Clostridium cellulolyticum est une bactérie anaérobie stricte et cellulolytique qui produit des complexes multienzymatiques (cellulosomes) très performants pour la dégradation des polysaccharides de la paroi végétale. C. cellulolyticum adhère à la cellulose et ce phénomène intervient dès les premiers stades de croissance. Pour de nombreuses bactéries cellulolytiques, les cellulosomes semblent impliqués dans le processus d'adhérence et alors que les mécanismes moléculaires mis en jeu pour l'adhérence à la cellulose sont connus ou proposés, celui ou ceux de C. cellulolyticum sont inconnus.Mon projet de thèse a consisté à étudier l'adhérence et la colonisation des fibres de cellulose par C. cellulolyticum et d'identifier le ou les facteurs moléculaires impliqués dans l'adhérence. J'ai ainsi mis en œuvre deux stratégies distinctes. D'une part, une approche par mutagénèse aléatoire qui a permis d'isoler deux clones à l'adhérence diminuée et d'autre part, une approche par mutagénèse ciblée visant à inactiver des gènes candidats, susceptibles d'intervenir dans l'adhérence.J'ai aussi étudié la colonisation des fibres de cellulose par C. cellulolyticum et observé que les cellules adhèrent avec une haute spécificité et affinité à la cellulose. La colonisation des fibres se ferait en mono-couche cellulaire et par successions d'événements d'adhésion-relarguage-réadhésion. Un mutant d'inactivation de CipC, la protéine d'échafaudage des cellulosomes, a mis en évidence l'implication de cette protéine dans l'adhérence, mais aussi que l'adhérence à la cellulose pourrait être multifactorielle. Enfin, j'ai étudié le rôle de HycP, une protéine à CBM3 dont la fonction est inconnue. / Clostridium cellulolyticum is a strict anaerobe, cellulolytic bacteria. It produces multienzymatic complexes, called cellulosomes, which are able to efficiently degrade the plant cell wall polysaccharides. Cellulolytic bacteria, including C. cellulolyticum do binds to cellulose since early growth stage. For most of the studied cellulolytic bacteria, adherence to cellulose seems to be mediated by their cellulosomes. However, molecular factors involved in C. cellulolyticum adherence to cellulose remain unknown.My Ph.D. aimed to implement different but complementary strategies to study adhesion and colonization of cellulose fibers by C. cellulolyticum and to identify the molecular mechanism(s) by which the bacteria bind to cellulose. In order to identify some proteins encoding genes involved in adhesion, I firstly developed random mutagenesis and isolated two adhesion deficient mutants. I also used a targeted mutagenesis tool to inactivate some candidate genes.My studies highlight C. cellulolyticum adheres with both high specificity and affinity to cellulose. Colonization of cellulose fibers by C. cellulolyticum forms a mono-layer of segregated cells on cellulose surface and may occur through cycles of adhesion-release-re-adhesion to substrate. Inactivation of the CipC encoding gene led to a short decrease of the mutant strain's adherence level. This result suggests some other proteins may be involved in C. cellulolyticum adhesion to cellulose. Finally, I studied HycP, a produced and secreted CBM3 encoding protein of unknown function. HycP is a unique protein among databases and may have a phagic origin.

La composition des cellulosomes (complexes multi-enzymatiques impliqués dans la dégradation des polysaccharides de la paroi végétale) produits par Clostridium cellulolyticum varie en fonction du substrat de croissance. En particulier, l’expression d’un regroupement de 14 gènes prédits comme codants pour des hémicellulases (appelés xyl-doc) est induite par la présence de paille et non de cellulose. L’hypothèse a été faite que le système à deux composants putatif, codé par les deux gènes en amont des gènes xyl-doc, est impliqué dans cette régulation. Mes résultats montrent que le régulateur de réponse (appelé XydR) est impliqué dans l’activation de la transcription des gènes xyl-doc et d’un gène additionnel codant pour une protéine de fonction inconnue. Cette protéine possède cependant un module de liaison aux sucres prédit comme ciblant les hémicelluloses. Les régions promotrices, incluant les sites potentiels de liaison de XydR, ont été identifiées en amont des gènes régulés et un lien transcriptionnel entre tous les gènes xyl-doc a été mis en évidence.Un deuxième objectif de mon travail a été d’identifier le signal inducteur présent dans la paille susceptible d’être capté par le senseur apparenté à XydR. Il a été montré que la transcription des gènes cibles est spécifiquement induite par l’arabinose et le xylose qui sont les résidus glucidiques les plus abondants dans les hémicelluloses et donc relargués lors de leur dégradation.Finalement, des études biochimiques des produits de certains des gènes régulés ont montré qu’au moins trois des gènes codaient pour des produits impliqués dans la dégradation des hémicelluloses. / The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) is induced by the presence of straw and not of cellulose. The hypothesis was made that the putative two-component regulatory system, encoded by the genes localized upstream of xyl-doc, was involved in this regulation.My results provided evidence that the response regulator (called XydR) is involved in the activation of the transcription of xyl-doc genes and of an additional gene encoding a protein of unknown function harboring a carbohydrate binding module predicted to target hemicelluloses. Promoter regions, including XydR binding sites, have been identified upstream of the regulated genes and the transcriptional link between all xyl-doc genes has been demonstrated. A second aim of my work has been to identify the inducing signal present in straw that could be sensed by the cognate sensor of XydR. It was shown that the transcription of the target genes is specifically induced by arabinose and xylose which are the most abundant sugar residues present in hemicellulose and thus released by its degradation.Finally, biochemical studies of the products of some of the regulated genes demonstrated that at least three genes encoded products involved in hemicellullose degradation.

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.