Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-19Bitstream added on 2014-06-13T20:10:49Z : No. of bitstreams: 1 monteiro_nk_me_sjrp.pdf: 687247 bytes, checksum: 5856bbc04fe8d3e52218193110c45156 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bactérias do gênero Rhizobium são conhecidas por manterem uma relação simbiótica com determinadas plantas leguminosas e atuam como fixadores biológicos de nitrogênio. Elas vêm sendo estudadas em relação à produção de exopolissacarídeos (EPS), o qual acredita-se estar relacionado com o processo de reconhecimento e invasão do microrganismo na planta hospedeira. Estas moléculas apresentam propriedades físico-químicas e reológicas interessantes para a indústria alimentícia, podendo ser aplicadas como estabilizantes, emulsificantes ou gelificantes. Estudos da estrutura química realizados em quatro EPS produzidos por Rhizobuim tropici (em dois meios de cultivos diferentes), Mesorhizobium e Rhizobium sp os caracterizaram como sendo heteropolímeros. Esses polímeros são constituídos principalmente por glucose e galactose, com traços de manose, apresentando ácido urônico, acetila e piruvato com substituintes. Por cromatografia de gel permeação e eletroforese em gel de poliacrilamida apenas os polímeros produzidos por Rhizobium tropici (R2) e Mesorhizobium (R3) comportaram-se como moléculas homogêneas com baixo grau de polidispersividade. Estes EPS foram paracialmente caracterizados utilizando-se técnicas de FT-IR, RMN de 1H e 13C / Bacteria of the Rhizobium genus are known to maintain a symbiotic relationship with certain leguminous plants and they act as biological nitrogen fixation. They have been studied as exopolysaccharides (EPS) producers, which may be related to the recognition process and to the microorganism invasion into the host plant. These molecules exhibit interesting physical-chemical and rheological properties for the food industry since they can be used as stabilizers, emulsifiers or gelling agents. Studies of the chemical structure of four EPS produced by Rhizobium tropici (in two different culture media), Mesorhizobium and Rhizobium sp have characterized them as heteropolymers. The EPS are constituted mainly by glucose and galactose with traces of mannose besides uronic acid, pyruvate and acetyl as substituents. Gel permeation chromatography and polyacrylamide gel electrophoresis showed that the polymers produced by Rhizobium tropici (R2) and Mesorhizobium (R3) behaved as homogeneous molecules with low polydispersity. Thus these two EPS were partially characterized by the techniques of FT-IR, 1H and 13C NMR

Deux légumineuses (Anthyllis vulneraria et Lotus corniculatus) adaptées aux métaux lourds constituent un matériel d’intérêt pour la phytostabilisation de sites miniers. Leur fonction de fixatrices biologiques d’azote grâce à leur symbiose avec des bactéries symbiotiques permet l’établissement efficace d’une couverture végétale durable limitant la dispersion des métaux dans l’environnement. Nos objectifs ont été d’étudier les effets des métaux lourds et de la plante hôte sur les populations symbiotiques naturellement associées à ces légumineuses en analysant (i) les populations symbiotiques associées à A. vulneraria sur 8 sites contaminés ou non (ii) les populations de rhizobia associées à L. corniculatus qui ont été comparées à celles d’Anthyllis. La distribution des souches de Mesorhizobium isolées de nodosités d’A. vulneraria et provenant de plusieurs sites contaminés ou non dépend des fortes teneurs en métaux lourds des sols qui sélectionnent fortement les souches symbiotiques résistantes et influencent leur composition taxonomique. Les souches appartenant à l’espèce M. metallidurans ont été retrouvées seulement dans les sites fortement contaminés. Deux nouvelles espèces potentielles et résistantes aux métaux semblent exister chacune sur un site minier distinct. L’une d’elle est proche de M. ciceri et de M. loti et tous ses membres présentent la particularité de ne pas posséder de gène cadA, un gène impliqué dans la tolérance aux métaux chez M. metallidurans. Par contre, les sites non contaminés révèlent une diversité taxonomique différente avec la présence de nouvelles espèces de Mesorhizobium sensibles aux métaux lourds. Quatre de ces nouvelles espèces ont été définies. A. vulneraria et L. corniculatus partagent la même diversité taxonomique dans les sites contaminés testés. Par contre, les propriétés symbiotiques des souches varient selon la plante hôte utilisée pour le piégeage. Les souches appartiennent soit au symbiovar (sv.) anthyllidis soit au sv. loti selon le site géographique d’origine et ceci indépendamment des teneurs en métaux lourds dans le sol. A. vulneraria s’associe avec les souches possédant les sv. anthyllidis ou sv. loti. En revanche, L. corniculatus ne s’associe qu’avec des souches du sv. loti. Dans tous les sols qu’ils soient contaminés ou non, A. vulneraria nodule préférentiellement avec le sv. anthyllidis. En conclusion, A. vulneraria et L. corniculatus établissent des symbioses avec les mêmes espèces de Mesorhizobium et s’associent préférentiellement avec un sv. Les taxons retrouvés dépendent fortement des sites d’isolement, ce qui pourrait traduire des adaptations particulières aux conditions environnementales. L’utilisation des ressources biologiques locales est une stratégie que nous recommandons pour la végétalisation d’anciens sites miniers. / Two legumes (Anthyllis vulneraria and Lotus corniculatus) adapted to heavy metals form an interesting material for phytostabilisation strategy in mining sites. As biological nitrogen fixators, these legumes associated with compatible symbiotic bacteria provide an efficient establishment of a sustainable cover vegetation limiting metal dispersion in the environment. Our objectives were to study the effects of heavy metals and the host plant on symbiotic populations naturally associated with these legumes by analyzing (i) symbiotic populations associated with A. vulneraria on 8 contaminated and uncontaminated sites (ii) rhizobial populations associated with L. corniculatus that were compared with those of Anthyllis. The distribution of mesorhizobial strains isolated from A. vulneraria root-nodules from several contaminated and uncontaminated sites depends on high levels of heavy metals in soils by selecting highly resistant strains and impacting the taxonomic composition. Strains belonging to M. metallidurans were only found in highly contaminated sites. Two new potential metal-tolerant species were detected in two distinct mines. One of them was closely related to M. ciceri and M. loti and its members had the feature of not -possessing the cadA gene, a gene involved in metal-tolerance among M. metallidurans strains. By contrast, uncontaminated sites revealed a different taxonomic diversity with new species sensitive to heavy metals. Four of these new species were defined. A. vulneraria and L. corniculatus share the same taxonomic diversity in the contaminated sites tested. By contrast, symbiotic properties of the strains vary depending on the host plant used for trapping. Strains belong either to symbiovar (sv.) anthyllidis or to sv. loti according to geographic origins and independently of heavy metal levels in soils. A. vulneraria associated with strains of sv. anthyllidis or sv. loti. In contrast, L. corniculatus only associated with strains of sv. loti. In contaminated or uncontaminated soils, A. vulneraria was preferentially nodulated by sv. anthyllidis. In conclusion, A. vulneraria and L. corniculatus established symbiotic relationships with the same taxonomic groups of Mesorhizobium but associated with different symbiovars. The finding of taxonomic groups strongly depends on geographical sites, suggesting special adaptations to environmental conditions. Use of local biological resources is the strategy we recommend for revegetation of old mines.

La symbiose entre Anthyllis vulneraria et Mesorhizobium metallidurans, mise en évidence dans l'ancienne mine Zn/Pb des Avinières à St Laurent-le-Minier (Gard) permet une entrée d'azote dans les sols grâce à la fixation biologique qui favorise l'installation d'une végétation pérenne d'intérêt pour limiter la pollution métallique via l'érosion éolienne et hydrique. M. metallidurans ayant la particularité de résister à de fortes concentrations en Zn et Cd, la recherche des gènes d'adaptation aux métaux lourds a été mise en œuvre pour mieux connaitre cette bactérie impliquée dans des opérations de phytostabilisation, par des approches de génétique moléculaire et de transcriptomie (RNAseq). Les gènes codant pour des systèmes de séquestration, d'exclusion, de réduction et d'efflux des métaux lourds, dont cadA1, codant pour une PIB-ATPase exportant Cd/Zn ont été identifiés chez deux souches métallicoles associées à Anthyllis ; M. metallidurans STM 2683T (mine des Avinières) et Mesorhizobium sp. STM 4661 (mine d'Eylie). Une étude fonctionnelle de cadA1 a permis de caractériser son rôle dans la résistance aux métaux lourds chez M. metallidurans. Ce gène a ensuite été testé comme marqueur de résistance pour étudier la diversité et la répartition des symbiotes d'Anthyllis sur des sites miniers et non pollués. Pour cela ce travail a été complété par des analyses phénotypiques de tolérance au Zn et Cd et des analyses phylogénétiques basées sur des marqueurs taxonomiques et symbiotiques. La contrainte métallique exercée par les environnements miniers semble influencer la composition et la diversité bactérienne avec une plus forte proportion (i) de phénotype métallicole lié à la présence du gène cadA1 et (ii) de rhizobia appartenant à M. metallidurans ou à une espèce très proche. La plante hôte semble quant à elle influencer la diversité symbiotique des rhizobia, indépendamment de la contrainte métallique. / Efficient nitrogen-fixing symbiosis between Anthyllis vulneraria and Mesorhizobium metallidurans, identified in the highly Zn/Pb polluted mining site of Avinières (St Laurent-le-Minier, Gard county, France) has recently been described as a potential key bioremediation agent for stimulating the growth of a sustainable plant cover and thus limit heavy metal dispersion from contaminated sites. M. metallidurans strains were shown to be resistant to high Zn and Cd concentrations. The aim of our work was to identify and characterize genes involved in heavy metal adaptation in M. metallidurans by using genetic and transcriptomic approaches (RNAseq technology). Putative genes involved in heavy metal adaptation mechanisms such as exclusion, binding, reduction and efflux, like cadA1, encoding an efflux system PIB-type ATPase involved in Zn and Cd export, were identified in two Mesorhizobium strains associated with Anthyllis: M. metallidurans STM 2683T (Avinières mine) and Mesorhizobium sp. STM 4661 (Eylie mine). Functional studies allowed us to characterize the cadA1 efflux protein as involved in metal tolerance in M. metallidurans. Then, cadA1 was used as a metal-resistance marker to study the diversity and the distribution of Anthyllis symbionts from mine soils and unpolluted soils. This work was completed by Zn- and Cd-tolerance phenotype assays and phylogenetic analyses using taxonomical and symbiotic markers. Metals in mine environments seemed to influence the bacterial composition and the diversity with a high proportion of (i) metal-tolerant phenotypes consistent with the detection of the cadA1 gene and (ii) strains belonging to the M. metallidurans species or to a bacterial species close to it. The plant-hosts seemed to impact symbiotic diversity independently of the metal-tolerant property.

Zugl.: Bielefeld, Univ., Diss., 2008

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

Made available in DSpace on 2014-06-11T19:23:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-21Bitstream added on 2014-06-13T18:50:36Z : No. of bitstreams: 1 arandaselverio_g_me_sjrp.pdf: 1103053 bytes, checksum: 82b446306de04f7e3cee32bff6293b57 (MD5) / Exopolissacarídeos (EPS) são polímeros produzidos por uma grande variedade de microrganismos e possuem diferentes propriedades estruturais, físicas e químicas. A investigação das propriedades reológicas de suas soluções é importante devido ao crescente interesse na aplicação comercial de polissacarídeos, principalmente na indústria de alimentos. Assim, o objetivo deste trabalho foi investigar as características reológicas dos exopolissacarídeos R1, R2, R3 e R4 produzidos por diferentes linhagens de Rhizobium e Mesorhizobium. Análises quantitativas mostraram que o teor de ácido urônico encontrado em R3 (8,4 %) foi maior que em R1 (2,4 %), R2 (1,7 %) e R4 (0,8 %). A cromatografia de filtração em gel mostrou que R2 e R3 são mais homogêneos e menos polidispersos. Hidrólise ácida total e análise por HPAEC/PAD mostrou glucose como constituinte básico majoritário dos EPS, além de galactose e manose. Todos os polissacarídeos apresentaram comportamento não-Newtoniano, com características de soluções pseudoplásticas nas concentrações de 2, 5 e 10 g/L. O modelo reológico de Ostwald-de-Waele (Lei da Potência) foi utilizado para representar os dados experimentais de tensão de cisalhamento versus taxa de deformação. Os EPS R1, R2 e R4 demonstraram pequeno aumento na viscosidade em presença de NaCl, e apresentaram comportamento viscoelástico de gel, sendo R1 o que apresenta características de gel mais forte. O EPS R3 manteve-se como o menos viscoso, tanto em meio aquoso quanto em solução salina, provavelmente devido ao maior percentual de ácidos urônicos em sua estrutura. Além disso, R3 exibiu comportamento de solução diluída a baixas concentrações, e viscoelástico de gel fraco em concentrações mais elevadas. A análise da influência da temperatura sobre o comportamento viscoelástico das soluções polissacarídicas mostrou... / Exopolysaccharides (EPS) are polymers produced by a great variety of microorganisms and possess different structural, physical and chemical properties. Investigation of rheological properties of these solutions is important due to an increasing interest in polysaccharides commercial applications, mainly in food sectors. The objective of this work was investigate rheological characteristics of exopolysaccharides R1, R2, R3 and R4 produced by different Rhizobium and Mesorhizobium strains. Quantitative analysis showed that uronic acid component found in R3 (8,4 %) was higher than R1 (2,4 %), R2 (1,7 %) and R4 (0,8 %). Gel filtration chromatography indicated that EPS R2 and R3 are more homogeneous and less polidisperse. Acid hydrolysis and HPAEC/PAD analysis revealed that glucose was the main monosaccharide, beyond galactose and mannose. All exopolysaccharides had non-Newtonian behavior, with pseudoplastic characteristics at concentrations of 2, 5 and 10 g/L. The Ostwald-de-Waele (Power Law) was the rheological model used to represent the experimental data of the shear stress versus shear rate. EPS R1, R2 and R4 demonstrated a slight increase in viscosity in presence of NaCl, and viscoelastic behavior, R1 had strong gelling characteristics. The EPS R3 was less viscous, in water solutions and presence of salt, probably due to a high percentual of uronic acids on its structure. Moreover, R3 exhibited diluted solution behavior at low concentration, and viscoelastic weak gelling at high concentrations. Analysis of temperature influence over polysaccharide solutions viscoelastic behavior showed that EPS R1, R2 e R4 had strong gelling characteristics, at concentrations of 5 g/L.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20438号 / 農博第2223号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5059(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM