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Proteobacteria from Brazilian Soils

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IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β- PROTEOBACTERIA FROM BRAZILIAN SOILS KRISLE DA SILVA 2009 KRISLE DA SILVA IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β-PROTEOBACTERIA FROM BRAZILIAN SOILS Thesis presented to the Federal University of Lavras as part of the requirementes of Agricultural Microbiology Program to the obtainment of the title of “Doctor”. Major Professor Prof. Fátima Maria de Souza Moreira LAVRAS MINAS GERAIS - BRASIL 2009 Ficha Catalográfica Preparada pela Divisão de Processos Técnicos da Biblioteca Central da UFLA Silva, Krisle da. Identification and functional characterization of diazotrophic β- proteobacteria from Brazilian soils / Krisle da Silva. – Lavras : UFLA, 2009. 124 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2009. Orientador: Fátima Maria de Souza Moreira. Bibliografia. 1. Fixação biológica de nitrogênio. 2. Betaproteobacteria. 3. Leguminosas. 4. Floresta Amazônica. 5. Microbiologia do solo. I. Universidade Federal de Lavras. II. Título. CDD – 631.46 KRISLE DA SILVA IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β-PROTEOBACTERIA FROM BRAZILIAN SOILS Thesis presented to the Federal University of Lavras as part of the requirementes of Agricultural Microbiology Program to the obtainment of the title of “Doctor”. APROVED on 26th August 2009 Prof. Peter Vandamme UGhent Prof. Emanuel de Souza Maltempi UFPR Prof. Fábio Lopes Olivares UENF Prof. Ricardo Magela de Souza UFLA Profa. Patrícia Gomes Cardoso UFLA Prof. Fátima Maria de Souza Moreira UFLA (Major Professor) LAVRAS MINAS GERAIS – BRASIL A Deus, pela presença, amor incondicional, pela força para enfrentar as dificuldades e por preencher minha vida com pessoas maravilhosas. OFEREÇO A Ires Aurora da Silva, pelo amor, dedicação e companherismo, presentes durante toda minha vida. DEDICO AGRADECIMENTOS Primeiramente, a Deus, pois cada conquista, seja ela grande ou pequena, não seria possível sem sua presença em minha vida. Essa é, sem dúvida, mais uma conquista com as bênçãos de Deus. À Universidade Federal de Lavras, em especial ao Programa de Pós- Graduação em Microbiologia Agrícola, pela oportunidade de realização do curso. À Fundação de Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), pela concessão da bolsa de estudos. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela concessão da bolsa de estudos para a realização do doutorado sanduíche. Ao projeto TSBF-CIAT/GEF-UNEP, “Conservation and Sustainable Management of Below Ground Biodiversity”, pelo financiamento para execução deste trabalho. À professora Fátima Maria de Souza Moreira, pela orientação durante o mestrado e doutorado, pela confiança, pelo exemplo e amizade. Ao Prof. Peter Vandamme, pela excelente acolhida e pela coorientação, que foi muito positiva para a realização deste trabalho. A todos os professores do Programa de Pós-Graduação em Microbiologia Agrícola. Um agradecimento especial à pesquisadora Diva de Souza Andrade, pela amizade e iniciação em Microbiologia do Solo. Aos funcionários Marlene Aparecida de Souza e Manuel Aparecido da Silva, pela contribuição na realização deste trabalho e pela amizade. À funcionária do Laboratório de Microbiologia da Universidade de Ghent Evie de Brandt, pela paciência e contribuição a este trabalho. A Magda Aparecida Melo, por toda a dedicação durante o doutorado e pela grande disposição. A todos os amigos do Laboratório de Microbiologia do Solo, em especial Ligiane, Adrianinha, Rogério, Pedro e Michele Rocha. Aos membros da banca examinadora, professores Peter Vandamme, Emanuel Maltempi de Souza, Fábio Lopes Olivares, Ricardo Magela e Patrícia, pelas sugestões e críticas construtivas dadas a este trabalho. Às amigas de república durante mestrado e doutorado: Patrícia, Taís, Gláucia, Sílvia, Fabiana e Ludmila. Vocês foram minha nova família durante o perído em que vivemos juntas. Às amigas que fiz na Bélgica, que fizeram com que os dias nublados e frios se tornassem mais alegres: Inês, Tatiana, Luana, Jéssica, Tânia, Margo e Luciane. Aos meus grandes amigos e/ou irmãos: Meire, Amanda, Gláucia, Cândido e Sandro. Agradeço por todos os momentos que dividimos e que, sem dúvida, foram inesquecíveis. Tenho certeza de a nossa amizade não há distância ou tempo que desgate. Amo vocês. As minhas queridas amigas e/ou irmãs de Londrina, Daiane e Cintia. Apesar da distância, vocês estão sempre perto, seja no meu coração ou na minha mente. Amos vocês! A minha querida mãe, Ires Aurora da Silva, obrigada, pelo amor, exemplo de ser humano, educação e confiança. Amo você demais! Aos meus queridos familiares, Cleonice, Kátia, Diego, Ivani, Ladislau, Pedro e Murilo, pelo grande carinho. APRESENTAÇÃO Nesta tese apresenta-se parte dos resultados do projeto internacional “Conservação e Manejo Sustentado da Biodiversidade do Solo”, implementado em sete países tropicais - Brasil, Costa do Marfim, India, Indonesia, Quênia, Mexico e Uganda. Este projeto é coordenado pelo Tropical Soil Biology and Fertility Institute do CIAT (TSBF-CIAT) com cofinanciamento do "Global Environmental Facility" (GEF) e suporte para implementação do "United Nations Environment Program" (UNEP). O componente brasileiro do projeto é denominado BiosBrasil e é coordenado pela Universidade Federal de Lavras (UFLA) (www.biosbrasil.ufla.br). As opiniões expressas nesta publicação são de responsabilidade de seus autores e não necessariamente refletem aquelas das instituições dos autores, do "United Nations Environment Programme" ou do "Global Environmental Facility". SUMMARY Page TABLE LISTS.................................................................................................. i FIGURE LISTS................................................................................................ iii GENERAL ABSTRACT.................................................................................. v RESUMO GERAL........................................................................................... vi CHAPTER 1..………………………………………………………………... 01 1 Introdução Geral…………………………………………………………… 02 2 Referencial Teórico........................................................................................ 04 2.1 O gênero Burkholderia............................................................................... 04 2.2 Burkholderia spp. como bactérias promotoras do crescimento vegetal (PGPB).............................................................................................................. 05 2.3 Estirpes de Burkholderia e fixação biológica de nitrogênio...................... 08 2.4 Complexo Burkholderia cepacia (Bcc)………………………………….. 13 2.5 Complexo B. cepacia X ambiente.............................................................. 14 2.6 Estudos de identificação de Burkholderia spp............................................ 18 2.7 O gênero Cupriavidus................................................................................. 21 2.8 O gênero Cupriavidus e a fixação biológica de nitrogênio........................ 23 3 Referências Bibliográficas............................................................................. 31 CHAPTER 2: Diazotrophic Burkholderia spp. (B. fungorum, B. contaminans and B. lata) isolated from the Amazon region exhibit phenotypical, functional and genetic diversity………………………………. 53 1 Resumo.......................................................................................................... 54 2 Abstract.......................................................................................................... 55 3 Introduction……………………………………………………………….... 56 4 Material and Mhetods……………………………………………................ 58 4.1 Origin of isolates…………………………………………………………. 58 4.2 Preliminary identification………………………………………………... 62 4.3 Phenotypic characterization….................................................................... 62 4.3.1 Cultural characterization………………………………………………. 62 4.3.2 Biochemical characterization………………………………………….. 63 4.3.3 SDS-PAGE of whole-cell proteins…………………………………….. 63 4.4 Plant growth promotion traits…………………………………………... 63 4.4.1 Phosphate solubilization assay…………………………………………. 63 4.4.2 Nitrogenase activity……………………………………………………. 64 4.4.3 Antifungal-activity of Burkholderia isolates…………………………... 64 4.5 Genetic characterization…………………………………………………. 65 4.5.1 16S rRNA sequencing…………………………………………………. 65 4.5.2 gyrB gene sequencing………………………………………………….. 66 4.5.3 Bcc-specifc recA gene and MLST……………………………………... 67 5 Results……………………………………………………………………… 69 5.1 Phenotypic characterization……………………………………………… 69 5.1.1 Cultural characterization……………………………………………….. 69 5.1.2 Biochemical characterization…………………………………………... 69 5.1.3 SDS-PAGE of whole-cell proteins…………………………………….. 69 5.2 Plant growth promotion traits……………………………………………. 73 5.2.1 Phosphate solubilization assay…………………………………………. 73 5.2.2 Nitrogenase activity…………………………………………………… 73 5.2.3 Antifungal-activity of Burkholderia isolates…………………………... 74 5.3 Genetic characterization…………………………………………………. 75 5.3.1 16S rRNA sequencing…………………………………………………. 75 5.3.2 gyrB genes amplification and sequencing……………………………... 76 5.3.3 Bcc-specifc recA gene and MLST……………………………………... 78 6 Discussion………………………………………………………………….. 80 6.1 Phenotypic characteristics and Plant growth promotion traits .…………. 80 6.2 Genotipic characterization……………………………………………….. 81 7 Conclusions.................................................................................................... 83 8 References.....................................................................................................
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    Table S5. The information of the bacteria annotated in the soil community at species level No. Phylum Class Order Family Genus Species The number of contigs Abundance(%) 1 Firmicutes Bacilli Bacillales Bacillaceae Bacillus Bacillus cereus 1749 5.145782459 2 Bacteroidetes Cytophagia Cytophagales Hymenobacteraceae Hymenobacter Hymenobacter sedentarius 1538 4.52499338 3 Gemmatimonadetes Gemmatimonadetes Gemmatimonadales Gemmatimonadaceae Gemmatirosa Gemmatirosa kalamazoonesis 1020 3.000970902 4 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas indica 797 2.344876284 5 Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus Lactococcus piscium 542 1.594633558 6 Actinobacteria Thermoleophilia Solirubrobacterales Conexibacteraceae Conexibacter Conexibacter woesei 471 1.385742446 7 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas taxi 430 1.265115184 8 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas wittichii 388 1.141545794 9 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas sp. FARSPH 298 0.876754244 10 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sorangium cellulosum 260 0.764953367 11 Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae Sorangium Sphingomonas sp. Cra20 260 0.764953367 12 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas panacis 252 0.741416341
  • Degradation of 2-Nitrobenzoate by Burkholderia Terrae Strain KU-15

    Degradation of 2-Nitrobenzoate by Burkholderia Terrae Strain KU-15

    Biosci. Biotechnol. Biochem., 71 (1), 145–151, 2007 Degradation of 2-Nitrobenzoate by Burkholderia terrae Strain KU-15 y Hiroaki IWAKI and Yoshie HASEGAWA Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan Received July 25, 2006; Accepted September 17, 2006; Online Publication, January 7, 2007 [doi:10.1271/bbb.60419] Bacterial strain KU-15, identified as a Burkholderia tified and characterized. This information has contrib- terrae by 16S rRNA gene sequence analysis, was one of uted to understanding of the nature of the biodegradation 11 new isolates that grew on 2-nitrobenzoate as sole of nitroaromatic compounds and the development of source of carbon and nitrogen. Strain KU-15 was also bioremediation solutions. Very little research, however, found to grow on anthranilate, 4-nitrobenzoate, and 4- has been done on the degradation of 2-nitrobenzoate aminobenzoate. Whole cells of strain KU-15 were found (2-NBA). to accumulate ammonia in the medium, indicating that The degradation pathway of 2-NBA has been estab- the degradation of 2-nitrobenzoate proceeds through a lished from only two microorganisms: Pseudomonas reductive route. Metabolite analyses by high-perform- fluorescens strain KU-7 and Arthrobacter protophor- ance liquid chromatography indicated that 3-hydrox- miae strain RKJ100 (Fig. 5).13–15) These strains share the yanthranilate, anthranilate, and catechol are intermedi- same 2-NBA degradation pathway, including the for- ates of 2-nitrobenzoate metabolism in strain KU-15. mation of 2-hydroxylaminobenzoate (2-HABA) by an Enzyme studies suggested that 2-nitrobenzoate degra- NADPH-dependent nitroreductase.