Les ADN topoisomérases sont des enzymes capables de moduler la torsion de la double hélice d’ADN afin de rendre compatible sa topologie avec les différents processus cellulaires impliquant l’ADN. Les hyperthermophiles possèdent au moins une topoisomérase particulière, la reverse gyrase qui est constituée à la fois d’un domaine topoisomérase IA etd’un domaine hélicase de type SF2. Mon sujet de thèse a eu pour objectif de déterminer principalement l’implication des ADN topoisomérases IA dans les différents processus cellulaires de Sulfolobus solfataricus. Ce crenarchaeon hyperthermophile possède, en plus, d’une ADN topoisomérase de type II (Topo VI), trois ADN-topoisomérases IA dont une « classique » (TopA) et deux reverse gyrases (TopR1 et TopR2). Notre approche a permis d’estimer, pour la première fois, le nombre de TopR1 et de TopR2 par cellule en fonction des différentes conditions testées. L’étude des variations quantitatives des ADN topoisomérases a clairement mis en évidence que TopR1 et TopR2 sont régulées différemment ce qui renforce l’hypothèse d’une spécialisation de leurs fonctions. Nous avons ainsi montré que TopR1 est responsable du maintien de l’homéostasie du surenroulement de l’ADN. Si la Topo VI de par son activité antagoniste est impliquée dansce même contrôle homéostatique, elle ne fait pas l’objet d’une régulation quantitative. De plus, nous avons mis en évidence que TopR1 était liée à la vie à haute température. Enfin, nos résultats suggèrent que TopR2 serait pour sa part impliquée dans la stabilité des génomes. L’identification des partenaires protéiques respectifs des quatre ADN topoisomérases de S. solfataricus permettra d’avoir une vision globale des réseaux de régulation permettant derésoudre les différentes des contraintes topologiques générées au cours de la vie de cet hyperthermophile. / DNA topoisomerases act in all DNA metabolism processes to control the DNA topology. Hyperthermophiles possess at least a particular topoisomerase, the reverse gyrase composed of a DNA topoisomerase IA domain and a helicase SF2 domain within the same polypeptide. The general objective of my thesis was to determine the involvement of each DNA topoisomerase in different cellular processes of S. solfataricus. This hyperthermophilic crenarchaeon possesses in addition to a type II DNA topoisomerase (Topo VI), three DNA topoisomerases IA : a classical one (TopA) and two reverse gyrases (TopR1 and TopR2). Our experimental approach allowed to estimate for the first time the number of TopR1 and TopR2 per cell in relation to different conditions. The study of quantitative variations of each DNA topoisomerase clearly showed that TopR1 and TopR2 are differently regulated suggesting that they are involved in distinct cellular processes. Indeed, we showed that TopR1 is the main actor of the homeostatic control of the DNA supercoiling. If the Topo VI with its antogonistic activity is involved in this homeostatic control, there is no regulation at the level of protein quantity. In addition we evidenced that TopR1 is somehow linked to the life at high temperature. Our results suggest that TopR2 is involved in genome stability. The identification of the respective potential partners of the four DNA topoisomerases of S. solfataricus will allow to get a more detailed understanding of the DNA topology regulation during the hyperthermophilic life style.

Hyperthermophiles are microorganisms that can grow at temperatures close to the boiling point of water or above. They are potential resources of thermostable enzymes including alcohol dehydrogenases (ADHs). Both Thermococcus guaymasensis and Thermotoga hypogea produce ethanol as an end product using glucose as substrate. However, the metabolic pathway and enzymes involved in alcohol production by these hyperthermophiles were not clear. ADH is a key enzyme responsible for alcohol metabolism, and the enzyme has been purified and characterized. T. hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90??C. The NADP+-dependent ADH from T. hypogea was purified to homogeneity and a homodimeric protein with a subunit size of 40 ?? 1 kDa. A part of its encoding gene was cloned and sequenced, from which a major part of the amino acid sequence of the enzyme was deduced and found to have high similarities to iron-containing ADHs from other Thermotoga species and harbored typical iron and NADP+-binding motifs. The conserved domain search showed that T. hypogea ADH was a member of the family of uncharacterized iron-containing ADHs. The iron content of the enzyme was determined to be 1.02 ?? 0.06 g-atoms per subunit. It is the first characterized iron-containing ADH from hyperthermophilic bacteria. Similar to known iron-containing ADHs, T. hypogea ADH was oxygen sensitive; however, the loss of enzyme activity upon exposure to oxygen could be recovered by incubation with dithiothreitol and Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70??C, and its catalytic activity increased along with the rise of temperatures up to 95??C. Optimal pH values for the production and oxidation of alcohol were determined to be 8.0 and 11.0, respectively. The enzyme had a broad specificity in utilizing primary alcohols and aldehydes as substrates. Apparent Km values for ethanol and 1-butanol were much higher than that for acetaldehyde and butyraldehyde and thus the enzyme was likely to catalyze the reduction of aldehydes to alcohols in vivo. T. guaymasensis is a hyperthermophilic anaerobic archaeon capable of catalyzing the starch degradation and producing ethanol and acetoin as end-products. The purified T. guaymasensis ADH was an NADP+-dependent homotetramer with a subunit of 40 ?? 1 kDa. The enzyme was a primary-secondary ADH, but it exhibited substrate preference on secondary alcohols and corresponding ketones. In particular, it catalyzed the reduction of diacetyl to 2, 3-butanediol via acetoin in which the reduction from diacetyl to acetoin was irreversible. For the oxidation of 2, 3-butanediol, the enzyme exhibited higher activities on (2R, 3R)-(-)-2, 3-butanediol and meso-2, 3-butanediol than (2S, 3S)-(+)-2, 3-butanediol while the stereoselective reduction of racemic (R/S)-acetoin produced (2R, 3R)-(-)-2, 3-butanediol and meso-butanediol but not (2S, 3S)-(+)-2, 3-butanediol. The optimal pHs for the oxidation and formation of alcohols were determined to be 10.5 and 7.5, respectively. The enzyme activity increased along with the rise of temperatures up to 95??C, and it was highly stable with a half-life of 24 hours at 95??C. The enzyme was resistant to 30% (v/v) methanol (retaining 40% of its full activity). NADPH for the ketone reduction was efficiently regenerated using isopropanol as a substrate. The apparent Km value for NADPH was 40 times lower than that of NADP+, and the specificity constant with NADPH were 5 times higher than that of NADP+. Therefore, the physiological role of the enzyme was likely to be responsible for the reactions involving the NADPH oxidation???coupled formation of ethanol and/or acetoin. The fully active T. guaymasensis ADH contained 0.9 ?? 0.03 g atom zinc per subunit determined by inductively coupled plasma mass spectrometry (ICP-MS) and was the first characterized zinc-containing ADH from Thermococcus species. The gene encoding this enzyme was cloned and sequenced, and the deduced amino acid sequence contained 364 amino acids showing high similarities (85%) to those ADHs from Thermoanaerobacter species which have only the catalytic zinc atom. The motif analyses also indicated the enzyme lacked of the structural zinc-binding motif; thus, zinc might play a catalytic role in the enzyme. Further analyses showed the presence of the conserved domains of L-threonine dehydrogenases; however, the enzyme could not oxidize L-threonine or L-serine. Distinct from most of zinc-containing ADHs, the enzyme activity was almost fully inhibited by 100 ??M Zn2+ in the assay mixture. Moreover, it was sensitive to oxygen. An NADP+-dependent ADH was purified from the hyperthermophilic anaerobic archaeon Thermococcus strain ES1, an ethanol producer. The recombinant enzyme over-expressed in Escherichia coli was purified using a two-step procedure including heat treatment, and characterized in comparison with the native enzyme. The purified recombinant enzyme exhibited a specific activity of 52.8 U mg-1, close to that of the native enzyme (57 U mg-1). Both native and recombinant enzymes were homotetramers with a subunit size of 45 ?? 1 kDa. Their optimal pHs for the ethanol oxidation and acetaldehyde reduction were determined to be 10.5 and 7.0, respectively. Both enzymes were able to oxidize a series of primary alcohols and diols. Metal contents of the fully active recombinant enzyme were determined by ICP-MS to be 1.0 ?? 0.04 g atom iron per subunit, and both iron-containing enzymes were oxygen sensitive. Their kinetic parameters showed lower Km-values of acetaldehyde and NADPH than those of ethanol and NADP+, suggesting the native enzyme could be involved in ethanol formation in vivo. The recombinant and native enzymes had almost identical characteristics and thus its encoding gene was successfully over-expressed in E. coli. The deduced amino acid sequence of the ADH from Thermococcus strain ES1 was a 406 amino acid polypeptide. Its amino acid sequence showed high identities (80%) to iron-containing ADHs from the related archaea Thermococcus zilligii and Thermococcus hydrothermalis. The conserved domain search revealed it belonged to the family of iron-containing ADHs. Moreover, the sequence of the enzyme had catalytic metal and dinucleotide-binding motifs typical for iron-containing ADHs. In conclusion, the results indicate that iron- and zinc-containing ADHs from hyperthermophiles have significant differences in terms of biophysical, biochemical and molecular properties. The hyperthermophilic bacterial and archaeal iron-containing ADHs are divergent while the zinc-containing ADH from T. guaymasensis has significant similarity to thermophilic bacterial ones.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23230号 / 工博第4874号 / 新制||工||1761(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20712号 / 工博第4409号 / 新制||工||1685(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21276号 / 工博第4504号 / 新制||工||1700(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

L'immense diversité génomique des microorganismes leur permet de vivre partout, même dans les environnements extrêmes tels que Ies sources hydrothermales profondes. Ces dernières, disséminées sur l’ensemble des fonds océaniques, sont un bon modèle pour étudier la biogéographie et la diversification des génomes. Une approche de génomique comparative a été employée sur des isolats du genre Thermococcus proches d'un point de vue évolutif. Ce travail visait à identifier des mécanismes ayant un rôle dans Ia diversification de ces génomes, et également d'identifier des gènes impliqués dans cette différenciation. A cette fin, deux groupes d'une vingtaine d'isolats ayant des origines géographiques diverses ont été sélectionnés et séquencés. L'éloignement géographique résultant de la colonisation de nouveaux systèmes hydrothermaux semble être un facteur de diversification et de spéciation pour certains isolats. Cependant, lorsque les sites hydrothermaux sont relativement proches, il semblerait qu'un transfert de gènes entre les isolats soit toujours possible. Dans ce cas, l’adaptation à de nouvelles niches écologiques serait un facteur de la diversification des génomes. L'approche de génomique comparative a permis d'identifier des gènes spécifiques à certains sous-groupes, apparentés à des espèces. Ces gènes sont notamment impliqués dans les métabolismes des acides aminés, de production d'énergie et de transport d'ions inorganiques. Ceci reflète les pressions de sélections que peuvent subir ces organismes dans ces environnements hostiles à de nombreuses formes de vie. / The immense genomic diversity of microorganisms allows them to live everywhere, even in extreme environments such as deep hydrothermal vents. Scattered over the seabed, these are a good model for studying the biogeography and genomes diversification. A comparative genomics approach has been used on closely related isolates, of the genus Thermococcus. This work aimed at identifying mechanisms that have a role in the diversification of these genomes, and also to identify genes involved in this differentiation. For this purpose, two groups of about 20 isolates with different geographical origins were selected and sequenced.The geographical isolation resulting from colonization of new hydrothermal systems is likely to be a diversification and speciation factor for some isolates. But when hydrothermal sites are relatively close, it would seem that gene transfer between isolates is still possible. In this case, adaptation to new ecological niches would be a factor contributing to the genomes diversification. The comparative genomics approach allowed highlighting genes specific to certain subgroups, related to species. These genes are involved in amino acid metabolism, energy production and the transport of inorganic ions. This reflects selection pressures that these organisms may experience in these environments, otherwise hostile to many forms of life.

Suite à la découverte récente des sources hydrothermales les plus profondes de la planète au niveau de laFosse des Caïmans, des échantillons hydrothermaux y ont été prélevés en vue de cultiver des microorganismes de ce site encore peu documenté. Des cultures d’enrichissement ont été réalisées à partir de ces échantillons en vue d’isoler de nouveaux taxons microbiens ayant des métabolismes clés des cycles biogéochimiques du soufre et du carbone ou une physiologie particulière (piézophilie). Parmi les isolats obtenus, il y avait notamment une nouvelle archée hyperthermophile anaérobie sulfo-réductrice, désignée comme CDGST, qui provenait du champ hydrothermal Beebe, à 4964 m de profondeur. Cette souche, affiliée au genre Thermococcus, présentait une certaine plasticité physiologique et se démarquait de ses plus proches parents du point de vue de sa physiologie.Elle a été caractérisée en détails aux niveaux métabolique, physiologique et génomique. Cette souche estpiézophile et possède la plus large gamme de pression de croissance jamais décrite pour un organisme. Elle se développe de manière optimale à 75°C, pH 6,0 et sous une pression hydrostatique de 50 MPa, la pression in situ de son habitat naturel. Elle appartient à une nouvelle espèce qui a été appelée Thermococcus piezophilus sp.nov. Son génome a été séquencé et annoté.La croissance de ce nouvel isolat est efficace de pression atmosphérique jusqu’à au moins 120 MPa, et la souche croît avec plus de difficultés jusqu'à 130 MPa. Aucun autre microorganisme, qu’il soit psychrophile, mésophile ou hyperthermophile ne possède une telle gamme de pression de croissance. Pour cette raison, les mécanismes d’adaptation de cette souche à la pression ont été étudiés par une approche de transcriptomique.Cette souche s’adapte aux variations de pression notamment en modulant sa production et sa conversion d’énergie (transporteurs, hydrogénases, etc.) en fonction de la pression. / Following the recent discovery of the world’s deepest hydrothermal vents at the Cayman Trough, hydrothermal samples were taken for culturing microorganisms of this site still poorly documented. Enrichment cultures were performed using these samples to isolate new microbial taxa having key metabolisms of biogeochemical cycles of carbon and sulfur or a particular physiology (piezophily). Among the isolates, there was a new hyperthermophilic and anaerobic sulfur-reducing archaea, designated as CDGST, originating from the hydrothermal field Beebe, at 4964 m depth. This strain belonged to the Thermococcus genus. It exhibited some physiological plasticity and was distinguishable from its closest relatives from the point of view of its physiology. It has been characterized in great details at metabolic, physiological and genomics levels. This strain is piezophilic and has the broadest range pressure for growth ever described for an organism. It grows optimally at 75°C, pH 6.0 and under a hydrostatic pressure of 50 MPa, the in situ pressure of its natural habitat. It belongs to a new species that was called Thermococcus piezophilus sp. nov. Its genome has been sequenced and annotated.The growth of this new isolate is effective from atmospheric pressure to at least 120 MPa, and the strain grows with more difficulties up to 130 MPa. No other organism, whether psychrophilic, mesophilic or hyperthermophilic has such a range of growth pressure. For this reason, the adaptation mechanisms to pressure of the strain were studied by a transcriptomic approach. This strain adapts to pressure variations, by modulating notably its energy production and energy conversion (carriers, hydrogenases, etc.) depending on the pressure.

Thesis advisor: Mary F. Roberts / Crystallization studies in presence of organic osmolytes were conducted to better understand the specific mechanism of compatible solute binding to the inositol monophosphatase of Archaeoglobus fulgidus. The synthesis of a-diglycerol phosphate, one of the natural osmolytes of A. fulgidus, was also completed for kinetic testing of its I-1-Pase thermoprotective properties and for crystallization trials. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Depuis la découverte des sources hydrothermales en 1977, un petit nombre d'études ont permis l'isolement et la caractérisation de micro-organismes pouvant résister à de hautes pressions et températures. Parmi ceux-ci, Pyrococcus yayanosii, une archée hyperthermophile de l'ordre des Thermococcales est issue du site Ashadze (dorsale medio-atlantique) à 4100 m de profondeur. Cette espèce représente le premier organisme à la fois hyperthermophile et piézophile strict décrit à ce jour. Elle ne peut en effet se diviser à des pressions inférieures à 20 MPa et sa pression optimale de croissance est de 52 MPa. Afin d'étudier les mécanismes que met en oeuvre cette espèce pour se développer sous hautes pressions, des expériences de transcriptomique (puces à ADN) et protéomique (LC-MS/MS) ont été entreprises à différentes pressions notamment sub- et supraoptimales. La distinction entre les effets « stress » et « adaptations » à la pression a été effectué en comparant les résultats obtenus chez une autre Thermoccocale proche, Pyrococcus furiosus, qui est piézosensible. La détermination des pressions sub et supra optimales a été préalablement effectuée sur une large gamme de pression hydrostatique. Des analyses génomiques ont aussi été effectuées sur les Thermococcales en général et ses deux espèces en particulier et montrent des différences importantes au niveau des voies de biosynthèse des acides aminés ainsi que des transporteurs membranaires. Les analyses transcriptomiques et protéomiquesmontrent que P. yayanosii joue essentiellement sur ses mécanismes de production d'énergie (métabolisme del'hydrogène), de mobilité (chimiotactisme), de traduction (protéines ribosomales) ainsi que sur ses mécanismes de défense (CRISPR/cas). P. furiosus met en place des mécanismes se basant aussi sur la traduction et la mobilité (archaellum). Il semble que ces derniers puissent ainsi être considérés comme des réponses aux stress, alors que la modulation énergétique uniquement présente chez P. yayanosii soit plus un « shift » métabolique permettant à la cellule de s'adapter aux différentes conditions de pression de son environnement. / No

Hyperthermophilic proteins are of great interest in both the academic and industrial world in understanding how these proteins are capable of retaining their biological activity under such harsh environmental conditions. This thesis studies a tetrabrachion stalk domain from Staphylothermus marinus, know as Right Handed Coiled Coil (RHCC). This protein is of interest due to its extreme thermostability and its affinity for heavy metals. We aim to better understand the reason for the extreme thermal stability of the protein and to take advantage of the proteins affinity for heavy metals with a view to developing a novel approach to bioremediate Hg2+, a major environmental pollutant. Our results clearly indicated that the protein is more thermostable in alkaline conditions in comparison to acidic conditions. This observation can be explained by careful inspection of the high resolution structure. Our data also clearly show that RHCC is able to bind ionic mercury compounds such as mercury nitrate and dipotassium mercury iodide.