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Doctor of Philosophy




R. japonicum nodulates and fixes nitrogen on soybeans. Recently, R. japonicum was reclassified in the genus Bradyrhizobium. In 1982, several unusual fast-growing R. japonicum strains were discovered in the People's Republic of China. Most of these strains will nodulate but do not fix nitrogen on North American soybean cultivars. However, one exception is R. japonicum strain USDA191 which will effectively fix nitrogen on several North American soybean cultivars. The goal of this study was the identification and isolation of the genes responsible for nodulation and nitrogen fixation in strain USDA191 and also to determine if these symbiosis genes could function in other bacteria.;R. japonicum strain USDA191 was mutagenized with ultraviolet light, nitrosoquanidine and the transposon Tn5. In this manner, several auxotrophic mutants of R. japonicum USDA191 were generated. Tn5 was also used to label a plasmid in strain USDA191 that carries the symbiotic genes. A "helper" plasmid was used to mobilize Tn5-labeled pSym191 to other strains of Rhizobium to determine if these genes were expressed in a foreign background. When pSym191 was transferred to Agrobacterium tumefaciens, ineffective nodules were formed on soybean plants. However, when pSym191 was transferred to a Sym plasmid cured strain of R. leguminosarum, no nodules were formed.;pSym191 was also transferred to E. coli strains, where cointegrate plasmids were identified containing deleted regions of pSym191 and the helper pRL180. When these R-prime plasmids were transferred back to a Nod('-) Nif('-) strain of R. japonicum USDA191, only some of the deleted plasmids were able to form nodules on soybeans. It was determined that only two out of four HindIII fragments from strain USDA191 that hybridized with nod genes from R. meliloti were necessary for nodulation. A cosmid clone bank of plasmid DNA was constructed in the broad host-range vector pVK102 and the nodulation fragments isolated and subcloned in pVK102. The two HindIII fragments were subcloned together and transferred to Sym plasmid cured strains of R. trifolii, R. leguminosarum, and R. phaseoli. Only the R. trifolii transconjugants that contained the two R. japonicum USDA191 HindIII nod fragments were able to form nodules on soybeans.



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Kim Jonelle Stutzman Engwall



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194 pages