Les systèmes moléculaires décrits chez les Archaea mettent en évidence un caractère primitif et une simplicité, comparativement à leurs homologues eucaryotes. Par ailleurs, leur caractère extrêmophile a pour conséquence une hyper-robustesse qui rend leur manipulation in vitro et les études structurales beaucoup plus aisées. Ainsi les Archaea représentent de bons modèles pour comprendre les fonctions cellulaires complexes, particulièrement celles qui mettent en jeu de grandes machineries moléculaires, comme celles impliquées dans la protéolyse. Mon travail de thèse a consisté à comprendre les mécanismes de résistance et l'importance des systèmes de protéolyse dans l'adaptation des Archaea halophiles aux stress environnementaux. Les Archaea halophiles accumulent des concentrations multi-molaires de KCl/NaCl dans leur cytosol (3.4M KCl / 1.1M NaCl chez Halobacterium salinarum). Ainsi, les protéines de ces organismes sont elles-mêmes halophiles et ne sont solubles et repliées que dans des conditions de salinité extrêmes (de 2 à 5M).Nous avons étudié la réponse de l'Archaea halophile stricte H. salinarum à des stress, dont les stress à basse salinité, en se focalisant en particulier sur les modifications de la dynamique moléculaire du protéome in vivo (diffusion neutronique). Au cours de ce travail, il a été mis en évidence un phénomène de survie à la basse salinité associé à des modifications morphologiques.Un autre objectif de ma thèse a été de contribuer à la compréhension du rôle dans la protéolyse intracellulaire du complexe aminopeptidasique TET, dans les conditions de stress mises en place dans les études précédentes. / Molecular systems described for Archaea show primitive and simple characteristics, compared to their homologous eukaryotes. In addition, extremophilic characteristic results in an hyper-robust which makes in vitro manipulation and structural studies much easier. Thus, Archaea represent good models for understanding complex cellular functions, particularly those that involve large molecular machines, such as those involved in proteolysis. My thesis consisted in understanding the resistance mechanisms and the importance of proteolytic systems in the adaptation of halophilic Archaea to environmental stresses. Halophilic Archaea accumulate multi-molar concentrations of KCl / NaCl in their cytosol (3.4M KCl / NaCl 1.1M for Halobacterium Salinarum). This requires a very special biochemistry that allows operation in solvents where free water is scarce. Thus, the proteins of these organisms are themselves halophilic and are soluble and folded only in extreme salinity conditions (2 to 5 M). This particular biochemistry partly explain the extraordinary ability of halophilic Archaea to resist physical and chemical stress (temperature, radiation, dehydration). We study the response of the halophilic Archaea strict H. salinarum at low-salinity stress. Indeed, beyond the osmotic shock, the fall of the environment salt concentration causes a decrease in the intracellular KCl concentration, which should have a direct effect on the folding state of intracellular protein, as in case of heat stress. In the first part of this thesis, a study was conduct to determine viability limits and cytosolic modifications, associated with a salinity decrease. These studies involve intracellular salt dosages, viability studies (microscopic counts, color live / dead), induction of chaperone proteins linked to stress response and biophysical neutron experiments, to evaluate the effect of stress on proteins folding. In this work, a phenomenon of survival at low salinity linked to morphological changes was revealed. To describe this phenomenon, this second study involves confocal microscopy experiences, fluorescence microscopy, viability tests, counting on box, scanning electron microscopy, electron microscopy by negative staining, salt intracellular dosages and proteins separation experiments, to study the overall proteome composition during low salinity stress. In this study, a fall of the intracellular K $^+$ concentration and the proteome clarification during stress was revealed. Low salt concentrations causes halophiles proteins denaturation, the accumulation of misfolded proteins in the cytoplasm involves chaperones systems and intracellular proteolysis machinery. In this context, another objective of my thesis was to contribute to the understanding of the intracellular proteolysis role in the PAN-proteasome system and in the aminopeptidase TET complex, in stress conditions established in previous studies. This part of the thesis involves experiments of endopeptidase activity assay, aminopeptidase activity assay, quantification of mRNA genic expression by Northern blot, immunoprecipitation, proteins separation by sucrose gradient and proteasome chemical inhibition (drug). We show the role of the PAN-proteasome system in stress response and we deepen our understanding of the aminopeptidase TET role in vivo. This protease appears to have an independent role of the proteasome complex. The protease TET seems to participate at the amino acids treatment in cells to maintain the metabolic activities in nutritional deficiencies.

Made available in DSpace on 2015-03-26T13:51:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1109551 bytes, checksum: 5f0d13927b00626f1a0d15b78536b7bc (MD5) Previous issue date: 2009-07-13 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This is the first study of genetic diversity of the Archaea community associated to coffee fruits (Coffea arabica L.). It was performed in cherry coffee fruits of cultivars Bourbon Amarelo, Bourbon Vermelho, Catuaí Amarelo, Catuaí Vermelho and Catucaí Vermelho in different altitudes. The archaeal diversity during natural drying of depulped grains in cement covered yard was also studied. The addition of proteases during the coffee fruits metagenomic DNA extraction cell lysis step provided better recovery of DNA from Archaea. Comparing different hypervariable regions of 16S rDNA by DGGE (Denaturing Gradiente Gel Electrophoresis), the highest diversity resolution was obtained with the V3 region, for the cultivar Catucaí Vermelho at an altitude of 936m. The Archaea endophytic community analysis in four C. arabica cultivars revealed varied genotypic profiles among the samples. Three samples that showed distinct DGGE profiles were chosen for 16S rDNAs libraries construction. Sequencing of 63 clones revealed 12 OTUs and the prevalence of sequences related to Euryarchaeota phylum, mainly the halophylic genera Halobacterium, Halococcus, and Haloferax. Sequences with high identity with Methanobrevibacter, with the thermophilic Thermoplasma, and sequences related to uncultivated Archaea from marine environment were also found in the Euryarchaeota phylum. Of the four sequences belonging to the Crenarchaeota phylum, three phylogenetically clustered with uncultivated archaea from soil, and one with sequences from a marine environment. Rarefaction curve analysis and the estimated Coberture pointed out that the libraries constructed were large enough to cover the most of Archaea diversity in cherry coffee. The archaeal diversity study during natural drying revealed higher increase in OTUs number beginning at the seventh day up to the last day. Cluster analysis of DGGE fingerprints distinguished the population of the first days of drying from those in the last ones. The absence of physiological studies of uncultivable Archaea, especially in mesophilic environments, limits the knowledge of metabolism of these microorganisms and the determination of endophytes role in coffee fruits. Although, metagenomic studies of microbial community associated to coffee fruits will help to identify archaeal genes and establish relations among the presence of certain microorganisms and precursors of those compounds that make up the aroma and the flavor in the final quality of the beverage. / Este é o primeiro estudo de diversidade genética da comunidade de Archaea associada a frutos de café (Coffea arabica L.). Ele foi realizado em amostras de frutos no estádio cereja das cultivares Bourbon Amarelo, Bourbon Vermelho, Catuaí Amarelo, Catuaí Vermelho e Catucaí Vermelho, em diferentes altitudes. A diversidade de arqueas presentes durante a secagem natural de grãos despolpados em terreiro revestido com cimento também foi estudada. A adição de proteases durante a etapa de lise celular para extração de DNA metagenômico de frutos de café propiciou melhor recuperação de DNA de Archaea. A maior resolução da diversidade na comparação de diferentes regiões hipervariáveis do rDNA 16S por DGGE foi obtida quando se utilizou a região V3 para a amostra da cultivar Catucaí Vermelho em altitude de 936m. A análise da comunidade endofítica de Archaea em quatro cultivares de C. arabica revelou um variado perfil genotípico entre as amostras. Três amostras que apresentaram perfis de DGGE distintos entre si foram escolhidas para construção de bibliotecas de rDNAs 16S. O sequenciamento de 63 clones revelou a existência de 12 UTOs e a prevalência de sequências relacionadas ao filo Euryarchaeota, principalmente de arqueas halofílicas dos gêneros Halobacterium, Halococcus e Haloferax. Ainda no filo Euryachaeota, foram identificadas sequências com alta identidade com Methanobrevibacter, com a hipertermófila Thermoplasma e com sequências relacionadas à arqueas não cultiváveis de ambiente marinho. Das quatro sequências pertencentes ao filo Crenarchaeota, três agruparam filogeneticamente com sequências de arqueas não cultiváveis do solo, e uma com sequências de ambiente marinho. A análise das curvas de rarefação e o cálculo das coberturasmostraram que as bibliotecas construídas foram grandes o suficiente para cobrir a maior parte da diversidade de Archaea presentes em frutos de café cereja. No estudo da diversidade de Archaea durante a seca natural observou-se um aumento do número de UTOs a partir do sétimo dia,permanecendo até o último dia do processo. A análise de agrupamento dos perfis distinguiu as populações presentes nos dias finais de secagem. A ausência de estudos fisiológicos de arqueas não cultiváveis, especialmente de ambientes mesófilos, limita o conhecimento do metabolismo destes micro-organismos e a determinação do papel das endofíticas dos frutos de café. Contudo, estudos metagenômicos da comunidade microbiana associada a frutos de café ajudarão a identificar genes de Archaea e a estabelecer relações entre a presença de determinados micro- organismos e de compostos precursores daqueles que compõe o aroma e sabor na qualidade final da bebida.

Archaea ainda não foi reconhecido como patógeno de doença humana. O objetivo deste estudo foi determinar a prevalência, diversidade, níveis e proporções de Archaea no biofilme subgengival de indivíduos com periodontite agressiva (PA) e saúde periodontal (SP). Sessenta indivíduos foram selecionados para este estudo (n=30/grupo). A análise de diversidade foi realizada em 10 indivíduos/grupo. Quatro sítios/indivíduo do grupo PA e 2 sítios/indivíduo do grupo SP foram analisados por qPCR. A freqüência de Archaea foi de 60% dos indivíduos/ 15,2% dos sítios em PA e de 63,3% dos indivíduos/ 15,6% dos sítios em SP (p>0,05). Um a três filotipos foi identificado por amostra. O número de cópias e a proporção de Archaea e Bacteria foram menores no grupo SP do que no grupo PA (p<0,05). Archaea são encontrados no biofilme subgengival de indivíduos com PA e SP. Methanobrevibacter oralis é o filotipo mais prevalente, podendo ser considerado residente da cavidade bucal. A alteração ecológica na microbiota de indivíduos com PA inclui o aumento dos níveis e proporções de Archaea. / Membrers of Archaea domain may be detected in the microbiota of mucous surfaces of human and animals, but their association with diesase have not been yet stablished Some studies have suggested that Archaea domain may be indirectly associated with pathogenesis of periodontitis, since they are found restrict to subgingival sites with severe periodontal destruction. The aim of this study was to determine the prevalence, diversity, levels and proportions of microorganisms of Archaea domain in subgingival biofilm of aggressive periodontitis and periodontally healthy subjects. Thirty generalized aggressive periodontitis (GAgP) and 30 periodontally healthy (PH) subjects were selected. Archaea detection was performed by PCR using domain-specific primers in 9 subgingival samples taken from each subject. Archaea diversity was determined by evaluating a single positive sample per subject, randomly selected from 10 GAgP and 10 PH subjects. Archaeal 16S rRNA gene library were constructed to each sample and the identity of phylotypes were determined for the comparison of unrecognized sequences with gene database. The levels and proportions of Archaea in relation to total microbial load were analysed by quantitative PCR (qPCR) in 4 sites per GAgP subject and 2 sites per PH subject. A total of 540 subgingival samples were analysed to Archaea presence. This domain were detected in 18 GAgP (60%) and in 19 PH (63.3%) subjects. Forty-one (15.2%) and 42 (15.6%) samples were positive for this domain in GAgP and PH subjects, respectively. There was not difference in prevalence of this domain between subjects from GAgP and PH groups, as well as there was not difference in prevalence between sites with different probing depthsin GAgP group. Thenumber of 16S rRNA clones available to identification per sample varies from 33 to 47 in GAgP group, and from 15 to 23 in PH group, with a mean of 42.8+3.9 e 20.2 +2.2, respectively. The analysis of 629 sequencies permits an identification of 1 to 3 phylotypes of Archaea domain per sample. Methanobrevibacter oralis was detected in all archaeal positive samples, being the single detected specie of this domain in 5 subjects from GAgP group, and in 3 subjects from PH group. Methanobacterium curvum/congolense was detected in 3/10 GAgP and 6/10 PH samples, whereas Methanosarcina mazeii was detected in 4/10 samples from both groups. Archaea analysis by qPCR was carried out in 103 sites and 28 GAgP subjects and in 60 sites and 30 PH individuals. Archaea has been detected in 27/28 subjects and in 68% of studied sites in GAgP group and in 26/30 subjects and 58,3% of total analysed sites in PH group. Archaeal and bacterial 16S rRNA mean levels were smaller in PH group than in GAgP group (Mann Whitney, p<0.05). There was no statistic significance of difference in the levels of Archaea in regard to probing depth categories in GAgP group (p>0.05). Moreover, the proportion of Archaea in relation to total microbial load (Archaea + Bacteria) was 0.02% and 0.08% in PH and GAgP group (p<0.05), respectively. These data suggest that Archaea is commonly found in the subgingival biofilm of humans, and that M. oralis may be considered a member of the resident microbiota of subgingival sites. The ecological shift in the microbiota of aggressive periodontitis subjects includes the increase of levels and proportions of Archaea domain.

Les Archaea methanogènes sont des organismes environnementaux ayant été également détectés dans certaines flores associées aux muqueuses des mammifères. Chez l’homme ces microorganismes ont été associés avec les muqueuses intestinale, vaginale et orale. Ces organismes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. En effet, uniquement trois Archaea methanogènes ont été cultivées à partir de prélèvements humains, Methanobrevibater smithii et Methanosphaera stadtmanae à partir des selles puis Methanobrevibater oralis à partir de la plaque dentaire. Récemment l’ADN d’autres Archaea methanogènes et d’Archaea non-methanogènes a été détecté dans des selles humaines, y compris des séquences indiquant la présence d’espèces appartenant à un nouvel ordre de méthanogènes n’ayant aucun représentant cultivé. La connaissance actuelle sur la diversité de ces methanogènes chez l’homme et sur leurs effets potentiels sur la santé humaine est en grande partie basée sur les techniques de détection de l’ADN par PCR et métagénomique. Ces techniques fondées sur la détection de l’ADN ribosomal 16S et du gène mcrA codant la sous-unité alpha du methyl-coenzyme M reductase, une enzyme clé dans le processus de méthanogenèse, ont montré dans un premier temps que M. smithii était détecté chez moins de 50% des individus et M. stadtmanae chez 0-20 % seulement. Ces résultats étaient contradictoires avec le rôle de la méthanogenèse dans l’élimination des acides et d’autres produits du processus digestion, et nous avons émis l’hypothèse que ces résultats pouvaient ne pas refléter la quantité réelle des méthanogènes dans le tube digestif humain, suggérant la mise au point de nouvelles méthodes de détection moléculaire et de culture adaptées aux caractéristiques de ces organismes fastidieux. Dans ce travail, nous nous sommes fixés comme premier objectif de mettre au point une méthode moléculaire permettant de détecter M. smithii chez tous les individus testés et nous avons mis au point un protocole d’extraction et de détection d’ADN d’Archaea à partir des selles en se basant sur les génomes séquencés de M. smithii et M. stadtmanae. Ce protocole nous a permis de détecter M. smithii chez 95,5% des individus et M. stadtmanae chez 29,4% des individus. En ce basant sur ce protocole et moyennant une approche moléculaire basée sur une PCR universelle de l’ADN ribosomal 16S des méthanogènes, le séquençage et le clonage, nous avons également détecté chez 4% de la population, une séquence correspondant à un phylotype (FJ823135) ayant déjà été rapporté comme représentant un nouvel ordre de méthanogènes. A partir de là, nous avons choisi un prélèvement de selle susceptible de contenir le plus fort ratio de FJ823135/ M. smithii et nous avons réussi à isoler et à cultiver une nouvelle Archaea que nous avons nommé Methanomassiliicoccus luminyensis, premier représentant cultivé d’un nouvel ordre de méthanogènes et la quatrième Archaea cultivée chez l’homme. M. luminyensis et M. stadtmanae présentent des métabolismes similaires en réduisant le méthanol en méthane en utilisant l’hydrogène comme donneur d’électrons, cette observation nous a incité à tester l’addition de tungstate de sélénium, requis pour la croissance de M. luminyensis, dans une culture M. stadtmanae, et nous avons observé une accélération de la vitesse de croissance de M. stadtmanae par un facteur 3. Nous avons ensuite étudié la sensibilité des méthanogènes isolés chez l’homme aux antibiotiques et établi qu’ils sont seulement sensibles à des molécules efficaces contre les bactéries et les eucaryotes, ceci étant en accord avec leur position phylogénétique en tant qu’un des quatre domaines de la vie. [...] / Methanogenic Archaea are environmental organisms which have also been associated to mammals mucosa. In humans these microorganisms have been detected in the vaginal, intestinal and oral mucosa. These organisms are strict anaerobes and their culture conditions remains fastidious and poorly known. In fact only three methanogens have been isolated from human samples, both Methanobrevibater smithii and Methanosphaera stadtmanae from stool and Methanobrevibater oralis from dental plaque. Current knowledge on the diversity of methanogens in humans and their potential effects on human health were largely based on DNA detection methods as PCR and metagenomics. These techniques based on 16S rDNA and mcrA gene (encoding the alpha subunit of methyl coenzyme-M-reductase, a key enzyme in methanogenesis process) detection, showed that M. smithii was the most present in man and that the presence of M. stadtmanae was transient. Recently, the DNA of other methanogenic and non- methanogenic Archaea, has been detected in human feces, including sequences indicating the presence of non-cultured species belonging to potential new order of methanogens with no cultured representative. However, these studies detected M. smithii with variable prevalence in less than half of the tested individuals and no M. stadtmanae; such results does not confirm the paramount role of methanogenesis in preventing the accumulation of acids and other reaction end products during the digestion process, and can not reflect the actual amount of these two methanogens in the human digestive tract because of their specific association with the intestinal mucosa. Therefore, these studies pointed that the diversity of methanogens in humans has been underestimated suggesting the development of new molecular detection methods and cultural approaches adapted these fastidious organisms. In this work, we preset as first criteria, the detection of M. smithii in all tested individuals, therefore we developed an improved protocol for archaeal DNA extraction and detection from stool based on sequenced genomes of M. smithii and M. stadtmanae, this protocol allowed us to detect the first one DNA in 95.5% tested individuals and the second in a prevalence of 29.4%. Based on this protocol and through molecular approach based on universal amplification of methanogenic 16S rDNA, sequencing and cloning, we detected in 4% of the tested population, a sequence corresponding to a new phylotype (FJ823135) that has been previously reported and proposed as a representative of a new order of methanogens. From there, we chose one stool specimen susceptible to contain the highest amount of FJ823135 and successfully isolated Methanomassiliicoccus luminyensis B10T clone, the first cultured representative of a new order of methanogens and the fourth Archaea cultured in humans.This archaeon exhibited a similar type of metabolism to that of M. stadtmanae by oxidizing H2 and reducing methanol to methane but require tungstate-selenite, an element essential for its growth, this fact prompted us testing tungstate-selenite addition on M. stadtmanae growth and establishing that it was strongly stimulatory with a growth rate three times faster. We have thereafter studied the sensitivity of methanogens isolated from humans to antibiotics and established that they are susceptible only to molecules also effective against both Bacteria and Eucarya, in agreement with their phylogenetic location as a unique domain of life. The aim of the latter part of this work was to test the effectiveness of MALDI-TOF mass spectrometry identification of environmental and host-associated Archaea. The obtained data indicated that that MALDI-TOF-MS protein profiling is an efficient first-line step for the rapid phenotypic identification of cultured Archaea organisms including host-associated ones. [...]

L’objectif de ce travail était de caractériser la variabilité spécifique (Bos taurus vs Oryctolagus cuniculus), individuelle, spatiale (inter- et intra- fermenteurs digestifs) et temporelle (en conditions non perturbée ou perturbée) des écosystèmes digestifs des mammifères herbivores (communautés procaryotiques et biotope) en comparant deux modèles animaux, la vache et le lapin, et en utilisant des outils moléculaires (CE-SSCP et qPCR). D’un point de vue méthodologique, nous avons développé un programme informatique, StatFingerprints, pour améliorer le traitement et l’analyse statistique des profils CE-SSCP et ainsi mieux extraire l’information écologique qu’ils contiennent en termes de structure et de diversité des communautés. Nous avons également mis au point des systèmes de qPCR plus spécifiques (Firmicutes et Bacteroides Prevotella) que ceux précédemment décrits. D’un point de vue cognitif, nos travaux démontrent une forte influence de l’espèce hôte sur les caractéristiques de son écosystème : les communautés sont plus riches (+8 %, +12 % pour les bactéries et les Archaea, respectivement), plus diverse (+19 % pour les bactéries) mais moins abondantes (-4.9 % de bactéries) dans le rumen de la vache que dans le caecum du lapin. Le biotope ruminal est moins acide (+0.6 unité pH), plus réducteur (-30 mV), et contient moins d’acides gras volatils (-19%) et plus de NH3-N (+39%) que le biotope caecal. Cela suggère un rôle déterminant de la physiologie digestive de l’hôte et/ou des phénomènes de coévolution hôte/microbiote. Nos travaux n’ont pas permis de mettre en évidence un effet de « l’individu hôte » sur les caractéristiques de sa communauté microbienne suggérant que la proximité génétique entre les individus (race) et/ou la forte standardisation des conditions d’élevage (logement, alimentation etc.) tendent à uniformiser l’influence des individus sur le déterminisme de leurs communautés procaryotiques. Nous avons montré qu’il existe une évolution des communautés microbiennes le long du tractus digestif en relation avec la physiologie et les conditions environnementales des différents compartiments à laquelle s’additionne, dans le rumen, une variabilité liée à la fraction, liquide ou solide, considérée. Nos données suggèrent qu’à l’état basal et en situation de perturbation, les communautés procaryotiques des deux espèces hôtes n’évoluent pas de la même façon dans le temps. Ainsi, dans le rumen de la vache, la communauté bactérienne fluctue de façon sporadique suggérant un état d’équilibre dynamique alors qu’elle reste dans un état d’équilibre statique dans le caecum du lapin. Les deux communautés réagissent de façon rapide (&#60; 2jours) et s’adaptent rapidement à une augmentation du ratio amidon/fibres pour atteindre un nouvel équilibre, dynamique dans le rumen et statique dans le caecum. En revanche, nos travaux n’ont pas mis en évidence de corrélations importantes entre les communautés bactériennes et les paramètres de leur environnement. D’un point de vue finalisé, ces données confirment que la nutrition est une voie d’action pertinente pour essayer de réorienter le fonctionnement des écosystèmes digestifs chez ces espèces d’intérêt zootechnique, vers une meilleure santé et/ou efficacité digestive. / The aim of this work was to characterize the specific (Bos taurus vs Oryctolagus cuniculus), individual, spatial (inter- and intra- digestive fermentors) and temporal (in undisturbed or disturbed conditions) variability of the digestive ecosystems (prokaryotic communities and biotope) in herbivorous mammals by comparing two animal models, cow and rabbit, and by using molecular methods (CE-SSCP and qPCR). Concerning methodology, we developed the StatFingerprints program to improve processing and statistical analysis of CE-SSCP profiles and better extract ecological information they contained about structure and diversity of communities. We also developed more specific (Firmicutes and Bacteroides Prevotella) primers than those available. From a cognitive point of view, our work demonstrated a strong effect of host species on ecosystem: communities presented a higher richness (+8 %, +12 % for bacteria and Archaea, respectively), a greater diversity (+19 % for bacteria) but are less abundant (-4.9 % bacteria) in cow rumen than in rabbit caecum. The rumen biotope is less acid (+0.6 pH unit), more reductive (-30 mV), and contains a lower concentration of fatty acids (-19%) and a higher concentration of NH3-N (+39%) than caecal biotope. Taken together, these results suggested a determining role of the digestive physiology of the host and of coevolution phenomena between the host and its microbiota. Our results did not permit to evidence an individual effect on the procaryotic communities suggesting that the genetic similarity between animals we used and/or the strong standardization of breeding conditions (housing, food etc) tended to reduce the influence of the individuals on their prokaryotic communities. We showed that the procaryotic communities evolved along the digestive tract in relation to the physiology and the environmental conditions of the various compartments in which they live. In addition, in the rumen, we evidenced a variability of the bacterial community related to the fraction considered, liquid or solid. Our data suggested that, both in basal and disturbed situations, the bacterial communities of the two host species did not evolve in the same way in time. Indeed, in the rumen of the cow and basal condition, the bacterial community fluctuated sporadically suggesting a dynamic balance whereas it remains in a stable state in the caecum of the rabbit. The two communities reacted quickly (&#60; 2 days) and adapted quickly to an increased ratio starch/fibres to reach a new balance, dynamic in the rumen and stable in the caecum. On the other hand, our work did not highlight important correlations between the bacterial communities and the parameters of their environments. From a finalized point of view, these data confirmed that the nutrition is a relevant way to try to reorientate the functioning of digestive ecosystems in these two species, toward a better digestive health and/or efficiency.

Bulge-helix-bulge (BHB) elements guide the enzymatic splicing machinery that in Archaea excises introns from tRNAs, rRNAs from their primary precursor, and accounts for the assembly of piece-wise encoded tRNAs. This processing pathway renders the intronic sequences as circularized RNA species. Although archaeal transcriptomes harbor a large number of circular small RNAs, it remains unknown whether most or all of them are produced through BHB-dependent splicing. We therefore conduct a genome-wide survey of BHB elements of a phylogenetically diverse set of archaeal species and complement this approach by searching for BHB-like structures in the vicinity of circularized transcripts. We find that besides tRNA introns, the majority of box C/D snoRNAs is associated with BHB elements. Not all circularized sRNAs, however, can be explained by BHB elements, suggesting that there is at least one other mechanism of RNA circularization at work in Archaea. Pattern search methods were unable, however, to identify common sequence and/or secondary structure features that could be characteristic for such a mechanism.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science

Background: Enteric methane from rumen methanogens is responsible for 25.9 % of total methane emissions in the United States. Rumen methanogens also contribute to decreased animal feed efficiency. For methane mitigation strategies to be successful, it is important to establish which factors influence the rumen methanogen community and rumen volatile fatty acids (VFA). In the present study, we used next-generation sequencing to determine if dairy breed and/or days in milk (DIM) (high-fiber periparturient versus high-starch postpartum diets) affect the rumen environment and methanogen community of primiparous Holstein, Jersey, and Holstein-Jersey crossbreeds. Results: When the 16S rRNA gene sequences were processed and assigned to operational taxonomic units (OTU), a core methanogen community was identified, consisting of Methanobrevibacter (Mbr.) smithii, Mbr. thaueri, Mbr. ruminantium, and Mbr. millerae. The 16S rRNA gene sequence reads clustered at 3 DIM, but not by breed. At 3 DIM, the mean % abundance of Mbr. thaueri was lower in Jerseys (26.9 %) and higher in Holsteins (30.7 %) and Holstein-Jersey crossbreeds (30.3 %) (P < 0.001). The molar concentrations of total VFA were higher at 3 DIM than at 93, 183, and 273 DIM, whereas the molar proportions of propionate were increased at 3 and 93 DIM, relative to 183 and 273 DIM. Rumen methanogen densities, distributions of the Mbr. species, and VFA molar proportions did not differ by breed. Conclusions: The data from the present study suggest that a core methanogen community is present among dairy breeds, through out a lactation. Furthermore, the methanogen communities were more influenced by DIM and the breed by DIM interactions than breed differences.

DNA is susceptible to various types of damage as a result of normal cellular metabolism or from environmental sources. In order to maintain genome stability a number of different, partially overlapping DNA repair pathways have evolved to tackle specific lesions or distortions in the DNA. Nucleotide excision repair (NER) is highly conserved throughout eukarya, bacteria and archaea and predominantly targets lesions that result from exposure to UV light, for example cyclobutane pyrimidine dimers and 6-4 photoproducts. The majority of archaea possess homologous of the eukaryotic repair genes and this thesis describes the isolation and the characterization of two XPB homologues identified in the crenarchaeon Sulfolobus solfataricus, SsoXPB1 and SsoXPB2. Human XPB is one of 10 proteins that make up the TFIIH transcription complex. The activity of XPB is tightly controlled by protein interactions, in particular with p52, which stimulates the ATPase activity of XPB. Rather than a conventional helicase, human XPB is thought to act as an ATP dependent conformational switch. Consistent with human XPB, however, the S. solfataricus proteins were unable to catalyse strand separation and the identification of an archaeal protein partner, Bax1, for SsoXPB2 was one of the focuses of this project. In order to maintain genome stability, the DNA must be replicated accurately with each cell cycle. When the advancing replication fork stalls at a lesion or a DNA break, it is crucial that the fork is reset and that replication continues to completion. The helicase Hel308 is thought to clear the lagging strand template of a stalled replication fork in order for replication restart to proceed via homologous recombination (HR). Although the specific function of Hel308 is not well understood, the possibilities are described in this thesis. Strand exchange proceeds to form a D-loop, followed by branch migration to increase regions of heterology during the synapsis stage of HR. No motors for branch migration have previously been recognised in archaea, although the identification of a possible candidate was investigated during this project.

DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea.