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DIVERSIDADE DE RIZÓBIOS ISOLADOS DE NÓDULOS DE Mimosa gymnas Barneby NATIVAS DOS CAMPOS GERAIS DO PARANÁ (BRASIL)Paulitsch, Fabiane 22 February 2017 (has links)
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Previous issue date: 2017-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Levantamentos florísticos realizados nos “Campos Gerais” (Paraná, Brasil) indicam que Fabaceae é uma das famílias com maior riqueza e abundância da região. Esses estudos também apontam a elevada diversidade e endemismo de Mimosa spp. Nosso estudo relata a diversidade de rizóbios isolados de nódulos radiculares de plantas nativas de Mimosa gymnas Barneby. Vinte e nove estirpes foram obtidas de M. gymnast nodulíferas em três áreas do Parque Estadual do Guartelá, uma das mais importantes unidades de conservação dos Campos Gerais; os solos dessas áreas foram caracterizados como arenosos, ácidos, pobre em nutrientes e matéria orgânica e com elevados teores de alumínio. A árvore filogenética construída com sequencias parciais do gene 16S rDNA agrupou as estirpes em um grande clado sendo que algumas estipes foram mais relacionados com a espécie Paraburkholderia nodosa enquanto outras foram mais relacionadas com P. bannensi. A análise de perfil genômico baseada em BOX-PCR revelou um elevado grau de variabilidade intraespecífica entre os isolados. Resultados baseados na filogenia de sequencias concatenadas dos genes recA-gyrB dividiram os isolados em dois grandes grupos. O clado II não agrupou nenhuma estirpe tipo e os isolados apresentaram uma identidade nucleotídica de no máximo 97% com P. nodosa. A filogenia do gene nodC agrupou todos os isolados em um grupo único com elevado suporte estatístico, não agrupando com a sequencia correspondente de nenhuma estirpe tipo do gênero Paraburkholderia. Nossos resultados reforçam a constatação de que rizóbios do gênero Paraburkholderia são os preferenciais simbiontes de Mimosa na América do Sul e a associação de estirpes nodulíferas do gênero com condições edáficas particulares. Ainda, os resultados das análises filogenéticas sugerem que os isolados agrupados no clado II na árvore filogenética dos genes recA-gyrB podem representar uma nova espécie de Paraburkholderia, reinterando a importância de estudos de diversidade com plantas leguminosas nativas e endêmicas.
. / Floristic surveys performed in the “Campos Gerais” (Paraná, Brazil) indicate that Fabaceae is one of the most species-rich and abundant families. These studies also pointed out the high diversity and endemism of Mimosa spp.
Our study report the diversity of rhizobia isolated from root nodules of native Mimosa gymnas Barneby. Twenty-nine strains were obtained from from nodulating M. gymnas in three areas of Guartelá State Park, one of the most important conservation unities of the “Campos Gerais”; soils of these areas were characterized as sandy, acid, poor in nutrients and organic matter and with high aluminum contents. The phylogenetic tree constructed with partial 16S rDNA sequences grouped the strains in a larger cluster and some strains were more related with Paraburkholderia nodosa, although some strains were more related to P. bannensi. The analysis of the genomic profile by BOX-PCR revelead a high degree of intraspecific variability among the isolates. Results based on phylogeny of concatened genes recA-gyrB divided the strains in two large clusters. The cluster II didn’t grouped with any type strain and the isolates showed a nucleotide identity of 16S rDNA of at most 97% with P. nodosa. The nodC phylogeny grouped all the strains into a well-supported clade, not grouping with any know strain of Paraburkholderia genus. Our results support that Paraburkholderia are the main symbionts of Mimosa in South America and the association of nodulating strains of this genera with particular edaphic conditions. Also, the results of the phylogenetic analysis suggest that the isolates grouped in cluster II in the recA-gyrB phylogenetic tree may represent a new species of Paraburkolderia, highlighting the importance of diversity studies with native and endemic leguminous plants.
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Análise da diversidade microbiana aquática em rios e lagos da região amazônicaToyama, Danyelle 07 November 2012 (has links)
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Previous issue date: 2012-11-07 / Financiadora de Estudos e Projetos / The Amazon region has the largest hydrographic basin on the planet, the one that includes Amazon River, and it also has the largest rainforest in the world. Moreover, it presents a great biological diversity, both related to the fauna and flora, and microbiological. Microorganisms are responsible for most biogeochemical cycles that shape the terrestrial environment and the freshwater and marine ecosystems, and they can be widely exploited biotechnologically. It is estimated that less than 1% of all bacterial species is known due to our inability to simulate the environment in which they live. However, new techniques have allowed the study of these microorganisms. Through Metagenomics it is possible to study complex environmental samples without the need for isolation and individual cultivation of these organisms. For this purpose, the16S ribosomal DNA (16S rDNA) is used in bacteria and archaea in order to study phylogeny and diversity. This sequence is used because it has been fairly maintained during the processes of biological evolution and it may serve as an indicator of how organisms are closely related. For these studies, this region was amplified by the Polymerase Chain Reaction (PCR) and cloned into vectors through the recombinant DNA technology, thus enabling the construction of 16S rRNA libraries. These libraries were then sequenced and the microorganisms were identified by comparison with databases. In this study it was used DNA extracted from Solimões River filtered water, in addition to water from other rivers and adjacent lakes, to the construction of libraries, in order to study the biodiversity through 16S rRNA analysis. In all libraries, phylum Proteobacteria was the most abundant, and most of the genera observed belong to the Betaproteobacteria class. The freshwater cosmopolitan taxa Candidatus Planktophila limnetica and Polynucleobacter were observed, as well as primary producers were represented by the genera Synechococcus and Cyanobium. Samples in which the construction of 16S rRNA libraries was possible for Archaea, the phylum Crenarchaeota was the most abundant in all libraries. / A Região Amazônica apresenta a maior bacia hidrográfica do planeta, a do rio Amazonas, e também a maior floresta tropical do mundo. Além disso, apresenta uma grande diversidade biológica, tanto relacionada à fauna e flora, quanto microbiológica. Os micro-organismos são responsáveis pela maioria dos ciclos biogeoquímicos que moldam o ambiente terrestre e os ecossistemas de água doce e marinhos, e podem ser amplamente explorados biotecnologicamente. Estima-se que menos de 1% de todas as espécies bacterianas seja conhecida, devido à incapacidade de simulação do ambiente em que vivem. Contudo, novas técnicas têm possibilitado o estudo desses micro-organismos. Através do Metagenoma é possível estudar amostras ambientais complexas sem a necessidade de isolamento e cultivo individual desses organismos. Para tanto, utiliza-se, em bactérias e arquéias, o DNA ribossomal 16S (16S rDNA), para fins de estudos de filogenia e diversidade. Esta sequência é utilizada por se ter mantido bastante conservada durante os processos de evolução biológica, podendo servir como um indicador de como os organismos estão intimamente relacionados. Para estes estudos, esta região foi amplificada pela Reação em Cadeia da Polimerase (PCR) e clonada em vetores através da tecnologia do DNA recombinante, possibilitando, assim, a construção de bibliotecas de 16S rRNA. Estas bibliotecas foram então sequenciadas e os micro-organismos identificados por comparação com bancos de dados. Neste trabalho foi utilizado DNA extraído de filtrados de água do Rio Solimões, além de outros rios e lagos adjacentes, para construção de bibliotecas, com a finalidade de estudar a biodiversidade por meio de análise do 16S rRNA. Em todas as bibliotecas o filo Proteobacteria foi o mais abundante, e a maioria dos gêneros observados pertence à classe Betaproteobacteria. Os taxa cosmopolitas de água doce Candidatus Planktophila limnetica e Polynucleobacter foram observados, assim como os produtores primários foram representados pelos gêneros Synechococcus e Cyanobium. Nas amostras em que a construção das bibliotecas de 16S rRNA foi possível para Archaea, obteve-se o filo Crenarchaeota como o mais abundante em todas as bibliotecas.
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Bioinformatics applied to natural products discovery processes: systematization, biosynthetic evidences, and isolation of promising speciesZANIN, João Luiz Baldim 06 May 2016 (has links)
Estratégias guiadas por genoma foram utilizadas a fim de examinar o potencial biossintético de microorganismos da classe Betaproteobacteria no âmbito de Produtos Naturais. Uma estratégia capaz de ser expandida para todos os tipos de microorganismos foi criada para estimar as reações enzimáticas das Peptideo Sintetases Não Ribossomais a fim de sistematizar a e analisar suas similaridades biossintéticas. Todas as bases de dados e software user-friendly foram adotadas a fim de tornar esta estratégia simples e mais abrangente. Elas foram NCBI, KEGG, NORINE, antiSMASH, Cystoscape, Gitools, MEGA e Clustal. Os resultados tornaram possível a criação de uma stratégia, chamada XPAIRT (eXPAndable Identification of amino acids in nonRibosomal peptides Tendencies) correlacionando pares de peptídeos e seus genomas similares via Jaccard Index e filogenia. Neste contexto, espécies Betaproteobacteria mostraram sintetizar produtos naturais seguindo certa similaridade biossintética na montagem de monômeros para a construção do esqueleto peptídico. Subunidades estruturais tais como asp.ser e orn.ser foram amplamente encontradas. Essas similaridades foram correlacionados gerando índice de similaridade entre espécies e sua distribuição entre genomas semelhantes, que foram nomeados como contribuíntes. Quanto maior a identidade genômica de um cluster de gene biossintético para um produto natural de forma geral, maior a chance de um contribuínte expressar pares similares relativos ao cluster em questão. A partir de análises de contagem de clusteres de genes biossintéticos, pôde-se eleger microorganismos promissores para isolamento de amostras ambientais. Essas análises mostraram que espécies do gênero Burkholderia são as mais promissoras quando comparadas a todos os genomas disponíveis da subclasse Betaproteobacteria. Análises genômicas da espécie padrão do gênero, Burkholderia thailandensis mostraram que cromossomos 1 e 2, em comparação a uma cepa produtora de antibióticos padrão, S. coelicolor, não apresentarem mesmas informações para biossíntese de compostos, mas apresentam similaridades de classes, sendo elas, Terpenos, T1PKS, Bacteriocinas e Peptídeos Não Ribossomais. Todos os resultados não tiveram correlações com os clusteres de S. coelicolor evidenciando que B. thailandensis apresenta-se promissora para a descoberta de novos compostos. Como espécies do gênero Burkholderia foram o principal alvo neste trabalho, um método guiado por genoma foi desenvolvido para isolar tanto quanto possível cepas de amostras ambientais. O método levou em consideração as necessidades básicas de um microorganismo para sobreviver: a) o tipo de microbioma que os microorganismos de interesse se encontram, analizados através de resultados de metagenômica, b) resistência à antibióticos e metais, c) capacidade de metabolizar compostos com papel biológico, d) crescimento celular e nutrientes, e e) variações de pH e crescimento celular. Todas as análises foram cruzadas e os melhores candidatos à composição de meios de culturas celular específicos para o isolamento de microorganismos do gênero Burkholderia foram selecionados. A estratégia foi bem-sucedida para diversos tipos de amostras. Estes experimentos excepcionais demonstraram a eficácia na resolução de problemas químico-biológicos auxiliando a análise posterior de novos produtos naturais. / Genome-guided strategies were applied to examine Betaproteobacteria species potential for the biosynthesis of nonribosomal peptides. A generalizable strategy was created to track similarities in enzymatic reactions of nonribosomal peptides synthetases in order to organize their capability of assembling monomers building the peptides backbones. Databases and user-friendly software were adopted making this strategy a comprehensive one. Databases and software adopted, as well as, NCBI, KEGG, NORINE, antiSMASH, Cystoscape, Gitools, MEGA e Clustal were used for this purpose. Betaproteobacteria species showed to possess biosynthetic similarities in assembling monomers for the peptide backbone of a nonribosomal peptide. These evidences were correlated giving similarities indexes between species and their distribution between similar genomes. Predictions were fragmented in several ways, for example, monomers, pairs and triads. Correlation analyses displayed that pairs it is the best way of tracking similarities. This result turned possible to create a strategy, named XPAIRT (eXPAndable Identification of amino acids in nonRibosomal peptides Tendencies) correlating pairs of peptides and their similar genomes via Jaccard Index and phylogeny. Thought these investigations it was noticed that Betaproteobacteria species generally assemble asp.orn and orn.ser, mainly Burkholderia species, among other pairs of peptides. Further analysis showed that species from the genera Burkholderia are the most promising ones due to their Biosynthetic Gene Cluster counting for all available Betaproteobacteria genomes. These species were further analyzed and a standard strain, Burkholderia thailandensis, was used to the identification of intraspecific variation for their biosynthetic potential. A specific study on Biosynthetic Gene Cluster variation was proceeded for discovering disparities between chromosomes 1 and 2, and a standard antibiotic producer strain, S. coelicolor. Results showed that B. thailandensis have different possibilities for biosynthesizing natural products. Even thought, common classes of compounds such as, Terpenes, Bacteriocins, T1PKS and Nonribosomal Peptides were identified for all strains. As Burkholderia species were the main target in this work, a genome-guided method was developed for isolating as much strains as possible from environmental samples. This very method took into account the basic needs for a microorganism to survive: a) the type of microbiome that microorganisms of interest coexist, analyzed through metagenomics, b) resistance to antibiotics and metals, c) ability to metabolize compounds with biological role, d) cell growth related to different nutrients, and e) cell growth under pH variations. The strategy was successful for diverse types of samples. These exceptional experiments are part of a novel way of working with Natural Products, using genomic, bioinformatics and visual statistical analysis in order to access common characteristics and uniqueness of species guiding the search of medically relevant natural products. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
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Ecophysiological characteristics of key members of Betaproteobacteria in freshwater bacterioplankton / Ecophysiological characteristics of key members of Betaproteobacteria in freshwater bacterioplanktonKASALICKÝ, Vojtěch January 2012 (has links)
This thesis primarily focuses on one segment of freshwater Betaproteobacteria, the Limnohabitans genus (including the RBT lineage). As opposed to other recent research directions, the major aim was to recover the members of the previously uncultured RBT lineage through their isolation from various freshwater habitats. However, the results presented in this thesis have also ambitions to go far beyond the taxonomic descriptions only; the dissertation intends to contribute significantly to unveiling of important ecophysiological characteristics of the studied lineage in a set of both laboratory and field research. Therefore, understanding of growth characteristics, mortality, diversity and life strategies of aquatic microbes is of highest importance regarding profound human impact on water quality and increasing need of drinking water supplies.
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Bactéries associées à l'éponge Méditerranéenne Crambe crambe : diversité et possible rôle dans la biosynthèse des alcaloïdes guanidiniques / Bacteria associated to the Mediterranean sponge Crambe crambe : diversity and possible role in the biosynthesis of guanidine alkaloidsCroué, Julie 18 September 2014 (has links)
Crambe crambe (Schmidt, 1862), espèce largement retrouvée en Méditerranée, est la source de nombreuxalcaloïdes guanidiniques (crambescines et crambescidines). Les voies de biosynthèse de ces métabolitessecondaires n’ont pas encore été démontrées. Il a cependant été proposé que les crambescidines seraientproduites à partir de polycétides, suggérant ainsi la possible implication de micro-organismes dans leurbiosynthèse. Cependant les quelques études portant sur la présence ou non de bactéries associées à cette épongesont contradictoires. Durant cette thèse, nous avons caractérisé la communauté bactérienne associée à C. crambepar l’utilisation de techniques à la fois moléculaires et microscopiques, mettant en évidence l’associationspécifique entre une bêtaprotéobactérie et cette éponge méditerranéenne. La présence d’un micro-organismespécifique au sein du mésohyle de l’éponge pose alors la question du rôle de ce dernier au sein de son hôte etplus particulièrement sa possible implication dans la synthèse des alcaloïdes guanidiniques. L’étude de ladiversité microbienne cultivable par la mise en place d’une procédure d’acclimatation et la conception de diversmilieux de cultures, a permis l’isolement de micro-organismes minoritairement associés à C. crambe, mais n’acependant pas conduit à l’isolement de la bêtaprotéobactérie spécifique. La comparaison des empreinteschimiques (CLHP/DAD/ELSD – CLHP/ESIMS) des extraits de l’éponge et des bactéries cultivées, a révélé queces micro-organismes minoritaires n’étaient pas à eux seuls producteurs de ces métabolites bioactifs. La bactériemajoritaire, principale candidate dans la synthèse des métabolites n’étant pas cultivable, nous avons développédeux études complémentaires afin d’apporter des éléments de réflexion quant à sa possible implication. Laculture ex situ de colonies C. crambe accompagnée d’un stress pH ainsi que l’étude de la distribution spatiale insitu des composés par imagerie par spectrométrie de masse se révèlent prometteuses bien qu’elles n’aient pas, àce jour permis d’identifier le possible rôle de la bêtaprotéobactérie dans la biosynthèse de ces métabolites. / Crambe crambe (Schmidt, 1862), is a marine sponge widely distributed in the Mediterranean Sea and known toproduce bioactive guanidine alkaloids (crambescins and crambescidins). While the biosynthetic pathways ofthese metabolites remains unknown, bio-mimetic chemical synthesis of crambescidins suggested a possiblecontribution of microorganisms in their biosynthesis. Contrastingly, it had been reported via electron microscopythat bacteria were absent in the tissues of this sponge. Thus to shed light onto these contrasting results I studiedthe microbial community associated with C. crambe using alternative approaches. Using molecular andmicroscopic techniques, we demonstrated that a single bacterial species affiliated to the Betaproteobacteria ispresent in abundances commonly found in low microbial abundance sponges, and dominates the bacterialcommunity associated with C. crambe. This finding suggests a possible implication of bacteria the biosynthesisof C. crambe’s guanidine alkaloids. The use of acclimatization procedure and diverse cultivation approachesallowed the isolation of associated microorganisms which were rare or absent among the community describedvia tag pyrosequencing but not isolation of the dominant betaproteobacterium. CLHP/DAD/ELSD andCLHP/ESIMS fingerprints of extracts from C. crambe and from the isolated bacteria showed no evidence of theimplication of these cultured micro-organismes in the synthesis of C. crambe’s metabolites. Finally in order toevaluate the potential role of the uncultivated betaproteobacterial symbiont in the biosynthesis of guanidiniumalkaloids, preliminary experiments using two alternative approaches were attempted. In the first I subjectedsponges to a pH stress and evaluated changes in secondary metabolite profiles, while collecting samples formicrobial community analysis. In the second I tried different mass spectrometry imaging techniques to localizeguanidinium alkaloids in C. crambe tissues. While these experiments did not allow to resolve the question of apossible implication of the dominant betaproteobacterium in the biosynthesis of the pentacyclic guanidinealkaloids, I was able to gather interesting results that will guide future studies towards resolving this question.
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