Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lipo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japonicum strain USDA110. Between 10(-7) M and 10(-6) M NodNGR factors permitted these NodABC- mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a nonhost. Detailed cytological investigations of V. unguiculata showed that the NodABC- mutant NGR delta nodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a Nlinked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

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