Moorella thermoacetica est une bactérie d’altération anaérobie thermophile sporulée produisant les spores les plus thermorésistantes isolées à ce jour en industrie agroalimentaire. Les spores de M. thermoacetica peuvent survivre aux traitements d’appertisation appliqués dans l’industrie de la conserve et représente la principale cause d’altération de conserves peu acides après incubation à 55 °C. Les mécanismes mis en place pendant la sporulation, ainsi que l’impact des facteurs environnementaux sur la sporulation et les propriétés des spores de Moorella sont largement méconnus.L’objectif de ces travaux était donc de caractériser l’impact de la température sur les caractéristiques de sporulation et de résistance des spores ainsi que de déterminer les mécanismes moléculaires impliqués dans la sporulation. Nous avons montré que la capacité de sporulation est meilleure lorsque la sporulation est effectuée à la température optimale (55 °C) en comparaison avec les températures limites basse et haute (45 °C et 65 °C, respectivement). De plus, les spores de M. thermoacetica produites à 45 °C sont plus sensibles à la chaleur humide et aux biocides que les spores produites à 55 °C. La structure des spores ainsi que leur composition protéique varient en fonction de la température de sporulation. Une étude RNAseq menée au cours de la sporulation de M. thermoacetica en conditions optimales régulées a montré que la plupart des 167 gènes de sporulation, identifiés in silico dans le génome de M. thermoacetica ATCC39073, est sur-exprimée pendant la sporulation. Ces résultats suggèrent que les mécanismes décrits chez d’autres espèces endosporulées sont conservés chez Moorella.L’ensemble des données acquises montrent que la température joue un rôle essentiel dans les caractéristiques de sporulation et de résistance des spores de M. thermoacetica mais également que les mécanismes moléculaires impliqués dans la sporulation semblent conservés au regard des données disponibles pour d’autres espèces. / Moorella thermoactica is a spoilage anaerobic and thermophilic spore-former producing the most highly heat-resistant spores isolated so far in food industry, which enables the bacteria to survive the sterilization process applied in cannery. M. thermoacetica is the main cause of low acid canned food spoilage after incubation at 55 °C. Little is known about sporulation mechanisms and spore properties according to environmental conditions.In this work, we aimed at characterizing the impact of environmental conditions on sporulation and spore resistance properties, as well as describing the molecular mechanisms underlying sporulation. We showed that sporulation capacities are higher when sporulation is performed at the optimal temperature (55 °C) that at limit temperatures (45 °C and 65 °C). Besides, spores are less resistant to wet heat and biocides when formed at 45 °C than at 55 °C. We showed that the ultrastructure and spore protein composition varied according to sporulation temperature. Moreover, the study of gene expression by RNAseq during sporulation in optimal regulated conditions showed that most of the 167 genes involved in the sporulation process and identified in silico in M. thermoacetica ATCC 39073 genome, were up-regulated during sporulation, suggeting that the mechanisms described in other endospore-formers are conserved in Moorella.Altogether, our results showed that sporulation temperature strongly impacts sporulation and spore properties of M. thermoacetica and that sporulation mechanisms tend to be conserved in Moorella considering data available on other endospore-formers.

The A-cluster of acetyl-CoA synthase (ACS) catalyzes the formation of acetyl- CoA from CO, coenzyme-A, and a methyl group donated by a corrinoid iron-sulfur protein. Recent crystal structures have exhibited three different metals, Zn, Cu, and Ni, in the proximal site, which bridges a square-planar nickel site and a [Fe4S4] cubane. Contradicting reports supported both the nickel and copper containing forms as representing active enzyme. The results presented here indicate that copper is not necessary or sufficient for catalysis and that copper addition to ACS is deleterious. Several proposed mechanisms exist for the synthesis of acetyl-CoA, the two most prominent are the ‘paramagnetic’ and ‘diamagnetic’ mechanisms. The ‘diamagnetic’ mechanism proposes a two electron activation that precedes methylation to produce an EPR silent Ni2+-CH3 species. This then reacts with CO and coenzyme-A to form acetyl- CoA and regenerate the starting species. The ‘paramagnetic’ mechanism assumes a one electron activation prior to the methylation of the paramagnetic Ni1+-CO state to form an unstable Ni3+-acetyl species. This is immediately reduced by an electron shuttle. Results are presented here that no shuttle or external redox mediator is necessary for catalysis. This supports the ‘diamagnetic’ mechanism, specifically that a two-electron reductive activation is necessary and that the Ni1+-CO species is not an intermediate. The two-electron reductive activation required by the ‘diamagnetic’ mechanism results in an unknown electronic state. Two proposals have been made to describe this form of the A-cluster. The first hypothesis from Brunold et al involves a one-electron reduction of the [Fe4S4]2+ cube and a one-electron reduction of the Nip 2+. This should result in a spin-coupled state that is S = integer. The Ni0 hypothesis requires both electrons to localize on the Nip 2+ forming a zero-valent proximal nickel. Mössbauer spectroscopy has been used to probe the oxidation state and spin state of the [Fe4S4] cube in the reduced active form. No integer spin system is found and this is interpreted as supporting the Ni0 hypothesis. Additionally, spectra are presented that indicate the heterogeneous nature of the A-cluster is not caused by the occupancy of the proximal site.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20479号 / エネ博第348号 / 新制||エネ||69(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻 / (主査)教授 坂 志朗, 教授 梅澤 俊明, 准教授 河本 晴雄 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM