Os microrganismos constituem dois terços da diversidade biológica na Terra, no entanto, muitos deles não podem ser cultivados por técnicas tradicionais. Portanto, o acesso a esta diversidade tem sido feita através da utilização de técnicas independentes de cultivo. Diante deste panorama, a metagenômica apresenta-se como uma alternativa, pois dispensa a necessidade de cultivo. Tal técnica possibilita inclusive a identificação e utilização do potencial metabólico destes organismos para o desenvolvimento de novos processos e produtos. Os polihidroxialcanoatos (PHAs) são poliésteres bacterianos, acumulados intracelularmente em forma de grânulos, cujas propriedades são similares a de alguns plásticos de origem petroquímica. O objetivo deste trabalho foi identificar e avaliar a diversidade de genes relacionados à produção de PHAs em bibliotecas metagenômicas de solo. A prospecção realizada resultou na identificação de clones contendo o gene phaC. De uma forma geral, pôde-se concluir que ainda há uma grande diversidade deste gene a ser descoberta no ambiente estudado. / Microorganisms constitute two third of the Earth\'s biological diversity, however, many of them cannot be cultured by standard techniques. Therefore, access to this diversity has been achieved through the use of culture-independent techniques. Facing this scenario, the metagenomic presents itself as an alternative, since it eliminates the need for cultivation. This technique also allows the identification and use of the metabolic pathways of these organisms to develop new processes and products. The polyhydroxyalkanoates (PHAs) are bacterial polyesters accumulated as granules, whose properties are similar to some plastics of petrochemical origin. The aim of this work was to identify and access the diversity of genes related to PHAs production in soil metagenomic libraries. The screening resulted in the identification of clones containing the phaC gene. In a general way, it was concluded that there is still a considerable diversity of this gene to be discovered in the study environment.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-11-23T13:00:29Z No. of bitstreams: 1 JoelmaMPL_DISSERT.pdf: 1767206 bytes, checksum: c5b6652a65db20c661f3ad0f021c09fe (MD5) / Made available in DSpace on 2017-11-23T13:00:30Z (GMT). No. of bitstreams: 1 JoelmaMPL_DISSERT.pdf: 1767206 bytes, checksum: c5b6652a65db20c661f3ad0f021c09fe (MD5) Previous issue date: 2017-07-21 / The coalho cheese is considered a cultural patrimony and has great social and economic importance for the northeastern Brazilian region. The evaluation of the microbiological conditions of this product becomes fundamental for the affirmation of food safety and investigation of the real characteristics that make this kind of cheese so peculiar. The objective of the study was to identify the microbial community of coalho cheese. Metagenomic target DNA was extracted from eight samples of coalho cheese, four of which were made from pasteurized milk and four from raw milk. The DNA of the samples was sequenced by the next generation sequencing technology using the 16S and 18S rDNA gene as the basis for the identification of the organisms. Sequencing analyzes revealed several prokaryotic pathogenic microorganisms such as Rothia dentocariosa, Elizabethkingia meningoseptica and Bacillus cereus, as well as bacterial genera belonging to the microbiota previously described as Lactobacillus, Lactococcus, Escherichia, Enterococcus. Several fungi, such as Candida tropicalis, Candida parapsilolis, Pichia membranifaciens, Tritirachium oryzae, Malassezia furfur and Kluyveromyces marxianus were identified in the eukaryotic research, as well as microorganisms that had not yet been described in rennet cheeses such as Vibrio rumoienses, Ruminococcus flavefaciens, Piscicoccus intestinalis and Dekkera bruxellensis / O queijo de coalho é considerado um patrimônio cultural e tem grande importância social e econômica para a região nordeste brasileira, por isso a avaliação das condições microbiológicas desse produto torna-se fundamental para a afirmação da segurança alimentar e investigação das reais características que fazem com que esse tipo de queijo apresente sabores, texturas e aromas tão peculiares. A partir disso, o objetivo do trabalho foi identificar a comunidade microbiana do queijo de coalho. Para isso, o DNA metagenômico do queijo foi extraído de oito amostras de queijo de coalho, sendo quatro feitas a partir do leite pasteurizado e quatro feitas a partir do leite cru. O DNA das amostras foi sequenciado pela tecnologia de próxima geração da Illumina utilizando como base para a identificação dos organismos o gene rDNA 16S e 18S. As análises do sequenciamento revelaram vários exemplares de microrganismos patógenos procarióticos como Rothia dentocariosa, Elizabethkingia meningoseptica e Bacillus cereus, assim como gêneros de bactérias próprias da sua microbiota previamente descrita como Lactobacillus, Lactococcus, Escherichia, Enterococcus. Na investigação dos eucarióticos foram identificados diversos fungos como a Candida tropicalis, Candida parapsilolis, Pichia membranifaciens, Tritirachium oryzae, Malassezia furfur e Kluyveromyces marxianus, além dos microrganismos que ainda não tinham sido descritos em queijos coalho como o Vibrio rumoienses, Ruminococcus flavefaciens, Piscicoccus intestinalis e Dekkera bruxellensis / 2017-11-23

Made available in DSpace on 2014-12-17T14:10:25Z (GMT). No. of bitstreams: 1 AbinadabeJM_DISSERT.pdf: 2202389 bytes, checksum: c6fb8bcc059666158ed3af8030f88987 (MD5) Previous issue date: 2012-07-19 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ? 118 bp) and 127.563 sequences (352 ? 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well / Atividades industriais, derramamentos de petr?leo e seus derivados, bem como a combust?o incompleta de combust?veis f?sseis t?m causado um grande ac?mulo de hidrocarbonetos no meio ambiente. O n?mero de microrganismos no planeta ? estimado em 1030, sendo os procariotos os mais abundantes. Eles colonizaram diversos ambientes durante milhares de anos, incluindo aqueles considerados extremos e representam uma fonte inexplorada de diversidade gen?tica e metab?lica com um grande potencial biotecnol?gico. Sabe-se que muitos microrganismos possuem vias metab?licas complexas atuando na biodegrada??o de hidrocarbonetos derivados de petr?leo e, em muitos ecossistemas, existe uma comunidade aut?ctone capaz de realizar essa fun??o. A metagen?mica tem revolucionado a Microbiologia permitindo o acesso ?s comunidades microbianas n?o cultiv?veis, sendo uma potente ferramenta para elucida??o de suas fun??es ecol?gicas, dos perfis metab?licos, bem como para identifica??o de novas biomol?culas. Assim, o presente estudo aplicou abordagens metagen?micas n?o apenas para sele??o funcional de genes envolvidos nos processos de biodegrada??o e biossurfacta??o de hidrocarbonetos derivados do petr?leo, mas tamb?m para descri??o da composi??o taxon?mica e metab?lica de dois metagenomas de microbiota aqu?tica. Foram analisadas 123.116 (365 ? 118 pb) e 127.563 sequ?ncias (352 ? 120 pb) dos metagenomas marinho e estuarino, respectivamente. Oito clones foram encontrados, sendo quatro envolvidos na biodegrada??o de petr?leo e quatro capazes de emulsificar querosene, indicando a habilidade de sintetizar biossurfactantes. Portanto, as an?lises metagen?micas realizadas foram eficientes n?o apenas na busca de bioprodutos de interesse biotecnol?gico como tamb?m na an?lise do perfil funcional e taxon?mico dos metagenomas estudados

Made available in DSpace on 2014-12-17T15:18:11Z (GMT). No. of bitstreams: 1 AmandaLSS.pdf: 2422936 bytes, checksum: d5a0f2bb2f42d87bacd7a1d2c3f5c6bc (MD5) Previous issue date: 2009-03-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Bacteria trom Shewanella and Geobacter ganera are the most studied iron-reducing microorganisms particularly due to their electron transport systems and contribution to some industrial and environmental problems, including steel corrosion, bioenergy and bioremediation of petroleum-impacted sites. The present study was focused in two ways: the first is an in silico comparative ecogenomic study of Shewanella spp. with sequenced genomes, and the second is an experimental metagenomic work to detect iron-reducing Shewanella through PCR-DGGE of a metabolic gene. The in silico study resulted in positive correIation between copy number of 16S rDNA and genome size in Shewanella spp., with clusters of rrn near lhe origin of replication. This way, the genus is inferred as opportunist. There are no compact genomes and their sequences length varied, ranging from 4306142 nt in S. amazonensis SB2B to 5935403 nt in S. woodyi ATCC 51908, without correIation to temperature range characteristic of each specie. Intragenomic 16S rDNA sequences possess little divergence, but reasonable to resuIt in different phyIogenetic trees, depending on the sequence that is chosen to compare. For moIecuIar detection of iron-reducing Shewanella, it is proposed the mtrB gene as new biomarker. because it codes to a fundamental protein at Fe (III)-reduction. The specific primers were designed and evaluated in silico and resulted in a fragment of 360 pb. In the second study, these primers were tested in a genomic sample from S. oneidensis MR-1, amplifying the expected region. After this successfuI resuIt, the primer set was used as a tool to assess the iron-reducing communities of ShewaneIla genus under an environmental stress, i.e. crude oil contamination in mangrove sediment in Rio Grande do Norte State (Brazil). The primers presented high specificity and the reactions performed resulted in one single band of ampIification in the metagenomic samples. The fingerprinting obtained at DGGE reveaIed temporal variation of Shewanella spp. in analyzed samples. The resuIts presented show the detection of a biotechnological important group of microorganisms, the iron-reducing Shewanella spp. using a metabolic gane as target. It is concluded there are eight or more 16S rDNA sequences in Shewanella genus, with little divergence among them that affects the phylogeny; the pair of primers designed to ampIify mtrB sequences is a viable alternative to detect iron-reducing ShewanelIa in metagenomic approaches; such bacteria are present in the mangrove sediment anaIyzed, with temporal variations in the samples. This is the first experimental study that screened the iron-reducing Shewanella genus in a metagenomic experiment of mangrove sediments subjected to oil contamination through a key metabolic gene / Bact?rias dos g?neros Shewanella e Geobacter s?o os microrganismos redutores de ferro mais estudados. Esse interesse ocorre particularmente devido aos seus sistemas de transporte de el?trons e contribui??o em alguns problemas industriais e ambientais, tais como corros?o de oIeodutos, bioenergia e biorremedia??o de locais contaminados com petr?leo. O presente estudo foi tocado em duas partes: a primeira ? um estudo ecogen?mico comparativo de ShewanelIa spp. com genomas seq?enciados, e a segunda ? um trabalho metagen?mico experimental para detectar Shewanella redutoras de ferro atrav?s de PCR-DGGE de um gene metab?lico. O estudo in silico resultou em correla??o positiva entre o n?mero de c?pias 16S rDNA e tamanho do genoma em ShewaneIla spp., com agrupamentos de rrn. pr?ximo ? origem de replica??o. Desta maneira, o g?nero ? inferido como oportunista. N?o existem genomas compactos e o tamanho de suas sequ?ncias variam de 4306142 nt em S. amazonensis SB2B at? 5935403 nt em S. woodyi ATCC 51908, sem correla??o com a faixa de temperatura caracter?stica de cada esp?cie. Sequ?ncias intragen?micas de 16S rDNA possuem pouca diverg?ncia. mas razo?vel para resultar em diferentes ?rvores filogen?ticas. dependendo da sequ?ncia que ? escolhida para compara??o. Para a detec??o moIecuIar de ShewanelIa redutoras de ferro, ? proposto o gene mtrB como um novo biomarcador, por ser codante de uma prote?na fundamental na redu?ao de Fe (III). Os primers espec?ficos foram desenhados e avaliados in silico e resultou em um fragmento de 360 pb. No segundo estudo, esses primers foram testados em . amostra gen?mica de S. oneidensis MR-1, amplificando a regi?o esperada. Depois desse resultado favor?vel, o par de primers foi utilizado como ferramenta para acessar as comunidades redutoras de ferro do g?nero ShewanelIa sob um stress ambiental - contamina??o com ?leo cru em sedimento de mangue, no Estado do Grande do Norte (Brasil). Os primers apresentaram alta especificidade e as rea??es resultaram em banda ?nica de amplifica??o das amostras metagen?micas. O perfil obtido no DGGE revelou varia??o temporal de ShewanelIa spp. nas amostras analisadas. Os resultados apresentados mostram a defec??o de um grupo de microrganismos biotecnologicamente importante. ShewaneIla spp. redutoras de ferro, usando um gene metab?lico como alvo. Concluiu-se que existem oito ou mais sequ?ncias 16S rDNA no g?nero ShewaneIla, com pouca diverg?ncia entre elas que afetam a filogenia; o par de primers desenhados para amplificar sequ?ncias mtrB ? uma alternativa vi?vel para detectar Shewanella redutoras de ferro em abordagens metagen?micas; tais bact?rias est?o presentes no sedimento de mangue analisado, com varia??es temporais nas amostras. Este ? o primeiro estudo experimental que examina Shewanella redutoras de ferro em um experimento metagen?mico de sedimento de mangue submetido a contamina??o por ?leo atrav?s de um gene metab?lico

O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos. / In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.

Au cours des dernières années, de nombreuses études ont porté sur les relations entre le microbiote intestinal et l'obésité. Des groupes bactériens ont été incriminés et des hypothèses proposées mais les mécanismes liant microbiote et obésité restent en grande partie inconnus. Le microbiote intestinal est constitué de plusieurs centaines d’espèces et est spécifique de chaque individu. Récemment, il a été révélé l’implication potentielle de ce microbiote dans le développement de l’obésité. Lors d’une étude précédente, notre équipe a ainsi montré que les souris sans germes (dites « axéniques ») sont résistantes à l’obésité et aux désordres métaboliques induits par un régime hypercalorique (Roy et al. 2012). Par ailleurs, nous avons observé que certaines souris conventionnelles soumises à un tel régime développent une obésité et une insulinorésistance importantes (phénotype dit répondeur) tandis que d'autres restent minces et tolérantes au glucose (phénotype dit non répondeur) indépendamment de la quantité de nourriture consommée. Les mécanismes expliquant cette hétérogénéité de phénotype sont actuellement peu connus. Ce projet de thèse vise donc à élucider les mécanismes liant le microbiote intestinal au développement de l'obésité et des désordres associés en partant du principe que l'hétérogénéité (en termes de composition et de fonctions) du microbiote intestinal, spécifique de chaque individu (aussi bien chez l'homme que chez le rongeur), joue un rôle dans ces réponses différentes à un même régime hypercalorique. Pour ce faire, des souris conventionnelles ont reçu un régime contrôle ou un régime hyperlipidique pendant 12 semaines. A l’issu de ce régime, des souris ont été sélectionnées en tant que répondeuses ou non-répondeuses Une approche par métagénomique à partir d’échantillons fécaux prélevés avant et après régime hyperlipidique et issus des souris répondeuses et non-répondeuses ont été analysés. Les profils de microbiotes avant et après régime hyperlipidique ont été comparés afin de déterminer les effets de ce régime sur le microbiote intestinal. De plus, le profil du microbiote des souris répondeuses a été comparé à celui des souris non-répondeuses afin de détecter des espèces bactériennes, des gènes ou des voies métaboliques sous- ou sur-représentés dans les deux phénotypes. Par ailleurs, des échantillons de fèces, d’urine et de plasma prélevés avant et après le régime hyperlipidique ont été analysés par métabolomique et nous avons ainsi analysé les fluctuations métaboliques induites par l’effet du régime. L’ensemble de ces travaux a eu pour but d’identifier s’il existe un microbiote de prédisposition au développement ou à la résistance du développement d’une obésité induite. Au cours de ce projet de thèse nous avons utilisé l’approche de métagénomique à partir d’échantillon fécaux afin d’identifier les profils du microbiote intestinal des souris NR et des souris R avant et après régime hyperlipidique en vue de mesurer l’impact de ce type de régime sur le microbiote, et surtout de détecter les espèces bactériennes, les gènes ou les voies métaboliques potentiellement sous- ou sur-représentés dans les deux phénotypes. D’autre part, l’approche de métabolomique a été utilisée sur des échantillons d’urine, de fèces et de plasma avant et après régime afin de visualiser les fluctuations métaboliques induites par l’effet du régime d’une part et d’identifier les métabolites (biomarqueurs) associés à chacun des phénotypes d’autre part. / In recent years, numerous studies have examined the relationship between the gut microbiota and obesity. Bacterial groups have been incriminated but the mechanisms linking microbiota and obesity remain largely unknown. Intestinal microbiota consists of several hundred species and is specific for each individual. Recently, it was revealed that the potential contribution of microbiota in the development of obesity. In a previous study, our team has shown that mice without germs (called "germ-free") are resistant to obesity and metabolic disorders induced by a high calorie diet (Roy et al. 2012). Furthermore, we observed that certain conventional mice subjected to such a plan develop an important obesity and insulin resistance (responder phenotype) while others remain thin and glucose tolerant (non responder phenotype) regardless of the amount of food consumed. The mechanisms explaining this phenotype heterogeneity are currently poorly known. This PhD project aims to elucidate the mechanisms linking the intestinal microbiota in the development of obesity and disorders associated with the assumption that heterogeneity (in terms of composition and functions) of the intestinal microbiota, specific for each individual ( both in humans than in rodents), plays a role in these different responses to the same high-fat diet. To do this, conventional mice received control diet or a high fat diet for 12 weeks. At the end of this diet, mice were selected as a responder or non-responder A metagenomics approach from fecal samples taken before and after high fat diet and from responder and non-responder mice were analyzed. Microbiota profiles before and after fat diet were compared to determine the effects of this diet on the intestinal microbiota. In addition, the profile of the microbiota of responder mice was compared to that of non-responder mice in order to detect bacterial species, genes or pathways under- or over-represented in both phenotypes. Furthermore, samples of feces, urine and plasma collected before and after fat diet were analyzed by metabolomics and we have analyzed the metabolic changes induced by the effect of diet. All of this work has been aimed to identify if there is a predisposition microbiota to the development or the resistance of induced obesity. In this PhD project we used the metagenomic approach from fecal sample to identify the profiles of the intestinal microbiota in mice NR and R mice before and after fat diet in order to measure the impact of this type of diet on the microbiota, and especially to detect bacterial species, genes or metabolic pathways potentially under- or over-represented in both phenotypes. On the other hand, the approach of metabolomics has been used on samples of urine, feces and plasma before and after diet in order to visualize the metabolic changes induced by the effect of diet on the one hand and to identify metabolites (biomarkers) associated with each other hand phenotypes.

>Magister Scientiae - MSc / Antarctica is known for its pristine environments. A variety of unsuitable environmental conditions were once thought to render the continent unsuitable for sustaining life. However, metagenomic data have revealed a wealth of species diversity in a range of biotopes.Hypolithons, photosynthetic communities which live under translucent rocks in climatically extreme environments, are an important input source for both carbon (C) and nitrogen (N) in this hyperarid desert environment. Microbial contribution to biogeochemical cycling resulting in fixation of both C and N remains poorly understood. Moreover, there is a reported close interplay between both cycles, with nitrogen being reported to be a limiting factor in carbon assimilation.In this study the diversity of C and N fixing organisms was investigated by using the cbbL and nifH genes as phylogenetic and functional markers. High Molecular weight metagenomic DNA and RNA was extracted from hypolithons. PCR amplification was carried out using cbbL (800 bp for red-like, 1,100 bp for green-like) and nifH (360 bp) gene specific primers.Resultant PCR products were used to construct libraries which were screened for correct sized inserts. Restriction Fragment Length Polymorphism (RFLP) was used to de-replicate clones prior to sequencing. Phylogenetic positions from both clone libraries were established by aligning nucleotide sequences and constructing similarity trees using NJ clustering methods.BLASTn results indicated the presence of previously uncultured organisms which contain cbbL and nifH genes. BLASTn results were characterized by low percentages of maximum identity (typically <95%), a potential indicator of novel taxa. Sequences from respective libraries clustered with cyanobacteria such as Nostoc, Scytonema, and Tolypothrix and α-, β-, and γ-Proteobacteria such as Azotobacter, Agrobacterium and Mesorhizobium. Generally sequence results indicate a largely homogenous, being dominated by specific taxa. Each group may contain potential keystone species, essential for both biogeochemical cycling in oligotrophic environment.

Os microrganismos constituem dois terços da diversidade biológica na Terra, no entanto, muitos deles não podem ser cultivados por técnicas tradicionais. Portanto, o acesso a esta diversidade tem sido feita através da utilização de técnicas independentes de cultivo. Diante deste panorama, a metagenômica apresenta-se como uma alternativa, pois dispensa a necessidade de cultivo. Tal técnica possibilita inclusive a identificação e utilização do potencial metabólico destes organismos para o desenvolvimento de novos processos e produtos. Os polihidroxialcanoatos (PHAs) são poliésteres bacterianos, acumulados intracelularmente em forma de grânulos, cujas propriedades são similares a de alguns plásticos de origem petroquímica. O objetivo deste trabalho foi identificar e avaliar a diversidade de genes relacionados à produção de PHAs em bibliotecas metagenômicas de solo. A prospecção realizada resultou na identificação de clones contendo o gene phaC. De uma forma geral, pôde-se concluir que ainda há uma grande diversidade deste gene a ser descoberta no ambiente estudado. / Microorganisms constitute two third of the Earth\'s biological diversity, however, many of them cannot be cultured by standard techniques. Therefore, access to this diversity has been achieved through the use of culture-independent techniques. Facing this scenario, the metagenomic presents itself as an alternative, since it eliminates the need for cultivation. This technique also allows the identification and use of the metabolic pathways of these organisms to develop new processes and products. The polyhydroxyalkanoates (PHAs) are bacterial polyesters accumulated as granules, whose properties are similar to some plastics of petrochemical origin. The aim of this work was to identify and access the diversity of genes related to PHAs production in soil metagenomic libraries. The screening resulted in the identification of clones containing the phaC gene. In a general way, it was concluded that there is still a considerable diversity of this gene to be discovered in the study environment.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The use of conventional culture-based approach results in vast majority of microbes (90 - 99%) unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and endonuclease), which are central in genetic engineering. They are usually grouped into four broad classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the extracted metagenomic DNA was used for sequencing and another for fosmid library construction. Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector. A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly, the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the developed DNA-manipulating enzymes and study the practical application and chart way for commercialization. Moreover, the constructed fosmid library could also be screened for potentially novel thermo-stable biomolecules of industrial and therapeutic importance.

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