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. melioti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.

Earlier, we showed that Rhizobium meliloi nod&f codes for glucosamine synthase and that nodM and nodN mutants produce strongly reduced root hair deformation activity and display delayed nodulation of Medicago sativa (Baev et al., Mol. Gen. Genet. 228:113-124, 1991). Here, we demonstrate that nodPf and nodN genes from Rhizobium leguminosarum biovar viciae restore the root hair deformation activity of exudates of the corresponding R. meliloti mutant strains. Partial restoration of the nodulation phenotypes of these two strains was also observed. In nodulation assays, galactosamine and N-acetylglucosamine could substitute for glucosamine in the suppression of the R. melilot nodM mutation, although N-acetylglucosamine was less efficient. We observed that in nodules induced by nodM mutants, the bacteroids did not show complete development or were deteriorated, resulting in decreased nitrogen fixation and, consequently, lower dry weights of the plants. This mutant phenotype could also be suppressed by exogenously supplied glucosamine, N-acetylglucosamine, and galactosamine and to a lesser extent by glucosamine-6-phosphate, indicating that the nodAf mutant bacteroids are limited for glucosamine. In addition, by using derivatives of the wild type and a nod&t mutant in which the nod genes are expressed at a high constitutive level, it was shown that the nodM mutant produces significantly fewer Nod factors than the wild-type strain but that their chemical structures are unchanged. However, the relative amounts of analogs of the cognate Nod signals were elevated, and this may explain the observed host range effects of the nodM mutation. Our data indicate that both the nod&f and nodN genes of the two species have common functions and confirm that NodM is a glucosamine synthase with the biochemical role of providing sufficient amounts of the sugar moiety for the synthesis of the glucosamine oligosaccharide signal molecules.Rhizobia are capable of establishing symbiotic associations with their legume hosts. During this process, the bacteria undergo rapid developmental changes in order to make the transition from a soil environment through the root surface environment to a new ecological niche inside the plant host cells. The partnership between a given plant host and its microsymbiont is highly specific; most leguminous plants can be nodulated by a more or less limited number of Rhizobium species. For example, Rhizobium meliloti nodulates species of the genera Medicago, Meliotus, and Trigonella (11).Numerous bacterial and plant genes are expressed specifically during nodule development (19 Lerouge and coworkers (17) identified a major alfalfa-specific signal molecule as an acylated and sulfated glucosamine tetrasaccharide. Using a similar approach, we showed that R meliloti produces a family of biologically active Nod signal molecules (26). Host-specific signal molecules secreted by Rhizobium leguminosarum have also been identified (30).In different rhizobia, common and host-specific nod genes have been identified (12). The b...

Until now more than thirty nodulation genes have been identified in different rhizobia which are induced by the regulatory protein NodD in conjunction with flavonoids secreted from the legume roots. In the promoter region of the flavonoid inducible transcriptional units, the nod box, a highly conserved sequence of 47 bp is present [ 11 ]. Using a synthetic oligonucleotide nod box probe we [ 11 ] identified six nod box copies in the megaplasmid pRme41b of Rhizobium meliloti strain AK631. Subsequently, it was demonstrated that all nod box copies are associated with inducible transcriptional units, forming the nod regulon [6,8,1]. Transcriptional units comprising the nodABCIJ, nodFEG, nodH genes are preceded by nod box copies nl, n2 and n3, respectively. By constructing lacZ fusions we showed that nod boxes n4 and n5 sponsor induction of downstream regions by flavonoids, and that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover [6,1]. Recently, the n4 locus was shown to contain six genes: nodM, noIFGHI and nodN [1].In this report we present the nucleotide sequence of the first open reading frame (ORF) associated with nod box n5 (Fig. 1). Comparison of the predicted protein sequence with translations of the SWISSPROT protein sequence database revealed strong homology (839/0 identity, Fig. 2) to NodE of Rhizobium leguminosarum bv. viciae [2,13]. On this basis this ORF was designated nodL. Homology between the NodL protein and two acetyltransferases from Escherichia coli was also detected, namely galactoside acetyltransferase (EC 2.3.1.18) encoded by the lacA gene [7] (37~o identity in 181 amino acid overlap, Fig. 2) and serine acetyltransferase (EC 2.3.1.30) encoded by cysE [4] (30~o identity in 110 amino acid overlap, Fig. 2). Homology of nodL of R. leguminosarum by. viciae with E. coli lacA and cysE was presented earlier [ 13]. In addition, we found that nodL shows weak homology to UDPacetylglucosamine acyltransferase encoded by gene IpxA from E. coli [3] (Fig. 2).In our previous work the n5 region was subjected to transposon mutagenesis and the mutants showed delayed nodulation ability on alfalfa and sweetclover [6]. These mutations were located within the nodL coding region identified in this study. A DNA region probably corresponding to the n5 locus ofR. meliloti AK631 was identified also in R. meliloti strain 1021 by Renalier et al. [ 10]. It was found that deletion of this reThe nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X61083.

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