京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21813号 / 農博第2326号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5185(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

>Magister Scientiae - MSc / The complete degradation of recalcitrant lignocellulose biomass into value-added products requires the efficient and synergistic action of lignocellulose degrading enzymes. This has resulted in a need for the discovery of new hydrolytic enzymes which are more effective than commonly used ones. β-xylosidases and β-glucosidases are key glycoside hydrolases (GHs) that catalyse the final hydrolytic steps of xylan and cellulose degradation, essential for the complete degradation of lignocellulose. Functional-based metagenomics has been employed successfully for the identification and discovery of novel GH genes from a metagenome library. Therefore, this approach was used in this study to increase the chances of discovering novel glycoside hydrolase genes from a horse manure metagenomic DNA library constructed in a previous study. Three fosmid clones P55E4, P81G1, and P89A4 exhibiting β-xylosidase activity were found to encode putative glycosyl hydrolases designated XylP55, XylP81, and BglP89. Amino acid sequence analysis revealed that XylP55, XylP81, and BglP89 are members of the GH43, GH39, and GH3 glycoside hydrolase families, respectively. Phylogenetic analysis of XylP81 and BglP89 indicated that these showed relatively low sequence similarities to other homologues in the respective GH families. The enzymes were expressed and purified, and only XylP81 and BglP89 were biochemically characterized. XylP81 (~58 kDa) and BglP89 (~84 kDa) both showed optimum activity at pH 6 and 50℃ and retained 100% residual activity at 55℃ after 1-hour indicating that they are moderately thermostable. XylP81 had high specific activity against 4-nitrophenyl-β-D-xylopyranoside (pNPX; 122 U/mg) with a KM value of 5.3 mM, kcat/KM of 20.3 s-1mM-1, and it showed enzyme activity against α-L-arabinofuranosidase, β-galactosidase, and β-glucosidase activity. BglP89 had a high specific activity for 4-nitrophenyl-β-D-glucopyranoside (pNPG; 133.5 U/mg) with a KM value of 8.4 mM, kcat/KM of 22 s-1mM-1 and also showed α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and low β-xylosidase activity. BglP89 also showed low hydrolytic activity on cellobiose, β-glucan, and lichenan indicating that it is a broad specificity β-glucosidase. XylP81 retained ~40% activity in the presence of 3 M xylose whilst BglP89 showed considerable glucose tolerance at 150 mM glucose and retained ~46% residual activity. This study reveals two metagenomic derived enzymes (β-xylosidase and β-glucosidase) showing characteristics that could make them potential candidates for lignocellulose biomass degradation in biotechnological and industrial applications.

abstract: This project was completed to understand the evolution of the ability to digest wood in termite symbiotic protists. Lower termites harbor bacterial and protist symbionts which are essential to the termite ability to use wood as a nutritional source, producing glycoside hydrolases to break down the polysaccharides found in lignocellulose. Yet, only a few molecular studies have been done to confirm the protist species responsible for particular enzymes. By mining publicly available and newly generated genomic and transcriptomic data, including three transcriptomes from isolated protist cells, I identify over 200 new glycoside hydrolase sequences and compute the phylogenies of eight glycoside hydrolase families (GHFs) reported to be expressed by termite hindgut protists. Of those families examined, the results are broadly consistent with Todaka et al. 2010, though none of the GHFs found were expressed in both termite-associated protist and non-termite-associated protist transcriptome data. This suggests that, rather than being inherited from their free-living protist ancestors, GHF genes were acquired by termite protists while within the termite gut, potentially via lateral gene transfer (LGT). For example one family, GHF10, implies a single acquisition of a bacterial xylanase into termite protists. The phylogenies from GHF5 and GHF11 each imply two distinct acquisitions in termite protist ancestors, each from bacteria. In eukaryote-dominated GHFs, GHF7 and GHF45, there are three apparent acquisitions by termite protists. Meanwhile, it appears prior reports of GHF62 in the termite gut may have been misidentified GHF43 sequences. GHF43 was the only GHF found to contain sequences from the protists not found in the termite gut. These findings generally all support the possibility termite-associated protists adapted to a lignocellulosic diet after colonization of the termite hindgut. Nonetheless, the poor resolution of GHF phylogeny and limited termite and protist sampling constrain interpretation. / Dissertation/Thesis / Masters Thesis Biological Design 2019

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by persistent sinonasal inflammation and sinus microbiome dysbiosis. The basis of this heterogeneity is poorly understood. We sought to address the hypothesis that a limited number of compositionally distinct pathogenic bacterial microbiota exist in CRS patients and invoke discrete immune responses and clinical phenotypes in CRS patients. Results: Sinus brushings from patients with CRS (n = 59) and healthy individuals (n = 10) collected during endoscopic sinus surgery were analyzed using 16S rRNA gene sequencing, predicted metagenomics, and RNA profiling of the mucosal immune response. We show that CRS patients cluster into distinct sub-groups (DSI-III), each defined by specific pattern of bacterial co-colonization (permutational multivariate analysis of variance (PERMANOVA); p = 0.001, r(2) = 0.318). Each sub-group was typically dominated by a pathogenic family: Streptococcaceae (DSI), Pseudomonadaceae (DSII), Corynebacteriaceae [DSIII(a)], or Staphylococcaceae [DSIII(b)]. Each pathogenic microbiota was predicted to be functionally distinct (PERMANOVA; p = 0.005, r(2) = 0.217) and encode uniquely enriched gene pathways including ansamycin biosynthesis (DSI), tryptophan metabolism (DSII), two-component response [DSIII(b)], and the PPAR-gamma signaling pathway [DSIII(a)]. Each is also associated with significantly distinct host immune responses; DSI, II, and III(b) invoked a variety of pro-inflammatory, T(H)1 responses, while DSIII(a), which exhibited significantly increased incidence of nasal polyps (Fisher's exact; p = 0.034, relative risk = 2.16), primarily induced IL-5 expression (Kruskal Wallis; q = 0.045). Conclusions: A large proportion of CRS patient heterogeneity may be explained by the composition of their sinus bacterial microbiota and related host immune response-features which may inform strategies for tailored therapy in this patient population.

This thesis describes the use of different DNA sequencing technologies to investigate the structure and function of microbial communities in two extreme environments, boreal acid sulfate soil and the terrestrial deep biosphere. The first of the two investigated environments was soils containing un-oxidized metal sulfides that are termed ‘potential acid sulfate soil’ (PASS) materials. If these materials are exposed to atmospheric oxygen by either natural phenomena (e.g., land uplift) or human activities (e.g., drainage) then the metal sulfides become oxidized and the PASS becomes acidic and is defined as an ‘acid sulfate soil’ (ASS). The resulting acid and metal release from metal sulfide oxidation can lead to severe environmental damage. Although acidophilic microorganisms capable of catalyzing acid and metal release have been identified from many sulfide mineral containing environments, the microbial community of boreal PASSs/ASSs remains unclear. This study investigated the physicochemical and microbial characteristics of PASSs and ASSs from the Risöfladan experimental field in Vasa, Finland. Sanger sequencing of 16S rRNA gene sequences of microorganisms present in the PASSs and ASSs were mostly assigned to acidophilic species and environmental clones previously identified from acid- and metal-contaminated environments. Enrichment cultures inoculated from the ASS demonstrated that the acidophilic microorganisms were responsible for catalyzing acid and metal release from PASSs/ASSs. Lastly, the study investigated how to mitigate metal sulfide oxidation and the concomitant formation of sulfuric acid by treating ASSs in situ with CaCO3 or Ca(OH)2 suspensions. The DNA sequencing still identified acidophilic microorganisms after the chemical treatments. However, the increased pH during and after treatment suggested that the activity of the acidophiles might be inhibited. This study was the first to identify the microbial community present in boreal PASSs/ASSs and suggested that treatment with basic compounds may inhibit microbial catalysis of metal sulfide dissolution. The second studied environment was the deep, dark terrestrial subsurface that is suggested to be both extremely stable and highly oligotrophic. Despite the scarcity of carbon and energy sources, the deep biosphere is estimated to constitute up to 20% of the total biomass on earth and thus, represents the largest microbial ecosystem. However, due to the difficulties of accessing this environment and our inability to cultivate the indigenous microbial populations, details of the diversity and metabolism of these communities remain largely unexplored. This study was carried out at Äspö Hard Rock Laboratory, Sweden and utilized second-generation sequencing to identify the taxonomic composition and genetic potential of planktonic and biofilm populations. Community DNA sequencing of planktonic cells from three water types at varied age and depth (‘modern marine’, ‘undefined mixed’, and ‘old saline’) showed the existence of ultra-small cells capable of passing through a 0.22 μm filter that were phylogenetically distinct communities from the >0.22 μm fraction. The reduced cell size and/or genome size suggested a potential adaptation to the oligotrophic environment in the terrestrial deep biosphere. The identified planktonic communities were dominated by Proteobacteria, Candidate divisions, unclassified archaea, and unclassified bacteria. Functional analysis of the assembled genomes showed that the planktonic population from the shallow modern marine water demonstrated a predominantly anaerobic and heterotrophic lifestyle. In contrast, the deeper, old saline water was more closely aligned with the hypothesis of a hydrogen-driven deep biosphere. Metagenomic analysis of subsurface biofilms from ‘modern marine’ and ‘old saline’ water types suggested only a subset of populations were involved in initial biofilm formation. The identified biofilm populations from both water types were distinct from the planktonic community and were suggested to be dominated by hydrogen fed, chemolithoautotrophic and diazotrophic populations.

Este trabalho teve como objetivo analisar, através de uma abordagem metagenômica/computacional, a variação do conteúdo gênico e do potencial metabólico das comunidades microbianas associadas a dois ambientes da Fundação Parque Zoológico de São Paulo: um reservatório artificial, o Lago São Francisco e o sistema de compostagem de resíduos do parque. Para o estudo do primeiro ambiente, amostras foram coletadas mensalmente no Lago São Francisco de outubro de 2012 a setembro de 2013 e submetidas ao sequenciamento metagenômico. Esse estudo mostrou que agrupamentos de amostras de uma mesma estação são estatisticamente suportados. As coocorrências de espécies, com suporte estatístico alto, foram estabelecidas e representadas em uma rede separada em 60 grupos ou módulos. A maioria dos grandes módulos foram formados quase exclusivamente por espécies do mesmo filo, indicando possíveis mecanismos de resposta a fatores ambientais e à presença de nutrientes ao invés da pura interação entre espécies. Os fatores que levaram à variação dos táxons também influenciaram o potencial metabólico da comunidade: embora os módulos metabólicos de forma geral estivessem distribuídos ao longo dos meses, alguns se destacaram pela variação intensa, principalmente na amostra de novembro de 2012. Para o estudo do segundo ambiente, foram analisados dois conjuntos de dados de sequenciamento metagenômico gerados a partir de amostras seriadas coletadas ao longo do processo de compostagem. Nos dias iniciais do processo de compostagem, os mais discrepantes, houve uma super-representação de módulos metabólicos relacionados principalmente ao metabolismo de carboidratos e à síntese de aminoácidos. Em conjunto, os dados obtidos nesse estudo indicaram uma comunidade microbiana com potencial metabólico variando direcionalmente em função dos compostos inicialmente presentes ou oriundos do substrato, em função da presença de oxigênio e em função da etapa do processo. / This project aimed to analyze, through a computational approach, the change of gene content and metabolic potential in metagenomes from two microbiomes from São Paulo Zoo Park Foundation: an artificial reservoir, Lake São Francisco, and the compost systems in the park. For the study on the first microbiome, samples were monthly collected in Lake São Francisco from October 2012 to September 2013 and submitted to metagenomic sequencing. This study showed that clustering of the samples from a same season is statistically supported. The species co-occorrences, with high statistical support, were established and represented in a network composed by 60 groups or modules. The most biggest modules were formed almost exclusively by species of the same phylum, pointing possible response mechanisms to environmental factors and to presence of nutrients instead of the simple species interaction. The factors which leaded to taxa variation also influenced the metabolic potential of the community: although the metabolic modules generally are spreaded through the months, some highlighted because the intense variation, mainly in the sample from November 2012. For the latter environment, two metagenomic datasets were analyzed from serial samples collected throughout the composting process. In the initial days of composting process, the most discrepant, there was an uprepresentation of metabolic modules related to carbohydrates metabolism and amino acids synthesis. All data in this study indicated a microbial community with metabolic potential varying according to nutrientes, oxygen presence and process stage.

O processo de domesticação e posterior melhoramento das plantas cultivadas foram essenciais para o sustento do crescimento populacional observado na recente história da humanidade. Porém, no processo de melhoramento das culturas não foi considerado o importante papel que o microbioma rizosférico desempenha nas plantas. Neste contexto, esta pesquisa foi direcionada ao estudo do microbioma rizosférico do feijoeiro, considerando um genótipo selvagem e outro cultivado, a fim de determinar a composição e funcionalidade do microbioma em cada rizosfera. Assim, para testar a hipótese de que materiais ancestrais têm maior capacidade de hospedar micro-organismos benéficos na rizosfera quando comparados a cultivares modernas, os genótipos de feijão cultivado IAC Alvorada e selvagem Wild Mex foram cultivados em Terra Preta da Amazônia (TPA), solo caracterizado por abrigar uma grande diversidade microbiana. O DNA total do solo rizosférico de cada genótipo de feijão e do bulk soil foi extraído para realizar o sequenciamento metagenômico usando a plataforma Illumina MiSeq. O solo rizosférico também foi usado para isolar e selecionar bactérias antagonistas a fungos radiculares patógenos do feijoeiro, Rhizoctonia solani e Fusarium oxysporum f. sp. phaseoli. A abordagem dependente de cultivo permitiu recuperar 11 bactérias que apresentaram atividade antagônica in vitro contra os patógenos avaliados, os isolados bacterianos foram identificados como pertencentes aos gêneros Streptomyces, Kitasatospora, Alcaligenes, Achromobacter, Pseudomonas, Stenotrophomonas, Brevibacillus e Paenibacillus. A abordagem independente de cultivo revelou que a composição da comunidade microbiana na rizosfera do feijão selvagem Wild Mex foi caracterizada pela maior abundância relativa dos filos bacterianos Acidobacteria, Verrucomicrobia, Gemmatimonadetes, e do filo fúngico Glomeromycota quando comparada com a composição da rizosfera do genótipo cultivado IAC Alvorada, a qual foi constituída em maior proporção pelos filos bacterianos Firmicutes, Planctomycetes, Deinococcus-Thermus e pelo filo fúngico Ascomycota. No nível taxonômico de gênero, a comunidade microbiana da rizosfera do feijão selvagem Wild Mex apresentou maior frequência relativa de gêneros fixadores do nitrogênio, nitrificadores, antagonistas e promotores do crescimento vegetal. A rizosfera do feijão selvagem Wild Mex apresentou maior abundância relativa de funções relacionadas à fixação do nitrogênio, produção de sideróforos e de ácido indol acético (AIA), quando comparada com o genótipo cultivado IAC Alvorada. O padrão de distribuição observado na análise de ordenação das funções no microbioma da rizosfera do feijão selvagem Wild Mex foi diferente do padrão encontrado no bulk soil e na rizosfera do feijão cultivado IAC Alvorada. Os resultados indicaram que o genótipo selvagem é mais seletivo no recrutamento de micro-organismos e funções na rizosfera quando comparado com o cultivar moderno. Em conclusão, os resultados revelaram que o processo de domesticação e melhoramento genético das plantas cultivadas potencialmente debilitou a capacidade do hospedeiro em selecionar e sustentar micro-organismos e funções benéficas na rizosfera. / The process of domestication and subsequent plant breeding were key to support human population growth over the last decades. However, plant breeding has neglected the important role of the rhizosphere microbiome on plant performance. In this context, this research aimed the study of common bean rhizosphere microbiome in a wild and in a cultivated genotype to determine the composition and functionality of their microbial community. We tested the hypothesis that ancestor materials have higher ability to host beneficial microorganisms in the rhizosphere when compared to modern cultivars. The common bean genotype IAC Alvorada and wild common bean Wild Mex were grown in highly biodiverse soil (Amazonian Dark Earth - ADE) and the total DNA from bulk soil and each common bean rhizosphere was extracted for sequencing by using Illumina MiSeq platform. In addition, rhizosphere soil was also used to isolate and select antagonistic bacteria against soil borne pathogens Rhizoctonia solani and Fusarium oxysporum f. sp. phaseoli. Using cultivation-dependent approach, 11 bacteria were isolated and showed antagonistic in vitro activity against the evaluated pathogens. The bacterial isolates were identified belonging to Streptomyces, Kitasatospora, Alcaligenes, Achromobacter, Pseudomonas, Stenotrophomonas, Brevibacillus and Paenibacillus genus. The cultivation- independent approach revealed that microbial community composition in the Wild Mex bean rhizosphere was characterized by higher relative abundance of bacterial phyla Acidobacteria, Verrucomicrobia, Gemmatimonadetes and fungal phylum Glomeromycota when compared with IAC Alvorada cultivated bean, which showed a higher relative abundance of bacterial phyla Firmicutes, Planctomycetes, Deinococcus-Thermus and fungal phylum Ascomycota. Wild Mex rhizosphere microbiome showed higher relative frequency of nitrogenfixing, nitrifying, antagonists and plant growth promoting microorganisms. The wild bean also showed higher relative abundance of functions related to nitrogen fixation, siderophore and indole acetic acid (IAA) production, when compared with IAC Alvorada bean. The distribut ion pattern observed in the functions ordination analysis of Wild Mex was different from the bulk soil and IAC Alvorada patterns. The results showed that wild genotype is more selective for recruiting microorganisms and functions in the rhizosphere when compared with modern cultivar. In conclusion, the results revealed that domestication and plant breeding potentially undermined rhizosphere microbiome composition and functions debilitating the host\'s abilit y to select and support beneficial microbes.

O processo de domesticação e posterior melhoramento das plantas cultivadas foram essenciais para o sustento do crescimento populacional observado na recente história da humanidade. Porém, no processo de melhoramento das culturas não foi considerado o importante papel que o microbioma rizosférico desempenha nas plantas. Neste contexto, esta pesquisa foi direcionada ao estudo do microbioma rizosférico do feijoeiro, considerando um genótipo selvagem e outro cultivado, a fim de determinar a composição e funcionalidade do microbioma em cada rizosfera. Assim, para testar a hipótese de que materiais ancestrais têm maior capacidade de hospedar micro-organismos benéficos na rizosfera quando comparados a cultivares modernas, os genótipos de feijão cultivado IAC Alvorada e selvagem Wild Mex foram cultivados em Terra Preta da Amazônia (TPA), solo caracterizado por abrigar uma grande diversidade microbiana. O DNA total do solo rizosférico de cada genótipo de feijão e do bulk soil foi extraído para realizar o sequenciamento metagenômico usando a plataforma Illumina MiSeq. O solo rizosférico também foi usado para isolar e selecionar bactérias antagonistas a fungos radiculares patógenos do feijoeiro, Rhizoctonia solani e Fusarium oxysporum f. sp. phaseoli. A abordagem dependente de cultivo permitiu recuperar 11 bactérias que apresentaram atividade antagônica in vitro contra os patógenos avaliados, os isolados bacterianos foram identificados como pertencentes aos gêneros Streptomyces, Kitasatospora, Alcaligenes, Achromobacter, Pseudomonas, Stenotrophomonas, Brevibacillus e Paenibacillus. A abordagem independente de cultivo revelou que a composição da comunidade microbiana na rizosfera do feijão selvagem Wild Mex foi caracterizada pela maior abundância relativa dos filos bacterianos Acidobacteria, Verrucomicrobia, Gemmatimonadetes, e do filo fúngico Glomeromycota quando comparada com a composição da rizosfera do genótipo cultivado IAC Alvorada, a qual foi constituída em maior proporção pelos filos bacterianos Firmicutes, Planctomycetes, Deinococcus-Thermus e pelo filo fúngico Ascomycota. No nível taxonômico de gênero, a comunidade microbiana da rizosfera do feijão selvagem Wild Mex apresentou maior frequência relativa de gêneros fixadores do nitrogênio, nitrificadores, antagonistas e promotores do crescimento vegetal. A rizosfera do feijão selvagem Wild Mex apresentou maior abundância relativa de funções relacionadas à fixação do nitrogênio, produção de sideróforos e de ácido indol acético (AIA), quando comparada com o genótipo cultivado IAC Alvorada. O padrão de distribuição observado na análise de ordenação das funções no microbioma da rizosfera do feijão selvagem Wild Mex foi diferente do padrão encontrado no bulk soil e na rizosfera do feijão cultivado IAC Alvorada. Os resultados indicaram que o genótipo selvagem é mais seletivo no recrutamento de micro-organismos e funções na rizosfera quando comparado com o cultivar moderno. Em conclusão, os resultados revelaram que o processo de domesticação e melhoramento genético das plantas cultivadas potencialmente debilitou a capacidade do hospedeiro em selecionar e sustentar micro-organismos e funções benéficas na rizosfera. / The process of domestication and subsequent plant breeding were key to support human population growth over the last decades. However, plant breeding has neglected the important role of the rhizosphere microbiome on plant performance. In this context, this research aimed the study of common bean rhizosphere microbiome in a wild and in a cultivated genotype to determine the composition and functionality of their microbial community. We tested the hypothesis that ancestor materials have higher ability to host beneficial microorganisms in the rhizosphere when compared to modern cultivars. The common bean genotype IAC Alvorada and wild common bean Wild Mex were grown in highly biodiverse soil (Amazonian Dark Earth - ADE) and the total DNA from bulk soil and each common bean rhizosphere was extracted for sequencing by using Illumina MiSeq platform. In addition, rhizosphere soil was also used to isolate and select antagonistic bacteria against soil borne pathogens Rhizoctonia solani and Fusarium oxysporum f. sp. phaseoli. Using cultivation-dependent approach, 11 bacteria were isolated and showed antagonistic in vitro activity against the evaluated pathogens. The bacterial isolates were identified belonging to Streptomyces, Kitasatospora, Alcaligenes, Achromobacter, Pseudomonas, Stenotrophomonas, Brevibacillus and Paenibacillus genus. The cultivation- independent approach revealed that microbial community composition in the Wild Mex bean rhizosphere was characterized by higher relative abundance of bacterial phyla Acidobacteria, Verrucomicrobia, Gemmatimonadetes and fungal phylum Glomeromycota when compared with IAC Alvorada cultivated bean, which showed a higher relative abundance of bacterial phyla Firmicutes, Planctomycetes, Deinococcus-Thermus and fungal phylum Ascomycota. Wild Mex rhizosphere microbiome showed higher relative frequency of nitrogenfixing, nitrifying, antagonists and plant growth promoting microorganisms. The wild bean also showed higher relative abundance of functions related to nitrogen fixation, siderophore and indole acetic acid (IAA) production, when compared with IAC Alvorada bean. The distribut ion pattern observed in the functions ordination analysis of Wild Mex was different from the bulk soil and IAC Alvorada patterns. The results showed that wild genotype is more selective for recruiting microorganisms and functions in the rhizosphere when compared with modern cultivar. In conclusion, the results revealed that domestication and plant breeding potentially undermined rhizosphere microbiome composition and functions debilitating the host\'s abilit y to select and support beneficial microbes.

Made available in DSpace on 2014-12-17T14:05:23Z (GMT). No. of bitstreams: 1 UaskaBS_TESE.pdf: 2951660 bytes, checksum: 93152a3c6ea30c7f4eaa465f3f5d2969 (MD5) Previous issue date: 2013-04-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / 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 Jundia?-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 Jundia? river / A busca por genes baseada na constru??o e an?lise de bibliotecas metagen?micas a partir de solo gera oportunidades para explorar uma enorme diversidade gen?tica e metab?lica de microrganismos. Os rios s?o ecossistemas com alta diversidade biol?gica, mas ainda pouco explorados por meio de metagen?mica. Com o objetivo de explorar a diversidade microbiana, uma biblioteca metagen?mica foi constru?da a partir de DNA extra?do de substrato de rio em tr?s pontos ao longo do rio Jundia? (Rio Grande do Norte-Brasil). Os pontos de amostragem s?o derivados de ?rea aberta, terreno acidentado e com a incid?ncia direta da luz solar. Esta biblioteca foi analisada funcionalmente e tamb?m com base em sequ?ncias. Para a an?lise funcional foi utilizado meio de cultura s?lido LB com concentra??o de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com caracter?sticas halotolerantes. Os DNAs recombinantes foram extra?dos e retransformados em cepa de Escherichia coli DH10B e curvas de sobreviv?ncia foram obtidas para confirma??o e quantifica??o da resist?ncia ao estresse abi?tico. As sequ?ncias dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam prote?nas hipot?ticas. Um dos clones apresentou uma ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na s?ntese do precursor de glicerol, sendo um soluto compat?vel para equilibrar a press?o osm?tica no interior e no exterior das c?lulas. Posteriormente, para identifica??o de genes que codificam osm?litos relacionados com halotoler?ncia e identifica??o da diversidade microbiol?gica, amostras de DNA ambiental do substrato do rio e da coluna d??gua do estu?rio e oceano foram coletadas e pirosequenciadas. As sequ?ncias de osm?litos de diferentes microrganismos foram obtidas a partir do UniProt e utilizadas como RefSeqs para a identifica??o por homologia (TBLASTN) nos bancos de dados metagen?micos. As sequ?ncias identificadas nos bancos de dados ambientais foram submetidas ao programa HMMER com o fim de identificar dom?nios funcionais. Foram identificadas as enzimas: alfa-trealose-fosfato sintase, L-ectoina sintase (ectC), transaminase do ?cido L-2,4-diaminobut?rico (EctB), ?cido L-2 ,4-diaminobut?rico acetiltransferase (EctA), L-treonina 3-desidrogenase (via de s?ntese do sorbitol), Glicerol-3-fosfato desidrogenase, inositol-3-fosfato desidrogenase, chaperonas, L-prolina glicina beta?na liga??o transportador ABC, mio-inositol-1-fosfato sintase, a prote?na simportadora de prolina/s?dio -PutP e trealose-6-fosfato fosfatase. Estas s?o enzimas que participam da s?ntese de osm?litos comumente relacionados a ambientes salinos, no entanto a identifica??o desses solutos em ambiente de rio ? justificada pela elevada concentra??o salina no solo durante prolongadas esta??es de seca neste rio. Quanto ? riqueza da microbiota foi identificado que o substrato do rio possui uma abund?ncia de halobact?rias semelhante a do mar e superior a do estu?rio. Esses dados confirmam a exist?ncia de uma resposta especializada contra o estresse salino por microrganismos no ambiente do rio Jundia?

Orientadora: Eliana Gertrudes Macedo Lemos / Banca: Lúcia Maria Carareto Alves / Banca: Vanderlei Rodrigues / Resumo: Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene. / Mestre