The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.

The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

R. cellulolyticum est une bactérie mésophile, anaérobie stricte et cellulolytique, qui sécrète des macro-complexes multienzymatiques (cellulosomes) très performants dans la dégradation des polysaccharides de la paroi végétale. Les Glycoside Hydrolases de famille 9 (GH9) sont toujours surreprésentées chez les bactéries à cellulosomes. Le génome de R. cellulolyticum code 13 GH9 dont 12 participent aux cellulosomes. Mon travail de thèse a consisté à étudier l’ensemble des GH9 de R. cellulolyticum, en déterminant leurs activités à l’état libre et en complexes, afin d’élucider leurs rôles dans la dégradation de la cellulose. Les GH9 ont chacune des activités et des spécificités de substrats différentes. Deux GH9 présentent des activités atypiques, puisque l’une d’elles est inactive et l’autre est une xyloglucanase. Les caractérisations en complexes ont souligné l’importance de la diversité des GH9 et ont montré qu’elles agissent en synergie dans la dégradation de la cellulose. De plus, l’élargissement du panel des GH9 de R. cellulolyticum par l’introduction d’une cellulase exogène de Lachnoclostridium phytofermentans a permis d’améliorer les capacités cellulolytiques de la clostridie. L’activité xyloglucanase d’une des GH9 m’a poussé à étudier le catabolisme du xyloglucane chez R. cellulolyticum. Ce travail a mis en exergue la présence d’un équipement spécialisé dans l’utilisation de ce sucre. Ainsi, après une dégradation du xyloglucane par les enzymes cellulosomales en xyloglucane dextrines, ces dernières sont importées dans le cytoplasme par un transporteur ABC spécifique puis hydrolysées séquentiellement par les enzymes cytoplasmiques en mono et disaccharides assimilables. / Ruminiclostridium cellulolyticum is a mesophilic and strictly anaerobic bacterium. It produces multienzymatic complexes called cellulosomes which efficiently degrade the plant cell wall polysaccharides. Family-9 Glycoside Hydrolases (GH9) are plethoric in cellulosome-producing bacteria. The genome of R. cellulolyticum thus encodes for 13 GH9 enzymes, 12 of them participate to the cellulosomes.My Ph. D. aimed at characterizing all GH9 enzymes from R. cellulolyticum, by determining their activities in a free and complexed states, in order to elucidate their role in cellulose degradation. All GH9 enzymes exhibit various activities and substrate specificities. Two of them have atypical activities, since one is inactive and one is a xyloglucanase. Results obtained when all GH9 are in complex highlighted the importance of GH9 diversity and revealed they act synergistically in cellulose depolymerization. Moreover, expanding the panel of GH9 enzymes by introducing an exogenous cellulase from Lachnoclostridium phytofermentans improved the cellulolytic capacities of R. cellulolyticum. The xyloglucanase activity of one GH9 enzyme prompted me to investigate the xyloglucan catabolism in R. cellulolyticum. This work uncovered the presence of a specialized equipment for xyloglucan utilization. After extracellular digestion of xyloglucan by cellulosomal enzymes, xyloglucan dextrins are imported into the cytoplasm via a specific ABC-transporter and sequentially hydrolyzed by cytoplasmic enzymes into fermentable mono and disaccharides.