6 NODULATION GENES OF NOD BOX LOCUS-4 IN RHIZOBIUM-MELILOTI ARE INVOLVED IN NODULATION SIGNAL PRODUCTION - NODM CODES FOR D-GLUCOSAMINE SYNTHETASE

The nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the D-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS- mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied D-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. meliloti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.