Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-12-15T17:45:12Z No. of bitstreams: 1 SinaraCarlaDaSilvaAraujo_DISSERT.pdf: 1885448 bytes, checksum: b4a8226a4f1778e4f626da3394657f9a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2015-12-29T20:51:37Z (GMT) No. of bitstreams: 1 SinaraCarlaDaSilvaAraujo_DISSERT.pdf: 1885448 bytes, checksum: b4a8226a4f1778e4f626da3394657f9a (MD5) / Made available in DSpace on 2015-12-29T20:51:37Z (GMT). No. of bitstreams: 1 SinaraCarlaDaSilvaAraujo_DISSERT.pdf: 1885448 bytes, checksum: b4a8226a4f1778e4f626da3394657f9a (MD5) Previous issue date: 2014-08-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Os microrganismos apresentam uma imensa diversidade gen?tica e est?o presentes em toda biosfera, no entanto cerca de 1% das esp?cies pode ser cultivada por t?cnicas laboratoriais padr?o. A metagen?mica tornou poss?vel o acesso direto ao genoma microbiano derivado de amostras ambientais, utilizando t?cnicas independentes de cultivo. A metodologia permite obter informa??o funcional de prote?nas, assim como a identifica??o de potenciais produtos com interesse biotecnol?gico e de novos recursos biol?gicos industrialmente explor?veis, a exemplo novas solu??es para impactos ambientais. ?reas contaminadas com petr?leo s?o caracterizadas por um grande ac?mulo de hidrocarbonetos e surfactantes s?o utilizados para sua biorremedia??o. Sendo assim, a abordagem metagen?nima foi utilizada para selecionar genes envolvidos no processo de degrada??o e biossurfacta??o de hidrocarbonetos. Em um trabalho anterior, o DNA ambiental (eDNA) foi extra?do de amostras de solo coletadas em duas diferentes ?reas (Caatinga e rio salino) do Rio Grande do Norte (Brasil), as bibliotecas metagen?micas foram constru?das e analisadas funcionalmente. Os clones capazes de degradar o ?leo foram avaliados quanto ? capacidade de sintetizar biossurfactante. O clone foi sequenciado e an?lise da sequencia revelou uma ORF com 897 pb, 298 amino?cidos e prote?na de peso molecular pr?ximo a 34 kDa. A busca por homologia no GenBank revelou similaridade com a sequ?ncia que codifica uma prote?na hipot?tica de representantes da fam?lia Halobacteriaceae, que foram mostradas recentemente como produtoras de biossurfactantes. A presen?a da sequ?ncia codificante inserida e do fen?tipo adquirido foram confirmadas. Primers foram desenhados e suas ORFs amplificadas por PCR. Em seguida, foram subclonadas em vetor de express?o pETDuet-1, contendo uma cauda de histidina, para express?o e posterior purifica??o da prote?na de interesse. Os testes foram realizados para confirma??o da atividade de biossurfactante e degrada??o de hidrocarbonetos e apresentaram resultados positivos. O ensaio de imunodetec??o (western blot) com a utiliza??o do anticorpo monoclonal Anti-His? confirmou a presen?a da prote?na ambiental. Esse estudo foi o primeiro a relatar uma poss?vel prote?na com atividade biossurfactante obtida a partir de uma abordagem metagen?mica. / The microorganisms have a vast genetic diversity and they are present throughout the biosphere, however, only about 1% of the species can be cultivated by traditional cultivation techniques. Within this diversity there is a huge pool genetic and biological being explored. The metagenomics has enabled direct access to microbial genome derived from environmental samples using independent methods of cultivation. The methodology enables to obtain functional information about the proteins, as well as identify potential products with biotechnological interest and new industrially exploitable biological resources, such as new solutions to environmental impacts. Oil-contaminated areas are characterized by a large accumulation of hydrocarbons and surfactants may be used for bioremediation. Thus, the metagenomic approach was used in this study in order to select genes involved in the degradation and hydrocarbon emulsification. In a previous work, the environmental DNA (eDNA) was extracted from soil samples collected from two different areas (Caatinga and Saline River) of Rio Grande do Norte (Brazil), the metagenomic libraries were constructed and functionally analyzed. The clone able to degrade the oil was evaluated for the ability to synthesize biosurfactants. The sequence analysis revealed an ORF with 897 bp, 298 amino acids and a protein with around 34 kDa. The search for homology in GenBank revealed sequence similarity with a hypothetical protein of representatives Halobacteriaceae family, who were recently shown as strains producing biosurfactants. The presence of the inserted coding sequence and the acquired phenotype was confirmed. Primers were designed and the ORF amplified by PCR. The ORF was subcloned into pETDuet-1 expression vector for subsequent purification of the protein of interest containing a histidine tail. The tests performed to confirm the biosurfactant activity and the ability of hydrocarbon degradation showed positive results. The immunodetection test (western blot) using the monoclonal AntiHis? confirmed the presence of the environmental protein. This study was the first to report a possible protein with biosurfactant activity obtained from a metagenomic approach

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

Submitted by Kelson Anthony de Menezes (kelson@ucb.br) on 2016-12-19T18:17:11Z No. of bitstreams: 1 LuisFelipeSchroederDissertacao2014.pdf: 2932891 bytes, checksum: 9867ac60d140318b946ef45528984bca (MD5) / Made available in DSpace on 2016-12-19T18:17:11Z (GMT). No. of bitstreams: 1 LuisFelipeSchroederDissertacao2014.pdf: 2932891 bytes, checksum: 9867ac60d140318b946ef45528984bca (MD5) Previous issue date: 2014-09-30 / There are different process being developed for cellulosic ethanol production, with possible different pretreatments with varying temperatures and pH, in addition to several biomasses can be used as the source of fermentable sugars. Among the important enzymes for deconstruction of plant biomass, stand out xylanases. These enzymes are responsible for deconstruction of the hemicellulose present in the structure of the plant cell walls. There are several ways to accomplish the identification of these enzymes: purification from an isolated microorganism is one. In this study, genomic and metagenomic approaches were used to carry out the prospection of the genes responsible for coding these enzymes. Clones from two libraries were used for detection and evaluation of activity on solid medium, supplemented with xylan and acid pretreated sugarcane bagasse. Nineteen clones of a goat rumen metagenomic library and five clones from an AB60 bacterium genomic library, with 15,000 clones constructed in this study, were selected initially. Fourteen clones from the metagenomic library were completely sequenced and their ORFs were analyzed. Four clones from the genomic library were partially sequenced and one clone had its sequence completely determined and 104 ORFs were obtained for all clones completely or partially sequenced ORFs were analyzed. Eleven ORFs showed some similarity to genes of importance for the degradation of complex polysaccharides. Among the most important ORFs and most likely to be related to the detected activity, are genes coding for ??-glucosidase, ??-xylosidase and ??-glucuronidase. Furthermore, also other ORFs with lower probability of relation with the activity or necessity to full sequencing of the clones for a few more conclusive analysis were identified. About 40% of the ORFs present in the rumen clones and 37.8% of the ORFs present in Acidobacteria clones showed similarity with hypothetical or uncharacterized proteins, which could be important in the activity detected. A ??-glucuronidase gene detected in a clone from the goat rumen metagenomic library was synthesized, its sequence was optimized for expression in Escherichia coli. However, in the present work, it was not possible to sub-cloning, made expression and purification of this enzyme. Some ORFs detected can be used for future studies of expression and characterization in order to improve knowledge about biotechnological potential present in the rumen and acidobacteria AB60, besides the ecological role of these microorganisms in their environment. / Existem diversos processos em desenvolvimento para a produ????o de etanol celul??sico, havendo diferentes poss??veis pr??-tratamentos, com temperaturas e pH variados, al??m das diversas biomassas que podem ser utilizadas como fonte de a????cares ferment??veis. Dentre as enzimas importantes para a desconstru????o de biomassa vegetal, destacam-se as xilanases. Estas enzimas s??o respons??veis pela desconstru????o da estrutura hemicelul??sica presente na parede celular das plantas. H?? diversas maneiras para alcan??ar a identifica????o destas enzimas: purifica????o a partir de micro-organismo isolado sendo uma delas. No presente trabalho, foram utilizadas abordagens gen??mica e metagen??mica a fim de realizar a prospec????o dos genes respons??veis pela codifica????o para estas enzimas. Foram utilizados clones oriundos de duas bibliotecas para a detec????o e avalia????o da atividade em meio s??lido suplementado com xilana e baga??o de cana-de-a????car pr??-tratado com ??cido. Dezenove clones de uma biblioteca metagen??mica de r??men de caprinos e cinco clones de uma biblioteca gen??mica da bact??ria AB60, com 15.000 clones constru??da no presente trabalho, foram selecionados inicialmente. Quatorze clones da biblioteca metagen??mica foram sequenciados completamente e tiveram as suas ORFs analisadas. Quatro clones da biblioteca gen??mica foram parcialmente sequenciados e um clone teve a sua sequ??ncia completa determinada e as ORFs analisadas. Das 104 ORFs obtidas de todos os clones completamente ou parcialmente sequenciados, onze ORFs apresentaram alguma similaridade com genes de import??ncia para a degrada????o de polissacar??deos complexos. Dentre as ORFs de maior import??ncia e com maior probabilidade de estarem relacionadas com a atividade detectada, est??o genes codificantes para ??-glicosidase, ??-xilosidase e ??-glicuronidase. Al??m disso, tamb??m foram identificadas outras ORFs com menor probabilidade de rela????o com a atividade ou necessidade de sequenciamento completo de alguns dos clones para uma an??lise mais conclusiva. Cerca de 40% das ORFs presentes nos clones selecionados de r??men e 37,8% das ORFs presentes nos clones selecionados de Acidobacteria apresentaram similaridade com prote??nas hipot??ticas ou prote??nas n??o caracterizadas que podem ter import??ncia na atividade detectada. Um gene de ??-glicuronidase detectado em um clone da biblioteca metagen??mica de r??men de caprino foi sintetizado, otimizando-se sua sequ??ncia para express??o em Escherichia coli . Entretanto, n??o foi poss??vel a sub-clonagem, express??o e purifica????o desta enzima no presente trabalho. Algumas ORFs detectadas podem ser utilizadas para estudos futuros de express??o e caracteriza????o a fim de aprimorar o conhecimento a respeito do potencial biotecnol??gico presente no r??men e na Acidobact??ria AB60, al??m do papel ecol??gico destes micro-organismos em seu ambiente.