Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules.

Nod factors are a group of biologically active oligosaccharide signals that are secreted by symbiotically competent bacteria of the family Rhizobiaceae. Their biosynthesis is determined by rhizobial nodulation (nod) genes, and is specifically induced in response to flavonoids secreted from the roots of host leguminous plants. The biological activity of Nod factors on these host legumes dramatically mimics the early developmental symptoms of the Rhizobium-legume symbiosis including, amongst other effects, root hair deformations and nodule initiation. Structurally, all Nod factors are short oligomers of beta-1,4-linked N-acetylglucosamine residues [usually degree of polymerization (dp) 4 or 5] that are N-acylated on the distal glucosamine. This common 'core' structure may be modified by a number of species-specific substituents on the distal or reducing sugars. These modifications are governed by rhizobial host specificity nod genes. The biological activity of purified Nod factors mirrors this host specificity, indicating that the symbiotic host range of individual Rhizobium species is, at least partially, determined by the variety of Nod factors they are able to produce. Here we describe techniques that are universally applicable to the extraction, chromatographic separation and identification of Nod factors. We have applied these techniques to Nod factors from the broad-host-range species Rhizobium fredii USDA257 and Rhizobium spp. NGR234, and the more narrow-host-range Bradyrhizobium japonicum USDA110, and have identified a group of novel, relatively hydrophilic Nod factors from the NGR234 species that may have implications for Nod factor biosynthesis.