Plasmid-linked nifand “nod” genes in fast-growing rhizobia that nodulate Glycine max, Psophocarpus tetragonolobus, and Vigna unguiculata

Forty-nine fast-growing Rhizobiumstrains from the nodules of 26 different tropical legume genera were screened to find isolates that would (i) nodulate, e.g., winged beans, so producing large nodules for RNA and protein isolation; (ii) also nodulate various small-seeded legumes, thus allowing screening of large numbers of mutants; and (iii) harbor plasmids containing nifstructural genes as well as other functions involved in nodulation. On the basis of six different criteria, this rhizobial group appeared intermediate between classical fast- and slow-growing organisms, yet all contained plasmids. Plasmid numbers varied from one to five. Hybridizations between DNA prepared from nifDHand the putatative “nod” region of R. melilotiand these plasmids bound to nitrocellulose filters suggested that nif-nodgenes are linked on a single symplasmid. A broad-host-range strain containing a single symplasmid was chosen for further study. Its plasmid, pMPIK3030a, was isolated on cesium chloride gradients and cloned in the cosmid pJB8, and the overlapping fragments were mapped by homology with the nifand nodregions of R. meliloti. As the wild-type plasmid pMPIK3030a was not self-transmissible, confirmation that the nodgenes detected by homology were responsible for nodulation was obtained by introducing the mobilization functions of RP4(together with Tn5) and selecting transconjugants resistant to kanamycin and neomycin. Transconjugants (obtained at a frequency of about 10-6per recipient) in Agrobacterium tumefacienscured of the Ti plasmid produced ineffective nodules on Vigna unguiculata, those in nonnodulating (Nod-) R. melilotiwere partially effective, while those in Nod-R. leguminosarumwere often fully effective.