STRUCTURALLY DIVERSE CHITOLIPOOLIGOSACCHARIDE NOD FACTORS ACCUMULATE PRIMARILY IN MEMBRANES OF WILD-TYPE RHIZOBIUM-LEGUMINOSARUM BIOVAR TRIFOLII

The general view on Rhizobium chitolipooligosaccharides (CLOS) is that they are made in very low levels as diffusible molecules and are prim arily secreted by the bacteria into the extracellular milieu where the y interact with the host. However, the structural and predicted physic ochemical properties of these amphiphilic molecules led us to postulat e that they should normally be targeted to bacterial membranes after s ynthesis. Thus, we analyzed membrane lipid extracts of Rhizobium legum inosarum bv. trifolii wild-type strain A(.)NU843 cells and the corresp onding culture supernatants for CLOS-type glycolipids. As predicted, f ractionation of the membrane extracts from pelleted cells led to the i solation of a diverse family of CLOS in high yield (greater than or eq ual to 15 mg/L of culture), whereas all attempts to isolate CLOS from the corresponding culture supernatant failed. Structural analyses reve al that the membrane CLOS of ANU843 consist of a complex mixture of O- acetylated or non-O-acetylated chito- tri-, -tetra-, and pentasacchari des bearing an N-acyl moiety at the nonreducing glucosamine residue. c is-Vaccenic acid was the predominant acyl substituent (> 70%), but sev eral other saturated, unsaturated, and 3-hydroxy fatty acids were foun d in the CLOS glycolipids. Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4',7-dihydroxyflavone and pSym nod genes. Potential host-selective biological activity of the purified membrane CLOS fraction from ANU843 was indicated by its ability to elicit meris tems resembling rudimentary nodule primordia in the root cortex of axe nic seedlings of the host legume, white clover, but not of the nonhost legumes hairy vetch or alfalfa. These results indicate that, as predi cted, a very diverse family of chitolipooligosaccharide Nod factors ac cumulate primarily in bacterial membranes of wild-type R. leguminosaru m bv. trifolii, and our protocol which readily isolates these biologic ally active glycolipids in high yield from this source eliminates the need to use recombinant ''overproducing'' strains to obtain sufficient quantities for structural analyses. These results lead us to predict that these glycolipids may perform important membrane functions for wi ld-type rhizobia in the host root environment, and that these bacteria l factors are likely to operate primarily at short range rather than a s freely diffusible extracellular molecules during development of the Rhizobium-legume symbiosis.