R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41 a) and more than 300 Mdal (pRme41 b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41 b was found. Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41 b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41 b.
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