Rizóbios são bactérias estritamente aeróbias, quimioorganotróficas, com a forma de bastonetes não formadoras de esporos, Gram-negativas, com um tamanho que varia de 0,5- 0,9 µm X 1,2-3,0 µm. Normalmente encontradas no solo, fixam nitrogênio atmosférico (N2) em simbiose com leguminosas e algumas plantas não leguminosas, induzindo a formação de nódulos nas raízes, permanecendo nestas como bacteróides fixadores de nitrogênio. No Brasil rizóbios são inoculados em lavouras de soja, pois a inoculação com bactérias fixadoras de nitrogênio supre totalmente a necessidade de utilização de adubos nitrogenados. No presente estudo foi realizada uma análise comparativa entre as espécies Bradyrhizobium japonicum (estirpe USDA 110) e Bradyrhizobium elkanii (estirpe SEMIA 587) através da aplicação da técnica de RDA (Representational Difference Analysis). RDA é uma técnica bastante útil para revelar seqüências únicas entre dois genomas ou transcritomas semelhantes. Após três ciclos de hibridizações subtrativas e amplificações dos fragmentos tester, fragmentos de aproximadamente 300 pb foram gerados. Estes fragmentos foram clonados em pUC18 e seqüênciados. Das 200 seqüências obtidas, 46 pertenceram exclusivamente à B. elkanii e 154 tiveram homologia com B. japonicum. Entre as 46 seqüências sem homologia com B. japonicum, 39 não demonstraram homologia com nenhuma seqüência depositada nos bancos de dados públicos e sete seqüências mostraram homologia com proteínas conhecidas. Estas sete seqüências foram divididas em três grupos: seqüências homólogas a outras estirpes ou espécies de Bradyrhizobium, seqüências homólogas a outras bactérias fixadoras de nitrogênio e seqüências homólogas a bactérias não fixadoras de nitrogênio. O grupo com homologia a estirpes de Bradyrhizobium foi composto por dois clones: clone i5 foi homólogo a um transportador ABC (ATP Binding Cassette, hlyB like protein) de Bradyrhizobium sp. ORS278, e o clone i29 foi homólogo à subunidade menor da carboxylase (tipo RuBisCO) da estirpe foto-organotrófica Bradyrhizobium sp. BTA1. O grupo com homologia a outras bactérias fixadoras de nitrogênio foi composto por três clones: clone i150 foi homólogo à subunidade alfa da 4-hydroxybenzoyl-CoA redutase de Mesorhizobium loti, clone i170 foi homólogo a uma proteína hipotética conservada de Rhodopseudomonas palustris, e o clone ii23 foi homólogo ao fator de virulência mviN de Xanthobacter autotrophicus Py2. O grupo com homologia a bactérias não fixadoras de nitrogênio foi também composto por dois clones: clone i65 foi homólogo à peptidase M19 de Sphingopyxis alaskensis RB2256, e o clone i157 foi homólogo a uma proteína hipotética conservada de Nitrobacter winogradsky. Esse conhecimento genômico de B. elkanii poderá ajudar na compreensão das diferenças fisiológicas encontradas entre essas duas espécies e servir como base na caracterização de estirpes isoladas de nódulos de soja. / Rhizobia are strictly aerobic chemoorganotrophic rod-shaped sporeless bacteria. They are a Gram-negative bacteria with a size that varies between 0.5-0.9 µm to 1.2-3.0 µm. Normally found in the ground, they fix atmospheric nitrogen (N2) in symbiosis with leguminous plants and some non leguminous plants, inducing the formation of nodules in the roots where the bacterium differentiates itself into nitrogen-fixing bacteroids. In Brazil, rhizobia are inoculated in soybean crops. This inoculation totally fulfills the crop need of nitrogen. In the present study a comparative analysis was carried out between Bradyrhizobium japonicum (USDA 110) and Bradyrhizobium elkanii (SEMIA 587) through the application of the RDA technique (Representational Difference Analysis). RDA is a quite useful technique to reveal the unique sequences between two genomes or transcriptomes. After three cycles of subtractive hybridizations and amplifications of the tester DNA, 300 pb fragments were obtained. These fragments were cloned into pUC18 vector and were sequenced. Two hundred RDA sequences were obtained. Forty six sequences among the 200 belonged exclusively to the tester strain B. elkanii SEMIA 587, and 154 had homology to the driver strain B. japonicum USDA110. From the 46 sequences with no homology to B. japonicum USDA 110 genome, 39 showed no homology with sequences in public databases and seven sequences showed homology with known proteins. These seven sequences were divided in three groups: homolog to other Bradyrhizobium strains, homolog to other nitrogen-fixing bacteria, and homolog to non nitrogen-fixing bacteria. The group of homolog to other Bradyrhizobium strains was composed by two clones: clone i5 was homolog to a putative toxin secretion ABC transporter from Bradyrhizobium sp. ORS278, and clone i29 was homolog to a putative carboxylase like RuBisCO small subunit from the photoorganotroph Bradyrhizobium sp. BTA1. The group of homolog to other nitrogen-fixing bacteria was composed by three clones: clone i150 was homolog to a 4-hydroxybenzoyl-CoA reductase alpha-subunit of Mesorhizobium loti, clone i170 was homolog to a conserved hypothetical protein of Rhodopseudomonas palustris, and clone ii23 was homolog to a virulence factor mviN of Xanthobacter autotrophicus Py2. The group of homolog to other non nitrogen-fixing bacteria was also composed by two clones: clone i65 was homolog to a peptidase M19 of Sphingopyxis alaskensis RB2256, and clone i157 was homolog to a conserved hypothetical protein of Nitrobacter winogradsky. This better understanding of B. elkanii genome could help us in the comprehension of physiological differences between these two species and it could be a useful tool to characterize Bradyrhizobia strains isolated from soybean nodules.

Ureases (EC 3.5.1.5.) catalisam a hidrólise de ureia em NH3 e CO2, sendo sintetizadas por plantas, fungos e bactérias. No solo, a urease é encontrada em microrganismos, raízes de plantas e como uma enzima extracelular ligada a compostos orgânicos e inorgânicos. Em plantas e fungos, as ureases consistem em trímeros ou hexâmeros formados por uma subunidade de 90 kDa, enquanto que enzimas bacterianas são complexos com duas ou três subunidades. A inserção de dois átomos de níquel no sítio ativo requer pelo menos três proteínas acessórias, UreD, UreF e UreG em bactérias, ou seus ortólogos em plantas e fungos. Bradyrhizobium japonicum é uma bactéria do solo que forma nódulos fixadores de nitrogênio em plantas de soja. Esse microrganismo produz uma urease, e seu papel na sinalização, tanto para a planta de soja quanto para outros organismos no complexo ambiente da rizosfera, ainda não foi investigado. Desta forma, o presente estudo objetivou purificar e caracterizar a urease de B. japonicum (BJU), bem como avaliar o papel desta enzima, tanto a de origem vegetal quanto a de origem bacteriana, no processo de nodulação da soja. A capacidade da enzima em induzir exocitose/secreção foi avaliada no teste de agregação plaquetária, utilizando-se plasma rico em plaquetas obtido de sangue de coelho e monitorando-se a agregação por turbidimetria. Observamos que a urease de B. japonicum possui a propriedade de agregar plaquetas, implicando em uma provável atividade indutora de exocitose. Ensaios de quimiotaxia demonstraram a atração exercida pela urease ubíqua recombinante de soja sobre células de B. japonicum. Para os ensaios de nodulação, sementes pré-germinadas de soja tiposelvagem (Williams 82) e de mutantes deficientes na proteína urease (eu1-sun/eu4) foram expostas a culturas de B. japonicum USDA110 (tipo selvagem), B. japonicum ΔureG (ausência de atividade ureásica) ou B. japonicum ΔureABC (ausência de urease), e semeadas em vasos de Leonard modificados. Os nódulos foram contados e pesados em diferentes tempos após a inoculação. Além disso, foi determinado o conteúdo de leghemoglobina destes nódulos e o conteúdo de nitrogênio na parte aérea das plantas, como uma maneira de estimar a eficiência da fixação biológica de nitrogênio. Plantas deficientes em urease formam nódulos maiores e em menor número que as selvagens, independente do fenótipo da bactéria. O pico de produção de leghemoglobina em plantas tipo-selvagem é maior e anterior ao pico observado nas plantas mutantes. Inibição de toda a atividade enzimática de urease nas plantas selvagens pelo inibidor fenilfosforodiamidato não causou as alterações observadas pela ausência da proteína urease nas plantas mutantes. Esses resultados sugerem que o desenvolvimento do nódulo em plantas requer a proteína urease, de maneira independente de sua atividade enzimática. Em contraste, a urease da bactéria parece não influenciar a nodulação ou a fixação biológica de N2 na planta. Concluímos que a urease da soja apresenta um papel relevante na simbiose planta - B. japonicum, independente de sua atividade ureolítica, e não compartilhado com a urease bacteriana. / Ureases (EC 3.5.1.5.) catalyze the hydrolysis of urea in NH3 and CO2, and are synthesized by plants, fungi and bacteria. In the soil, urease occurs in microorganisms and plant roots, and as an extracellular enzyme bound to organic and inorganic compounds. In plants and fungi, ureases consist of trimers or hexamers formed by a subunit of 90 kDa, whereas bacterial enzymes are complexes with two or three subunits. The insertion of two nickel atoms into the active site requires at least three accessory proteins, ureD, ureF, and ureG in bacteria, or their orthologs in plants and fungi. Bradyrhizobium japonicum is a soil bacterium that forms nitrogen fixing nodules on soybean plants. This bacterium produces a urease, and its role in signaling for both the soybean plant and other organisms in the complex environment of the rhizosphere, has not yet been investigated. Thus, the present study aimed to purify and characterize B. japonicum urease (BJU), and to evaluate the role of this enzyme, from both plant and bacteria, in the process of soybean nodulation. The induction of secretion was assessed by the ability of the enzyme to induce platelet aggregation in rabbit platelet-rich plasma monitored by turbidimetry. We found that the urease of B. japonicum possesses the property of aggregating platelets, implying a secretion inducing activity. Chemotaxis assays demonstrated the attraction of recombinant soybean ubiquitous urease upon B. japonicum cells. For nodulation assays, pre-germinated seeds of wild-type soybeans (Williams 82) and of mutants deficient in the urease protein (eu1-sun/eu4) were exposed to cultures of B. japonicum USDA110 (wild-type), B. japonicum ΔureG (lack of urease activity) or B. japonicum ΔureABC (no urease), and planted in modified Leonard jars. The nodules were counted and weighed at different times after inoculation. Additionally, we determined the leghemoglobin content of nodules and the nitrogen content in the shoots, as a way to estimate the efficiency of biological nitrogen fixation. Plants deficient in urease (eu1-sun/eu4) form fewer but larger nodules than wildtype plants, regardless of the phenotype of the bacteria. The peak of leghemoglobin production in wild-type plants is higher and earlier than the peak observed in mutant plants. Inhibition of all the enzymatic activity of urease in wild-type plants by phenylphosphorodiamidate did not result in the alterations seen in mutant plants lacking urease. These results suggest that the development of nodule requires the protein urease, but not its enzyme activity. In contrast, the bacterial urease seems to play no roles in the nodulation and biological N2 fixation in the plant. We conclude that the soybean urease plays an important role in the soybean - B. japonicum symbiosis, which is independent of its ureolytic activity and is not shared by the bacterial urease.

Rizóbios são bactérias estritamente aeróbias, quimioorganotróficas, com a forma de bastonetes não formadoras de esporos, Gram-negativas, com um tamanho que varia de 0,5- 0,9 µm X 1,2-3,0 µm. Normalmente encontradas no solo, fixam nitrogênio atmosférico (N2) em simbiose com leguminosas e algumas plantas não leguminosas, induzindo a formação de nódulos nas raízes, permanecendo nestas como bacteróides fixadores de nitrogênio. No Brasil rizóbios são inoculados em lavouras de soja, pois a inoculação com bactérias fixadoras de nitrogênio supre totalmente a necessidade de utilização de adubos nitrogenados. No presente estudo foi realizada uma análise comparativa entre as espécies Bradyrhizobium japonicum (estirpe USDA 110) e Bradyrhizobium elkanii (estirpe SEMIA 587) através da aplicação da técnica de RDA (Representational Difference Analysis). RDA é uma técnica bastante útil para revelar seqüências únicas entre dois genomas ou transcritomas semelhantes. Após três ciclos de hibridizações subtrativas e amplificações dos fragmentos tester, fragmentos de aproximadamente 300 pb foram gerados. Estes fragmentos foram clonados em pUC18 e seqüênciados. Das 200 seqüências obtidas, 46 pertenceram exclusivamente à B. elkanii e 154 tiveram homologia com B. japonicum. Entre as 46 seqüências sem homologia com B. japonicum, 39 não demonstraram homologia com nenhuma seqüência depositada nos bancos de dados públicos e sete seqüências mostraram homologia com proteínas conhecidas. Estas sete seqüências foram divididas em três grupos: seqüências homólogas a outras estirpes ou espécies de Bradyrhizobium, seqüências homólogas a outras bactérias fixadoras de nitrogênio e seqüências homólogas a bactérias não fixadoras de nitrogênio. O grupo com homologia a estirpes de Bradyrhizobium foi composto por dois clones: clone i5 foi homólogo a um transportador ABC (ATP Binding Cassette, hlyB like protein) de Bradyrhizobium sp. ORS278, e o clone i29 foi homólogo à subunidade menor da carboxylase (tipo RuBisCO) da estirpe foto-organotrófica Bradyrhizobium sp. BTA1. O grupo com homologia a outras bactérias fixadoras de nitrogênio foi composto por três clones: clone i150 foi homólogo à subunidade alfa da 4-hydroxybenzoyl-CoA redutase de Mesorhizobium loti, clone i170 foi homólogo a uma proteína hipotética conservada de Rhodopseudomonas palustris, e o clone ii23 foi homólogo ao fator de virulência mviN de Xanthobacter autotrophicus Py2. O grupo com homologia a bactérias não fixadoras de nitrogênio foi também composto por dois clones: clone i65 foi homólogo à peptidase M19 de Sphingopyxis alaskensis RB2256, e o clone i157 foi homólogo a uma proteína hipotética conservada de Nitrobacter winogradsky. Esse conhecimento genômico de B. elkanii poderá ajudar na compreensão das diferenças fisiológicas encontradas entre essas duas espécies e servir como base na caracterização de estirpes isoladas de nódulos de soja. / Rhizobia are strictly aerobic chemoorganotrophic rod-shaped sporeless bacteria. They are a Gram-negative bacteria with a size that varies between 0.5-0.9 µm to 1.2-3.0 µm. Normally found in the ground, they fix atmospheric nitrogen (N2) in symbiosis with leguminous plants and some non leguminous plants, inducing the formation of nodules in the roots where the bacterium differentiates itself into nitrogen-fixing bacteroids. In Brazil, rhizobia are inoculated in soybean crops. This inoculation totally fulfills the crop need of nitrogen. In the present study a comparative analysis was carried out between Bradyrhizobium japonicum (USDA 110) and Bradyrhizobium elkanii (SEMIA 587) through the application of the RDA technique (Representational Difference Analysis). RDA is a quite useful technique to reveal the unique sequences between two genomes or transcriptomes. After three cycles of subtractive hybridizations and amplifications of the tester DNA, 300 pb fragments were obtained. These fragments were cloned into pUC18 vector and were sequenced. Two hundred RDA sequences were obtained. Forty six sequences among the 200 belonged exclusively to the tester strain B. elkanii SEMIA 587, and 154 had homology to the driver strain B. japonicum USDA110. From the 46 sequences with no homology to B. japonicum USDA 110 genome, 39 showed no homology with sequences in public databases and seven sequences showed homology with known proteins. These seven sequences were divided in three groups: homolog to other Bradyrhizobium strains, homolog to other nitrogen-fixing bacteria, and homolog to non nitrogen-fixing bacteria. The group of homolog to other Bradyrhizobium strains was composed by two clones: clone i5 was homolog to a putative toxin secretion ABC transporter from Bradyrhizobium sp. ORS278, and clone i29 was homolog to a putative carboxylase like RuBisCO small subunit from the photoorganotroph Bradyrhizobium sp. BTA1. The group of homolog to other nitrogen-fixing bacteria was composed by three clones: clone i150 was homolog to a 4-hydroxybenzoyl-CoA reductase alpha-subunit of Mesorhizobium loti, clone i170 was homolog to a conserved hypothetical protein of Rhodopseudomonas palustris, and clone ii23 was homolog to a virulence factor mviN of Xanthobacter autotrophicus Py2. The group of homolog to other non nitrogen-fixing bacteria was also composed by two clones: clone i65 was homolog to a peptidase M19 of Sphingopyxis alaskensis RB2256, and clone i157 was homolog to a conserved hypothetical protein of Nitrobacter winogradsky. This better understanding of B. elkanii genome could help us in the comprehension of physiological differences between these two species and it could be a useful tool to characterize Bradyrhizobia strains isolated from soybean nodules.

Ureases (EC 3.5.1.5.) catalisam a hidrólise de ureia em NH3 e CO2, sendo sintetizadas por plantas, fungos e bactérias. No solo, a urease é encontrada em microrganismos, raízes de plantas e como uma enzima extracelular ligada a compostos orgânicos e inorgânicos. Em plantas e fungos, as ureases consistem em trímeros ou hexâmeros formados por uma subunidade de 90 kDa, enquanto que enzimas bacterianas são complexos com duas ou três subunidades. A inserção de dois átomos de níquel no sítio ativo requer pelo menos três proteínas acessórias, UreD, UreF e UreG em bactérias, ou seus ortólogos em plantas e fungos. Bradyrhizobium japonicum é uma bactéria do solo que forma nódulos fixadores de nitrogênio em plantas de soja. Esse microrganismo produz uma urease, e seu papel na sinalização, tanto para a planta de soja quanto para outros organismos no complexo ambiente da rizosfera, ainda não foi investigado. Desta forma, o presente estudo objetivou purificar e caracterizar a urease de B. japonicum (BJU), bem como avaliar o papel desta enzima, tanto a de origem vegetal quanto a de origem bacteriana, no processo de nodulação da soja. A capacidade da enzima em induzir exocitose/secreção foi avaliada no teste de agregação plaquetária, utilizando-se plasma rico em plaquetas obtido de sangue de coelho e monitorando-se a agregação por turbidimetria. Observamos que a urease de B. japonicum possui a propriedade de agregar plaquetas, implicando em uma provável atividade indutora de exocitose. Ensaios de quimiotaxia demonstraram a atração exercida pela urease ubíqua recombinante de soja sobre células de B. japonicum. Para os ensaios de nodulação, sementes pré-germinadas de soja tiposelvagem (Williams 82) e de mutantes deficientes na proteína urease (eu1-sun/eu4) foram expostas a culturas de B. japonicum USDA110 (tipo selvagem), B. japonicum ΔureG (ausência de atividade ureásica) ou B. japonicum ΔureABC (ausência de urease), e semeadas em vasos de Leonard modificados. Os nódulos foram contados e pesados em diferentes tempos após a inoculação. Além disso, foi determinado o conteúdo de leghemoglobina destes nódulos e o conteúdo de nitrogênio na parte aérea das plantas, como uma maneira de estimar a eficiência da fixação biológica de nitrogênio. Plantas deficientes em urease formam nódulos maiores e em menor número que as selvagens, independente do fenótipo da bactéria. O pico de produção de leghemoglobina em plantas tipo-selvagem é maior e anterior ao pico observado nas plantas mutantes. Inibição de toda a atividade enzimática de urease nas plantas selvagens pelo inibidor fenilfosforodiamidato não causou as alterações observadas pela ausência da proteína urease nas plantas mutantes. Esses resultados sugerem que o desenvolvimento do nódulo em plantas requer a proteína urease, de maneira independente de sua atividade enzimática. Em contraste, a urease da bactéria parece não influenciar a nodulação ou a fixação biológica de N2 na planta. Concluímos que a urease da soja apresenta um papel relevante na simbiose planta - B. japonicum, independente de sua atividade ureolítica, e não compartilhado com a urease bacteriana. / Ureases (EC 3.5.1.5.) catalyze the hydrolysis of urea in NH3 and CO2, and are synthesized by plants, fungi and bacteria. In the soil, urease occurs in microorganisms and plant roots, and as an extracellular enzyme bound to organic and inorganic compounds. In plants and fungi, ureases consist of trimers or hexamers formed by a subunit of 90 kDa, whereas bacterial enzymes are complexes with two or three subunits. The insertion of two nickel atoms into the active site requires at least three accessory proteins, ureD, ureF, and ureG in bacteria, or their orthologs in plants and fungi. Bradyrhizobium japonicum is a soil bacterium that forms nitrogen fixing nodules on soybean plants. This bacterium produces a urease, and its role in signaling for both the soybean plant and other organisms in the complex environment of the rhizosphere, has not yet been investigated. Thus, the present study aimed to purify and characterize B. japonicum urease (BJU), and to evaluate the role of this enzyme, from both plant and bacteria, in the process of soybean nodulation. The induction of secretion was assessed by the ability of the enzyme to induce platelet aggregation in rabbit platelet-rich plasma monitored by turbidimetry. We found that the urease of B. japonicum possesses the property of aggregating platelets, implying a secretion inducing activity. Chemotaxis assays demonstrated the attraction of recombinant soybean ubiquitous urease upon B. japonicum cells. For nodulation assays, pre-germinated seeds of wild-type soybeans (Williams 82) and of mutants deficient in the urease protein (eu1-sun/eu4) were exposed to cultures of B. japonicum USDA110 (wild-type), B. japonicum ΔureG (lack of urease activity) or B. japonicum ΔureABC (no urease), and planted in modified Leonard jars. The nodules were counted and weighed at different times after inoculation. Additionally, we determined the leghemoglobin content of nodules and the nitrogen content in the shoots, as a way to estimate the efficiency of biological nitrogen fixation. Plants deficient in urease (eu1-sun/eu4) form fewer but larger nodules than wildtype plants, regardless of the phenotype of the bacteria. The peak of leghemoglobin production in wild-type plants is higher and earlier than the peak observed in mutant plants. Inhibition of all the enzymatic activity of urease in wild-type plants by phenylphosphorodiamidate did not result in the alterations seen in mutant plants lacking urease. These results suggest that the development of nodule requires the protein urease, but not its enzyme activity. In contrast, the bacterial urease seems to play no roles in the nodulation and biological N2 fixation in the plant. We conclude that the soybean urease plays an important role in the soybean - B. japonicum symbiosis, which is independent of its ureolytic activity and is not shared by the bacterial urease.

Lok Chui-Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgement --- p.II / Abstract --- p.IV / Chinese Abstract (摘要） --- p.VI / Abbreviation --- p.VIII / Table of Contents --- p.XIII / List of Tables --- p.XVII / List of Figures --- p.XVIII / Chapter Chapter One : --- Literature Review --- p.1 / Chapter 1.1 --- The Schistosoma Species --- p.1 / Chapter 1.1.1 --- The Schistosoma Gene Discovery --- p.3 / Chapter 1.1.2 --- Schistosome Transcriptome --- p.4 / Chapter 1.2 --- Schistosomiasis --- p.4 / Chapter 1.2.1 --- Immunopathology of Schistosomiasis --- p.5 / Chapter 1.2.2 --- Diagnosis of Schistosomiasis --- p.7 / Chapter 1.2.3 --- Treatment and Control for Schistosomiasis --- p.7 / Chapter 1.2.4 --- Vaccine Development for Schistosomiasis --- p.8 / Chapter 1.3 --- "The Species, Schistosoma japonicum" --- p.9 / Chapter 1.3.1 --- The Life Cycle of Schistosoma japonicum --- p.10 / Chapter 1.3.1.1 --- "The Egg, Miracidium Phase of the Life Cycle" --- p.12 / Chapter 1.3.1.2 --- Developmental Cycle within Mollusc Host --- p.12 / Chapter 1.3.1.3 --- The Cercaria Phase of Life Cycle --- p.13 / Chapter 1.3.1.4 --- Adult Schistosome in Definitive Host --- p.14 / Chapter 1.4 --- Invasion by Schistosome Cercariae --- p.15 / Chapter 1.5 --- "The Anti-inflammatory Protein, Sml6" --- p.16 / Chapter 1.5.1 --- Discovery of Sm 16 --- p.16 / Chapter 1.5.2 --- Cloning and Expression of Gene-encoding Sm 16 --- p.17 / Chapter 1.5.3 --- Potential Anti-inflammatory Therapy using Sm 16 --- p.18 / Chapter 1.6 --- Innate Immunity and Adaptive Immunity --- p.18 / Chapter 1.6.1 --- Macrophage --- p.18 / Chapter 1.6.2 --- Major Histocompatiblity Complex (MHC) --- p.20 / Chapter 1.6.3 --- Adaptive Immunity to Parasites --- p.20 / Chapter 1.7 --- Inflammation --- p.21 / Chapter 1.7.1 --- Cells of the Inflammatory Process --- p.23 / Chapter 1.7.2 --- Cytokines --- p.24 / Chapter 1.7.2.1 --- Interleukin-1 (IL-1) System --- p.26 / Chapter 1.7.2.2 --- Interferon (IFN) System --- p.27 / Chapter 1.7.3 --- Anti-inflammatory Therapy --- p.28 / Chapter 1.8 --- Aim of Study --- p.29 / Chapter Chapter Two : --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Cell Lines, Mouse Strain and Bacterial Strains" --- p.30 / Chapter 2.1.2 --- Plasmids --- p.31 / Chapter 2.1.3 --- Chemicals --- p.31 / Chapter 2.1.4 --- "Kits, Nucleic Acids and Reagents" --- p.34 / Chapter 2.1.5 --- Antibodies and Immunoglobins --- p.35 / Chapter 2.1.6 --- Cell Culture Reagents --- p.35 / Chapter 2.1.7 --- Solutions --- p.36 / Chapter 2.1.8 --- Solutions of Reaction Kits --- p.39 / Chapter 2.1.9 --- Enzymes --- p.41 / Chapter 2.1.10 --- Major Equipments and Materials --- p.41 / Chapter 2.1.11 --- Primers --- p.43 / Chapter 2.1.11.1 --- Sequencing and Sj 16 Gene-coding Specific Primers --- p.43 / Chapter 2.1.11.2 --- Primers for Cytokines --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Amplification of Sjl6 cDNA from Schistosoma japonicum Cercariae --- p.45 / Chapter 2.2.1.1 --- Isolation of Cercariae total RNA by Guanidinium Thiocyanate - Cesium Chloride Ultracentrifugation --- p.45 / Chapter 2.2.1.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.1.2.1 --- Reverse Transcription (RT) --- p.46 / Chapter 2.2.1.2.2 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2 --- Cloning and Subcloning of Sj 16 --- p.47 / Chapter 2.2.2.1 --- Preparation of DH5a Competent Cells --- p.47 / Chapter 2.2.2.2 --- Purification of Plasmid DNA --- p.48 / Chapter 2.2.2.3 --- Restriction Enzyme Digestion of DNA --- p.49 / Chapter 2.2.2.4 --- Purification of DNA Fragments from Agarose Gel --- p.50 / Chapter 2.2.2.5 --- Ligation of Purified DNA Fragments --- p.51 / Chapter 2.2.2.6 --- Transformation of Recombinant Plasmid --- p.52 / Chapter 2.2.2.7 --- Selection of Transformed Clones --- p.52 / Chapter 2.2.2.7.1 --- Screening by X-gal and IPTG : a-complementation --- p.52 / Chapter 2.2.2.7.2 --- Screening by Polymerase Chain Reaction --- p.53 / Chapter 2.2.2.8 --- Cycle Sequencing --- p.53 / Chapter 2.2.3 --- Expression of the rSj 16 in Eukaryotic System --- p.55 / Chapter 2.2.3.1 --- Transfection of pSecTag2B/Sj 16 Plasmid into Animal Cells --- p.55 / Chapter 2.2.3.2 --- PCR Screening of Transfected Cells --- p.56 / Chapter 2.2.3.3 --- Analysis of mRNA Transcript by RT-PCR --- p.56 / Chapter 2.2.3.4 --- Concentration of the Condition Medium --- p.57 / Chapter 2.2.3.5 --- Western Blot analysis of rSjl6 Expression --- p.58 / Chapter 2.2.4 --- Expression of rSjl6 in Bacterial System --- p.59 / Chapter 2.2.4.1 --- Transformation of pET30a+/Sjl6 Plasmid into BL21 --- p.59 / Chapter 2.2.4.2 --- Optimization of rSj 16 Expression --- p.60 / Chapter 2.2.4.3 --- Solubility of the rSjl6 --- p.60 / Chapter 2.2.4.4 --- Estimation of rSj 16 Concentration --- p.62 / Chapter 2.2.4.5 --- Western Blot Analysis of rSj 16 --- p.62 / Chapter 2.2.5 --- Recombinant Protein Purification --- p.63 / Chapter 2.2.5.1 --- Affinity Chromatography of Recombinant Protein --- p.63 / Chapter 2.2.5.2 --- Dialysis of Eluted Recombinant Protein in PBS --- p.64 / Chapter 2.2.5.3 --- Estimation of Recombinant Protein Concentration --- p.65 / Chapter 2.2.6 --- Demonstrate the Anti-inflammatory Activity of rSj 16 --- p.65 / Chapter 2.2.6.1 --- Thioglycollate Induced Macrophage Recruitment --- p.65 / Chapter 2.2.6.2 --- Cytospin and Hemacolor Staining of PECs --- p.66 / Chapter 2.2.6.3 --- FACS Analysis of PECs --- p.67 / Chapter 2.2.6.4 --- Isolation of total RNA by TRIZOL Reagent --- p.67 / Chapter 2.2.7 --- Immunogenicity and Antigenicity of rSjl6 --- p.68 / Chapter 2.2.7.1 --- Western Blot of rSjl6 with Schistosoma japonicum infected rabbit serum --- p.69 / Chapter 2.2.7.2 --- Preparation of Anti-Sj 16 Serum --- p.69 / Chapter 2.2.7.3 --- Western Blot of rSjl6 with immunized mice serum --- p.70 / Chapter 2.2.8 --- FACS analysis of MHC (I) Expression --- p.71 / Chapter 2.2.9 --- Anti-proliferative Assay using BrdU Kit --- p.72 / Chapter Chapter Three : --- Results --- p.73 / Chapter 3.1 --- Amplification of Sj 16 cDNA from Schistosoma japonicum Cercariae total RNA --- p.73 / Chapter 3.2 --- Construction of pBluescript II SK(-) / Sjl6 --- p.75 / Chapter 3.3 --- Analysis of Sj 16 Nucleotide and Amino Acid Sequence --- p.78 / Chapter 3.3.1 --- Blastn Search Analysis --- p.80 / Chapter 3.3.2 --- Blastx Search Analysis --- p.82 / Chapter 3.3.3 --- Structural Analysis --- p.84 / Chapter 3.4 --- Subcloning of Sjl6 cDNA into pET30a+ and pSecTag2B Expression Vector --- p.88 / Chapter 3.5 --- Expression of the rSj 16 --- p.92 / Chapter 3.5.1 --- Animal Cell Expression --- p.92 / Chapter 3.5.1.1 --- Analysis of mRNA Transcript by RT-PCR --- p.93 / Chapter 3.5.1.2 --- Western Blot of Condition Medium --- p.95 / Chapter 3.5.2 --- Bacterial Cell Expression --- p.97 / Chapter 3.5.2.1 --- Optimization of rSjl6 Expression --- p.97 / Chapter 3.5.2.2 --- Estimation of rSjl6 Concentration --- p.98 / Chapter 3.5.2.3 --- Solubility of rSj16 --- p.99 / Chapter 3.5.2.4 --- Western Blot Analysis of rSjl6 --- p.100 / Chapter 3.6 --- Purification of Recombinant Protein --- p.101 / Chapter 3.6.1 --- Purification of rSj16 --- p.101 / Chapter 3.6.2 --- Purification of rSjCa8 --- p.104 / Chapter 3.7 --- Anti-inflammatory Activity of rSj 16 --- p.107 / Chapter 3.7.1 --- Analysis of PECs in Thioglycollate Induced Inflammation --- p.107 / Chapter 3.7.2 --- Hemacolor Staining of PECs --- p.110 / Chapter 3.7.3 --- FACS Analysis of PECs --- p.110 / Chapter 3.7.4 --- RT-PCR of RNA Isolated from PECs --- p.115 / Chapter 3.8 --- Immunogenicity and Antigenicity of rSjl6 --- p.117 / Chapter 3.8.1 --- Immunogenicity of rSj 16 --- p.117 / Chapter 3.8.2 --- Antigenicity of rSj16 --- p.117 / Chapter 3.9 --- Inhibitory Effect of rSj 16 on rMuIFN-a4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.1 --- Time Course of rMuIFN-α4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.2 --- Inhibitory Effect of rSjl6 on rMuIFN-α4 Induced MHC (I) Up-regulation --- p.120 / Chapter 3.9.3 --- "Anti-proliferation Effect of rMuIFN-a4, rSj 16 and rSjCa 8" --- p.124 / Chapter 3.9.4 --- Effect of Signal Transduction Inhibitors on rMuIFN-a4 Induced MHC (I) Up-regulation --- p.126 / Chapter Chapter Four : --- Discussion and Conclusion --- p.129 / Chapter 4.1 --- Discussion --- p.129 / Chapter 4.1.1 --- Overview --- p.129 / Chapter 4.1.2 --- Molecular and Structural Analysis of rSj 16 --- p.130 / Chapter 4.1.3 --- Relationship between Sml6 and Sjl6 --- p.131 / Chapter 4.1.4 --- Anti-inflammatory Activity of rSj 16 --- p.132 / Chapter 4.1.5 --- Immunogenicity and Antigenicity of rSjl6 --- p.137 / Chapter 4.1.6 --- Inhibitory Effect of rSjl6 on rMuIFN-a4 Induced Up-regulation of MHC (I) Expression --- p.138 / Chapter 4.1.7 --- Relation between Sj 16 and the Innate Immune System --- p.139 / Chapter 4.1.8 --- Further Study and Significance --- p.140 / Chapter 4.2 --- Conclusion --- p.141 / References --- p.142

Das Bakterium Bradyrhizobium japonicum ist wie andere Rhizobien in der Lage mit Pflanzen der Familie Fabales (Leguminosen) eine Symbiose einzugehen. Im symbiontischen Zustand fixieren die Mikrosymbionten atmosphärischen, molekularen Stickstoff und stellen diesen den Pflanzen in verwertbarer Form zur Verfügung. Im Gegenzug erhalten die Bakterien von den Pflanzen verschiedene Verbindungen als Kohlenstoff- und Energiequelle. Dem geht ein komplexer Signalaustausch voraus um die gegenseitige Erkennung der Partner und die Spezies-spezifische Symbiose zu ermöglichen. Auf Seite der Bakterien erfolgt die Reaktion auf die Gegenwart pflanzlicher Signalmoleküle. In B. japonicum induziert das Flavonoid Genistein die Transkription einer Reihe von Genen. Durch die Expression der nod-Gene erfolgt eine Synthese von Lipochitooligosacchariden. Diese sogenannten Nodulationsfaktoren rufen wiederum eine Reaktion in der Wirtspflanze hervor. Um die zugrunde liegende Genexpression und deren Regulationsmechanismen zu erforschen, standen bis dato nur Methoden für die Untersuchung einzelner Gene zur Verfügung. Die erstmals 1995 publizierte Microarraytechnologie eröffnete die Möglichkeit zu einem Zeitpunkt die Gentranskription eines gesamten Organismus zu untersuchen (Schena et al. 1995). Um mit dieser Technologie die Gentranskription bei B. japonicum zu untersuchen, wurde ein auf PCR-Produkten basierender Microarray hergestellt. Grundlage für die Synthese genspezifischer PCR-Produkte war die symbiontische Region von B. japonicum und andere zu diesem Zeitpunkt bekannte Gensequenzen (Göttfert et al. 2001). Nach der 2002 erfolgten Veröffentlichung des Genoms von B. japonicum erfolgte ein Abgleich der bisher verwendeten Sequenzen mit der vollständigen Sequenzinformation (Kaneko et al. 2002a). Nach Synthese der PCR-Produkte und deren Kontrolle durch Sequenzierung erfolgte die Herstellung des Microarrays unter Einsatz eines Microarrayspotters. Geeignete Techniken der cDNA-Markierung und die Microarray-Hybridisierung wurden mit Total-RNA von B. japonicum-Kulturen ausgetestet und etabliert. Eine differentielle Expremierung war eine Stunde nach Genistein- bzw. Methanolgabe zum Kulturmedium nicht nachweisbar. Zum darauffolgenden Zeitpunkt konnte die bekannte Induktion der nod-Gene durch dass Flavonoid Genistein beobachtet werden. Diese Induktion fiel in den verbleibenden zwei Zeitpunkten stark ab. Dieser Abfall der Induktionsrate ließ sich nicht mit dem Genisteingehalt im Kulturmedium erklären, da dieser Zeitraum konstant blieb. Vier Stunden nach Genisteingabe war die Induktion der Gene nolA und nodD2, deren Produkte sind an der negativen Regulation der nod-Gene beteiligt, nachweisbar. Dies wäre eine mögliche Erklärung für den Abfall der Induktion der nod-Gene. Im gleichen untersuchten Zeitraum wurde die Transkription verschiedener Gene der Stickstofffixierung und Denitrifikation durch das Flavonoid Genistein induziert. Auch für die bekannten Regulatoren dieser Gene war eine Induktion nachweisbar. Nach bisherigen Arbeiten war die Expression dieser Gene auf mikroaerobe und anaerobe Zustände, wie z.B. dem symbiontischen Stadium, beschränkt. Eine Induktion durch Flavonoide ist bisher nicht beobachtet worden. Um die Verwendbarkeit des Microarrays auch für den symbiontischen Zustand von B. japonicum zu testen, wurden Versuche mit Wurzelknöllchen von Sojabohne (Glycine max) durchgeführt. Dafür wurden Keimlinge der Sojabohne (G. max) mit B. japonicum inokuliert und die Total-RNA der entstehenden Wurzelknöllchen isoliert. Jedoch war eine Kreuzhybridisierung mit pflanzlicher Total-RNA zu beobachten. Der auf PCR-Produkten basierende Microarray ist für die Untersuchung des symbiontischen Stadiums von B. japonicum nicht einsetzbar. Im Vergleich dazu wurde ein auf Oligomeren basierender, kommerzieller Microarray für diesen Versuch getestet. Die Kreuzhybridisierung mit pflanzlicher Total-RNA war auf die Oligomere für die 16S rDNA und 23S rDNA reduziert.

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No description available.

This study was undertaken to characterize the nod and sdh operons of Bradyrhizobium japonicum and Sinorhizobium meliloti. Ten putative B. japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. The mutants were found to have higher nodY expression than the wild-type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1muM) and/or temperature (15°C). The expression of nodY gene induction was determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochitooligosaccharide than the wild-type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. Based on nodY gene expression and symbiotic phenotype the B. japonicum mutants were classified into three groups. / A molecular genetic approach was taken to investigate the regulation of expression of succinate dehydrogenase (SDH) in S. meliloti. The sdhCDAB genes encoding SDH were shown by RT-PCR to be co-transcribed and thus constitute an operon. The transcriptional start site and putative promoter region of the first gene in the operon, sdhC , were identified by 5'-RACE and DNA sequence analysis. Transcriptional lacZ fusions to sdhC indicated that expression of the operon is regulated by carbon source in the growth medium but not by growth phase. The highest expression of the sdh operon was observed in cells grown with acetate, arabinose and glutamate, as sole carbon sources, and the lowest expression was observed in cells grown with glucose and pyruvate as sole carbon sources. / Also presented is the isolation and characterization of the first defined sdh mutant in a rhizobial species. The mutants helped demonstrate that the total lack of SDH activity would be lethal to S. meliloti cells. Symbiotic phenotype of the mutants indicated that SDH is required for N2-fixation.

In the N2 fixing legume symbiosis, mineral nitrogen (N) not only decreases N2 fixation, but also delays and inhibits the formation and development of nodules. The purposes of this thesis were to elucidate the role of signaling in the mineral N effects on nodulation and nitrogen fixation in soybean [Glycine max (L.) Merr.] and to attempt to find ways to overcome this inhibition. The responses of soybean plants, in terms of daidzein and genistein synthesis and exudation, to different mineral N levels were studied. Daidzein and genistein distribution patterns varied with plant organs, mineral N levels, and plant development stages. Mineral N inhibited daidzein and genistein contents and concentrations in soybean root and shoot extracts, but did not affect root daidzein and genistein excretion in the same way. In both synthesis and excretion, daidzein and genistein were not affected equally by mineral N treatments. Variability existed among soybean cultivars in the responses of root daidzein and genistein contents and concentrations to mineral N levels. The amount of daidzein and genistein excreted by soybean roots did not always correspond to the daidzein and genistein contents and concentrations inside the roots. On the Bradyrhizobium japonicum side, nod gene expression was inhibited by mineral nitrogen. Genistein was used to pre-incubate B. japonicum cells or was applied directly into the plant growing medium. The results showed that genistein manipulation increased nodule weight and nodule nitrogen fixation under greenhouse conditions, but interactions existed among soybean cultivars, genistein concentrations and nitrate levels. Similar results were found under field conditions. Soybean yield was increased on sandy-loam soil by preincubation of B. japonicum cells with genistein. Addition of genistein beginning at the onset of nitrogen fixation also improved soybean nodulation and nitrogen fixation. Soybean cultivars had different sensitivities to genistein additi / Other studies also show that temperature affected genistein and daidzein content and concentration in soybean roots. The effect of temperature varied among soybean cultivars. Some PGPR strains can mitigate the negative effects of nitrate on soybean nodulation and nitrogen fixation, however, this is influenced by soybean genotype. Applying PGPR together with genistein preincubation of B. japonicum cells improved soybean nodulation and increased yield. The level of improvement varied among soybean cultivars and PGPR strains. Preincubation of B. japonicum cells with genistein improved strain competitiveness under greenhouse, but not field conditions. / Overall, these findings suggested that both plant-to-Bradyrhizobium and Bradyrhizobium-to-plant signals play important roles in the effects of mineral N on nodulation and N fixation. Signal manipulation could partially overcome the inhibitory effects of mineral N on soybean- B. japonicum N fixation symbiosis.