RHIZOBIUM-LEGUMINOSARUM HAS 2 GLUCOSAMINE SYNTHASES, GLMS AND NODM, REQUIRED FOR NODULATION AND DEVELOPMENT OF NITROGEN-FIXING NODULES

The Rhizobium leguminosarum nodM gene product shows strong homology to the Escherichia coli glmS gene product that catalyses the formation of glucosamine 6-P from fructose 6-P and glutamine. DNA hybridization with nodM indicated that, in addition to nodM on the symbiotic plasmid, another homologous gene was present elsewhere in the R. leguminosarum genome. A glucosamine-requiring mutant was isolated and its auxotrophy could be corrected by two different genetic loci. It could grow without glucosamine when the nodM gene on the symbiotic plasmid was induced or if the cloned nodM gene was expressed from a vector promoter. Alternatively, it could be complemented by a second fragment of R. leguminosarum DNA that carries a region homologous to E. coli glmS. Biochemical assays of glucosamine 6-P formation confirmed that the two R. leguminosarum genes nodM and glmS have interchangeable functions. No nodulation of peas or vetch was observed with a double nodM glmS mutant, and this block occurred at a very early stage since no root-hair deformation or infection threads were seen. Nodulation and root-hair deformation did occur with either the nodM or the glmS mutant, showing that the gene products of either of these genes can be involved in the formation of the lipo-oligosaccharide nodulation signal. However, the glmS mutant formed nodules that had greatly reduced nitrogen fixation. Constitutive expression of nodM restored nitrogen fixation to the glmS mutant. Therefore the reduced nitrogen fixation probably occurs because glmS is absent and nodM is not normally expressed in nodules and, in the absence of glucosamine precursors, normal bacteroid maturation is blocked.