Synthesis of an opine-like compound, a rhizopine, in alfalfa nodules is symbiotically regulated.

We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodule-specific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA. The 5'-regulatory region and amino-terminal end of the first open reading frame of the mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium meliloti nifH gene. The coordinate regulation of mos and nif genes suggests that the mos locus plays a symbiotic role. We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.

Genes for the catabolism and synthesis of an opine-like compound in Rhizobium meliloti are closely linked and on the Sym plasmid.

In alfalfa nodules induced by Rhizobium meliloti strain L5-30 the compound L-3-O-methyl-scyllo-inosamine (3-O-MSI) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the Agrobacterium opines. To answer the question whether opine-like compounds ("Rhizopines") play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-O-MSI (moc genes) and for the induction of its synthesis in the nodule [mos gene(s)]. moc and mos genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other R. meliloti strains where they are expressed. The location of the mos genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.

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

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 Tn5) 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.