The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191.

R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod(-) Nif(-) derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.