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Stefan Schwab

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Stefan Schwab STEFAN SCHWAB IDENTIFICAÇÃO E ANÁLISE DE GENES DE Herbaspirillum seropedicae REGULADOS PELA DISPONIBILIDADE DE AMÔNIO Tese apresentada ao Curso de Pós-graduação em Bioquímica, Setor de Ciências Biológicas, Universidade Federal do Paraná, como requisito parcial à obtenção do título de Doutor em Ciências (Bioquímica). Orientador: Profa. Dra. Liu Un Rigo Co-orientador: Prof. Dr. Emanuel Maltempi de Souza Curitiba Novembro 2006 STEFAN SCHWAB IDENTIFICAÇÃO E ANÁLISE DE GENES DE Herbaspirillum seropedicae REGULADOS PELA DISPONIBILIDADE DE AMÔNIO Tese apresentada ao Curso de Pós-graduação em Bioquímica, Setor de Ciências Biológicas, Universidade Federal do Paraná, como requisito parcial à obtenção do título de Doutor em Ciências (Bioquímica). Orientador: Profa. Dra. Liu Un Rigo Co-orientador: Prof. Dr. Emanuel Maltempi de Souza Curitiba Novembro 2006 i Orientador: Profa. Dra. Liu Un Rigo Co-orientador: Prof. Dr. Emanuel Maltempi de Souza ii AGRADECIMENTOS À Profa. Dra. Liu Un Rigo pela orientação neste trabalho e amizade. Ao Prof. Dr. Emanuel M. de Souza pela co-orientação e participação. Ao Prof. Dr. Fábio de O. Pedrosa e ao Núcleo de Fixação de Nitrogênio pela oportunidade do trabalho de doutorado em seu laboratório e contribuição. Aos Profs. Drs. Leda S. Chubatsu, M. Berenice R. Steffens, Leonardo M. Cruz e Rose A. Monteiro por toda sua colaboração. Ao Dr. M. Geoffrey Yates pela revisão crítica do manuscrito. Aos Drs. Humberto J. de O. Ramos, Carolina W. Galvão, Salah M. AlJanabi, Guilherme L. Sassaki e Alan G. Gonçalves pelo suporte técnico. Ao Dr. Ray Dixon pela discussão. Ao Programa Genopar por ceder dados de seqüências de DNA da bactéria H. seropedicae. Aos Drs. J. J. Dennis e G. Z. Zylstra por ceder o plasmídeo precursor pTnMod- OGm. A Valter Baura, Roseli Prado e Julieta Pie pela assistência técnica. Ao Dr. David Galbraith pela oportunidade do estágio de doutorando em seu laboratório, aos Drs. Jeremy D. Edwards, Ambika Gaikwad, Jaroslav Janda, Chanqing Zhang e Roger Barthelson por sua contribuição no trabalho de estágio, e a Georgina Lambert e Leukena Cheam pela assistência técnica durante o mesmo. Às agências CNPq (Demanda Espontânea), CAPES (PDEE), PRONEX (FINEP/CNPq/MCT), CNPq/PADCT III, Paraná Tecnologia e Fundação Araucária pelo financiamento deste trabalho. Aos colegas, amigos e familiares por todo o suporte à execução deste trabalho. iii SUMÁRIO LISTA DE FIGURAS ............................................................................................................................v LISTA DE TABELAS......................................................................................................................... vii LISTA DE EQUAÇÕES..................................................................................................................... vii LISTA DE ABREVIATURAS .......................................................................................................... viii RESUMO .............................................................................................................................................. ix 1 INTRODUÇÃO ................................................................................................................................1 1.1 FIXAÇÃO BIOLÓGICA DE NITROGÊNIO..............................................................................1 1.2 COLONIZAÇÃO ENDOFÍTICA POR DIAZOTROFOS ...........................................................6 1.3 MECANISMOS DE REGULAÇÃO DA TRANSCRIÇÃO EM BACTÉRIAS........................10 1.4 ASSIMILAÇÃO DE NITROGÊNIO EM PROTEOBACTÉRIAS ...........................................12 1.5 Herbaspirillum seropedicae .......................................................................................................23 1.6 OBJETIVOS...............................................................................................................................27 1.6.1 Objetivo Geral......................................................................................................................27 1.6.2 Objetivos Específicos ...........................................................................................................27 2 MATERIAL E MÉTODOS...........................................................................................................28 2.1 ESTIRPES, MEIOS E CONDIÇÕES DE CULTIVO................................................................28 2.1.1 Escherichia coli.....................................................................................................................28 2.1.2 Herbaspirillum seropedicae..................................................................................................28 2.2 PREPARAÇÃO DE PLASMÍDEOS PELA TÉCNICA DA LISE ALCALINA.......................29 2.3 CLIVAGEM DE DNA POR ENZIMAS DE RESTRIÇÃO ......................................................30 2.4 ELETROFORESE EM GEL DE ÁGAR OU AGAROSE .........................................................30 2.5 LIGAÇÃO DE DNA ..................................................................................................................30 2.6 TRANSFORMAÇÃO POR ELETROPORAÇÃO ....................................................................31 2.6.1 Preparação de células eletrocompetentes ..........................................................................31 2.6.2 Eletroporação.......................................................................................................................31 2.7 ENSAIO DE ATIVIDADE DE β-GALACTOSIDASE.............................................................32 2.8 DETERMINAÇÃO DA CONCENTRAÇÃO DE PROTEÍNA.................................................33 2.9 PREPARAÇÃO DE DNA CROMOSSÔMICO DE Herbaspirillum seropedicae ....................34 2.10 CONSTRUÇÃO E CLONAGEM DO PLASMÍDEO MUTAGÊNICO pTnMod-OGmKmlacZ .....................................................................................................................................................35 2.11 IDENTIFICAÇÃO DOS GENES ALVOS DE INSERÇÃO ...................................................37 2.11.1 Preparação da amostra de DNA.......................................................................................37 2.11.2 Reação de seqüenciamento................................................................................................38 2.11.3 Edição e análise das seqüências ........................................................................................39 2.12 TESTES DE FISIOLOGIA ......................................................................................................40 2.13 INVESTIGAÇÃO DA PRESENÇA DE ÁCIDO INDOL-3-ACÉTICO NO SOBRENADANTE DE CULTURAS DE Herbaspirillum seropedicae.....................................40 2.13.1 Quantificação de compostos de indol ...............................................................................40 2.13.2 Detecção de ácido indol-3-acético por cromatografia em camada delgada ..................41 3 RESULTADOS...............................................................................................................................43 3.1 OBTENÇÃO DO PLASMÍDEO pTnMod-OGmKmlacZ PARA A MUTAGÊNESE ALEATÓRIA DE Herbaspirillum seropedicae ..........................................................................43 3.2 CONSTRUÇÃO E ANÁLISE ESTATÍSTICA DE UMA BIBLIOTECA DE MUTANTES ALEATÓRIOS DE Herbaspirillum seropedicae ........................................................................44 3.3 OBTENÇÃO DE UMA COLEÇÃO DE MUTANTES Herbaspirillum seropedicae Z78 COM PADRÃO DE EXPRESSÃO lacZ DIFERENCIAL....................................................................45 3.4 IDENTIFICAÇÃO DAS ORFS CONTENDO INSERÇÃO OGmKmlacZ COM PADRÃO DE EXPRESSÃO lacZ DIFERENCIAL ...........................................................................................51 3.5 EFEITOS FISIOLÓGICOS DAS MUTAÇÕES E EXPRESSÃO DOS GENES ALVOS DE INSERÇÃO EM FONTES VARIADAS DE NITROGÊNIO.....................................................62 iv 3.6 PARTICIPAÇÃO DA PROTEÍNA NITRILA HIDRATASE NO METABOLISMO DE ÁCIDO INDOL-3-ACÉTICO DE Herbaspirillum seropedicae .................................................70 3.7 IDENTIFICAÇÃO DE PROVÁVEIS ELEMENTOS DE REGULAÇÃO NAS SEQÜÊNCIAS A MONTANTE DOS GENES ALVOS DE INSERÇÃO REGULADOS POR AMÔNIO.......72 4 DISCUSSÃO...................................................................................................................................81 5 CONCLUSÕES ..............................................................................................................................93 ADENDO ..............................................................................................................................................94 A1 INTRODUÇÃO ...........................................................................................................................95 A1.1 JUSTIFICATIVA ....................................................................................................................95 A1.2 EXPERIMENTOS EM MICROARRANJO DE OLIGONUCLEOTÍDEOS E MÉTODOS DE DETECÇÃO DE HIBRIDIZAÇÃO ............................................................................................97 A1.3 OBJETIVOS ..........................................................................................................................102 A1.3.1 Objetivo geral ..................................................................................................................102 A1.3.2 Objetivos específicos .......................................................................................................102
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