Abstract
A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.
Highlights
NGR234 is a rough LPS (R-LPS), which lacks the polysaccharide O-chain [8, 49]. This is typical of many Sinorhizobium strains examined to date [8, 9, 14, 32, 35] and contrasts with R. leguminosarum and R. etli, which synthesize a major smooth LPS (S-LPS, containing O-chain) when grown as free-living cultures [14, 16]
In free-living culture, the major LPS synthesized by Sinorhizobium sp
Over 90% of these R-lipid A molecules contain a single copy of an long chain fatty acid (LCFA), carried as the secondary fatty acid of an acyloxyacyl substituent on the distal glucosamine residue
Summary
Growth of Bacteria—Agar slants of Sinorhizobium sp. strain NGR234 were provided by Dr Peter van Berkum at the National Rhizobium Germplasm Collection Center in Beltsville, MD. Water layer extracts containing LPS were dialyzed and treated sequentially with ribonuclease, deoxyribonuclease, and proteinase K [5] and redialyzed and subjected to size exclusion chromatography under dissociative conditions (0.25% sodium deoxycholate, 0.2 M NaCl, 1.0 mM EDTA, 10 mM Tris, pH 9.2) on a column of Sephadex G-150 (2.2 ϫ 100 cm). This procedure separates the R-LPS from the S-LPS. Column eluants were monitored by colorimetric assays for neutral carbohydrate [41] and Kdo [48] and by refractive index detection
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