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Caracterização De Novos Microrganismos Cultivados a Partir De Solos Do Cerrado

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Caracterização De Novos Microrganismos Cultivados a Partir De Solos Do Cerrado Universidade de Brasília Instituto de Ciências Biológicas Departamento de Biologia Celular Programa de Pós-Graduação em Biologia Molecular Caracterização de novos microrganismos cultivados a partir de solos do Cerrado ALINE BELMOK DE ARAÚJO DIAS IOCCA Tese apresentada ao Programa de Pós- Graduação em Biologia Molecular do Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, para a obtenção do título de Doutora em Biologia Molecular. Orientadora: Dra. Ildinete Silva-Pereira Co-orientadora: Dra. Cynthia Maria Kyaw Brasília, outubro de 2020 Dedico essa tese à memória de meu querido avô Nestor Belmok, de quem herdei a paixão por livros e pelo conhecimento e que com tanto orgulho falava de sua neta que um dia seria doutora. ii SUMÁRIO Agradecimentos................................................................................................................ v Lista de Tabelas e Figuras..............................................................................................viii Lista de Abreviações.......................................................................................................xii RESUMO ........................................................................................................................................ 1 ABSTRACT ...................................................................................................................................... 2 INTRODUÇÃO GERAL ..................................................................................................................... 3 CAPÍTULO 1 – Estabelecimento e caracterização de co-cultivos de archaeas e bactérias obtidos a partir de amostras de solo de Cerrado ....................................................................................... 4 1.1. Introdução ......................................................................................................................... 4 1.1.1. O cultivo laboratorial de microrganismos e o início da microbiologia ............................. 4 1.1.2. A microbiologia na era das abordagens moleculares e a proposta do domínio Archaea 6 1.1.3. A importância do cultivo na era das “ômicas” ............................................................... 10 1.1.4. A grande diversidade microbiana nos solos do bioma Cerrado ..................................... 13 1.2. Objetivos ......................................................................................................................... 16 1.2.1. Objetivo geral ................................................................................................................. 16 1.2.2. Objetivos específicos ...................................................................................................... 16 1.3. Material e Métodos ......................................................................................................... 17 1.3.1. Coleta dos solos .............................................................................................................. 17 1.3.2. Confecção do meio de cultivo, inóculo inicial e manutenção das culturas ................... 17 1.3.3. Extração de DNA genômico total ................................................................................... 18 1.3.4. Ensaios de PCR ................................................................................................................ 19 1.3.7. Transformação por choque térmico, seleção e estoque em glicerol de clones recombinantes ............................................................................................................................ 22 1.3.8. Extração de DNA plasmidial por lise alcalina ................................................................. 22 1.4. Resultados e discussão .................................................................................................... 29 1.4.1. Características químicas do solo e obtenção de culturas em meio sólido ..................... 29 1.4.2. Obtenção de culturas em meio líquido .......................................................................... 32 1.4.3. Caracterização filogenética dos organismos cultivados a partir da análise dos produtos obtidos nos ensaios de PCR dirigidos aos genes de rRNA 16S .................................................... 32 1.4.4. Análises microscópicas das células presentes no co-cultivo .......................................... 41 1.4.5. Sequenciamento genômico total dos cultivos de solo de Cerrado ................................ 49 CAPÍTULO 2 - Caracterização morfo-fisiológica e genômica da bactéria Novosphingobium terrae GeG2T, isolada a partir de solo de Cerrado ................................................................................. 53 2.1. Introdução ....................................................................................................................... 53 2.1. Objetivos ......................................................................................................................... 55 2.1.1. Objetivo geral ................................................................................................................. 55 2.1.2. Objetivos específicos ...................................................................................................... 55 2.2. Material e Métodos ......................................................................................................... 56 2.2.1. Isolamento e cultivo da bactéria em meios de cultura definidos .................................. 56 iii 2.2.2. Observação das células bacterianas por microscopia óptica ......................................... 57 2.2.3. Extração de DNA genômico, amplificação, clonagem, sequenciamento e análises do gene de rRNA 16S da cultura bacteriana .................................................................................... 57 2.2.4. Comparações fisiológicas entre o isolado bacteriano GeG2 e Novosphingobium rosa DSM 7285 .................................................................................................................................... 59 2.2.5. Caracterizações químicas de GeG2 ................................................................................ 61 2.2.6. Comparação do perfil proteico das bactérias GeG2 e N. rosa DSM 7285 por espectrometria de massa ............................................................................................................ 62 2.2.7. Avaliação do perfil de susceptibilidade de GeG2 a antibióticos .................................... 64 2.2.8. Observação das células bacterianas por microscopia eletrônica de varredura (MEV) e de transmissão (MET) ....................................................................................................................... 64 2.2.9. Sequenciamento, montagem e anotação do genoma completo do isolado bacteriano GeG2............................................................................................................................................65 2.2.10. Análises taxonômicas baseadas no genoma ................................................................ 67 2.3.11. Análises preliminares do crescimento de GeG2 em MM contendo o corante aromático verde malaquita .......................................................................................................................... 68 2.3. Resultados e Discussão ................................................................................................... 69 2.3.1. Isolamento e cultivo da bactéria presente no cultivo do solo de Cerrado em meio definido ....................................................................................................................................... 69 2.3.2. Avaliação das células bacterianas GeG2 por microscopia óptica .................................. 70 2.3.3. Análises do gene rRNA 16S ............................................................................................. 72 2.3.4. Caracterizações fisiológicas de GeG2 e comparações com N. rosa DSM 7285 .............. 74 2.3.5. Caracterizações químicas de GeG2 ................................................................................ 78 2.3.6. Caracterização do perfil proteico de GeG2 por espectrometria de massa e comparação com o perfil de N. rosa DSM 7285 .............................................................................................. 81 2.3.7. Perfil de susceptibilidade a antibióticos de GeG2 .......................................................... 82 2.3.8. Observação das células de GeG2 e N. rosa DSM 7285 por MEV e MET......................... 84 2.3.9. Dimorfismo planctônico/séssil do isolado GeG2 ........................................................... 89 2.3.10. Sequenciamento, montagem, anotação geral e arquitetura do genoma da linhagem GeG2............................................................................................................................................ 96 2.3.11. Classificação taxonômica da linhagem GeG2 baseada no genoma completo ........... 104 2.3.12. Análises funcionais do genoma da linhagem GeG2 .................................................... 108 2.3.13. Potencial do isolado GeG2 na degradação de compostos aromáticos ...................... 125 4.3.14. Proposta da espécie Novosphingobium terrae sp. nov. ............................................. 129 CONCLUSÕES E PERSPECTIVAS.................................................................................................. 130 REFERÊNCIAS BIBLIOGRÁFICAS ................................................................................................
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