Structural characterisation of lipo-chitin oligosaccharides isolated from Bradyrhizobium aspalati, microsymbionts of commercially important South African legumes.

The shoots of the South African legume Aspalathus linearis spp. linearis (A. linearis) are used in the manufacture of an increasingly popular beverage that has acclaimed beneficial effects on health; this important export product is known as Rooibos (or Redbush) tea. Three strains of Bradyrhizobium aspalati, which are the nitrogen-fixing symbionts of Aspalathus carnosa, A. hispida and A. linearis, were tested for the production of lipo-chitin oligosaccharide signal molecules using thin-layer chromatographic analysis after induction with different inducers, including Rooibos tea extract, and radioactive labelling. Large-scale separation, using high-performance liquid chromatography, of lipo-chitin oligosaccharides from B. aspalati isolated from A. carnosa was performed for structural characterisation using fast-atom bombardment mass spectrometry and chemical modifications followed by gas chromatography-mass spectrometric analysis. The strain was shown to secrete a family of unusual lipo-chitin oligosaccharides that are highly substituted on the nonreducing-terminal residue but unsubstituted on the reducing-terminal residue. They have a backbone of three to five beta-(1-->4)-linked N-acetyl-D-glucosamine residues substituted on the nonreducing terminus with a C16:0, C16:1, C18:0, C18:1, C19:1cy, or C20:1 fatty acyl chain, and are both N-methylated and 4,6-dicarbamoylated.

Novel branched nod factor structure results from alpha-(1-->3) fucosyl transferase activity: the major lipo-chitin oligosaccharides from Mesorhizobium loti strain NZP2213 bear an alpha-(1-->3) fucosyl substituent on a nonterminal backbone residue.

Mesorhizobium loti has been described as a microsymbiont of plants of the genus Lotus. Lipo-chitin oligosaccharides (LCOs), or Nod factors, produced by several representative M. loti strains all have similar structures. Using fast-atom-bombardment tandem mass spectrometry and NMR spectroscopy, we have now examined the LCOs from the type strain NZP2213 and observed a much greater variety of structures than has been described for the strains of M.loti studied previously. Interestingly, we have identified as the major LCO a structure that bears a fucose residue alpha-1,3-linked to the GlcNAc residue proximal to the nonreducing terminal GlcNAc residue. This is the first time, to our knowledge, that substitution on an internal GlcNAc residue of the LCO backbone has been observed. This novel LCO structure suggests the presence of a novel fucosyltransferase activity in strain NZP2213. Since the presence of this extra structure does not have the effect of broadening the host range, we suggest that the modification of the LCOs with a fucose residue linked to a nonterminal GlcNAc residue might provide protection against degradation by a particular host plant enzyme (e.g., a chitinase) or alternatively represents adaptation to a particular host-specific receptor. The action of the alpha-(1-->3) fucosyltransferase seems to reduce significantly the activity of NodS, the methyltransferase involved in the addition of the N-methyl substituent to the nonreducing terminal GlcNAc residue. An additional novel LCO structure has been identified having only a GlcNAc2 backbone. This is to our knowledge the first description of such a minimal LCO structure.

Mass spectrometric analysis of lipo-chitin oligosaccharides--signal molecules mediating the host-specific legume-rhizobium symbiosis.

Lipo-chitin oligosaccharides (LCOs) are novel bacterial glycolipid signal molecules that mediate the species--specific symbiosis between rhizobial bacteria and leguminous plants. Nodulation of the legume roots and nitrogen-fixation in the resulting nodules by Rhizobia is controlled by the bacterial nodulation genes that encode the LCO biosynthetic enzymes. The length of the LCO chitin backbone, the length and degree of unsaturation of the fatty acyl chain attached to it, and the combination of different chemical substituents on the reducing- and nonreducing-terminal residues all contribute to the species--specificity of the signal. LCOs are bioactive in the nanomolar and subnanomolar concentration range and are produced as heterogeneous mixtures, making determination of their structures a difficult task, most successfully approached by the application of modern mass spectrometric methods in combination with specific chemical treatments aimed at identifying specific chemical moieties. This review presents an overview of these methods as they are being used for the structural elucidation of LCOs, and discusses the role of structural diversity in mediating species-specificity.