No description available.

Thesis (Ph. D.)--University of Washington, 1983. / Vita. Bibliography: leaves [118]-133.

Debido a sus características particulares fenotípicas y taxonómicas, los rizobios tipo Oregon han atraído la atención de los rizobiólogos desde su aislamiento original por Eardly et al. (1985) en Oregon, USA. Estos rizobios son tolerantes a la acidez, tienen un rango de hospedadores amplio (nodulan varias leguminosas), son muy competitivos para la nodulación de alfalfa en suelos ácidos y son ineficientes para la fijación de nitrógeno en asociación con la alfalfa. Todos estos factores señalan a este grupo de rizobios como un potencial factor de riesgo en los suelos agrícolas en los que coexisten y compiten con E. meliloti, el simbionte eficiente de alfalfa. Nueva información sobre la genética y fisiología de las bacterias tipo Oregon es necesaria para desarrollar mejores estrategias y herramientas (moleculares) para su detección, para comprender de modo más acabado su ecología en suelos de cultivo, y para minimizar los impactos sobre la simbiosis eficiente entre alfalfa y E. meliloti. Objetivos Generales: Abordar estudios genómicos y caracterizar funcionalmente a los rizobios tipo Oregon, noduladores de alfalfa y otras leguminosas, tolerantes a la acidez e ineficientes en la fijación biológica de nitrógeno. Objetivos Específicos: - Obtener el genoma completo de la cepa Rhizobium sp. LPU83, como el representante mejor caracterizado de los rizobios tipo Oregon. - Realizar el análisis funcional in silico del nuevo genoma y evaluar, comparativamente, marcadores cromosomales y plasmídicos respecto de sus homólogos en otros rizobios con el fin de establecer relaciones funcionales y filogenéticas con otras rizobacterias. - Analizar la información contenida en plásmidos, y su transmisibilidad por vía conjugativa. - Determinar la estructura de los factores de nodulación de las rizobios tipo Oregon como determinantes de especificidad para la nodulación de alfalfa. Explorar las relaciones estructurales de los factores de nodulación con los de otros rizobios, especialmente los noduladores de Medicago spp. y Phaseolus vulgaris.

Graduation date: 1993

No description available.

Symbiotic interaction of Rhizobium with roots of legume plants leads to the formation of root nodules that are capable of reducing atmospheric nitrogen. In nodule cells, bacteria are always enclosed inside the peribacteroid membrane (pbm) which is derived from the plant cell plasma membrane. The pbm mediates all the molecular exchanges between the host plant and bacteria. The pbm of soybean (Glycine max) nodules induced by Bradyrhizobium japonicum was characterized for the presence of nodule-specific proteins (nodulins), which may be involved in some of the nodule-specific roles the pbm plays. The pbm was found to contain a number of nodulins, and most of them were shown to be of plant origin. The peribacteroid fluid (pbf) was also purified and shown to contain nodulins. Some of the pbf proteins were highly glycosylated. Immunological localization at the electron microscopic level has shown that pbm nodulins are inserted specifically in the pbm; almost none are detected in the plasma membrane (pm) of infected cells even though the pbm is derived from it. The amino acid sequences of three different pbm nodulins (nodulin-23, -24 and -26) do not share significant primary structure homology, neither do their tentative secondary structures. Our results do not preclude targeting based on pbm-nodulin-specific feature(s) of the proteins; but it is possible that pbm-specific localization is achieved by differential rates of membrane synthesis, or turnover, in pbm as compared to the plasma membrane.

Phenotypic characterizations demonstrated that diversity among 19 naturalized North American and 5 commercial Kura clover ( Trifolium ambiguum M.B.) rhizobial strains was limited. Growth chamber and field evaluations indicated the superiority of North American isolates, increasing foliage accumulation by 30% when compared to commercial inoculant strains. Nitrogen fertilization, however, produced greater accumulations in all evaluations. Genetic diversity among 128 isolates from the lower Caucasus was significant. Nodulation specificity of rhizobia from the lower Caucasus was demonstrated to be more complex than what was reported in the literature, as plants of different ploidy levels and even of different species were, in some cases, nodulated by the same isolate. Specificity of a given rhizobial strain varied depending on the isolate. This study identified naturalized North American rhizobial isolates that are more efficient than currently used commercial strains and increased the genetic diversity of Kura clover rhizobia currently available.

A Sinorhizobium meliloti (S. meliloti ) mutant, Rm30O49, deficient in malate dehydrogenase (MDH) activity was isolated via random Tn5tac1 mutagenesis. DNA sequence analyses revealed 60 the inaction is within the mdh gene. Rm30049 lacks MDH activity under all growth conditions, but shows increased or decreased activities of the TCA cycle enzymes 2-oxoglutarate dehydrogenase and succinate dehydrogenase in the presence or absence, respectively, of IPTG (isopropyl beta-D-thiogalactoside). The symbiotic phenotype of the mutant is an inability to fix nitrogen. Alfalfa seedlings inoculated with Rm30049 produced small white root nodules, but were chlorotic and failed to reach a wild-type shoot dry weight. Cosmid clone pDS15 was isolated by heterologous complementation of a Rhizobium leguminosarum sucD mutant by the S. meliloti pLAFR1 clone bank. This cosmid also restored MDH activity to Rm30049, and complemented the mutant growth and symbiotic phenotypes. Three Tn5 insertions isolated in pDS15 within sucA failed to complement Rm30049. DNA sequence analyses indicate that the mdh gene is part of the TCA cycle operon with sucCD, and that downstream and upstream of this, are operons encoding sucAB and sdhCDAB, respectively.

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.

Transcriptional fusion mutants and Tn5-B20 transposon mutants were isolated where the only genes affected are believed to either be involved in the hypothetical GABA shunt or code for subunits of the alpha-ketoglutarate dehydrogenase enzyme complex of Sinorhizobium meliloti. The growth phenotypes of Rm30222 (gabT) and eight mutants in gabD1, 2, 3, and 4 on minimal media were comparable to that of the wild-type. Compared to wild-type, Rm30222 (gabT) lacked alpha-ketoglutarate-dependent gamma-aminobutyrate transaminase activity showed high induction of gabT on GABA but produced green plants indicative of being Fix+. Mutants in gabD alleles maintained wild-type levels of succinic semialdehyde dehydrogenase activity and could fix nitrogen as well as the wild-type in symbiosis. / Mutation of sucB encoding a subunit of a-ketoglutarate dehydrogenase produced a mutant, Rm30230, that initially had difficulty growing on minimal media supplemented with either arabinose or glutamate. In symbiosis with alfalfa, Rm30230 had a fix- phenotype and was also devoid of alpha-ketoglutarate dehydrogenase activity. The ability of Rm30230 to grow on arabinose or glutamate, without alpha-ketoglutarate dehydrogenase activity, strengthens the hypothesis that S. meliloti has a functional GABA shunt allowing it to circumvent the forward-direction TCA cycle from alpha-ketoglutarate to succinate. Mutation of the second potential dihydrolipoamide succinyltransferase component (E2) of alpha-ketoglutarate dehydrogenase yielded Rm30267 (SMb20019) with wild-type growth on minimal media and a Fix+ phenotype in plants. The introduction or a sucB mutation into the SMb20019 mutant background (Rm30275) was comparable to the sole sucB mutation. This finding shows that the locus SMb20019 cannot be substituted for sucB in the alpha-ketoglutarate dehydrogenase enzyme complex.