Purification and mass spectrometry of six lipid A species from the bacterial endosymbiont Rhizobium etli - Demonstration of a conserved distal unit and a variable proximal portion

Lipid A of Rhizobium etli CE3 differs dramatically from that of other Gram- negative bacteria. Key features include the presence of an unusual C28 acyl chain, a galacturonic acid moiety at position 4', and an acylated aminoglu conate unit in place of the proximal glucosamine. In addition, R. etli lipi d A is reported to lack phosphate and acyloxyacyl residues. Most of these r emarkable structural claims are consistent with our recent enzymatic studie s. However, the proposed R. etli lipid A structure is inconsistent with the ability of the precursor (3-deoxy-D-manno-octulosonic acid)(2)-4'-P-32-lip id IVA to accept a C28 chain in vitro (Brozek, K. A., Carlson, R. W., and R aetz, C. R. H. (1996) J. Biol. Chern. 271., 32126-32136). To re-evaluate th e structure, CE3 lipid A was isolated by new chromatographic procedures. CE 3 Lipid A is now resolved into six related components. Aminogluconate is pr esent in D-1, D-2, and E, whereas B and C contain the typical glucosamine d isaccharide seen in lipid A of most other bacteria. All the components poss ess a peculiar acyloxyacyl moiety at position 2', which includes the ester- linked C28 chain. As judged by mass spectrometry, the distal glucosamine un its of A through E are the same, but the proximal units are variable. As de scribed in the accompanying article (Que, N. L. S., Ribeiro, A. A., and Rae tz, C. R. H. (2000) J. Biol Chem. 275, XXXX-XXXX), the discovery of compone nt B suggests a plausible enzymatic pathway for the biosynthesis of the ami nogluconate residue found in species D-l, D-2, and E of R. etli Lipid A. We suggest that the unusual Lipid A species of R. etli might be essential dur ing symbiosis with leguminous host plants.