Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-08T13:51:47Z No. of bitstreams: 1 OtavioHenriqueB.PintoDissertacao2017.pdf: 3191289 bytes, checksum: b7b005d77190f465fe0685851ff5f090 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-08T13:52:01Z (GMT) No. of bitstreams: 1 OtavioHenriqueB.PintoDissertacao2017.pdf: 3191289 bytes, checksum: b7b005d77190f465fe0685851ff5f090 (MD5) / Made available in DSpace on 2017-09-08T13:52:02Z (GMT). No. of bitstreams: 1 OtavioHenriqueB.PintoDissertacao2017.pdf: 3191289 bytes, checksum: b7b005d77190f465fe0685851ff5f090 (MD5) Previous issue date: 2017-03-29 / Acidobacteria is one of the most abundant phyla in soil; however, it is not yet known the reasons for their success in terrestrial habitats. This characteristic may be due to mechanisms of resistance as well as the large amount of glycosyl hydrolases and transferases present in Acidobacteria. Therefore, this study aimed to investigate mechanisms of resistance that may contribute to the abundance of Acidobacteria in soil. In addition, the degradation of cellulose was investigated, to understand its participation in processes of plant biomass degradation. In this study, a member of subdivision 1 of Acidobacteria, Occallatibacter savannae AB23 was used as model. This isolate was taxonomically characterized and cellulose degradation and carotenoid production were evaluated. The degradation of cellulose was evaluated using carboxymethylcellulose (CMC). The pigments were identified since this bacterium produced several carotenoids under different environmental stimuli. The results showed that the isolate AB23 can assimilate CMC, however in a very slow way, requiring 2 months for the visualization of colonies and 1 year to visualization of biomass in liquid medium. For this isolate, endoglucanases were found associated with the cell fraction of culture. In addition, cellobiose has been shown to be a repressor and lactose an inducer of endoglucanases expression. It was possible to observe the production of carotenoids in AB23. By mass spectrometry, it was possible to find ions related to carotenoids: ?? -carotene, neurosporene, lycopene, ??-carotene, ??-carotene, phytoene and phytofluene. These carotenoids might be used as a resistance mechanism since they are produced in response to stress caused by light. However, the presence of vitamins, oxygen and trace elements are essential for carotenoid production. The characteristics found in this work may be one of the mechanisms of persistence and / or resistance that confer abundance for Acidobacteria in soil. / Acidobacteria ?? um dos filos mais abundantes no solo, entretanto, n??o se conhece as raz??es que levam a essa abund??ncia. Estudos indicam que essa caracter??stica pode ser devido a mecanismos de resist??ncia e a grande quantidade de glicosil hidrolases e transferases presentes em Acidobacteria. Assim, esse estudo teve como objetivo investigar mecanismos de resist??ncia que podem contribuir para a abund??ncia de Acidobacteria no solo. Al??m disso, foi investigada a degrada????o de celulose em Acidobacteria, procurando entender sua participa????o em processos de degrada????o de material originado de plantas. Neste estudo foi utilizado como modelo Occallatibacter savannae AB23. Este isolado foi caracterizado taxonomicamente e foram avaliadas a degrada????o de celulose e produ????o de caroten??ides. A degrada????o de celulose foi avaliada utilizando-se carboximetilcelulose (CMC). Para os pigmentos, foram identificados quais carotenoides essa bact??ria produz e os est??mulos que levam a sua produ????o. Os resultados mostraram que o isolado AB23 consegue assimilar CMC, no entanto de uma forma muito lenta, sendo necess??rios 2 meses para a visualiza????o de col??nias. Para este isolado, as endoglucanasases foram encontradas associadas ?? fra????o celular da cultura. Al??m disso, celobiose mostrou ser um repressor e a lactose um indutor da express??o de endocglucanases. Foi poss??vel observar a produ????o de pigmentos em AB23. Por espectrometria de massa, foi poss??vel encontrar ??ons relacionados aos carotenoides: ??-caroteno, neurosporeno, licopeno, ??-caroteno, ??-caroteno, fitoeno e fitoflueno. Estes devem estar sendo empregados como um mecanismo de resist??ncia, uma vez que eles s??o produzidos em resposta ao estresse causado pela luz. No entanto a presen??a de vitaminas, oxig??nio e elementos tra??o s??o essenciais para produ????o desses pigmentos. As caracter??sticas encontradas neste trabalho podem ser um dos mecanismos de persist??ncia e/ou resist??ncia que conferem abund??ncia para Acidobacteria no solo.

Soil microbiomes play pivotal roles to the health of the environment by maintaining metabolic cycles. One question is how will climate change affect soil bacteria over time and what could the repercussions be. To answer these questions, the Harvard Forest Long-Term Warming Experiment was established to mimic predicted climate change by warming plots of land 5℃ above ambient conditions. In 2017, 14 soil core samples were collected from Barre Woods warming experiment to mark 15 years since the establishment of the soil warming in that location. These samples underwent traditional metatranscriptomics to generate an mRNA library as well as a process coined cell-sorted or mini-metagenomics involving the sorting of single bacterial cells from the environment using FACS. This was followed by pooling into groups of 100 cells for more cost efficient genome recovery. 200 high-quality genomes were compiled, 12 of which were taxonomically identified as Acidobacteria. Acidobacteria are an extremely abundant and diverse phylum of bacteria that were found to be very well represented in the soil samples. Due to their abundance in many different soil environments as well as their known importance in many metabolic cycles, they were chosen as the candidate phylum to further investigate. Using a reference-based read mapping approach with the 12 Acidobacteria genomes and metatranscriptomic data, we identified over 3,000 differentially expressed genes within these organisms as a result of soil warming. Due to the diversity within the phylum itself, many of the genomes indicated different patterns of expression making it difficult to identify phylum-wide differential expression trends. However, the sigma70 factor, an important housekeeping gene used as a transcription regulator, was found to be up-regulated in a majority of the genomes. Over 30 different glycoside hydrolase encoding genes and glycosyltransferases were also found to be differentially expressed across the Acidobacteria reference genomes as well as 23 chemotaxis-related genes. Despite identifying four different groups of genes that showed statistically significant differences in expression levels, there may be more changes occurring in these soil bacteria and the soil microbiome as a whole due to climate change than previously measured by read-based analyses of metatranscriptomic data.

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.