Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:34:33Z No. of bitstreams: 1 HenriqueCesarTeixeiraVerasDissertacao2013.pdf: 26147875 bytes, checksum: 3014f6ec0d791f46e4e1ee8c49894c2f (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:35:09Z (GMT) No. of bitstreams: 1 HenriqueCesarTeixeiraVerasDissertacao2013.pdf: 26147875 bytes, checksum: 3014f6ec0d791f46e4e1ee8c49894c2f (MD5) / Made available in DSpace on 2018-11-07T17:35:09Z (GMT). No. of bitstreams: 1 HenriqueCesarTeixeiraVerasDissertacao2013.pdf: 26147875 bytes, checksum: 3014f6ec0d791f46e4e1ee8c49894c2f (MD5) Previous issue date: 2013-09-06 / Humans live in constant association with microorganims. The amount of microorganims present in the human body exceeds our own cell number. That community of microorganisms has deep influence in health and disease. The use of high-throughput DNA sequencing technologies and culture independent approaches have been enlarging the understanding concerning the communities of microorganisms and the association of these with the host. The human gastrointestinal tract contains one of the most complex bacterial communities. It was proposed recently that the microbiome can be classified in three enterotypes. In our study, we used data metagenomics quantitative search to identify phylogenetic patterns in the intestinal microbiome to develop prediction models for the enterotypes. To reach this aim, statistical tests were applied to the data regarding abundance of bacteria in level taxonomic corresponding to genus. We identified genus significantly with the abundance different and important correlations. Besides the ratio among genus to be used as parameter bioindicator of the respectives enterotypes. Through the logistic regression test we identified that the prediction model for ET1 was influenced significantly by the ratio of Bacteroides / (Prevotella + Ruminococcus). In the model for prediction of ET2, it was the ratio of Prevotella / Bacteroides with such as characteristic significance. And for the model of ET3, we identified the ratio of (Akkermansia + Alistipes) / (Bacteroides + Prevotella) as significant parameter. These models were assessed against two groups of independent data and associated with the value of cut-off 5%; 20% and 95% respectively. Besides the value of cutoff for each models, the crossed validation allowed the association of the model with the measures of PPV for ET1, specificity for ET2 and PNV for ET3. We propose the experimental validation of these models for the qPCR technique. And with that methodology established, it would be possible to do the diagnosis of the enterotype individually. / Humanos vivem em constante associação com microrganismos. A quantidade de microrganismos presentes no corpo humano ultrapassa o nosso próprio número de células. Essa comunidade de microrganismos tem profunda influência na saúde e doenças. As tecnologias de sequenciamento de DNA de alta capacidade e abordagens moleculares independente de cultura têm ampliado a compreensão acerca das comunidades de microrganismos e a associação destes com o hospedeiro. O trato gastrointestinal humano abriga uma das mais complexas comunidades bacterianas. Foi proposto recentemente que o microbioma pode ser categorizado em três enterotipos. No nosso estudo, utilizamos dados metagenômicos quantitativos buncando identificar padrões filogenéticos no microbioma intestinal para desenvolver modelos de predição para os enterotipos. Para alcançar este objetivo, testes estatísticos foram aplicados aos dados referente a abundância de bactérias em nível taxonômico correspondente a gênero. Identificamos gêneros com a abundância significativamente diferente e correlações importantes. Além da razão entre gêneros para ser utilizada como parâmetro bioindicativo dos respectivos enterotipos. Através do teste de regressão logística identificamos que o modelo de predição para o ET1 foi influenciado significativamente pela razão de Bacteroides / (Prevotella + Ruminococcus). No modelo para predição do ET2, foi a razão de Prevotella / Bacteroides que apresentou significância. E para o modelo do ET3, identificamos a razão de (Akkermansia + Alistipes) / (Bacteroides + Prevotella) como parâmetro significativo. Estes modelos foram avaliados contra dois conjuntos de dados independentes e associados com o valor de cut-off 5%; 20% e; 95% respectivamente. Além do valor de cut-off para cada modelos, a validação cruzada permitiu a associação do modelo com as medidas de PPV para o ET1, especificidade para o ET2 e PNV para o ET3. Propomos a validação experimental destes modelos pela técnica de qPCR. E com essa metodologia estabelecida, seria possível fazer o diagnóstico do enterotipo individualmente.

A crescente resistência dos micro-organismos patogênicos aos fármacos já existentes gera intensa demanda por novos agentes terapêuticos. Em contrapartida, a eficiência na descoberta de compostos com novas estruturas químicas diminuiu nos últimos anos. Tendo em vista a vasta biodiversidade existente de micro-organismos, a redução da eficiência na descoberta de novos produtos naturais não indica que todos compostos existentes já foram descritos, mas sim que as metodologias para isolamento dos mesmos devem ser aperfeiçoadas e diversificadas, e novos nichos devem ser explorados. Esta tese compreende três capítulos, que trazem abordagens que podem ser utilizadas na busca por produtos naturais. O capítulo 1 aborda a aplicação de conhecimentos de biologia molecular na pesquisa de produtos naturais, através da metagenômica. O capítulo 2 aborda o conceito de química ecológica para estimular o metabolismo secundário, através da utilização de interações microbianas. O capítulo 3 aborda o uso de genome mining para entender a capacidade metabólica de uma linhagem bacteriana, bem como o uso de variações nos parâmetros da cultura para alterar o metabolismo desta. Metagenômica: a triagem anti-parasitária das bibliotecas metagenômicas detectou clones bioativos contra Leishmania major. As análises químicas e biológicas das culturas destes clones não permitiram a identificação dos compostos responsáveis pelas atividades observadas. Esta abordagem apresenta grandes desafios técnicos e tem passado por ajustes envolvendo sequenciamento e bioinformática para aumentar a taxa de sucesso em seu uso. Interações microbianas: esta metodologia mostrou-se promissora para a busca por compostos bioativos, tendo em vista que diversos pares exibiram nova atividade antibiótica, corroborando a hipótese que interações microbianas podem levar à expressão diferenciada do metabolismo secundário. Foi escolhida para caracterização química e biológica a interação entre uma actinobactéria rara (Krasilnikovia sp. T082) e uma actinobactéria endossimbionte de besouro (Streptomyces sp. SPB78). Esta interação é robusta e estimula a biossíntese de um antibiótico polar capaz de inibir o crescimento de uma bactéria multirresistente. Diversas técnicas foram testadas para o isolamento do composto indutor e do antibiótico induzido. Este processo foi desafiador devido ao caráter polar de ambos compostos e pelo fato da atividade antibiótica ser instável. Foi demonstrado que o antibiótico induzido é capaz de inibir o crescimento do micro-organismo indutor, sugerindo importância ecológica deste composto. A utilização desta abordagem metodológica permitiu a identificação de uma linhagem pertencente a um gênero de actinobactéria rara que nunca teve seu metabolismo secundário estudado (Krasilnikovia). Isto foi realizado através da investigação de seu genoma e também através do isolamento de compostos produzidos por esta linhagem. Esta linhagem demonstrou potencial para a produção de metabólitos secundários, apresentando pelo menos 21 potenciais clusters gênicos biossintéticos detectados pelo antiSMASH em seu genoma. Esta linhagem foi cultivada em dois meios de cultura (ISP2 e TSB) e diferentes metodologias de extração foram empregadas. O metabolismo secundário desta linhagem é expresso de maneira diferente de acordo com a metodologia de cultivo, evidenciando a importância da variação da composição do meio de cultura para o acesso da real capacidade metabólica de microorganismos. Foram identificadas algumas dicetopiperazinas, que são conhecidas por seu amplo espectro de atividades biológicas, e três compostos pertencentes a uma classe de peptídeos nãoribossomais não usuais com atividade antibiótica, que possuem estrutura química complexa e incomum para produtos naturais / Increasing drug resistance among microbial pathogens is a public health threat; therefore, new antibiotics are needed. On the other hand, the rate of discovering new compounds has diminished. Considering the wide biodiversity of microorganisms, reduced efficiency on the discovery of new natural products does not indicate that all existing compounds have been described, but that methods for isolation should be improved and diversified and new niches should be explored. This thesis comprises three chapters that demonstrate approaches that can be used in the search for natural products. Chapter 1 demonstrates the application of molecular biology tools on the search for natural products through metagenomics. Chapter 2 discusses the concept of chemical ecology for the stimulation of secondary metabolism by the use of microbial interactions. Chapter 3 discusses the use of genome mining to understand the metabolic capacity of a bacterial strain as well as the use of different culture parameters to alter bacterial metabolism. The anti-parasitic metagenomic screening on metagenomic libraries detected bioactive clones against L. major. Chemical and biological analysis of cultures of these clones did not permit identifying the compounds responsible for the observed activities. This approach faces diverse technical challenges and is currently being improved by the use of sequencing and bioinformatics analysis in order to increase its hit rate. Microbial interactions: this approach has shown to be promising in the search for bioactive compounds, considering that several pairs exhibited new antibiotic activity, supporting the hypothesis that microbial interactions can stimulate differential expression of secondary metabolism. It was chosen for chemical and biological characterization the interaction between a rare actinobacteria (Krasilnikovia sp. T082), which belongs to a genus that its secondary metabolism has not yet been studied in the literature, and an endosymbiont actinobacteria of beetle (Streptomyces sp. SPB78). This interaction is robust and stimulates the biosynthesis of a polar antibiotic capable of inhibiting the growth of multi-resistant bacteria. Several techniques have been tested for the isolation process of the inducer compound and the induced antibiotic. This process has been particularly challenging due to the polar character of both compounds, and because the antibiotic activity is unstable. It has been shown that the induced antibiotic is capable of inhibiting the growth of the inducer microorganism, suggesting an ecological importance of this compound. The use of this co-culture approach led to the identification of a strain belonging to a rare actinobacteria genus whose secondary metabolism has never been studied before (Krasilnikovia). This was accomplished through sequencing and analysis of its genome and also by isolating compounds produced by this strain (chapter 3). This strain has shown great potential for the production of secondary metabolites, exhibiting at least 21 biosynthetic gene clusters detected by antiSMASH in its genome, and only four of them showed high similarity to any known gene cluster. This strain was cultured in two different culture media (ISP2 and TSB), and different methods of extraction were used. The secondary metabolism of this strain is expressed differently according to the method of cultivation, showing the importance of variation in the composition of the culture medium to access the actual metabolic capacity of microorganisms. Some diketopiperazine were isolated, which are known for their wide spectrum of biological activities, and also three compounds belonging to a class of non-ribosomal peptides known for their high bioactivity, which have complex and unusual chemical structures for natural products

Cubatão, o maior pólo industrial da américa latina também já foi uma das cidades mais poluídas do mundo. Os 30 anos de intensa atividade industrial vêm pressionando o meio ambiente com substâncias tóxicas e afetando gravemente a saúde da população. Dentre as substâncias contaminantes mais importantes da região estão os derivados de petróleo como o benzeno, tolueno, etilbenzeno e xilenos. Conhecidos como BTEX, eles são produzidos e utilizados em larga escala e a contaminação ocorre frequentemente através de vazamentos. Nos solos, devido à sua solubilidade em água, essas substâncias podem se espalhar por longas distâncias a partir do ponto afetado contaminando locais distantes. Já foi comprovada a capacidade de micro-organismos de sobreviver e até utilizar BTEX como fonte de carbono. Os micro-organismos adaptados catabolizam os contaminantes transformando-os em substâncias menos tóxicas e até mesmo eliminando-os do ambiente, capacidade de grande interesse econômico e ambiental. Nessa linha, nossa proposta visa o estudo das comunidades microbianas de solos afetados e não afetados por BTEX. Para isso foi utilizada a metagenômica como abordagem de estudo identificando-se diferenças qualitativas e quantitativas nas estruturas microbianas de três diferentes locais do município de Cubatão, sendo um deles afetado diretamente por BTEX. Pelo método utilizado e aqui desenvolvido, foi possível identificar um panorama metabólico geral identificando-se genes relevantes e o potencial de degradação de hidrocarbonetos aromáticos de micro-organismos conhecidos e desconhecidos, revelando melhor o potencial metabólico dos solos identificados. Os resultados apresentados podem contribuir para um melhor entendimento da dinâmica in situ de uma comunidade microbiana afetada por BTEX assim como melhorar o conhecimento sobre a comunidade microbiana de um local altamente impactado como Cubatão. / Cubatão is the largest industrial site in Latin America and was in the past one of the most polluted cities in the world. 30 years of intense industrial activity has pushed environmental limits with toxic substances and has severely affected the inhabitants\' health. Among the contaminants found in the region, the petroleum derivatives benzene, toluene, ethylbenzene and xylenes are the most important. Known collectively as BTEX, they are produced and used at a large scale and contamination frequently occurs. Because it is highly soluble in water, when in soil BTEX can spread long distances from the original contamination site, thus affecting large areas. Some microorganisms are known to live in contaminated environments and use contaminants such as BTEX as a unique carbon source for energy production. They catabolize contaminants into less dangerous products or even eliminate them from environment, a feature which has great commercial and environmental interest. We therefore compared the microbial communities in soils which were affected and un-affected by BTEX contamination. To this end, we used a metagenomics approach and developed a comparison method to identify microorganisms and degradation potential of soils studied. We found qualitative and quantitative differences in microbial structures from three different sites in Cubatão County, one of which is contaminated with BTEX. We constructed a metabolic overview identifying important genes, degradation potential and microorganisms related to BTEX degradation. The results presented here could contribute to understanding the in situ dynamics of a BTEX affected microbial community as well as improving our knowledge of the microbial community of Cubatão, a highly environmentally impacted place.

Viral sequence data has great potential for answering questions about the epidemiological dynamics and evolution of viruses. Classical approaches have sought amino acid changes that alter pathogenesis or transmissibility by influencing a virus's ability to enter or replicate within cells. However, this approach rarely recognises the fundamental impact of heterogeneous host contact structures and existing immunological responses on viral transmission. This thesis draws heavily on ecological and immunological concepts to explore the epidemiological dynamics, diversity and evolution of viruses using molecular sequence data. A number of different research approaches and study systems are used in this thesis. I begin by describing a novel polyomavirus in a European badger, and apply phylogenetic techniques to analyze the evolutionary history of the Polyomaviridae. I subsequently describe a large metaviromic study in a population of wild mute swans, for which host demographic data are available. I describe nine new viral species and test whether age and season are associated with differences in abundance and prevalence of different viral taxonomic groups. The study highlights the potential of metaviromics for investigating viral epidemiological dynamics in natural populations. Influenza A viruses of avian origin (AIV) threaten human and animal health. Using phylogeographic methods, I reconstruct the spatial spread of an H5N8 virus at a regional scale, and investigate how bird density and migration shaped this dispersal. Despite the importance of acquisition of humoral immunity to different strains throughout the lifespan of wild birds for epidemiological dynamics, this topic is poorly understood. I assess the accumulation of immune responses to AIV with age in mute swans. I consider how ecological factors, including age-structured immunity, might have affected the epidemiology of an H5N8 outbreak in the population.

Les virus sont omniprésents dans la biosphère et infectent vraisemblablement l'ensemble des êtres vivants. Au sein des écosystèmes, ils ont ainsi un impact sur la diversité des populations microbiennes, l'évolution des génomes de ces populations, et directement ou indirectement sur les cycles biogéochimiques majeurs. Leur caractère protéiforme et l'absence de marqueur unique (tant génétique que physique) font toutefois de l'exploration de la diversité virale une tâche complexe, de telle sorte que nos connaissances sur ces communautés virales environnementales sont encore très limitées. La métagénomique, ou séquençage massif et aléatoire de fragments nucléotidiques extraits d'un prélèvement, offre un point de vue unique sur les génomes viraux. Ce type d'approche, récemment développé, a ainsi mis en évidence la richesse extraordinaire des populations virales environnementales, tant du point de vue des gènes que des génotypes. C'est dans ce cadre de l'étude des communautés virales de l'environnement par métagénomique que se sont inscrits les travaux de cette thèse, organisée autour de quatre axes principaux : • Le développement de nouvelles méthodes d'analyses adaptées aux spécificités des génomes et métagénomes viraux par la mise en place du serveur web Metavir, premier serveur dédié à l'analyse des viromes. Proposant aujourd'hui un ensemble cohérent d'outils pour différents types de viromes, Metavir compte plus de 300 utilisateurs pour plus de 2000 viromes analysés. • Le potentiel fonctionnel des génomes viraux a pu être approché par l'étude conjointe d'un ensemble de viromes. Après une analyse rigoureuse des contaminations potentielles, nous avons pu confirmer que les génomes viraux comprenaient un ensemble limité mais non négligeable de gènes associés au métabolisme cellulaire. La plupart des virus agissent ainsi certainement directement sur le métabolisme de la cellule hôte durant l'infection. • La prépondérance des paramètres environnementaux, et particulièrement de la salinité, en tant que facteurs structurant les communautés virales aquatiques a également pu être mise en avant. La distance géographique entre prélèvements semble n'avoir qu'une influence secondaire, confirmant la capacité importante de dispersion des capsides virales. Une adaptation locale semble toutefois exister dans certains cas, notamment en cas de compétition importante entre les résistances développées par les hôtes et les capacités d'infection des virus. • Enfin, différentes familles de petits virus à ADN simple brin ont pu être caractérisées par une méta-analyse de viromes. Leur apparente simplicité a ainsi révélé des mécanismes d'évolution plus complexes que prévus, impliquant différents cycles et capacités de transfert de gènes jusqu’ici plutôt considérés comme l'apanage des virus à ADN double brin, et remettant en cause les séparations admises entre les différents groupes de virus sur la base de la nature de leur génome. En permettant une étude depuis l'échelle de la communauté jusqu'à des génotypes spécifiques, les viromes constituent des outils de choix pour caractériser la diversité virale, appréhender les différents facteurs régulant ces communautés, et ainsi mieux comprendre la place des virus dans la biosphère. De plus, ces études ont confirmé l'existence d'interactions étroites entre virus et organismes cellulaires, ces interactions semblant nombreuses, multiples dans leurs natures et conséquences, et présentes tout au long de l'histoire du vivant. Ces nouvelles connaissances apportées par l'analyse de viromes permettent donc d'aborder certaines questions fondamentales concernant l'origine des grandes innovations évolutives ou le fonctionnement global des écosystèmes. / Viruses likely infect every organism on Earth (in some cases even other viruses!), and represent vast morphological and genetic diversity. Not surprisingly given their numerical dominance, viruses significantly impact ecosystems through regulating microbial populations, driving major biogeochemical cycles, and shaping the evolution of hosts genomes. However, our understanding of viruses in nature is primitive, especially because the majority of environmental viral genomes remains uncharacterized. Metagenomics (i.e. random and massive sequencing of genomic fragments isolated from a sample) applied to encapsidated genetic templates provides a unique perspective on the viral pangenome. The first viral metagenomes (or viromes) generated entire sets of new questions about viral diversity, especially concerning their genetic and species richness. This work was set within this frame of viral diversity study through metagenomics, and organized into four main themes : • The development of bioinformatics tools adapted to the specific features of viral genomes and metagenomes led to the release of Metavir, the first web server dedicated to virome analysis. Providing a comprehensive set of connected tools, Metavir has now been used by more than 300 users in the analysis of more than 2000 viromes. • The functions encoded within viral genomes were for the first time thoroughly examined, following a rigorous examination of a set of published viromes toward contamination by cellular DNA. A new picture of the viral functional potential could thus be drawn, which confirmed that the range of cellular functions encoded in viral genomes is wider than the one retrieved from the complete genomes currently available, though not as great as previously estimated. • The study of the aquatic viral metagenomes also revealed the importance of salinity in the distribution of viral communities across the globe. The ubiquitous distribution of most viral genotypes confirmed that viral particles seem to be able to move across any distance on Earth. Viruses are thus likely selected based on factors such as the presence of their host in the samples and the competition with other parasites, which can still drive local adaptations. • Finally, viromes were used to better characterize the diversity of different ssDNA viral families. Despite their small size and relative simplicity, these viruses were found to harbor unexpectedly complex cycles and evolutionary mechanisms, in particular a great potential of recombination and gene transfer. Overall, the new genomes assembled from viromes notably challenge the separation between viruses based on the nature of their genome. Eventually, as illustrated by these different works and analyses, viromes are unique and extremely powerful tool to assess and characterize viral genetic diversity. Moreover, considering the tight links between viral and cellular worlds, insights into the viral communities provided by metagenomics make it possible to address fundamental questions such as the origin of important evolutive innovations or the functioning of ecosystems, so that these results are of interest for the whole field of biology.

Os micro-organismos apresentam uma imensa diversidade genética e desempenham funções únicas e cruciais na manutenção de ecossistemas. Uma dessas funções é a produção de enzimas extracelulares, que ajudam na degradação da matéria orgânica e são cada vez mais procuradas e exploradas pela indústria. Essa propriedade aumenta a busca por enzimas que possam ser utilizadas nos diversos setores industriais com maior aproveitamento e baixo custo. A celulase pertence a essa classe de enzimas e é formada por um complexo multienzimático capaz de hidrolisar celulose por meio da quebra da ligação β,1-4. A partir dessa característica da celulase, foi realizada uma expressão heteróloga relacionada com a hidrólise da celulose em biblioteca metagenômica de solo da Caatinga. Foram realizados testes de produção enzimática por meio dos quais selecionamos os clones 283/A8 e 307/E11 como melhores produtores de endoglicanases. Com o objetivo de analisar a cinética de produção de celulases pelos clones, estes foram inoculados em diferentes fontes de celulose, pH e temperatura. A faixa ideal de pH foi 5,0 e de temperatura, 50º C, verificada para as enzimas Celulase Total, Endoglicanase e β-glicosidase. Quanto à termoestabilidade, as enzimas presentes mantiveram mais de 60% da atividade inicial após 2 horas de incubação a 50º C. O perfil de proteínas analisado por SDS-PAGE demonstrou que os clones secretam um conjunto de enzimas celulolíticas com 25 a 100 KDa, quando cultivado em farelo de trigo, e 30 a 60 KDa, quando cultivados em CMC. No ensaio de cromatografia para o clone 307/E11, foram selecionadas 5 frações que obtiveram melhores resultados na dosagem enzimática e testados frente ao pH e à temperatura. O resultado obtido foi que o complexo enzimático bruto, extraído do sobrenadante produzido pelo clone, possui melhor atividade frente ao pH e à temperatura do que as frações parcialmente purificadas. / Microorganisms are distinguished by a wide genetic diversity and they perform unique and crucial functions concerning the maintenance of ecosystems. One of those functions is the production of extracellular enzymes, which help in the degradation of organic matter and which are increasingly wanted and explored by industry. Such feature boosts the search of enzymes that can be exploited in several industrial sectors with improved full use and low cost. Cellulase belongs to such class of enzymes and it is composed of a multi-enzymatic complex which is able to hydrolyse cellulose through the breaking of the chemical bond β,1-4. Considering such attribute of the cellulase, a heterologous expression, related to cellulose hydrolysis in a metagenomic inventory of the soil of Caatinga, was realized. Tests of enzymatic production were performed, and through them, we could elect the clones 283/A8 and 307/E11 as the best endoglicanase producers. Those clones were inoculated in different cellulose sources, pH and temperature, so that we could analyse the kinetic of cellulase production between them. The ideal pH range was 5.0 and the ideal temperature range was 50º C (122º F), verified for the enzymes Total Cellulase, Endoglicanase and β-glucosidase. With regard to thermostability, the present enzymes kept more than 60% of the initial activity after 2 hours of incubation at 50º C (122º F). The proteins profile analysed with the help of SDS-PAGE proved that the clones secrete a group of cellulolytic enzymes with a weight average of 25 to 100 KDa, when cultivated in wheat bran, and of 30 to 60 KDa, when cultivated in CMC. Five fractions with the best results, regarding enzymatic dosage and tested before pH and temperature, were chosen by the chromatography research for the clone 307/E11. The achieved result proved that the raw enzymatic complex, extracted from the supernatant produced by the clone, develops a better activity before pH and temperature than the fractions partially purified.

Microbial biodegradation is the primary mechanism by which petrogenic hydrocarbons (PHCs) are removed from the environment. Though hydrocarbon biodegradation is widely studied in marine systems, knowledge of how it occurs in freshwater systems is still lacking. The Peace-Athabasca Delta (PAD), located in northeastern Alberta, is an ideal location to study microbial hydrocarbon degradation since it has a long history of exposure to PHCs. What’s more, these PHCs are predominately sourced from bituminous deposits and are therefore relevant to the Canadian Oil Sands Industry. This thesis investigated the genetic potential for hydrocarbon degradation of PHCs via metagenomic reconstruction of microbial communities in lakes of the Peace and Athabasca Deltas, as well as reference lakes in the nearby boreal uplands. In order to properly evaluate the microbial community and its potential for hydrocarbon degradation, a comprehensive analysis of PHCs (including n-alkanes, polycyclic aromatic compounds (PACs), and petroleum biomarkers of terpanes, hopanes, and steranes) was performed. PHC analysis showed that n-alkanes in lake sediments from all three regions were highly similar and predominately biogenic, while PAC composition was significantly different in each region. Restricted-drainage lakes of the Athabasca Delta had the highest concentrations of PACs from petrogenic sources. Closed-drainage lakes in the Peace Delta had lower concentrations of PACs that likely originated from a mixture of pyrogenic and petrogenic sources. Closed-drainage lakes in the boreal upland region had the lowest concentrations of PACs likely sourced from pyrogenic wood combustion with traces of petrogenic PACs, possibly from atmospheric deposition of dust. Petroleum biomarkers of terpanes, hopanes, and steranes were successfully used to identify the long-range fluvial, and possibly atmospheric, transport of bituminous compounds more than one hundred kilometers from their potential source. This validates the future use of these biomarkers in environmental forensics. Microbial communities in all three regions under study were highly diverse, and their composition was significantly different in both sediment and water. Targeted gene analysis identified a total of 3885 genes involved in the degradation of n-alkanes and PACs in sediment and water. The results show that organic carbon, nitrogen, and sulfur content, as well as PAC and short-chain alkane concentrations were important chemical predictors of change in degradation gene composition. Furthermore, genes for anaerobic degradation of PHCs were identified in syntrophic bacteria, methanogens, nitrate and sulfate reducers, demonstrating the potential for syntrophic hydrocarbon degradation in PAD lakes. Though this thesis confirms the genetic potential for hydrocarbon degradation in PAD and boreal upland lakes, further research is necessary to determine whether these microbial communities can actively degrade the PHCs present in these lakes.

The Upper Mississippi River (UMR) basin contributes over 50,000 metric tons of nitrogen (N) to the Gulf of Mexico each year, resulting in a “dead zone” inhospitable to aquatic life. Land-applied N (fertilizer) in the corn-belt is attributed with a majority of the N-load reaching the Gulf and is difficult to treat as run-off is considered a non-point source of pollution (i.e. not from a pipe). One solution to this “grand challenge” of intercepting N pollution is utilizing filter-feeding organisms native to the UMR. Freshwater mussel (order Unionidae) assemblages collectively filter over 14 billion gallons of water, remove tons of biomass from overlying water, and sequester tons of N each day. Our previous research showed mussel excretions increased the sediment porewater concentrations of ammonium by 160%, and indirectly increased nitrate and nitrite by 40%, presumably from microbial degradation of ammonium. In response, the goal of this research was to characterize how mussels influenced microbial communities (microbiome) to determine the fate of N in UMR sediment. First, we used qPCR and non-targeted amplicon sequencing within sediment layers to identify the N-cycling microbiome and characterized microbial community changes attributable to freshwater mussels. qPCR identified that anaerobic ammonium oxidizing (anammox) bacteria were increased by a factor of 2.2 at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced microbial species richness and diversity and indicated that sediment below mussels harbored distinct microbial communities. Furthermore, mussels increased the abundance of ammonia oxidizing bacteria (family Nitrosomonadaceae), nitrite oxidizing bacteria (genus Nitrospira), but decreased the abundance of ammonia oxidizing archaea (genus Candidatus Nitrososphaera), and microorganisms which couple denitrification with methane oxidation. These findings suggested that mussels may enhance microbial niches at the interface of oxic and anoxic conditions, presumably through excretion of N and burrowing activity. In response, we performed metagenomic shotgun sequencing to identify which genes of the microbiome were most impacted by mussels. We hypothesized that genes responsible for ammonia and nitrite oxidation would be greater in the sediment with mussel assemblages. We found the largest abundance of N-cycling genes were responsible for nitrate reduction and nitrite oxidation, which is corroborated by the high concentration of nitrates in UMR water. Linear discriminant analysis statistical analyses showed nitrification genes were most impacted by mussels, and this presented an opposing effect on genes responsible for producing nitrous oxide, a potent greenhouse gas. Further investigation showed an increased abundance of a novel organism capable of completely oxidizing ammonia to nitrate (Candidatus Nitrospira inopinata) and coexisted with metabolically flexible Nitrospira (sp. moscoviensis), likely enhancing both carbon and N-cycling. We demonstrated that native mussels harbor a unique niche for N-cycling microorganisms with large metabolic potentials to degrade mussel excretion products. Our findings suggest the ecosystem services of mussels extend beyond water filtration, and includes enhanced biogeochemical cycling of carbon, N, and reduces the potential for a potent microbially-produced greenhouse gas. Ultimately, this research could be used to advocate for mussel habitat restoration in the UMR to lessen the impacts of non-point pollution.

Western Oregon is home to native Rhododendron species and is the center for cultivated Rhododendron production in the United States. These Rhododendron spp. are known to be infected with fungal endophytes. However, the community structure of these endophytes in native and cultivated Rhododendron is poorly understood. Our study targeted the foliar fungal endophyte communities of two native Rhododendron spp. and two non-native commercially cultivated Rhododendron varieties. Parallel culturedependent (fungal isolation and identification based on ITS sequencing) and cultureindependent sequencing approaches (metagenomic sequencing of the ITS region using 454 pyrosequencing) were employed, and results provided evidence of distinctly different community structure in each host species. Additionally, results indicated higher diversity among cultivated and nursery grown Rhododendron. This suggests that the close proximity of the nurseries sampled to the forest environment allows exposure to two distinct sources of endophyte infection. Together, our results show the importance of host identity and environment in structuring the associated endophyte communities. / Graduation date: 2013

The application of second generation sequencing technology to the characterization of complex microbial communities has profoundly affected our appreciation of microbial diversity. The explosive growth of microbial sequence data has also necessitated advances in bioinformatic methods for profiling microbial communities. Data aggregation strategies should allow the relation of metagenomic sequence data to our understanding of microbial taxonomy, while also facilitating the discovery of novel taxa. For eukaryotes, a method has been established that links DNA sequences to the identification of organisms: DNA Barcoding. A similar approach has been developed for prokaryotes using target genic regions as markers for species identification and to profile communities. A key difference in these efforts is that within DNA barcoding there is a formalized framework for the evaluation of barcoding targets, whereas for prokaryotes the 16S rRNA gene target has become the de facto barcode without formal evaluation. Using the framework developed for evaluating DNA barcodes in eukaryotes, a study was undertaken to formally evaluate 16S rRNA and cpn60 as DNA barcodes for Bacteria. Both 16S rRNA and cpn60 were found to meet the criteria for DNA barcodes, with cpn60 a preferred barcode based on its superior resolution of closely related taxa. The high resolution of cpn60 enabled a method of sequence data aggregation through sequence assembly: microbial profiling using metagenomic assembly (mPUMA). The scoring of metagenomic assemblies in terms of sensitivity and specificity of the operational taxonomic units formed was used to evaluate and optimize the assembly of cpn60 barcodes. Using optimized parameters, mPUMA was demonstrated to faithfully reconstruct a synthetic community in terms of richness and abundance. To facilitate the use of mPUMA, a software package was developed and released under an open source license. The utility of mPUMA was further examined through the characterization of the epiphytic seed microbiomes of Triticum and Brassica species. A microbiome shared across both crop genera including fungi and bacteria was detected: a particularly important observation as it implies that seeds may serve as a vector for microbes that could include both pathogenic and beneficial organisms. The relative abundances of taxa identified by mPUMA were confirmed by qPCR for multiple cases of both fungal and bacterial taxa. By culturing isolates of both bacteria and fungi from the seed surfaces it was demonstrated that mPUMA faithfully assembled consensus sequences for OTUs that were 100% identical to isolated fungi and bacteria. Patterns observed in the relative abundances of the shared microbiome OTUs were used to generate the hypothesis that an Pantoea-like bacterium and an Alternaria-like fungus had an antagonistic relationship, since sequences corresponding to these organisms showed reciprocal abundance patterns on Triticum and Brassica seeds. Studies of the interactions of cultured isolates revealed fungistatic interactions that could account for their reciprocal abundances. These interactions could be directly relevant to plant health, given that Alternaria-like fungi are linked to grain spoilage in wheat, and diseases in canola. Taken together, results of this thesis demonstrate the superiority of the cpn60 universal target as a barcode for Bacteria, forming the basis for an assembly-based strategy for microbial profiling of bacterial and eukaryotic microbial communities that can lead to the discovery of novel taxa and microbial interactions.