Microorganisms are an indispensable part of our ecosystem, yet the natural metabolic and ecological diversity of these organisms is poorly understood due to a historical reliance of microbiology on laboratory grown cultures. The awareness that this diversity cannot be studied by laboratory isolation, together with recent advances in low cost scalable sequencing technology, have enabled the foundation of culture-independent microbiology, or metagenomics. The study of environmental microbial samples with metagenomics has led to many advances, but a number of technological and methodological challenges still remain. A potentially diverse set of taxa may be represented in anyone environmental sample. Existing tools for representing the genetic composition of such samples sequenced with short-read data, and tools for identifying variation amongst them, are still in their infancy. This thesis makes the case that a new framework based on a joint-genome graph can constitute a powerful tool for representing and manipulating the joint genomes of population samples. I present the development of a collection of methods, called SCRAPS, to construct these efficient graphs in small communities without the availability or bias of a reference genome. A key novelty is that genetic variation is identified from the data structure using a probabilistic algorithm that can provide a measure of the confidence in each call. SCRAPS is first tested on simulated short read data for accuracy and efficiency. At least 95% of non-repetitive small-scale genetic variation with a minor allele read depth greater than 10x is correctly identified; the number false positives per conserved nucleotide is consistently better than 1 part in 333 x 103. SCRAPS is then applied to artificially pooled experimental datasets. As part of this study, SCRAPS is used to identify genetic variation in an epidemiological 11 sample Neisseria meningitidis dataset collected from the African meningitis belt". In total 14,000 sites of genetic variation are identified from 48 million Illumina/Solexa reads. The results clearly show the genetic differences between two waves of infection that has plagued northern Ghana and Burkina Faso.

Os produtos naturais microbianos (NP) tem demonstrado ser inestimáveis pontos de partida na descoberta e desenvolvimento de medicamentos aprovados pelo FDA. A abordagem tradicional para a identificação de produtos naturais microbianos exige a cultura em laboratório. Infelizmente, os métodos convencionais baseados nesta metodologia foram desestimulados devido a altas taxas de redescoberta de moléculas. Os métodos independentes de cultura que se baseiam no sequenciamento do metagenoma microbiano sugerem a ocorrência de um enorme reservatório inexplorado de clusters biossintéticos de produtos naturais (BGCs) no meio ambiente. Neste trabalho utilizamos uma metodologia baseada em PCR e barcoding amplicon-sequencing para buscar importantes famílias de produtos naturais como peptídeos não ribossomais (NRP), ácido 3-amino-5-hidroxibenzóico (AHBA), dímeros de triptofano (TD), policetídeos, aminoglicosídeos e outros. Para isto desenvolvemos um script chamado SecMetPrimer que nos permitiu bioinformaticamente desenhar conjuntos de primers contendo um gradiente de degenerâncias. No total, desenhamos 165 conjuntos de primers. Os amplicons foram obtidos por PCR padrão, tendo sido concatenados barcodes específicos por amostra e sequenciados através de Illumina MiSeq. Para validar, utilizamos eDNA (environmental DNA) de bibliotecas metagenômicas, totalizando 223 milhões de clones. Através das análises bioinformáticas, as curvas de rarefação foram calculadas e a diversidade para cada família foi determinada. Foi realizada uma reamplificação dos domínios de adenilação de peptídeo não ribossomal e domínios de cetosintase de policetídeos utilizando eDNA isolado de 25 amostras diferentes coletadas em Mata Atlântica, Cerrado e ambiente marinho. Nossos dados indicaram a correlação entre distância geográfica e o tipo ecológico dos biomas. Deste modo, foi possível assim atribuir genes relacionados à clusters biossintéticos que codificam importantes produtos naturais à informações taxonômicas e metabólicas. Deste modo identificamos os melhores hotspots para busca de diversidade biossintética dentre as amostras analisadas. / Microbial natural products (NP) have proven to be invaluable starting points in the discovery and development of many drugs approved by FDA. The traditional approach to identify microbial natural products requires the culturing in the laboratory. Unfortunately, conventional culture-based methods have been deemphasized due to high rediscovery rates. Culture-independent methods applying microbial (meta)genome sequencing suggest the occurrence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters (BGCs) in the environment. Here we have used a PCR-based approach and barcoding ampliconsequencing derived from important families of microbial natural products such as nonribosomal peptides (NRP), polyketides (PK), 3-amino-5-hydroxybenzoic acidcontaining NPs (AHBA), tryptophan dimmers (TD), aminoglycosides, phosphonocontaining NPs and others. We have written an internal script called SecMetPrimer that allowed us to bioinformatically design sets of primers containing a range of degeneracy to amplify these genes. At the total, we designed 165 different sets of primers. The amplicons were obtained by standard PCR containing double-barcodedtarget primers and sequenced by Illumina MiSeq platform. The validation process was conducted using eDNA from metagenomic libraries containing a 223 millions of clones. The rarefaction and diversity analyses were assigned, and the best-hit primer for each family was chosen. We have re-amplified the nonribosomal peptide adenylation domains and polyketide ketosynthase domains, using as substrate environmental DNA isolated from 25 different samples collected in Atlantic Forest, Cerrado and marine environment. Our data indicate a correlation between geographic distance and biome-type, and the biosynthetic diversity found in these environments. Thus, by assigning reads to known BGCs against taxonomic and metabolic profiles, we have identified the hotspots of relevant biosynthetic diversity among the analyzed samples.

Selon les projections démographiques de la FAO, la population mondiale atteindra 9 milliards de personnes d'ici 2050. Cette augmentation sera associée à une demande accrue de produits agricoles et à une augmentation de la production de déchets. Par conséquent, des approches alternatives dans les pratiques agricoles, tels que l'utilisation permanente de la couverture végétale et/ou l'application de boues d'épuration, sont envisagées pour répondre aux exigences mondiales et préserver l'environnement. Ces nouvelles pratiques pourraient influencer le fonctionnement et les propriétés du sol et des organismes microbiens présents dans cet environnement. Par conséquent, passer de l'agriculture intensive à une agriculture écologiquement intensive pourrait entraîner des modifications de la biodiversité des sols. En utilisant différents systèmes expérimentaux permettant une comparaison entre différentes pratiques agricoles, des études de la diversité microbienne taxonomique et fonctionnelle du sol (bactéries et champignons) ont été entreprises. La diversité taxonomique des organismes microbiens a été obtenue par séquençage à haut débit des régions hypervariables des gènes codant l'ARN16S et l'ITS1. Nous avons évalué les rôles écologiques des microorganismes du sol en utilisant des identifications taxonomiques, puis des études permettant d'examiner leur physiologie et leurs fonctions par rapport à différentes propriétés physicochimiques du sol. Nous avons constaté que la fertilisation azotée avait une incidence négative sur la diversité microbienne du sol et modifié leur fonctionnalité. Ces effets peuvent être modulés par l'utilisation de PPC ou l'application de boues. Ces travaux indiquent que les pratiques agricoles conventionnelles ont un impact sur la biodiversité microbienne du sol et peuvent être remplacées par des pratiques agricoles plus respectueuses de l'environnement afin de préserver l'écosystème et ses services / According to demographic projections, world population will reach 9 billion people by 2050. This increase will be associated with higher demand of agricultural products and an increase in wastes production. Therefore, alternative approaches in agricultural practices; such as permanent plant cover usage and/or sewage sludge application, are envisaged to meet global demands and preserve the environment. These new practices could therefore influence the properties of the soil and its functioning. Therefore moving from intensive to ecologically intensive agriculture could lead to modifications in soil biodiversity. Using different experimental systems allowing comparison between different agricultural practices, studies of the taxonomic and functional soil microbial diversity (bacteria and fungi) had been undertaken. This was achieved by next generation high-throughput sequencing of the hypervariable regions of the genes encoding RNA16S and ITS1. Sequencing was performed using an Illumina platform and the sequences obtained were analyzed using various bioinformatic tools. We inferred the ecological roles of soil micro-organisms by using taxonomic identifications, moving on to the examination of their physiology and functions in comparison with different soil physiochemical properties. We found that nitrogen fertilization negatively impacted the soil microbial diversity and altered their functionality. These negative effects have been modulated by the PPC usage or SS application. Proving that conventional agricultural practices effects the soil biodiversity and can be replaced by ecofriendly farming applications in order to preserve the ecosystem and its services

Na última década tem sido possível reconstruir o genoma de bactérias e arquéias presentes em comunidades microbianas de ambientes naturais a partir de dados metagenômicos. Isso tem revolucionado nosso entendimento sobre a topologia da árvore da vida e a descoberta de novas capacidades metabólicas, bem como auxiliado na identificação mais acurada de genes de interesse industrial, visto que os dados estão mais completos e menos fragmentados. Com base neste contexto, o objetivo geral deste projeto foi reconstruir o genoma de bactérias ligadas a degradação de biomassa vegetal em comunidades microbianas da compostagem, focando em análises de diversidade de enzimas de Glicosil Hidrolases (GHs), a partir de dados de sequências metagenômicas gerados no projeto temático processo 11/50870-6. Para alcançar os nossos objetivos, foram desenvolvidos pipelines computacionais com softwares já disponíveis na literatura e foram utilizados dois conjuntos principais de dados de sequenciamento massivo (um conjunto de dados seriados que engloba inúmeros estágios do processamento da compostagem e um conjunto de dados do metagenoma de um consórcio microbiano celulolítico e termofílico construído a partir de amostras da compostagem). Foram reconstruídos 13 genomas (sete genomas em amostras dos dados seriados e seis genomas na amostra do consórcio microbiano), sendo identificado no mínimo quatro novas espécies. As análises baseadas em filogenômica indicam a presença de pelo menos uma nova classe dentro do filo Firmicutes, uma nova espécie da família Paenibacillaceae e a reconstrução pela primeira vez do genoma da espécie Bacillus thermozeamaize. Também foram identificadas 33 lacunas/ilhas metagenômicas (IMs). Essas regiões apresentaram genes diretamente ligados a biossíntese de polissacarídeos do envelope celular, pseudogenes e proteínas hipotéticas. Algumas dessas proteínas estão diretamente ligadas ao reconhecimento de bacteríofagos durante a fase de infecção viral. A presença de IMs também indica uma divergência entre as populações microbianas presentes na compostagem com a espécie de referência. Quanto ao potencial de degradação de biomassa vegetal, todos os microrganismos apresentam genes com potencial para degradação de material lignocelulolítico durante o processamento de diferentes estágios da compostagem, indicando a importância do papel funcional dessas bactérias na compostagem. / In the last decade it has been possible to reconstruct Bacteria and Archaea genomes that are in natural microbial communities from metagenomic samples. This has revolutionized our understanding of the topology of the tree of life and the discovery of new metabolic functions, as well as aided in more accurate identification of industrial bioprospecting genes, since the genomic data are more complete and less fragmented. Based on this background, the aim of this project was to reconstruct the bacterial genomes linked to plant biomass degradation in composting communities, focusing on diversity analysis of Glycosyl Hydrolases (GHs) from metagenomic sequence data generated in the Thematic Project (Process 11/50870-6). To achieve our objectives, computational pipelines have been developed (this pipelines were based on software already available in the literature) and we use these pipelines in two massive data sets generated by high-throughput sequencing (one data set of time series compost sample which includes several stages of the composting process and other data set from a cellu- lolytic and thermophilic microbial consortium). Thirteen genomes were reconstructed (seven genomes from time series metagenomic data and six genomes from microbial consortium). At least four new species have been identified, and the analyzes based on phylogenomic inferences indicate the presence of at least one new class of Firmicutes phylum, and a new Paenibacillaceae family and the reconstruction for the first time the Bacillus thermozeamaize genome. They also identified 33 gaps/metagenomic Islands (IMs). These gaps had genes directly linked to polysaccharide biosynthesis of the cell envelope, pseudogenes and hypothetical proteins. Some of these proteins are directly linked to the bacteriophage during the recognition phase of viral infection. The presence of gaps also indicates a divergence between microbial populations present in the compost with the reference genome. All microbial genomes reconstructed in this studyhave genes linked to lignocellulolytic potential degradation during the different stages of composting process, indicating the functional role this bactéria in this environment.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:46:25Z No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:46:36Z (GMT) No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) / Made available in DSpace on 2017-09-04T20:46:36Z (GMT). No. of bitstreams: 1 MuhammadFaheemTese2016.pdf: 102451329 bytes, checksum: 190df4ef7dc2a9c25576fabb32681db8 (MD5) Previous issue date: 2016-02-12 / Metagenomics techniques are now widely used for the search of new valuable enzymes of interest and other biotechnological products. Sophistication in the second-generation sequencing has significantly facilitated metagenomics technique for collection of huge amount of microbial genomic data. One of the current focuses in science is to seek the interpretation and transformation of the collected genomic data into functional proteomics data. Combination of structural biology and genomic data is one way to achieve such goal. In this study we have assessed a novel bacterial protein selected on a screen for activity on carbohydrates in a microbial metagenomic library from the gut of Capra hircus. Initial sequence analysis of the open reading frame (ORF) for this selected novel bacterial protein indicated that it could be annotated as an uncharacterized novel bacterial cell wall modifying enzyme. Sequence analysis of the protein has shown that it carries three domains: an N-terminus cysteine protease, a peptidoglycan binding (PGBD) and a C-terminus Src-Homology 3 (SH3) bacterial domain. Later with homology modeling we have observed that it carries an additional N-terminus domain with LCI fold. We have successfully cloned, expressed and purified this Capra hircus putative cysteine protease (PCP). Autoproteolytic activity has been observed for PCP, which was inhibited with protease inhibitors cocktail. We have observed that the autoproteolytic activity is carried either by the second or third domain of PCP. This protein has shown cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most of bacterial cell wall modifying enzymes. Ampicillin binding to PCP was further evaluated with fluorimetric analysis. PCP structure was modeled by homology modeling with good validation statistics and in agreement with circular dichroism data. The domains of PCP have conserved LCI, Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), PGBD and SH3 folds. It has a conserved active site dyad, Cys100 and His161, which is a signature of cysteine proteases. Furthermore, the overall architecture of the model was assembled in SAXS generated density map. Initial protein crystals are also obtained for the last two domains, which diffracted to very low resolution. / ***

Na última década tem sido possível reconstruir o genoma de bactérias e arquéias presentes em comunidades microbianas de ambientes naturais a partir de dados metagenômicos. Isso tem revolucionado nosso entendimento sobre a topologia da árvore da vida e a descoberta de novas capacidades metabólicas, bem como auxiliado na identificação mais acurada de genes de interesse industrial, visto que os dados estão mais completos e menos fragmentados. Com base neste contexto, o objetivo geral deste projeto foi reconstruir o genoma de bactérias ligadas a degradação de biomassa vegetal em comunidades microbianas da compostagem, focando em análises de diversidade de enzimas de Glicosil Hidrolases (GHs), a partir de dados de sequências metagenômicas gerados no projeto temático processo 11/50870-6. Para alcançar os nossos objetivos, foram desenvolvidos pipelines computacionais com softwares já disponíveis na literatura e foram utilizados dois conjuntos principais de dados de sequenciamento massivo (um conjunto de dados seriados que engloba inúmeros estágios do processamento da compostagem e um conjunto de dados do metagenoma de um consórcio microbiano celulolítico e termofílico construído a partir de amostras da compostagem). Foram reconstruídos 13 genomas (sete genomas em amostras dos dados seriados e seis genomas na amostra do consórcio microbiano), sendo identificado no mínimo quatro novas espécies. As análises baseadas em filogenômica indicam a presença de pelo menos uma nova classe dentro do filo Firmicutes, uma nova espécie da família Paenibacillaceae e a reconstrução pela primeira vez do genoma da espécie Bacillus thermozeamaize. Também foram identificadas 33 lacunas/ilhas metagenômicas (IMs). Essas regiões apresentaram genes diretamente ligados a biossíntese de polissacarídeos do envelope celular, pseudogenes e proteínas hipotéticas. Algumas dessas proteínas estão diretamente ligadas ao reconhecimento de bacteríofagos durante a fase de infecção viral. A presença de IMs também indica uma divergência entre as populações microbianas presentes na compostagem com a espécie de referência. Quanto ao potencial de degradação de biomassa vegetal, todos os microrganismos apresentam genes com potencial para degradação de material lignocelulolítico durante o processamento de diferentes estágios da compostagem, indicando a importância do papel funcional dessas bactérias na compostagem. / In the last decade it has been possible to reconstruct Bacteria and Archaea genomes that are in natural microbial communities from metagenomic samples. This has revolutionized our understanding of the topology of the tree of life and the discovery of new metabolic functions, as well as aided in more accurate identification of industrial bioprospecting genes, since the genomic data are more complete and less fragmented. Based on this background, the aim of this project was to reconstruct the bacterial genomes linked to plant biomass degradation in composting communities, focusing on diversity analysis of Glycosyl Hydrolases (GHs) from metagenomic sequence data generated in the Thematic Project (Process 11/50870-6). To achieve our objectives, computational pipelines have been developed (this pipelines were based on software already available in the literature) and we use these pipelines in two massive data sets generated by high-throughput sequencing (one data set of time series compost sample which includes several stages of the composting process and other data set from a cellu- lolytic and thermophilic microbial consortium). Thirteen genomes were reconstructed (seven genomes from time series metagenomic data and six genomes from microbial consortium). At least four new species have been identified, and the analyzes based on phylogenomic inferences indicate the presence of at least one new class of Firmicutes phylum, and a new Paenibacillaceae family and the reconstruction for the first time the Bacillus thermozeamaize genome. They also identified 33 gaps/metagenomic Islands (IMs). These gaps had genes directly linked to polysaccharide biosynthesis of the cell envelope, pseudogenes and hypothetical proteins. Some of these proteins are directly linked to the bacteriophage during the recognition phase of viral infection. The presence of gaps also indicates a divergence between microbial populations present in the compost with the reference genome. All microbial genomes reconstructed in this studyhave genes linked to lignocellulolytic potential degradation during the different stages of composting process, indicating the functional role this bactéria in this environment.

Metagenomics, or genomic sequence of the community of microbiota (bacteria, fungi, virus), enables an investigation of the full complement of genetic material, including virulence, antibiotic resistance, and strain differentiating markers. The granularity to distinguish between closely related strains is important as within one species, these strains possess distinct functions and relationships to a host. To analyze metagenomic samples, I developed a reference-based approach that utilizes both single nucleotide variants and genetic content to assign species and strain-level designations. After refining this approach with complex simulated communities, I utilized it to analyze the microbial communities present in skin samples from healthy and diseased individuals. First, to investigate strain-level heterogeneity in healthy adults, I focused on the common skin commensals Propionibacterium acnes and Staphylococcus epidermidis with well-documented sequence variation. Results indicated that an individual’s strains of P. acnes are shared across multiple sites of his or her body, and that those strains are more similar within than between individuals. For S. epidermidis, in addition to individual site similarities, there were also site-specific strains. Overall these results emphasize that both individuality and site specificity shape our bodies’ microbial communities. Based on longitudinal data, an individual’s strain signatures remain stable for up to a year despite external, environmental perturbations. I then used metagenomic data to explore microbial temporal dynamics in atopic dermatitis (AD; eczema), an inflammatory skin disease commonly associated with Staphylococcal species. Species-level investigation of AD flares demonstrated a microbial dichotomy in which S. aureus predominated on more severely affected patients while S. epidermidis predominated on less severely affected patients. Strain-level analysis determined that S. aureus-predominant patients were monocolonized with distinct S. aureus strains, while all patients had heterogeneous S. epidermidis strain communities. To assess the host immunologic effects of these species, I topically applied patient-derived strains to mice. AD strains of S. aureus were sufficient to elicit a skin immune response, characteristic of AD patients. This suggests a model whereby staphylococcal strains contribute to AD progression through activation of the host immune system. Overall, this strain-level analysis of healthy and disease communities provides previously unexplored resolution of human skin microbiome. / 2018-03-24T00:00:00Z

Since the Industrial Revolution at the end of the 18th century, anthropogenic changes in the environment have shifted from the local to the global scale. Even remote environments such as the high Arctic are vulnerable to the effects of climate change. Similarly, anthropogenic mercury (Hg) has had a global reach because of atmospheric transport and deposition far from emission point sources. Whereas some effects of climate change are visible through melting permafrost, or toxic effects of Hg at higher trophic levels, the often-invisible changes in microbial community structures and functions have received much less attention. With recent and drastic warming-related changes in Arctic watersheds, previously uncharacterized phylogenetic and functional diversity in the sediment communities might be lost forever. The main objectives of my thesis were to uncover how microbial community structure, functional potential and the evolution of mercury specific functions in lake sediments in northern latitudes (>54ºN) are affected by increasing temperatures and Hg deposition. To address these questions, I examined environmental DNA from sediment core samples and high-throughput sequencing to reconstruct the community composition, functional potential, and evolutionary responses to historical Hg loading. In my thesis I show that the microbial community in Lake Hazen (NU, Canada) sediments is structured by redox gradients and pH. Furthermore, the microbes in this phylogenetically diverse community contain genomic features which might represent adaptations to the cold and oligotrophic conditions. Finally, historical Hg pollution from anthropogenic sources has likely affected the evolution of microbial Hg resistance and this deposition can be tracked using sediment DNA on the Northern Hemisphere. My thesis underscores the importance of using culture-independent methods to reconstruct the structure, functional potential and evolution of environmental microbial communities.

Global energy requirements are heavily dependent on fossil fuels such as oil, coal, and natural gas. With the expectation of fossil fuels being exhausted in the future, novel strategies need to be discovered for alternative energy generation. Biofuels such as acetone, butanol, ethanol, and hydrogen gas are gaining interest as high value energy sources. These fuels can be produced by anaerobic clostridia as metabolic byproducts of fermentation. The capability to produce these biofuels has been widely studied using glucose or other common feedstocks. Biofuels from renewable and industrial waste feedstocks such as algae and cheese whey may have significant implications on the efficiency of biofuel production, where the price associated with feedstocks is considered a major bottleneck in biotechnology processes. Algae and cheese whey are both rich in organic nutrients and can be utilized by clostridia to produce not only biofuels, but also bioacids, which are considered fuel intermediate compounds. Additionally, understanding microbial communities both in the biosphere and within bioreactors can provide knowledge on microbial relationships and novel microbes, and provide knowledge to optimize engineered systems for biofuels and bioremediation strategies. In this study, a comprehensive investigation of the Logan City Wastewater Lagoon System at the microbial level was executed. Microalgae were utilized for the production of acetone, butanol, and ethanol using Clostridium saccharoperbutylacetonicum. High-throughput 454 pyrosequencing technology was utilized to understand the biogas-producing microbial consortium within an algal-fed anaerobic digester inoculated with lagoon sludge. This technology platform was also utilized to study the microbial diversity of a municipal waste remediating community while probing for clostridia capable of producing biofuels. Bioproduct producing clostridia from this system were isolated and employed using cheese whey as feedstock for the production of hydrogen, ethanol, acetic acid, butyric acid, and lactic acid. Integrating fundamental science with engineering strategies was demonstrated using this lagoon system. To optimize and fully understand and manage anaerobic microbial systems, an understanding of their phylogeny and their capabilities are vital for success at the industrial level for the production of high value bioproducts.

The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.