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

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Abstract
Forty-nine fast-growing Rhizobium strains 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 nif structural 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 nifDH and the putatative “ nod ” region of R. meliloti and these plasmids bound to nitrocellulose filters suggested that nif-nod genes are linked on a single sym plasmid. A broad-host-range strain containing a single sym plasmid 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 nif and nod regions of R. meliloti . As the wild-type plasmid pMPIK3030a was not self-transmissible, confirmation that the nod genes detected by homology were responsible for nodulation was obtained by introducing the mobilization functions of RP 4 (together with Tn 5 ) and selecting transconjugants resistant to kanamycin and neomycin. Transconjugants (obtained at a frequency of about 10 -6 per recipient) in Agrobacterium tumefaciens cured of the Ti plasmid produced ineffective nodules on Vigna unguiculata , those in nonnodulating (Nod - ) R. meliloti were partially effective, while those in Nod - R. leguminosarum were often fully effective.