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.

Les microorganismes du domaine du vivant Archaea sont très divers sur le plan biologique et sont présents dans de nombreux types d'écosystèmes. Ils sont majoritaires dans les environnements dits extrêmes. Parmi les virus d'archées, ceux infectant les espèces d'un phylum majeur des archées, Crenarchaeota, constitué d'hyperthermophiles, forment un groupe exceptionnel. En effet, leurs morphotypes sont uniques, variés, et complexes. Le contenu de leur génome est également unique. Enfin, la plupart de ces virus se maintiennent dans la cellule hôte en état porteur, une relation chronique qui permet un équilibre entre production de virions et division cellulaire. J'ai d'abord démontré que le virus de crenarchée Sulfolobus islandicus rod-shaped virus 2 est un virus virulent, et non chronique comme il avait été suggéré. Un mécanisme de lyse unique a été découvert. La paroi cellulaire est modifiée en plusieurs points, avec l'apparition de structures pyramidales saillantes. Celles-ci s'ouvrent en fin de cycle infectieux, permettant aux virions, assemblés auparavant dans le cytoplasme, de quitter la cellule. Puis j'ai travaillé sur des échantillons de sources géothermiques de la péninsule de Kamchatka (Russie) et contribué à l'isolement et la caractérisation d'un virus de morphotype filamenteux. Des protéines structurales supplémentaires ont ainsi été identifiées. Enfin, des mots courts exceptionnels ont été identifiés dans un grand nombre de génomes d'archées et de leurs éléments extra-chromosomiques. Ce sont potentiellement des motifs fonctionnels non-codants, impliqués dans des mécanismes biologiques importants. Typiquement, les motifs palindromiques sont évités dans les génomes

Les Archaea sont des micro-organismes rencontrés dans tous les écosystèmes, mais qui apparaissent comme majoritaires dans les environnements dits extrêmes. Les archaea hyperthermophiles, comme Pyrococcus abyssi sont en permanence exposées à des températures qui peuvent augmenter le taux de dommages de l'ADN, pourtant, le taux de mutations spontanés chez ces micro-organismes est similaire à celui des espèces modèles mésophiles. Il est ainsi probable que les hyperthermophiles possèdent des systèmes particulièrement efficaces pour dupliquer, maintenir et stabiliser leur génome. L'objectif de ce projet était d'explorer le réseau d'interaction impliqué dans les processus de réplication et de réparation de l'ADN. L'approche méthodologique mise en oeuvre a consisté à coupler la capture de partenaires d'interaction par pull-down avec leur identification par spectrométrie de masse. J'ai pu ainsi mettre en évidence, au sein l'extrait cellulaire de P. abyssi, un réseau préliminaire reliant des protéines de la maintenance génomique. Nous avons non seulement mis en évidence de nouvelles protéines impliquées probablement dans des mécanismes de réparation, mais également des nouvelles interactions non suspectées entre des composants déjà caractérisés. Les principaux résultats sont les suivants : (1) La nucléase Pab2263, partenaire du PCNA, est un nouvel acteur du métabolisme de l'ADN. (2) Le PCNA forme un macrocomplexe avec les protéines ubiquitaires Mre11 et Rad50 suggérant un rôle de ce complexe dans la réparation des cassures double brin de l'ADN lors de la réplication. (3) Les protéines Fen1 et l'ADN primase interagissent physiquement et peuvent collaborer in vitro pour résoudre une étape intermédiaire de la voie de réparation par excision de base. Ces résultats enrichissent notre compréhension des processus de réparation de l'ADN chez les archées.

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DNA replication is essential for the propagation of all living organisms. The ability of a cell to accurately duplicate its entire genome is dependent upon the activity of numerous proteins. Identifying the molecular mechanisms by which these proteins act, and determining how they are physically and functionally coordinated at sites of active DNA replication, is central to understanding this essential cellular process. Archaea possess a DNA replication machinery which is ancestral to the one present in eukaryotes, and thus these organisms serve as simplified model systems for understanding the complexities of eukaryotic DNA replication. This thesis investigates the molecular mechanisms underlying Okazaki fragment maturation in the crenarchaeon Sulfolobus solfataricus, which is essential to the completion of lagging strand DNA replication. Reconstitution of Okazaki fragment maturation in vitro demonstrated that the activities of three enzymes – PolB1, Fen1, and Lig1 – are required for this process in S. solfataricus. Furthermore, it was shown that optimum coordination of their three distinct activities is dependent on the ability of PolB1, Fen1 and Lig1 to simultaneously interact with a single PCNA ring, providing evidence for a mechanism of multi-enzyme coordination which may be universally employed by DNA sliding clamp proteins. The importance of protein flexibility in the accommodation of multiple proteins around a single PCNA was also investigated. Finally, the physical coordination of one of these key maturation enzymes – PolB1 – with other replisome proteins was examined. It was demonstrated that PolB1 exists in a trimeric complex in vivo with two previously unidentified factors, raising the possibility of uncharacterised activities and interactions for this crucial enzyme. Taken together, these data provide new insights into functionally important protein-protein interactions within the archaeal replisome, and facilitate a greater understanding of the DNA replication machinery in both archaea and eukaryotes.

Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge to dairy farmers, maintaining ruminant health, and enhancing bioactive FA in milk. The first objective was to use next-generation sequencing techniques to determine if lactation stage and dairy breed affect rumen methanogen and protozoal community structures and protozoa cell FA compositions in Jersey, Holstein, and Holstein-Jersey crossbred cows at 3, 93, 183, and 273 days in milk (DIM). A core methanogen community persisted by lactation stage and breed. At 3 DIM, methanogen 16S rRNA gene sequences formed distinct clusters apart from 93, 183, and 273 DIM, reflective of the dietary transition period post-partum. The starch-utilizing protozoal genus Entodinium, was more abundant in Holsteins than in Jerseys and Holstein-Jersey crossbred cows and positively correlated with milk yield. Jerseys had greater iso-BCFA contents in protozoa and milk and protozoa of the genus Metadinium. The second objective was to determine if supplementation of mixed cool-season grasses with annual forages (AF) alters the forage, microbial, and milk FA contents during typical periods of decreased pasture growth in Northeastern US. In short-term grazing (21d) of AF, ruminal VFA and major rumen-derived FA were not altered in bacterial and protozoal cells, suggesting little alteration of biohydrogenation and maintenance of ruminant health. In spring, milk contents of iso-15:0 and 17:0 per serving of whole milk were greater in control (CON)-fed cows, while contents of 12:0 and 14:0 per serving were greater in AF-fed cows. Contents of de novo FA and OCFA per serving of whole milk were greater in summer AF-fed cows than CON-fed cows, while total contents and BCFA did not differ, suggesting post-ruminal FA modifications in adipose tissue and the mammary gland. The third objective was to characterize and relate the rumen microbiota from CON- and AF-fed cows to animal performance. Rumen protozoal taxa were not altered, while less abundant bacterial taxa (< 5%) were different in both periods. The protozoal genus Diplodinium was positively correlated with feed efficiency and milk fat yield. In spring, AF-fed cows had greater abundances of the methanogen species Methanobrevibacter millerae, whereas CON-fed cows had greater abundances of the methanogen species Methanobrevibacter ruminantium, potentially as a result of differences in substrate availability. In conclusion, the work presented identifies several factors that influence rumen microbiota, rumen microbial FA, and milk FA, while providing new information to dairy farmers, researchers, and consumers.

Soils, and the microbial communities contained within them, are vital for most chemical, physical, and biological processes. This study investigated how microbial community structure responded to environmental changes, such as hydrology, across vertical space (depth) and time in an emergent fresh water wetland. Research was conducted in a non-tidal freshwater wetland along the James River (Charles City County, Virginia) by establishing plots in two areas that experienced different hydrologic regimes and plant communities. Soil cores (30 cm) were collected monthly from January 2008 to February 2009, and then every two to three months thereafter until October 2009, for a total of 17 sampling events. The soil cores were divided by depth (Top: 0 – 10 cm, Bottom: 20 – 30 cm) and analyzed for a variety of soil properties including: pH, organic matter (OM), water content (WC), C:N, redox, and root biomass. Additionally, above-ground plant communities were monitored during the growing seasons. Based on preliminary analysis, one date from each season (Winter, Spring, Summer, and Fall) from both sampling years were selected for in depth analysis of the microbial community structure via Terminal Restriction Fragment Length Polymorphism (T-RFLP) of 16S-rRNA. Analysis of variance (ANOVA) found significant differences were found between the environmental parameters in regards to site, depth, and season. Three physical-chemical variables (WC, OM, and redox) were different between sites, but the majority of environmental parameters were significantly different between depths and seasons. The dominant environmental effect on microbial communities was soil depth and, overall, no seasonal patterns were observed in the microbial communities. Further, archaeal communities were most strongly correlated to changes in water content, while redox was strongly correlated to changes across depth in the bacterial communities. Collectively, these results demonstrate that wetland microbial communities are not a product of one separate variable or spatial scale, but result from various factors interlinked to shape microbial communities. More long-term studies are needed to investigate interactions between microbial community structure and environmental variables in these dynamic ecosystems.

Through complex metabolic interactions aquatic microbial life is essential as a driver of ecosystem functions and hence a prerequisite for sustaining plant and animal life in the sea and on Earth. Despite its ecological importance, infor­mation on the complexity of microbial functions and how these are related to environmental conditions is limited. Due to climate change and eutrophication, marine areas facing oxygen depletion are increasing and predicted to continue to do so in the future. Vertically steep oxygen gradients are particularly pronoun­ced in the Baltic Sea. In this thesis, therefore, the ecosystem functions of micro­bial communities were investigated, using metagenomics, to understand how they were distributed along the steep oxygen gradient at the Landsort Deep, the deepest point of the Baltic Sea. Furthermore, microbial communities from the Lands­ort Deep transect were compared to microbial communities of other marine environments to establish whether the environment at this site resulted in a characteristic community. To reveal what microbial community functions and taxa were active in the anoxic sediment a metatranscriptomic approach was used. Results showed a marked effect of the coupled environmental parameters dissolved oxygen, salinity and temperature on distribution of taxa and par­ti­cularly community functions. Microbial communities showed functional capa­cities consistent with a copiotrophic life-style dependent on organic ma­terial sinking through the water column. The eutrophic condition with high organic load was further reflected in the metatranscriptome of the anoxic sedi­ment com­munity, which indicated active carbon mineralisation through ana­erobic hetero­trophic-autotrophic community synergism. New putative linkages between nitro­gen and- sulphur metabolisms were identified at anoxic depths. Further­more, viable Cyanobacteria in the anoxic sediment was evident from the tran­script analyses as another reflection of marine snow. High abundance and expres­­sion of integron integrases were identified as a charac­teristic feature of the Lands­ort Deep communities, and may provide these communities with a mech­an­ism for short-term-adaptation to environmental change. In summary, this thesis clearly documents what impact eutrophication and oxygen depletion have on microbial community functions. Furthermore, it specifically advances the mechanistic insight into microbial processes in anoxic deep-water sediment at both genomic and transcriptional level. Given the predicted progress of oxygen depletion in marine and brackish environments, this work advances information necessary to estimate effects on marine and in particular brackish ecosystem functions where anoxic conditions prevail. / Mikroorganismer är essentiella för fungerande ekosystemfunktioner i akvatiska miljöer och därmed en förutsättning för övrigt växt- och djurliv på vår planet. Trots deras ekologiska nyckelroll är kunskapen om mikroorganismernas funk­tion och komplexitet samt hur dessa är relaterade till miljön begränsad. På grund av eutrofiering och klimatförändringar har marina områden som lider av syrebrist ökat och en ytterligare utbredning av marina och bräckta områden med syrebrist är predicerad i framtiden. Stora områden av Östersjön kännetecknas av vertikala syregradienter med syresatt ytvatten och anoxiskt bottenvatten. I denna avhandling undersöktes därför med metagenomik hur mikrobiella ekosystems funktioner var utbredda längs den vertikala syregradienten i Östersjöns djupaste del, Landsortsdjupet. Dessutom jämfördes de mikrobiella samhällena från Lands­­­ortsdjupet med mikrobiella samhällen från andra marina miljöer för att utröna om den karakteristiska miljön i Landsortsdjupet återspeglade de mikro­biella samhällen som lever där. För att undersöka vilka mikroorganismer samt vilka mikrobiella ekosystemfunktioner som var aktiva i det anoxiska sedimentet i Lands­ortsdjupet användes metatranskriptomik. Resultaten visade en stark kor­re­lation mellan miljöparametrarna syrehalt, salinitet och temperatur och för­del­ningen av mikrobiell taxa och i synnerhet mikrobiell funktion längs Lands­orts­djupets transekt. De mikrobiella samhällena uppvisade en funktionell kapa­citet förenlig med en livsstrategi beroende av organiskt material som sjunker genom vattenkolonnen som en konsekvens av eutrofiering. Eutrofa förhållanden med hög halt av organiskt material var även återspeglad i metatranskriptomet från det anoxiska sedimentet, som indikerade aktiv mineralisering av organiskt kol genom anaerob heterotrof-autotrof synergism. Nya möjliga kopplingar mellan kväve- och svavelmetabolism identifierades i det anoxiska vattnet. Vidare visade resultat från metatranskriptom-analys att livsdugliga cyanobakterier var abun­danta i det mörka och anoxiska sedimentet, vilket även detta kan vara en konse­kvens av sjunkande organiskt material. Hög abundans och hög transkribering av integrongener kunde identifieras som ett karakteristiskt kännetecken hos de mikro­biella samhällena i Landsortsdjupet vilket skulle kunna förse dem med en me­kanism för anpassning till miljöförändringar. Sammanfattningsvis dokumen­terar denna avhandling tydligt vilken påverkan eutrofiering och syrebrist har på mikrobiella funktioner. Dessutom för den specifikt kunskapen om mikrobiella processer i anoxiska djupvattensediment framåt på både genom- och transkrip­tions­nivå. Mot bakgrund av en predicerad ökning av syrebristen i marina mil­jöer, bidrar denna avhandling med information som är viktig för att kunna förutse vilka effekter anoxiska förhållanden kan komma att få på ekosystemfunktioner i marina miljöer och i brackvattenmiljöer i synnerhet.

Resumo: Estratégias para reduzir o aquecimento da Terra e aumentar a produção animal requerem novos sistemas, onde devem ser consideradas as emissões de metano e de outros gases que possam provocar danos ao meio ambiente. O objetivo deste trabalho foi a mensuração da produção de metano por arquéias e aplicação da metagenômica para detecção destas na fração sólida do conteúdo ruminal. Para a análise da produção de metano foi colhido o conteúdo ruminal seguido do preparo da solução a ser fermentada e armazenamento dos gases. A amplificação da região 168 rDNA foi obtida por PCR e a seguir foram feitas clonagem, seqüenciamento e análise das seqüências pelos programas "Sequencing Analysis 3.4" e Phred/Phrap/Consed, submetendo-as ao programa BLA8T. Maiores produções de metano e relações acetato:propionato foram observadas nos tratamentos contendo 70% de volumoso. As análises do BLA8T permitiram identificar nos tratamentos com 70% e 30% de feno, 96 seqüências relacionadas à família Methanobacteriaceae, 47 seqüências a arquéias não cultiváveis e 60 seqüências foram de arquéias desconhecidas (T1), 125 seqüências relacionadas à família Methanobacteriaceae, 42 seqüências a arquéias não cultiváveis e 32 seqüências foram de arquéias não conhecidas (T2). Para os tratamentos feitos com 70% e 30% de silagem de milho, foram observadas 30 seqüências referentes à família Methanobacteriacea, 18 seqüências à família Methanomicrobiacea, 43 seqüências a arquéias não cultiváveis e 118 seqüências foram de arquéias desconhecidas (T3) e 173 seqüências referentes à família Methanobacteriacea, 31 seqüências a arquéias não cultiváveis e 25 seqüências foram de arquéias desconhecidas (T4). As análises deste experimento mostraram variação na produção de metano quanto às diferentes proporções V:C e a metagenômic... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Strategies to reduce the Earth worming and raise animal production require new systems, where it must be considered methane and other gases emission that might cause environmental damages. The aim of this work was to evaluate the archaea methane production and the metagenomic evaluation of these bacteria present on the solid phase of the bovine ruminal content. For methane production analysis the ruminar content was collected followed by the proper manipulation for the fermentation process to take place and produced gas storage. The ribosomal 16S rRNA region was obtained by PCR amplification which was followed by cloning and DNA sequencing. The data was later analyzed by the software Sequencing Analysis 3.4, Phred/Phrap/Consed and BLAST. The highest methane production and acetate:propionate ratios were observed for the treatments containing 70% of roughage. The BLAST analysis allowed to identify 96 DNA sequences related to the Methanobacteriaceae family, 47 DNA sequences related to unculturable archaea and 60 DNA sequences were related to unknown archaea (T1), 125 DNA sequences related to Methanobacteriaceae, 42 DNA sequences do unculturable archaea and 32 DNA sequences were considered related to unknown archaea (T2) for the treatments containing 70% and 30% of hay. For those containing 70% and 30% of com silage it was possible to detect 30 DNA sequences related to Methanobacteriaceae, 18 DNA sequences related to Methanomicrobiaceae, 43 sequences related to unculturable archaea, 118 DNA sequences related to unknown archaea (T3) and 173 DNA sequences related to Methanobacteriaceae, 31 sequences related to unculturable archaea, and 25 to unknown archaea (T4). The analysis carried out in this experiment have shown a variation of the methane production as different R:C ratio were used and metagenomic with the rDNA conserved region together with archaea taken from the ruminal content DNA... (Complete abstract click electronic access below) / Orientador: Manoel Victor Franco Lemos / Coorientadora: Jane Maria Bertocco Ezequiel / Banca: Maria Isabel da Silva / Banca: Raul Franzolin Neto / Banca: Janete Apparecida Desidério Sena / Banca: Mauro Dal Secco de Oliveira / Doutor