Orientadores: Wanderley Dias da Silveira, Eliana Gertrudes de Macedo Lemos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:23:49Z (GMT). No. of bitstreams: 1 Paganelli_FernandaLaroza_M.pdf: 1101767 bytes, checksum: e8882c8b6ec40ea617dd997584b239c3 (MD5) Previous issue date: 2009 / Resumo: Polihidroxialcanoatos (PHAs) são polímeros de hidroxialcarioatos produzidos, e acumulados, intracelularmente, como fonte de carbono e/ou outros materiais energéticos, em vários microorganismos. Freqüentemente, o acúmulo dos PHAs ocorrem em condições abaixo do limite nutricional de elementos como N, P, S, O ou Mg e excesso de carbono, podendo representar até 80% da massa seca total da célula. Mais de 300 diferentes microorganismos podem sintetizar e acumular PHAs. O polihidroxibutirato (PHB) é o mais conhecido dentre os polímeros bacterianos biodegradáveis denominados polihidroxialcanoatos. Por ter propriedades semelhantes ao polipropileno, o PHB pode ser usado na fabricação do plástico biodegradável. Além da busca por maior produção de tal polímero, pouco é conhecido sobre seu papel biológico, em especial nos rizóbios. Estudos revelaram que há variação na capacidade da produção e acúmulo de PHB nessas bactérias quando em simbiose, dependendo da espécie em questão e das condições de cultivo das mesmas, observando-se bactérias incapazes de acumular PHB quando bacterióides, como é o caso do Rhizobium meliloti, ou capazes de produzir e acumular PHB, nessas condições, como é o caso de Rhizobium etli, Bradyrhizobium japonicum e Bradyrhizobium elkanii. Desta forma, este trabalho teve por objetivo a identificação dos genes responsáveis pela síntese de PHB em Bradyrhizobium elkanii, clonagem e expressão dos mesmos em Escherichia coli, uma vez que esta bactéria é bem conhecida como ferramenta molecular e se multiplica rapidamente, podendo atingir uma alta produção do polímero esperado, em um curto período de tempo. Além disso, objetivou-se, também, aumentar a produção de PHB em B. elkanii através de mutações aleatórias (através da inserção do transposon TnphoA), já que esta é uma bactéria naturalmente produtora de PHB. Para isso, os genes phbA, phbB e phbC foram isolados através da técnica de PCR, amplificando-se os genes inteiros. Estes foram clonados em vetores de expressão tipo pET (NOVAGEN), sendo os genes phbA e phbB clonados em "operon " em um mesmo vetor e o gene phbC clonado separadamente. A expressão dos genes foi analisada, bem como sua capacidade de produzir PHB. Os mutantes de B. eíkanii obtidos através da inserção do transposon TnphoA foram analisados com o uso do corante Sudan Black, procurando-se selecionar linhagens maiores produtoras de PHB. A produção dos mutantes selecionados foi, posteriormente, analisada por cromatografia gasosa. Observou-se que a linhagem de E.coli com os três genes clonados teve a capacidade de produzir PHB, porém com baixa eficiência. Já os mutantes aleatórios de B. elkanii apresentação diferentes acúmulos em relação ao selvagem, com destaque para o MUT33 que teve 72% da sua massa seca acumulada na forma de PHB, enquanto o selvagem acumulou 51 % de PHB, nas mesmas condições. / Abstract: Polyhydroxyalkanoates (PHAs) are polymers of hydroxyalkanoate acids, produced and accumulated intracellularly as a source of carbon and energy storage material, in prokaryotic cells. Often, the PHAs accumulation occurs in conditions when the carbon source is in excess but one or several other nutrients are limited, and may represent up to 80% of the cell dry weight. More than 300 different microorganisms can synthesize and accumulate PHAs. The polyhydroxybutyrate (PHB) is the most studied polymer among the bacterial biodegradable polymers (PHAs). By having properties similar to polypropylene, PHB can be used in the manufacture of biodegradable plastic. On the search for greater production of such polymer, little is known about its biological role, especially in the genus Rhizobium. Studies have shown that there is a variation in the PHB capacity production and accumulation when these bacteria are in symbiosis. Depending on the species and cultivation conditions it has been observed either incapacity of PHB production and accumulation, when Bacteroides, as Rhizobium meliloti; or capacity and accumulation of PHB under bacteroides fase, as Rhizobium etli, Bradyrhizobium japonicum and Bradyrhizobium elkanii. Therefore, the present work aimed the identification, cloning and expression, in Escherichia coli, of responsible genes for PHB synthesis in Bradyrhizobium elkanii. Moreover, it was also expected to increase the production of PHB in B. elkanii, through random mutations (insertion of the TnphoA transposon), since B. elkanii is a good natural producer of PHB. For this, the entier phbA, phbB and phbC genes were isolated by PCR. Those genes were cloned in expression vectors such pET (NOVAGEN), where phbA and phbB genes were cloned in operon, in a single vector, whereas phbC gene was cloned separately, in another vector. The expression of those genes was analyzed, as well as its ability to produce PHB. Mutants B. elkanii, obtained by insertion of the TnphoA transposon, were analyzed using the dye Sudan Black, in order to select different strains that might produce higher quantities of PHB. The production of PHB by mutants was then analyzed by gas chromatography. It was observed that the E. coli with the three cloned genes had the ability to produce PHB, but with low efficiency. The B. elkanii random mutants show different accumulation compared to the wild, especially MUT33 that had 72% of its dry mass accumulated in the form of PHB, while the wild 51% of accumulated PHB under the same conditions. / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-21T17:26:17Z No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-21T18:17:40Z (GMT) No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-21T18:17:40Z (GMT). No. of bitstreams: 2 Dissertação - Aline Assis Cardoso - 2014.pdf: 1764081 bytes, checksum: e25295d0c53ab008ce92f6d68f628db7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Research on biological nitrogen fixation (BNF) in common bean had a great progress in recent years, especially in the knowledge of microsimbiont and exploring new approaches seeking greater variability in macrosimbiont for efficiency of BNF. Studies of bacterial diversity and taxonomy, especially applied to common bean symbionts showed a great evolution due to new molecular methodologies for evaluation and characterization. This study aimed to evaluate the tolerance to salinity and temperature, to characterize based on molecular markers and to evaluate the symbiotic efficiency of bacterial isolates obtained from nodules of common bean cultivated on soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were evaluated for salinity and temperature on YMA medium with different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28ºC, 33ºC, 38ºC, 43ºC and 48ºC). For molecular characterization based on BOX-PCR and REP-PCR profiles the isolates were grown in liquid YMA for 24 hours and then DNA extraction was performed. Evaluation of symbiotic efficiency of the isolates was conducted under greenhouse conditions in Leonard jars. Seeds of common bean (var. Pérola) were inoculated with different isolates selected in the previous analysis. Nodule number (NN), dry mass of nodules (DMN), specific mass of nodules (SMN), root dry weight (RDW), dry matter of aerial part (DMAP), relation root/shoot (R/S), total nitrogen (N) and leaf area (LA) were evaluated. It was observed that 41.12% of the isolates grew in more restrictive conditions than standard strains SEMIA 4077, SEMIA 4080 and SEMIA 4088, and 29.90% of the isolates grew in less restrictive conditions than SEMIAs strains.BOX-PCR and REP-PCR profiles showed high genetic diversity among the evaluated isolates, demonstrating a high degree of polymorphism. JPrG8A7 and JPrG8A6 isolates exhibited superior performance compared to standard strains when compared the NN , SMN and DMN. The latter showed a positive correlation with the DMAP, Total-N and LA. It was observed that some isolates showed competitive features equal or superior than commercial standards strains, with results that can improve the process of symbiosis between plant and bacteria, thereby generating greater productivity for the common bean cultivation. / A pesquisa sobre a fixação biológica de nitrogênio (FBN) no feijoeiro teve bastante progresso nos últimos anos, especialmente no conhecimento do microsimbionte e no estudo de novas abordagens buscando maior variabilidade no macrosimbionte para maior eficiência da FBN. Os estudos da diversidade e taxonomia bacteriana, especialmente aplicados aos simbiontes do feijoeiro-comum apresentou uma grande evolução devido às novas metodologias moleculares de avaliação e caracterização. Este trabalho teve como objetivo avaliar a resistência à salinidade e temperatura, caracterizar molecularmente e avaliar a eficiência simbiótica de isolados de nódulos de feijoeiro-comum oriundos dos estados de GO, MG e PR. Os isolados foram avaliadas quanto à salinidade e temperatura em meio YMA com diferentes concentrações de NaCl (0%; 1%; 2%; 4% e 6%) em diferentes temperaturas (28ºC; 33ºC; 38ºC; 43ºC e 48ºC). Para a caracterização molecular os isolados foram crescidos em meio YMA líquido por 24 horas e logo em seguida foi realizada a extração do DNA. Foram avaliados perfis BOX-PCR e REP-PCR. A avaliação da eficiência simbiótica dos isolados foi conduzida em casa-de-vegetação com vasos tipo Leonard com a cultivar Pérola inoculada com diferentes isolados selecionados na análise anterior. Foi avaliado o número de nódulos (NN), massa seca de nódulos (MSN), massa específica de nódulos (MEN), massa seca de raiz (MSR), matéria seca da parte aérea (MSPA), relação raiz/parte aérea (R/PA), nitrogênio total (N) e área foliar (AF). Observou-se que 41,12% dos isolados cresceram em condições mais restritivas que as estirpes padrão SEMIA 4077, SEMIA 4080 e SEMIA 4088, e 29,90% dos isolados cresceram em condições menos restritivas que as SEMIAs. Os perfis BOX-PCR e REP-PCR apresentaram grande diversidade genética entre os isolados avaliados, demonstrando um alto grau de polimorfismo. Os isolados JPrG8A7 e JPrG8A6 apresentaram desempenho superior as estirpes padrão quando comparados o NN, MEN e MSN. Esta última apresentou correlação positiva com a MSPA, N-Total e AF. Foi observado que alguns isolados apresentaram características competitivas iguais ou superiores as estirpes-padrões comerciais, apresentando resultados que podem melhorar o processo de simbiose entre a planta e a bactéria, gerando assim uma maior produtividade para a cultura do feijoeiro-comum.

The association between root-nodulating bacteria and leguminous plants is advantageous due to their ability to alter atmospheric nitrogen into a useful form in a process known as biological nitrogen fixation (BNF). Research has shown that BNF is the most efficient way to supply the large amounts of nitrogen needed by plants to produce high-yielding crops. As a result, there have been numerous studies into the diversity and identity of the associated nitrogen-fixing bacterial symbionts. Recent advances in molecular microbiology together with the isolation of rhizobia from previously uninvestigated legumes have led to major revisions of rhizobial taxonomy, most notably the inclusion of bacteria from the ß-Proteobacteria in the genera Burkholderia and Ralstonia. In this study, the diversity of root nodule bacteria associated with Phaseolus coccineus and Phaseolus vulgaris species in South Africa was investigated. A selection of rhizobial isolates were characterised by SDS-PAGE of whole cell proteins and rep-PCR DNA fingerprint analyses. These results were supplemented by partial 16S rDNA sequencing of a select number of isolates to confirm their identity. Where isolates displayed unexpected genus associations, partial nodA sequencing was performed to determine whether these were incidental contaminants or true nodulators. Based on 16S rDNA sequence analysis, the majority of isolates investigated were fast-growers belonging to the genus Rhizobium. A few isolates showed close relationship to species of the ß-Proteobacteria genus, Burkholderia. Both the SDS-PAGE analyses and the combined rep-PCR analyses were able to resolve isolates down to strain level, but the comparison of the SDS-PAGE and 16S rDNA sequencing data confirmed that bacterial discrimination using SDS-PAGE is not useful at the genus level and higher, as isolates showing affinity to Burkholderia were mingled with isolates showing similarity to Rhizobium. These isolates were separate from the Rhizobium isolates in the combined rep-PCR dendrogram. While there were discrepancies between results obtained from SDS-PAGE and rep-PCR analyses, results from the combined rep-PCR analysis correlated with many of the results obtained in the SDS-PAGE analysis. Both geographic location and host plant species appear to have affected the grouping of isolates. Many clusters consisted of isolates from the same location or the same host plant species in both the SDS-PAGE dendrogram as well as the combined rep-PCR dendrogram. The nodA sequencing demonstrated that the majority of isolates tested contain the nodA gene indicating that they are capable of nodulation. There was a large strain diversity observed for the isolates of this study and a number of the root-nodulating bacteria of the Phaseolus spp. appear to constitute several novel nodulating genotypes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Orientador: Leandro Borges Lemos / Resumo: A utilização de manejos sustentáveis para maximizar a produtividade das culturas sem aumentar os custos de produção é um dos grandes desafios da agricultura atual. Assim, objetivou-se avaliar o efeito de manejos de fornecimento de nitrogênio, associando formas de inoculação com Rhizobium tropici, co-inoculação com Azospirillum brasilense e adubação mineral de cobertura, no desempenho agronômico de cultivares de feijão comum do grupo comercial carioca de ciclo precoce. O experimento foi instalado em delineamento em blocos casualizados (DBC), no esquema fatorial 2x12 (2 cultivares e 12 manejos de fornecimento de N), com quatro repetições. Para os manejos de fornecimento de N foram associados adubação mineral de cobertura com N (45 e 90 kg ha-1 de N), inoculação de sementes com Rhizobium tropici e coinoculação via semente e foliar com Azospirillum brasilense. Foram avaliados o número de trifólios por planta, área foliar, índice de clorofila, teor de nitrogênio foliar e nos grãos, massa seca de plantas, número de vagens por planta, número de grãos por planta e produtividade, número de nódulos por planta, massa seca de nódulos por planta, massa seca de raízes e eficiência de nodulação. Os dados foram submetidos à análise de variância (teste F) e, quando necessário, as médias foram agrupadas pelo teste de Scott-Knott a 5% de probabilidade. Com exceção da massa seca de plantas, todas as variáveis analisadas apresentaram máxima resposta no tratamento com inoculação de sementes com Rh... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of sustainable managements techniques to maximize crop productivity without increasing production costs is one of the great challenges of modern agriculture. The objective of this study was to assess the effect of different nitrogen management by testing the inoculation with Rhizobium tropici, co-inoculation with Azospirillum brasilense, and topdressing N fertilization on the agronomic performance of common bean cultivars of early cycle. The experiment was carried out in a randomized block design (RBD), in a 2x12 factorial scheme (2 cultivars and 12 N supply management), with four replications. For N supply management, it was used topdressing N fertilization (45 and 90 kg ha-1 N), seed inoculation with Rhizobium tropici, and seed and leaf co-inoculation with Azospirillum brasilense. It was evaluated: the number of trifolium per plant, leaf area, chlorophyll index, leaf and grain nitrogen content, dry mass of plants, number of pods per plant, number of grains per plant and yield, number of nodules per plant, dry matter of nodules per plant, dry matter of root, and nodulation efficiency. Data were subjected to analysis of variance (F test) and, when necessary, means were grouped by Scott-Knott test at 5% probability. Except for the dry mass of plants, all variables analyzed showed maximum response in the treatment with seed inoculation with Rhizobium tropici, foliar application of Azospirillum brasilense, and topdressing fertilization with 90 kg ha-1 of N. In general, t... (Complete abstract click electronic access below) / Mestre

La culture de Légumineuses présente le double intérêt de permettre une production de graines à haute valeur nutritionnelle sans nécessité d’un apport d’engrais azoté. La nutrition azotée des légumineuses dépend en effet majoritairement de la fixation symbiotique de l’azote atmosphérique réalisée par des bactéries du sol, les rhizobia, au sein des nodosités, et dans une moindre mesure, de l’assimilation de l’azote minéral du sol par les racines.Une meilleure compréhension a été acquise sur le contrôle génétique de la mise en place des racines et des nodosités et sur leur impact sur la nutrition azotée. Une grande variabilité génétique pour ces caractères a été mise en évidence, ainsi que l’existence de corrélations génétiques entre eux. Une approche de génétique quantitative a permis d’identifier des régions génomiques pouvant être impliquées dans leurs variations. Deux pistes d’amélioration de la nutrition azotée ont aussi été étudiées : l’amélioration de l’acquisition d’azote par les racines à partir d’une étude détaillée d’un mutant de développement racinaire, et l’amélioration de la symbiose via l’étude de la capacité des pois à favoriser les associations symbiotiques avec les rhizobia les plus performants.Les résultats obtenus apportent des bases de réflexion concernant la conception d’un idéotype de nutrition azotée. Au-delà de la complémentarité indispensable entre les deux voies d’acquisition d’azote, il convient d’optimiser l’interaction entre les deux partenaires symbiotiques, mécanisme complexe mettant en jeu la formation et le fonctionnement des nodosités, en lien avec une signalétique et des interactions trophiques entre partenaires et intra-plante. / Grain legume pulse crops are of great interest to allow a production of seeds high nutritional value without any contribution of nitrate fertilizer. The nitrogen nutrition of legumes depends indeed mainly on the fixation in nodules of atmospheric dinitrogen through the plant-rhizobium symbiosis, and to a lesser extent, absorption by roots of soil mineral nitrogen.A better understanding has been obtained on the genetic control of the development of roots and nodules and on their impact on nitrogen nutrition. High genetic variability of these characters has been detected, and the existence of genetic correlations between them demonstrated. A quantitative genetic approach has identified several genomic regions that may be involved in their variations. The two different ways to improve nitrogen nutrition were also studied: the improvement of nitrogen acquisition by roots through a detailed study of a root architecture mutant, and the improvement of symbiosis via the study of the ability of peas to promote symbiotic associations with the most effective rhizobia.The results provide interesting bases for the design of a pea nitrogen-nutrition ‘ideotype’. Beyond the essential complementarity between the two pathways of nitrogen acquisition, it is necessary to optimize the interaction between the two symbiotic partners, which is a complex mechanism involving nodules formation and functioning in connection with complex signaling and trophic interactions between the partners and intra-plant.

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

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No description available.

Iron is essential to the majority of microorganisms; it is an important cofactor in many cellular processes and enzymes. However in an aerobic environment and at biological pH, iron is primarily found as insoluble oxyhydroxides and is unavailable to microorganisms. Many bacteria have the ability to produce siderophores, low molecular weight compounds that have a high affinity for Fe3+. Siderophores are part of a multi-component system that actively transports the iron-siderophore complex into the cytoplasm. Rhizobia are characterized by their ability to form symbiotic relationships with leguminous plants, where they can fix nitrogen for the host plant and the plant provides the bacteria with nutrients. Under iron-limiting conditions, Rhizobia are known to produce siderophores. Rhizobium leguminosarum IARI 917 produces one dihydroxamate-type siderophore. This siderophore has been purified and chemically characterized. Results indicate that this strain is producing schizokinen, which has not been described in a member of the Rhizobia family.

Broomrapes, <i>Orobanche</i> spp., are holoparasites that affect the growth of a variety of broadleaf crops. One of the distinct characteristics of the family Orobanchaceae is the lack of chlorophyll, and hence inability to synthesize their own food. Broomrapes subsist on the roots of the host plant from which they derive the carbon, water, and nutrients needed for further growth. Parasitism as such leads to yield reductions, and in case of heavy infestations, complete crop failure. Among other plants parasitized by broomrapes are several legumes, some of which are also the world's most economically important crops. As part of their unique biology, legumes provide an ecological niche for diazotropic soil bacteria, which belong to the family Rhizobiaceae. In return, the host plant receives fixed nitrogen from the nodules, specialized structures produced on the roots of most legume plants upon inoculation with bacteria. <i>Orobanche</i> spp. germination depends on the presence of chemical stimulant in host root exudates. It has been reported that inoculation of some legumes resulted in greater infestation by parasitic weeds. In addition, bacterial nodules were assumed to provide a place for broomrape invasion of host legume. Furthermore, infestations were observed to be more intense in aerobic conditions when rhizobia are most active. It is possible that production of the stimulant could be correlated with the infection of roots with <i>Rhizobium</i>, and that the nodules formed on the roots could play a role in broomrape parasitism; however, others have not confirmed this. Studies were conducted to investigate the possibility of interaction between <i>Orobanche</i> spp. attack and <i>Rhizobium</i> nodulation in legumes. Seed germination, number of parasitic attachments, as well as the morphology of two broomrape species, small broomrape (<i>Orobanche minor</i> Sm.) and Egyptian broomrape (<i>Orobanche aegyptiaca</i> Pers.), were studied. <i>O. minor</i> showed a greater percent seed germination, and formed a greater number of attachments on red clover (<i>Trifolium pratense</i> L.) inoculated with <i>Rhizobium leguminosarum</i> bv. <i>trifolii</i> in comparison with non-inoculated plants. However, the addition of the inoculum did not appear to enhance <i>O. aegyptiaca</i> seed germination or the number of its attachments on the host roots compared with the controls. Morphological observations of <i>O. minor</i> attachments on red clover suggest that parasitic attachments were not situated over the bacterial nodules, but perhaps involve parasite-induced enzymatic degradation followed by mechanical protrusion of host plant root cortex, possibly utilizing host plant-rhizobacteria interactions as well. / Master of Science