STRUCTURE OF LIPID-A COMPONENT OF RHIZOBIUM-LEGUMINOSARUM BV PHASEOLILIPOPOLYSACCHARIDE - UNIQUE NONPHOSPHORYLATED LIPID-A CONTAINING 2-AMINO-8-DEOXYGLUCONATE, GALACTURONATE, AND GLUCOSAMINE

The structure of lipid A hom the lipopolysaccharide of Rhizobium legum inosarum bv. phaseoli (wild type strain CE3) was investigated by alkyl ation analysis, nuclear magnetic resonance spectroscopy, and electrosp ray and fast atom bombardment mass spectrometry of the de-O-acylated l ipid A. The lipid A carbohydrate backbone was shown to be a trisacchar ide containing galacturonic acid, glucosamine, and the unique sugar 2- amino-2-deoxygluconic acid, previously unreported in lipopolysaccharid es. Nuclear magnetic resonance spectroscopy and ethylation analyses re vealed that the galacturonic acid is alpha-1,4-linked to the glucosami ne, while the amino aldonic acid residue, which may exist as the 1,5-l actone, is attached as an aglycone to the glucosamine and, thus, occup ies the reducing end of the molecule. The resulting backbone is hydrop hilic and analogous to the commonly observed bisphosphorylated glucosa mine disaccharide from enteric bacterial lipopolysaccharides in that b oth the nonreducing and reducing ends carry negatively charged substit uents. The fatty acids of the R. legumino-sarum lipid A are attached b oth as O- and N-acyl substituents to glucosamine and 2-aminogluconate. All fatty acids are hydroxylated consisting of 3-hydroxymyristate (3- OH-C-14.0), 3-hydroxypentadecanoate (3-OH-C-15.0), 3-hydroxypalmitate (3-OH-C-16.0), 3-hydroxystearate (3-OH-C-18.0), and 27-hydroxyoctacosa noate (27-OH-C-28.0) in the approximate mole ratio 3:0.2:1:0.6:1. Unli ke lipid As from enteric bacteria, the R. leguminosarum lipid A lacks 3-acyloxyacyl substituents; however, the long chain 27-hydroxy fatty a cid carries ester-linked beta-hydroxybutyrate at the 27-hydroxy positi on. Fast atom bombardment mass spectrometry of the de-O-acylated lipid A demonstrated the presence of 2 molecular species that differ by 28 mass units due to fatty acid heterogeneity at the two amide linkages. One species carries amide-linked 3-OH-C-14.0 and 3-OH-C-16.0; the seco nd species carries 3-OH-C-14.0 and 3-OH-C-18.0. Each molecular species also exists as the aldonolactone, yielding molecular ions at ((M + H) (+))-18. The heterogeneity in the amide-linked fatty acids further dis tinguishes the Rhizobium lipid A from enteric lipid As.