Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic l...

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Autor principal: Silva, Uaska Bezerra e
Outros Autores: Lima, Lucymara Fassarela Agnez
Formato: doctoralThesis
Idioma:por
Publicado em: Universidade Federal do Rio Grande do Norte
Assuntos:
Estresse salino. Osmólito. Halobactéria e metagenoma
Salt stress. Osmolytes. Halobacteria and metagenome
CNPQ::CIENCIAS BIOLOGICAS
Endereço do item:https://repositorio.ufrn.br/jspui/handle/123456789/12654
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id ri-123456789-12654
record_format dspace
institution Repositório Institucional
collection RI - UFRN
language por
topic Estresse salino. Osmólito. Halobactéria e metagenoma
Salt stress. Osmolytes. Halobacteria and metagenome
CNPQ::CIENCIAS BIOLOGICAS
spellingShingle Estresse salino. Osmólito. Halobactéria e metagenoma
Salt stress. Osmolytes. Halobacteria and metagenome
CNPQ::CIENCIAS BIOLOGICAS
Silva, Uaska Bezerra e
Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
description The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river
author2 Lima, Lucymara Fassarela Agnez
author_facet Lima, Lucymara Fassarela Agnez
Silva, Uaska Bezerra e
format doctoralThesis
author Silva, Uaska Bezerra e
author_sort Silva, Uaska Bezerra e
title Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
title_short Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
title_full Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
title_fullStr Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
title_full_unstemmed Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil
title_sort análise metagenômica da microbiota de ambientes aquáticos do estado do rio grande do norte - brasil
publisher Universidade Federal do Rio Grande do Norte
publishDate 2014
url https://repositorio.ufrn.br/jspui/handle/123456789/12654
work_keys_str_mv AT silvauaskabezerrae analisemetagenomicadamicrobiotadeambientesaquaticosdoestadodoriograndedonortebrasil
AT silvauaskabezerrae metagenomicanalysisofmicrobiotafromaquaticenvironmentsinthestateofriograndedonortebrazil
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spelling ri-123456789-126542019-01-30T07:59:55Z Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil Metagenomic analysis of microbiota from aquatic environments in the state of Rio Grande do Norte Brazil Silva, Uaska Bezerra e Lima, Lucymara Fassarela Agnez http://lattes.cnpq.br/9309987119591098 http://lattes.cnpq.br/1083882171718362 Quirino, Betania Ferraz http://lattes.cnpq.br/3916535995785654 Uchoa, Adriana Ferreira http://lattes.cnpq.br/6644671747055211 Theodoro, Raquel Cordeiro http://lattes.cnpq.br/0977453259767928 Thompson, Claudia Elizabeth http://lattes.cnpq.br/8413464882207319 Estresse salino. Osmólito. Halobactéria e metagenoma Salt stress. Osmolytes. Halobacteria and metagenome CNPQ::CIENCIAS BIOLOGICAS The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river Conselho Nacional de Desenvolvimento Científico e Tecnológico A busca por genes baseada na construção e análise de bibliotecas metagenômicas a partir de solo gera oportunidades para explorar uma enorme diversidade genética e metabólica de microrganismos. Os rios são ecossistemas com alta diversidade biológica, mas ainda pouco explorados por meio de metagenômica. Com o objetivo de explorar a diversidade microbiana, uma biblioteca metagenômica foi construída a partir de DNA extraído de substrato de rio em três pontos ao longo do rio Jundiaí (Rio Grande do Norte-Brasil). Os pontos de amostragem são derivados de área aberta, terreno acidentado e com a incidência direta da luz solar. Esta biblioteca foi analisada funcionalmente e também com base em sequências. Para a análise funcional foi utilizado meio de cultura sólido LB com concentração de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com características halotolerantes. Os DNAs recombinantes foram extraídos e retransformados em cepa de Escherichia coli DH10B e curvas de sobrevivência foram obtidas para confirmação e quantificação da resistência ao estresse abiótico. As sequências dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam proteínas hipotéticas. Um dos clones apresentou uma ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na síntese do precursor de glicerol, sendo um soluto compatível para equilibrar a pressão osmótica no interior e no exterior das células. Posteriormente, para identificação de genes que codificam osmólitos relacionados com halotolerância e identificação da diversidade microbiológica, amostras de DNA ambiental do substrato do rio e da coluna d´água do estuário e oceano foram coletadas e pirosequenciadas. As sequências de osmólitos de diferentes microrganismos foram obtidas a partir do UniProt e utilizadas como RefSeqs para a identificação por homologia (TBLASTN) nos bancos de dados metagenômicos. As sequências identificadas nos bancos de dados ambientais foram submetidas ao programa HMMER com o fim de identificar domínios funcionais. Foram identificadas as enzimas: alfa-trealose-fosfato sintase, L-ectoina sintase (ectC), transaminase do ácido L-2,4-diaminobutírico (EctB), ácido L-2 ,4-diaminobutírico acetiltransferase (EctA), L-treonina 3-desidrogenase (via de síntese do sorbitol), Glicerol-3-fosfato desidrogenase, inositol-3-fosfato desidrogenase, chaperonas, L-prolina glicina betaína ligação transportador ABC, mio-inositol-1-fosfato sintase, a proteína simportadora de prolina/sódio -PutP e trealose-6-fosfato fosfatase. Estas são enzimas que participam da síntese de osmólitos comumente relacionados a ambientes salinos, no entanto a identificação desses solutos em ambiente de rio é justificada pela elevada concentração salina no solo durante prolongadas estações de seca neste rio. Quanto à riqueza da microbiota foi identificado que o substrato do rio possui uma abundância de halobactérias semelhante a do mar e superior a do estuário. Esses dados confirmam a existência de uma resposta especializada contra o estresse salino por microrganismos no ambiente do rio Jundiaí 2014-12-17T14:05:23Z 2014-11-05 2014-12-17T14:05:23Z 2013-04-16 doctoralThesis SILVA, Uaska Bezerra e. Metagenomic analysis of microbiota from aquatic environments in the state of Rio Grande do Norte Brazil. 2013. 113 f. Tese (Doutorado em Biotecnologia Industrial; Biotecnologia em Agropecuária; Biotecnologia em Recursos Naturais; Biotecn) - Universidade Federal do Rio Grande do Norte, Natal, 2013. https://repositorio.ufrn.br/jspui/handle/123456789/12654 por Acesso Aberto application/pdf application/pdf Universidade Federal do Rio Grande do Norte BR UFRN Programa de Pós-Graduação em Biotecnologia Biotecnologia Industrial; Biotecnologia em Agropecuária; Biotecnologia em Recursos Naturais; Biotecn

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