The Rhizobium-legume symbiosis is an opportunistic association between two symbiotic partners that results in the formation of the root nodule. The process depends on the expression of a number of plant and bacterial genes that are considered critical for the establishment and maintainance of the symbiotic state. The merits of a mutational approach to the analysis of symbiosis have been recognized for several years and transposon Tn5 mutagenesis of Rhizobium has led to the identification of several symbiotic genes. This study describes the use of Tn5 mutagenesis for the isolation of symbiotically defective mutants of Bradyrhizobium japonicum. Two classes, auxotrophic and cell surface-altered mutants defective in nodule formation, have been characterized. In B. japonicum USDA 122, histidine auxotrophs that are defective in nodulation have been studied. The mutagenized DNA region has been cloned and the wild-type DNA region isolated by hybridization and complementation. In B. japonicum 61A76, Tn5-induced cell surface-altered mutants have been isolated by selecting for bacteriophage resistance. Several parameters have been used to demonstrate alterations in cell surface components. It has been shown that the Tn5 insertion is not the primary cause of the mutation in two of the characterized mutants. Complementation tests have led to the isolation of a wild-type DNA-containing cosmid, pPS23A, that overcomes the symbiotic defect in one of the mutants. Analysis of the cell surface showed a partial restoration of surface components in the complemented mutant.

Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Universiẗat, Diss., 2002--Marburg.

No description available.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 180-211).

published_or_final_version / Microbiology / Master / Master of Philosophy

Soybean [Glycine max (L.) Merr.] is a tropical to subtropical legume that requires root zone temperatures (RZTs) in the 25 to 30°C range for optimal symbiotic activity. The inability of soybean to adapt to cool soil conditions limits its development and yield in short season areas. In particular, nodulation and nitrogen fixation by this crop species is sensitive to cool RZTs. The isoflavone genistein, which is the most effective plant-to-bacterium signal in the soybean nitrogen fixation symbiosis, has been used to pretreat Bradyrhizobium japonicum inocula. This resulted in increased soybean nodulation and nitrogen fixation in several studies, indicating that genistein preincubated inocula could overcome low RZT inhibition of plant growth and yield. The effectiveness of isoflavones was found to vary among soybean cultivars. Some legume cultivars apparently supply limiting amounts of the flavonoids. The objective of this thesis was to determine how soybean cultivars of different maturity groups would respond to genistein incubation of B. japonicum prior to inoculation. Two field experiments were conducted in 1997 and 1998 involving eleven soybean cultivars of three maturity groups organized in a randomized complete block design. Cells of B. japonicum, treated with genistein or not, were applied onto seeds in the furrow at the time of planting. The results of this study indicated that genistein application increased nodule number and nodule dry matter per plant, increased plant nitrogen content, grain protein and grain yield of all cultivars. There was no interaction between maturity group and genistein application, and there was no correlation between maturity groups and increase in nodulation, total biomass, nitrogen content and yield due to genistein treatment. Thus, responses of soybean cultivars of different maturity groups to genistein treatment are not related to maturity and/or yield potential.

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.