudier le mécanisme moléculaire du transport TonB-dépendant, nous nous sommes attardés aux changements de conformation que FhuA, le récepteur de sidérophores hydroxamiques situé dans la membrane externe, subit en réponse à sa liaison avec TonB. Des épitopes pour anticorps monoclonaux contre FhuA ont été sélectionnés et leur liaison avec FhuA, en présence et en absence de TonB, a été testée. Les changements les plus notables ont été remarqués avec les anticorps se liant aux boucles extracellulaires de FhuA. Pour confirmer ces changements, un fluorophore, le MDCC, a été conjugué aux boucles externes de FhuA et l'intéraction entre les deux protéines engendre une extinction de fluorescence. / En étudiant d'autres étapes du transport TonB-dépendant, des intéractions entre TonB et FhuD, une protéine périplasmique de liaison de sidérophores hydroxamiques, ont été découvertes. Pour vérifier si des intéractions similaires existaient dans d'autres systèmes d'acquisition TonB-dépendant, nous avons investigué les intéractions entre TonB et BtuF, la protéine périplasmique de liaison de la cyanocobalamine. L'identification, par phage display, de résidus clés sur TonB et BtuF nous a permis de prédire une intéraction entre ces deux protéines, que nous avons confirmée par la suite in vitro. La stoechiométrie de liaison a été établie à 1 :1 et est indépendante de la présence de cyanocobalamine. / En conclusion, la liaison de TonB à la face périplasmique de FhuA induit des changements de conformation dans les boucles extracellulaires du récepteur qui pourraient servir au transport unidirectionnel de sidérophores. De plus, nous avons découvert une nouvelle intéraction entre TonB et BtuF qui est indépendante de la présence de cyanocobalamine. Cette intéraction positionnerait BtuF de façon à maximiser la rencontre, la liaison et le transport de son substrat jusqu'au cytoplasme.

Group II introns are ribozymes that self-splice from pre-mRNA transcripts, concurrently ligating their flanking exons. From an evolutionary perspective, they are considered to be the progenitors of eukaryotic nuclear introns and their splicing machinery, the spliceosome. It is hypothesized that the fragmentation of an ancestral group II intron gave rise to the five small nuclear RNAs (snRNAs - U1, U2, U4, U5, U6) that constitute the catalytic core of the spliceosome. One factor lending support to this hypothesis is the ability of fragmented organellar group II introns to splice in trans. We subjected the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to an EZ-Tn5-based fragmentation screen and selected for proficient tripartite trans-splicers through a trans-splicing/conjugation assay. By demonstrating the first tripartite trans-splicing Ll.LtrB variants in vivo, we provide further experimental support for the evolutionary link between group II introns and the snRNAs of the eukaryotic spliceosome. / Les introns de groupe II sont des introns autocatalytiques qui s'épissent des transcrits de pré-mARN, et liguent concurremment les exons flanquant. D'un point de vue évolutif, ils sont considérés comme les ancêtres des introns nucléaires eucaryotes et de leur appareil d'épissage, le spliceosome. On présume que le fractionnement d'un intron de groupe II ancestral a donné naissance aux cinq petits ARN nucléaires (snRNAs - U1, U2, U4, U5, U6) qui constituent le noyau catalytique du spliceosome. Cette hypothèse est supportée par la découverte d'introns de groupe II fragmentés qui s'épissent en trans dans certaines organelles. Nous avons soumis l'intron de groupe II Ll.LtrB de la bactérie Gram-positive Lactococcus lactis à un essai génétique basé sur le transposon EZ-Tn5, suivi par une analyse de conjugaison, afin d'isoler des introns tripartites capable de s'épisser en trans. En démontrant les premières variantes tripartites de Ll.LtrB in vivo, nous fournissons un support expérimental pour le lien évolutif entre les introns de groupe II et les snRNAs du spliceosome eucaryote.

The mycobacteria genus comprises bacteria pathogenic for humans and animals, including M. tuberculosis complex (MTC). In the past decade, considerable research has focused on genomic studies differences between MTC strains and sub-species. These data largely address the devolution of MTC organisms, through a process of reductive genomics and single nucleotide polymorphisms. We hypothesized that these findings present an incomplete evolutionary scenario for the pathogen M. tuberculosis. To test this possibility, we have conducted comparative genomic analysis among sequenced mycobacteria to look for evidence of horizontal gene transfers during the evolution of the genus. Using a bioinformatic screen, we predicted a number of foreign genes acquisitions during the step-wise genesis of M. tuberculosis. Along with others, this study emphasizes another side of the evolution of M. tuberculosis and demonstrates that before the process of genomic decay, M. tuberculosis had acquired foreign genes. / The description of the M. tuberculosis evolution is strengthened when accompanied by functional characterization of these evolutionary events. Therefore, we have focused on an already-known example of micro-evolution to determine its role in M. tuberculosis biology. Our laboratory had previously established that MTC members exhibit variable production of eleven proteins due to mutations in the anti-sigma factor of SigK (RskA). As a consequence, in M. tuberculosis, their expression is low in vitro but strongly induced during infection; in contrast, M. bovis and the Oryx bacillus constitutively express these proteins. We first determined which genes are SigK-regulated and described the SigK promoters using luciferase technology. We then used these tools for a detailed study of the function of RskA from different MTC organisms. These experiments demonstrated that the anti-sigma factor RskA is not only an inhibitor of SigK but also presents an activator function. Finally, the activating property of RskA was used to generate M. tuberculosis strains over-expressing the SigK regulon, enabling us to test the effect of over-expression on the host pathogen relationship during in vivo infections. These experiments reproducibly demonstrated that over-expressing SigK-regulated genes results in increased bacterial dissemination from the site of infection along with increased survival of the host. / Over-all, this study has provided new and complementary understanding on M. tuberculosis evolution. Additionally, our evolutionary approach has contributed to a better understanding of two major areas of research, specifically Sigma factor signaling and M. tuberculosis pathogenesis. / Le genre bactérien des mycobactéries comprend des espèces pathogènes pour les animaux mais aussi pour les humains comme les bactéries du groupe de Mycobacterium tuberculosis (MTC). Dans les dernières années, la recherche dans ce domaine à largement donné la priorité aux études des différences génomiques entre les sous-espèces et souches du MTC. Celles-ci ont alors permis d'observer que les organismes du MTC avaient évolué grâce à des délétions géniques et des mutations ponctuelles. Cependant, nous avons émis l'hypothèse que ces observations ne décrivent qu'en partie l'évolution de M. tuberculosis. En effet, en utilisant la comparaison bioinformatique des génomes des mycobactéries séquencées, nous avons décrit l'acquisition séquentielle de gènes étrangers par l'ancêtre de M. tuberculosis durant l'évolution du genre. Cette étude, additionnée à d'autres, démontre que l'évolution de M. tuberculosis a été ponctuée d'acquisition de gènes étrangers avant d'être suivi d'une période de dévolution génomique. / L'étude de l'évolution de M. tuberculosis ne serait pas complète sans la caractérisation fonctionnelle de ces évènements évolutifs. C'est pourquoi nous avons tenté de comprendre un exemple déjà connu de microévolution et de déterminer le rôle de cet évènement dans la biologie de M. tuberculosis. Notre laboratoire a déjà établi que les membres du MTC expriment de façon variable onze protéines suite à des mutations dans l'anti-sigma facteur de SigK (RskA). Par conséquent, si M. tuberculosis n'exprime pas ces protéines in vitro mais durant l'infection, M. bovis et le Bacille de l'antilope les expriment de façon constitutive incluant in vitro. Nous avons commencé par décrire les promoteurs régulés par SigK en utilisant la luciférase comme rapporteur. Ces données et ces outils nous ont alors permis d'étudier plus en détail la fonction des différentes versions de RskA provenant des membres du MTC. Nous avons démontré que RskA est non seulement un inhibiteur de SigK mais aussi un activateur. La découverte des propriétés activatrices de RskA nous a permis de construire des souches de M. tuberculosis qui surexpriment le regulon SigK et donc de tester l'effet de cette surexpression sur la relation hôte-pathogène lors d'une infection. Ces expériences ont démontré de façon répétée que la surexpression du regulon SigK accroit d'une part les capacités disséminatrices des bactéries et d'autre part le temps de survie de l'hôte. / En conclusion, cette étude a permis une meilleure description de l'évolution de M. tuberculosis. De plus, notre approche de l'évolution a contribué à une meilleure compréhension de deux domaines de recherche majeure à savoir la régulation de gènes par les Sigma facteurs mais aussi la pathogenèse de M. tuberculosis.

Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are live attenuated vaccines. These strains are genetically heterogeneous and the accumulation of deletions and mutations, particularly those affecting regulatory and antigenic proteins, has been proposed to have contributed to overly-attenuated vaccines. To examine this possibility, the effects of the two mutations incurred by BCG vaccines was formally studied through gene-deletion and complementation studies. First, the effect of deletion of the RD2 region was examined by over-expressing the antigenic protein MPB64 in a late strain of BCG and by creating an RD2-knockout strain in M. tuberculosis. These two approaches revealed minimal changes attributable to the RD2 region, but stimulated further investigation for other differences contemporaneous with the loss of RD2. By examining differences in gene expression between RD2+ and RD2- strains of BCG, two regions, in addition to the RD2 region itself, that were significantly down-regulated in the latter strains were identified. One region contained genes encoding the highly antigenic proteins MPB70 and MPB83, and the second region contained the gene encoding the extracellular function (ECF) sigma factor, sigK. Sequencing of sigK across all BCG strains revealed a start codon single nucleotide polymorphism (SNP) in strains with low production of MPB70 and MPB83. Complementation of BCG Pasteur with wild-type sigK resulted in up-regulation of mpb70/mpb83 and increased production of MPB70 and MPB83 proteins. Further studies revealed that the in vitro growth rate of BCG Pasteur::sigK was slower than that of BCG Pasteur::pMV306 and that cytotoxicity to macrophage-like THP-1 cells was greater following infection with BCG Pasteur::sigK than with BCG Pasteur::pMV306. Infection of C57BU6J mice demonstrated differences between BCG Pasteur::sigK and BCG Pasteur::pMV306 in initial deposition, growth rates and persistence. Vaccination of C57BU6J mice with BCG Pasteur::sigK stimulated an MPB70-specific immune response, indicating that restored protein production resulted in increased immunogenicity. Finally, in challenge experiments, mice vaccinated with BCG Pasteur::sigK demonstrated equal, or slightly greater, protection than vaccination with BCG Pasteur::pMV306 against subsequent challenge with M. tuberculosis. Together, these findings provide an explanation for the loss of MPB70 and MPB83 production by BCG vaccines during prolonged in vitro passaging, and provide the oasis for further studies aimed at understanding the role of the sigK-mpb70 regulon in tuberculosis pathogenesis.

The fate of soil organic carbon stocked in permafrost environments is a major concern in the context of global warming. In this thesis, the bacterial populations implicated in two important aerobic microbially-driven processes of the carbon cycle, aerobic methane oxidation and carbon dioxide production, were studied in different soils from the Canadian high Arctic. A protocol for the safe and sensitive detection of DNA in cesium chloride density gradients for stable isotope probing of DNA, a recent and widely used technique in microbial ecology allowing for the identification of microorganisms implicated in the degradation of a specific substrate, was developed. Using this protocol, active methanotrophic bacteria from the genera Methylobacter and Methylomonas were identified in active layer soils from Eureka, in the Canadian high Arctic. These soils had the capacity to oxidize methane at 4°C and at room temperature (RT), but the oxidation rates were greater at RT and were significantly enhanced by nutrient amendment. Bacterial populations implicated in aerobic methane oxidation and carbon dioxide production were studied in three different soils with highly distinctive physico-chemical characteristics from Axel Heiberg Island, in the Canadian high Arctic. Using microarray and clone library analyses of the particulate methane monooxygenase gene (pmoA), putative atmospheric methane oxidizers from the uncultured genotypes "upland soil cluster gamma" and "upland soil cluster alpha" were detected for the first time in Arctic soils and were associated with near neutral and acidic pH conditions, respectively. The overall methanotrophic bacterial diversity in these soils was higher than previously described for other Arctic soils and the community composition differed depending on the soil type. Potential methane oxidation rates of the soils at low and high methane concentrations were positively correlated to the relative abundance of genotype "upland soil cluster gamma". Differences in the bacterial community structure in the three soils from Axel Heiberg Island were detected at the genera/species levels using microarrays of the 16S rRNA gene and were related to soil pH and seasonal changes. Shifts in community structure were also detected at the phyla/classes levels by real-time PCR (qPCR) of the 16S rRNA gene, with the soil carbon dioxide production rate being positively correlated to the relative abundance of bacterial groups previously described as copiotrophs (Alphaproteobacteria, Bacteroidetes, and Betaproteobacteria). The results from this study indicated that bacterial communities in high Arctic soils play an important role in two aerobic processes of the carbon cycle, methane oxidation and carbon dioxide production. Methanotrophic bacteria and methane oxidation were detected in these soils and might be implicated in the reduction of methane emissions from the melting permafrost in the context of global warming. Beside, the relatively higher abundance of copiotrophic bacterial taxa in high Arctic soils with high organic matter content might lead, upon warming, to a rapid increase in soil carbon dioxide production. Further research is needed to assess the relevance of these findings under in situ conditions in a warming climate. / Le sort du carbone contenu dans le pergélisol est une source de préoccupation majeure dans le contexte des changements climatiques. Dans cette thèse, les populations bactériennes impliquées dans deux processus du cycle du carbone, l'oxydation du méthane et la production du dioxyde de carbone, ont été étudiées dans différents sols du haut Arctique canadien. Un protocole pour la détection sensible et sécuritaire de l'ADN dans les gradients de chlorure de césium, étape cruciale d'une technique appelée « stable isotope probing of DNA » qui permet d'identifier les bactéries impliquées dans la dégradation d'un substrat, a d'abord été développé. Ce protocole a par la suite permis d'identifier des bactéries méthanotrophes actives appartenant aux genres Methylobacter et Methylosarcina dans des sols provenant d'Eureka, dans le haut Arctique canadien. La capacité de ces sols à dégrader le méthane a été observée à 4°C et à température ambiante, mais les taux de dégradation étaient nettement plus élevés à température ambiante tout en étant grandement stimulés par l'ajout de nutriments. Les bactéries impliquées dans l'oxydation du méthane et la production de dioxyde de carbone ont été étudiées dans trois sols présentant des caractéristiques physico-chimiques distinctes provenant de l'île d'Axel Heiberg, dans le haut Arctique canadien. Grâce à l'utilisation de biopuces à ADN et de librairies de clones ciblant le gène codant pour l'enzyme méthane monooxygénase particulaire (pmoA), des bactéries potentiellement impliquées dans l'oxydation du méthane atmosphérique appartenant aux génotypes « upland soil cluster gamma » et « upland soil cluster alpha » ont été identifiées pour la première fois dans des sols de l'Arctique. La présence de ces deux génotypes était respectivement associée à des sols de pH neutres et acides. De manière générale, la diversité des populations méthanotrophes détectées dans les sols de l'île d'Axel Heiberg était supérieure à ce qui est généralement observé dans d'autres sols de l'Arctique et variait selon le type de sol. La capacité des sols à dégrader le méthane à des concentrations faibles et élevées était positivement corrélée à la présence dans les sols du génotype « upland soil cluster gamma ».L'utilisation d'une biopuce à ADN ciblant le gène codant pour l'ARN ribosomal 16S a permis la détection de différences dans la structure des communautés bactériennes au niveau genre/espèce dans les trois sols de l'île d'Axel Heiberg. Ces différences étaient associées au pH du sol et aux changements de saisons. Des différences dans la structure des communautés bactériennes ont également été détectées au niveau phylum/classe par PCR en temps réel ciblant le gène codant pour l'ARN ribosomal 16S de différents taxons bactériens. La capacité de production du dioxyde de carbone des sols était positivement corrélée à l'abondance relative de taxons bactériens présentant des attributs copiotrophes (Alphaproteobacteria, Bacteroidetes, and Betaproteobacteria). Les résultats de cette étude indiquent que les communautés bactériennes jouent un rôle important dans deux processus du cycle du carbone, l'oxydation du méthane et la production de dioxyde de carbone, dans les sols du haut Arctique canadien. L'oxydation du méthane, de même que les bactéries méthanotrophes associées à ce processus, ont été détectées dans ces sols, et pourraient donc jouer un rôle important dans la réduction des émissions de méthane liées au dégel du pergélisol suite au réchauffement climatique. D'autre part, l'abondance relative plus élevée de bactéries copiotrophes dans des sols du haut Arctique canadien contenant de grandes quantités de carbone pourrait conduire, suite à un réchauffement du climat, à une augmentation de la production de dioxyde de carbone. Des recherches plus approfondies seront nécessaires afin de déterminer si les résultats observés dans cette étude auront une incidence en conditions in situ dans le contexte des changements climatiques.

The general purpose of the present study was to further the understanding of the TCA cycle in the nitrogen-fixing soil bacterium Sinorhizobium meliloti, by characterizing the phenotype of three previously obtained succinate:ubiquinone oxidoreductase mutant strains. The succinate:ubiquinone oxidoreductase enzyme complex, also known as the succinate dehydrogenase (SDH) enzyme complex, lies at the intersection of the tricarboxilic acid (TCA) cycle, the electron transport chain (ETC), possibly the production and distribution of lipopolysaccharides (LPS) and exopolysaccharides (EPS), and possibly succinate-mediated catabolite repression. Previous workers had established that the SDH enzyme activity of the mutant strains was 25% of the activity measured in the wild-type (D'Aoust 2005). The pleiotrophic phenotype of the mutant strains includes a lack of nitrogen fixation and of bacteroid formation, a reduced carbohydrate metabolism, perturbations in the ETC, reduced motility, reduced growth in acidic conditions, increased requirements for calcium and/or magnesium, and increased sensitivity to hydrogen peroxide and to Polymyxin B. A basic mechanism for the differentiation of bacteria to bacteroid that occurs at IDR and shortly after, is proposed. / Le bût global de la présente étude était d'avancer notre compréhension du cycle de Krebbs chez la bactérie fixeuse d'azote Sinorhizobium meliloti, en caractérisant davantage le phénotype de trois souches mutantes isolées auparavant dans ce laboratoire. Ces souches sont affectées au niveau du complexe enzymatique nommé "succinate:ubiquinone oxidoréductase" et démontrent seulement 25% de l'activité enzymatique mesurée chez la souche-mère. Aussi connu sous le nom "succinate dehydrogenase", ce complexe enzymatique se trouve à la croisée des chemins du cycle de Krebbs; du transport d'éléctrons dans la membrane interne; possiblement de la production et la distribution des lipopolysaccharides (LPS) et des exopolysaccharides (EPS); et possiblement de la répression catabolite médiée par le succinate. Le phénotype pléiotrophique qu'expriment ces souches mutantes inclut une absence de fixation d'azote et de formation de bactéroïdes, un métabolism d'hydrocarbones diminué, des perturbations au niveau du transport d'éléctrons dans la membrane interne, une mobilité réduite, une croissance réduite en milieu acide, une exigence plus élevée en calcium et/ou magnésium, et une sensibilité accrue au péroxide d'hydrogène et à la Polymyxin B. Un méchanisme d'action permettant la transition de bactérie à bactéroïde survenant à la relâche de la gouttelette d'infection et peu après, est proposé dans cette étude.