R-prime plasmids carrying 100-200 kb regions of R. meliloti DNA coding for symbiotic functions were isolated from a Km8 derivative of R68.45. The R-primes were obtained from matings between R. meliloti strains containing Tn 5-induced symbiotic mutations and E. coli recipients by selecting for the kanamycin resistance marker of Tn 5. It was demonstrated that the regions inserted into the R-primes were identical with those DNA segments where Tn 5 was located in the respective parental R. meliloti. R-primes were generated from both the R. meliloti chromosome and from the megaplasmid pRme41 b. A set of R-primes, carrying the nitrogen fixation (nif) gene cluster, also carried genes required for nodulation (nod genes) of alfalfa. Transfer of these R-primes into different Nod- mutants restored the Nod+ phenotype. When they were introduced into A. tumefaciens the transconjugants formed small nodules on alfalfa. This indicates that nodulation and nitrogen fixation genes of the R. meliloti megaplasmid are clustered on a relatively short DNA segment.
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