It is a microbial world we live in: microbes outnumber other organisms by several orders of magnitude, and they have great importance for the environment. However, environmental microbes are notoriously difficult to grow in the laboratory, and using culture independent techniques is necessary to expand our view. In this thesis, I apply metagenomics and single-cell genomics to environmental samples from ancient human remains and lakes. First, I used metagenomics to learn about bacteria from a Neanderthal’s bone and the gut of Ötzi, a frozen natural mummy. Both were exploratory studies where the main question was what kind of bacteria are present. I found out that Streptomyces dominated this particular Neanderthal fossil, and the DNA lacked the damage that is often seen in ancient samples. Ötzi's gut sample was dominated by Clostridia and fungi belonging to Basidiomycota. Second, ten single-cell amplified genomes of freshwater Alphaproteobacterium LD12 and three metagenomes from Swedish lakes were sequenced. Comparative metagenomics allowed hypothesizing about which functions are important for microbe proliferation in freshwater, pointing to osmoregulation and transport proteins and, possibly, to different strategies of metabolizing sugars. I also focused on SAR11 sister-groups in oceans and lakes. Phylogenies and sequence evolutionary distance estimates indicated the existence of microclusters within LD12, showing variation in abundance between lakes. The most striking difference was the relative amount of recombination compared to mutation, the estimated r/m ratio. SAR11 marine and their freshwater cousins are found at the opposite extremes of the r/m range, lowest and highest, respectively. The genetic background or sequence diversity did not explain the observed dramatic difference, so it is possibly connected to environmental adaptation or population dynamics. In addition, I have spent a substantial amount of effort benchmarking available metagenomic methods, for example fragment recruitment of metagenomes to reference genomes. In conclusion, my exploratory metagenomic studies have shed some light on the bacteria present in ancient human remains; comparative metagenomics has suggested the importance of substrate preference on functional differences between lakes and oceans; finally, single-cell genomes have allowed some insight into molecular evolutionary processes taking place in the freshwater LD12 bacterium.

Orientador: Eliana Gertrudes de Macedo Lemos / Banca: Gabriel Padilla / Banca: Rodrigo Matheus Pereira / Banca: Lucia Maria Carareto Alves / Banca: Jackson Antonio Marcondes de Souza / Resumo: Neste trabalho foi realizada a prospecção de novos genes de interesse biotecnológico em bibliotecas metagenômicas e em linhagens de Burkholderia através da técnica de reação em cadeia da polimerase. Nas bibliotecas metagenômicas, foi possível identificar genes que codificam álcool desidrogenase, oxirredutase, acil-CoA desidrogenase, luciferase, transportadores de membrana, thiolase, aminoglicosídeo fosfotransferase, desidrogenase de cadeia curta, proteínas repressoras TetR, enoil-CoA hidratase/isomerase, entre outras. Nas treze linhagens de Burkholderia testadas, seis apresentaram amplificação positiva para o gene de xilose isomerase. Estes genes foram completamente sequenciados e as sequências foram utilizadas em análises computacionais, que permitiram estabelecer a identidade entre as sequências e a dedução da função das proteínas baseado em similaridades. Foi realizada a medida do índice de adaptação de códons, com a finalidade de se encontrar um hospedeiro onde a expressão seja maximizada, baseando-se na presença de códons preferenciais e raros. Está análise mostrou que os genes encontrados possuem boas possibilidades de expressão em Escherichia coli, mas que podem apresentar uma taxa de expressão ineficiente em Saccharomyces cerevisiae. Foi realizado um teste de complementação gênica utilizando um dos genes descobertos e uma linhagem de Escherichia coli que possui o gene de xilose isomerase nocauteado. Os transformantes foram capazes de crescer em meio cuja única fonte de carbono era o açúcar xilose, mostrando que o gene é funcional. Estes genes serão utilizados futuramente em ensaios de expressão para caracterização da enzima / Abstract: In this work was carried out prospection for new genes of biotechnological interest in metagenomic libraries and strains of Burkholderia through the technique of polymerase chain reaction. In metagenomics libraries, we could identify genes encoding alcohol dehydrogenase, oxidoreductase, acyl-CoA dehydrogenase, luciferase, membrane transporters, thiolase, aminoglycoside phosphotransferase, short-chain dehydrogenase, TetR repressor protein, enoyl-CoA hydratase/isomerase, among others. Of the 13 strains of Burkholderia tested, 6 showed positive amplification for the xylose isomerase gene. The genes were completely sequenced and the sequences were used in computational analysis, which allowed to establish the identity between the sequences and deducing protein function based on similarities. We evaluated the codon adaptation index, with the aim of finding a host in which the expression is maximized, based on the presence of preferred codons and rare. This analysis showed that the genes found have good possibilities of expression in Escherichia coli, but that may have an inefficient rate of expression in Saccharomyces cerevisiae. We conducted a genetic complementation test using one of the discovered genes and one strain of Escherichia coli that has the xylose isomerase gene knocked out. Transformants were able to grow in a medium whose sole source of carbon was the sugar xylose, showing that the gene is functional. These genes will be used in expression assays for characterization of new enzymes / Doutor

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-21Bitstream added on 2014-06-13T20:44:15Z : No. of bitstreams: 1 schuch_v_dr_jabo.pdf: 940291 bytes, checksum: b12a082987505c45883c2a6c161d5861 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste trabalho foi realizada a prospecção de novos genes de interesse biotecnológico em bibliotecas metagenômicas e em linhagens de Burkholderia através da técnica de reação em cadeia da polimerase. Nas bibliotecas metagenômicas, foi possível identificar genes que codificam álcool desidrogenase, oxirredutase, acil-CoA desidrogenase, luciferase, transportadores de membrana, thiolase, aminoglicosídeo fosfotransferase, desidrogenase de cadeia curta, proteínas repressoras TetR, enoil-CoA hidratase/isomerase, entre outras. Nas treze linhagens de Burkholderia testadas, seis apresentaram amplificação positiva para o gene de xilose isomerase. Estes genes foram completamente sequenciados e as sequências foram utilizadas em análises computacionais, que permitiram estabelecer a identidade entre as sequências e a dedução da função das proteínas baseado em similaridades. Foi realizada a medida do índice de adaptação de códons, com a finalidade de se encontrar um hospedeiro onde a expressão seja maximizada, baseando-se na presença de códons preferenciais e raros. Está análise mostrou que os genes encontrados possuem boas possibilidades de expressão em Escherichia coli, mas que podem apresentar uma taxa de expressão ineficiente em Saccharomyces cerevisiae. Foi realizado um teste de complementação gênica utilizando um dos genes descobertos e uma linhagem de Escherichia coli que possui o gene de xilose isomerase nocauteado. Os transformantes foram capazes de crescer em meio cuja única fonte de carbono era o açúcar xilose, mostrando que o gene é funcional. Estes genes serão utilizados futuramente em ensaios de expressão para caracterização da enzima / In this work was carried out prospection for new genes of biotechnological interest in metagenomic libraries and strains of Burkholderia through the technique of polymerase chain reaction. In metagenomics libraries, we could identify genes encoding alcohol dehydrogenase, oxidoreductase, acyl-CoA dehydrogenase, luciferase, membrane transporters, thiolase, aminoglycoside phosphotransferase, short-chain dehydrogenase, TetR repressor protein, enoyl-CoA hydratase/isomerase, among others. Of the 13 strains of Burkholderia tested, 6 showed positive amplification for the xylose isomerase gene. The genes were completely sequenced and the sequences were used in computational analysis, which allowed to establish the identity between the sequences and deducing protein function based on similarities. We evaluated the codon adaptation index, with the aim of finding a host in which the expression is maximized, based on the presence of preferred codons and rare. This analysis showed that the genes found have good possibilities of expression in Escherichia coli, but that may have an inefficient rate of expression in Saccharomyces cerevisiae. We conducted a genetic complementation test using one of the discovered genes and one strain of Escherichia coli that has the xylose isomerase gene knocked out. Transformants were able to grow in a medium whose sole source of carbon was the sugar xylose, showing that the gene is functional. These genes will be used in expression assays for characterization of new enzymes

Cellulolytic microorganisms are considered to be key players in biorefinery, especially for the utilization of plant biomass. These organisms have been isolated from various environments. The Red Sea is one of the seas with high biodiversity and a unique environment, characterized by high water temperature and high salinity . However, there is little information regarding cellulases in Red Sea environments. The aim of the present study is to evaluate the Red Sea as a gene resource for microbial cellulase. I first surveyed microbial cellulases in the Red Sea using a method called metagenomes, and then investigated their abundance and diversity. My survey revealed that the Red Sea biome has a substantial abundance and a wide range of cellulase enzymes with substantial abundance, when compared with those in other environments. Next, I tried to isolate cellulase-active microorganisms from the Red Sea and I successfully obtained seven strains of four different taxonomic groups. These strains showed a similarity of 99% identity to Aspergillus ustus, 99% to Staphylococcus pasteuri, 99% to Bacillus aerius and 99% to Bacillus subtilis. The enzyme assay I conducted, revealed that these strains actually secreted active cellulases. These results suggest that the Red Sea environment can be, indeed, an excellent gene resource of microbial cellulases.

A Amazônia hospeda a maior floresta tropical do planeta, considerada um \"hotspot\" de biodiversidade. O aumento das ações antropogênicas nas ultimas décadas (desmatamento e queimadas) tem ocasionado perdas irreparáveis na diversidade biológica, contribuindo significativamente nas mudanças climáticas por meio de alterações dos fluxos hidrológicos e emissão excessiva de gases relacionados ao efeito estufa, dentre os quais o metano. Assim, na busca de uma melhor compreensão dessas alterações é de fundamental importância o estudo das comunidades microbianas associadas ao consumo do metano. Este trabalho teve como objetivo avaliar o impacto das alterações no uso da terra sobre a estrutura genômica e das comunidades bacterianas associadas a oxidação do metano presentes no solo, buscando a combinação de análises independentes (de comunidades) e dependentes de cultivo (enriquecimento e cultivo), juntamente com o acesso aos genomas montados de bactérias metanotróficas. Amostras provenientes de diferentes usos do solo e regiões da floresta amazônica foram coletadas, em sistemas de florestas primárias, florestas secundárias e pastagens na região de Santarém (PA), e florestas primárias e pastagens em Ariquemes (RO). Os solos coletados foram adicionados ao meio enriquecido com metano para estimular o metabolismo das bacterias metanotróficas. Tanto nas amostras originais como nas oriundas dos enriquecimentos, foram realizadas quantificações do gene pmoA pela técnica de PCR quantitativo (qPCR) e caracterização da comunidade total (bactérias e arquéias) e metanotróficas por meio do sequênciamento da região V4-V5 do gene 16S rRNA. Foram também realizadas análises de network e isolamentos de bactérias, na busca de uma maior compreensão sobre as correlações e associações microbianas das amostras. Por fim, o acesso ao metagenoma de amostras enriquecidas e montagens de genomas por metagenoma (MAG - Metagenome Assembled Genome), permitiu comparar a estrutura genômica do operon pmoCAB oriunda de diferentes solos. Os resultados mostraram que estrutura da comunidade total e metanotrófica se alteram com a conversão do solo florestal em pastagem, de maneiras distintas nas áreas amostradas, e com comportamento diferenciado ao longo dos enriquecimentos. O filo Acidobacteria mostrou-se característico de solos florestais, enquanto Actinobacteria, Firmicutes e Bacteoidetes de pastagens, ao passo que a abundância relativa e a riqueza de metanotróficos foi mais elevada em solos de pastagens. Também ocorrem alterações nas interações microbianas, mostrando que florestas possuem correlações mais estáveis que solos de pastagem, porém diferentes entre as áreas estudadas. Estas correlações também são notadas na associação de bactérias metanotróficas com outros grupos, como os gêneros Burkholderia, Pseudomonas, Flavisobacter e Cupriavidus. Em resumo, observa-se que há diferenciação no enriquecimento de bactérias metanotróficas nos diferentes solos e áreas, e os genomas montados indicam a possibilidade de metabolismos distintos. Estes dados contribuem para a compreensão das alterações, em diferentes níveis, da comunidade microbiana nos ambientes de conversão do uso do solo. / The Amazon hosts the largest rainforest on the planet, considered a biodiversity \"hotspot\". The increase in anthropogenic actions in the last decades (deforestation and burning) has caused irreparable losses in biological diversity, contributing significantly to climate change through changes in hydrological flows and excessive emissions of greenhouse gases, including methane. Thus, in the search for the best understanding of these changes, it is of fundamental importance to study the microbial communities associated with methane consumption. This work aimed to evaluate the impact of changes in land use on the genomic structure and bacterial communities associated with the oxidation of methane in the soil, seeking the combination of independent (community) and dependent analyzes (enrichment and growth), along with access to the genomes assembled from methanotrophic bacteria. Samples from different soil uses and regions of the Amazon rainforest were collected, such as primary forests, secondary forests and pastures in the Santarém region (PA) and primary forests and pastures in Ariquemes (RO). The collected soils were added to the methane-enriched medium to stimulate the metabolism of the methanotrophic bacteria. In both the original and the enriched samples, were performed quantifications of the pmoA gene by the quantitative PCR technique (qPCR) and characterization of the total community (bacteria and archaea) and methanotrophic by the sequencing of the V4-V5 region of the 16S rRNA gene. Network analyzes and bacterial isolations were also carried out in order to obtain a better understanding of the microbial correlations and associations of the samples. Finally, the access to the metagenome of enriched samples and genome assemblies by metagenome (MAG - Metagenome Assembled Genome) allowed comparing the genomic structure of the pmoCAB operon from different soils. The results showed that the structure of the total and methanotrophic community analyzed changes with the conversion of the forest soil to pasture, in different ways in the sampled areas and in enriched soils. The phyla Acidobacteria showed to be characteristic of forest soils, while Actinobacteria, Firmicutes, and Bacteroides of pastures and the relative abundance and richness of methanotrophic are higher in pastures soils. The changes were observed for microbial interactions, showing that forests have more stable correlations than pasture soils, but different between the areas studied. These correlations are also noted in the association of methanotrophic bacteria with other groups, such as the genera Burkholderia, Pseudomonas, Flavisobacter and Cupriavidus. In summary, it was observed differentiation in the enrichment of methanotrophic bacteria in the different soils and areas, and the assembled genomes indicate the possibility of different metabolisms. These data contribute to the understanding of the changes, at different levels, of the microbial community in the land use conversion environments.

O atual cenário mundial das mudanças climáticas, somado ao aquecimento global e ao aumento das áreas em processo de desertificação tem impactado diretamente nos padrões de produção agrícola. A Caatinga é um bioma que só ocorre no Brasil e possui um clima semiárido, quente e de baixa pluviosidade, sendo que na estação seca a temperatura do solo pode chegar até 60ºC. A Caatinga apresenta uma grande riqueza de ambientes e espécies, e boa parte dessa diversidade não é encontrada em nenhum outro bioma. Uma característica muito peculiar da Caatinga é a existência de duas estações bem contrastantes durante o ano, o inverno caracterizado por ser a estação da chuva e o verão a época da seca. A vegetação é composta por Euforbiáceas, Bromeliáceas e Cactáceas, dentre as quais destacam-se o Cereus jamacaru (mandacaru), Pilosocereus gounellei (xique-xique) e Melocactus sp. (cabeça-de-frade). O mandacaru planta que sobrevive às altas temperaturas e baixa disponibilidade de água da Caatinga possui adaptações morfológicas estruturais que contribuem para a sobrevivência da mesma. Além dessas adaptações a comunidade microbiana da rizosfera foi estudada para descobrir quais micro-organismos presentes nesse ambiente auxiliam na manutenção do hospedeiro frente a essas condições adversas. Assim como, quais grupos e funções são mais abundantes nessas condições. Nesse estudo foi feito o sequenciamento parcial do gene 16S rRNA e do DNA total da rizosfera de mandacaru. A comunidade bacteriana foi bem representada pelos filos Actinobacteria, Proteobacteria e Acidobacteria, sendo que o filo Actinobacteria foi mais abundante na seca de acordo com o sequenciamento metagenômico e o filo Acidobacteria foi mais abundante no período de chuva. Em geral o sequenciamento do gene 16S rRNA, indicou que Actinobacteria e Proteobacteria são os filos mais abundantes e os genes relacionados às funções de resistência a doenças foram mais abundantes na estação seca, enquanto genes relacionados ao metabolismo do nitrogênio foram mais abundantes durante o período chuvoso, revelando assim, um pouco do potencial que o microbioma da rizosfera de mandacaru possui para auxiliar a planta hospedeira. / The present world scenario of climate change, global warming and the increase in areas undergoing desertification, have directly impacted on current patterns of agricultural crop production. The Caatinga is a specific Brazilian biome because of its semi-arid climate, hot and low rainfall, and the temperature that reaches the 60°C in the dry season. The Caatinga has a huge biodiversity and much of its diversity is not found in any other biome. A peculiar characteristic of the Caatinga biome is the occurrence of two very contrasting seasons during the year, the winter which is characterized by a rainy season and summer the dry season. The vegetation is composed by Euphorbiaceae , Bromeliaceae and Cactaceae, represented by Cereus jamacaru (Mandacaru) Pilosocereus gounellei (xique-xique) and Melocactus sp. (head-to-brother). Mandacaru is the plant that can survive through the specifics climate conditions of the Caatinga biome such as high temperatures and low water availability and this is probably due to some structural and morphological adaptations that contribute to its survival. Therefore, we assessed which microorganisms are associated with the plant rhizosphere, and which microbial groups contribute to the maintenance of the host throughout these adverse conditions. Also, we identified which are the most abundant microbial groups in these conditions and which microbial functions are more abundant in both evaluated seasons. Thus the present study assessed the mandacaru rhizosphere microbiome through a partial 16S rRNA gene sequencing and metagenomic sequencing. The bacterial community was well represented by the phyla Actinobacteria, Proteobacteria and Acidobacteria. The Actinobacteria was the most abundant microbial phyla in the dry season according to shotgun sequencing while the Acidobacteria was the most abundant microbial phyla in the rainy season. Overall, the 16S rRNA sequencing indicated that Actinobacteria and Proteobacteria were the most abundant groups and additionally, and genes related to disease resistance functions were more abundant in the dry season. Genes related to nitrogen metabolism were more abundant during the rainy season revealing some of the potential traits that the mandacaru can explore from its microbiome.

O atual cenário mundial das mudanças climáticas, somado ao aquecimento global e ao aumento das áreas em processo de desertificação tem impactado diretamente nos padrões de produção agrícola. A Caatinga é um bioma que só ocorre no Brasil e possui um clima semiárido, quente e de baixa pluviosidade, sendo que na estação seca a temperatura do solo pode chegar até 60ºC. A Caatinga apresenta uma grande riqueza de ambientes e espécies, e boa parte dessa diversidade não é encontrada em nenhum outro bioma. Uma característica muito peculiar da Caatinga é a existência de duas estações bem contrastantes durante o ano, o inverno caracterizado por ser a estação da chuva e o verão a época da seca. A vegetação é composta por Euforbiáceas, Bromeliáceas e Cactáceas, dentre as quais destacam-se o Cereus jamacaru (mandacaru), Pilosocereus gounellei (xique-xique) e Melocactus sp. (cabeça-de-frade). O mandacaru planta que sobrevive às altas temperaturas e baixa disponibilidade de água da Caatinga possui adaptações morfológicas estruturais que contribuem para a sobrevivência da mesma. Além dessas adaptações a comunidade microbiana da rizosfera foi estudada para descobrir quais micro-organismos presentes nesse ambiente auxiliam na manutenção do hospedeiro frente a essas condições adversas. Assim como, quais grupos e funções são mais abundantes nessas condições. Nesse estudo foi feito o sequenciamento parcial do gene 16S rRNA e do DNA total da rizosfera de mandacaru. A comunidade bacteriana foi bem representada pelos filos Actinobacteria, Proteobacteria e Acidobacteria, sendo que o filo Actinobacteria foi mais abundante na seca de acordo com o sequenciamento metagenômico e o filo Acidobacteria foi mais abundante no período de chuva. Em geral o sequenciamento do gene 16S rRNA, indicou que Actinobacteria e Proteobacteria são os filos mais abundantes e os genes relacionados às funções de resistência a doenças foram mais abundantes na estação seca, enquanto genes relacionados ao metabolismo do nitrogênio foram mais abundantes durante o período chuvoso, revelando assim, um pouco do potencial que o microbioma da rizosfera de mandacaru possui para auxiliar a planta hospedeira. / The present world scenario of climate change, global warming and the increase in areas undergoing desertification, have directly impacted on current patterns of agricultural crop production. The Caatinga is a specific Brazilian biome because of its semi-arid climate, hot and low rainfall, and the temperature that reaches the 60°C in the dry season. The Caatinga has a huge biodiversity and much of its diversity is not found in any other biome. A peculiar characteristic of the Caatinga biome is the occurrence of two very contrasting seasons during the year, the winter which is characterized by a rainy season and summer the dry season. The vegetation is composed by Euphorbiaceae , Bromeliaceae and Cactaceae, represented by Cereus jamacaru (Mandacaru) Pilosocereus gounellei (xique-xique) and Melocactus sp. (head-to-brother). Mandacaru is the plant that can survive through the specifics climate conditions of the Caatinga biome such as high temperatures and low water availability and this is probably due to some structural and morphological adaptations that contribute to its survival. Therefore, we assessed which microorganisms are associated with the plant rhizosphere, and which microbial groups contribute to the maintenance of the host throughout these adverse conditions. Also, we identified which are the most abundant microbial groups in these conditions and which microbial functions are more abundant in both evaluated seasons. Thus the present study assessed the mandacaru rhizosphere microbiome through a partial 16S rRNA gene sequencing and metagenomic sequencing. The bacterial community was well represented by the phyla Actinobacteria, Proteobacteria and Acidobacteria. The Actinobacteria was the most abundant microbial phyla in the dry season according to shotgun sequencing while the Acidobacteria was the most abundant microbial phyla in the rainy season. Overall, the 16S rRNA sequencing indicated that Actinobacteria and Proteobacteria were the most abundant groups and additionally, and genes related to disease resistance functions were more abundant in the dry season. Genes related to nitrogen metabolism were more abundant during the rainy season revealing some of the potential traits that the mandacaru can explore from its microbiome.

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

abstract: This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2013

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.