The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS- mutants of R. meliloti 2011, which produced ineffective (Fix-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.