Structural Characterization of the Lipid A Component of Sinorhizobium sp. NGR234 Rough and Smooth Form Lipopolysaccharide

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 ␤-(1 3 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, amidelinked 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, ␤-hydroxybutyrate, is esterified to ( ؊ 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.The family Rhizobiaceae includes the Rhizobium and Sinorhizobium, Gram-negative bacteria able to form nitrogen-fixing symbioses with legumes in a host-specific manner. Sinorhizobium sp. NGR234 is a fast growing, broad-host-range symbiont able to colonize a diverse range of commercially important legumes (1, 2), including both indeterminate and determinate nodule-forming hosts. Partly because of its agricultural role, the molecular genetics of NGR234 are of interest, and the symbiotic plasmid was recently sequenced (3). However, there is little complementary structural information on the cell surface macromolecules or the alterations that occur in these molecules during symbiotic infection and bacteroid differentiation.Lipopolysaccharides (LPS) 1 are the major structural and antigenic components of the rhizobial outer membrane (4 -8) and are proposed to contribute to the biochemical processes that result in symbiotic infection (8 -16). Rhizobial LPS structural mutants typically yield phenotypes with underdeveloped nodules (Ndv Ϫ phenotype) in which nitrogen fixation is absent or diminished (Fix Ϫ ) (5, 9, 15, 17-23).The lipid A moieties of rhizobial LPS are of interest, because of their ...

Structure and Biological Activity of the Short-chain Lipopolysaccharide from Bartonella henselae ATCC 49882T

“…A 1 H, 31 P HMQC experiment showed correlations of the former with H-1 of GlcN3N I at ␦ P /␦ H 4.72/5.26 and the latter with H-4 of GlcN3N II at ␦ P /␦ H 4.94/3.45. Therefore, the lipid A disaccharide backbone is bisphosphorylated at positions 1 and 4Ј, and the compound 1 has the structure shown in Fig.…”

“…Interestingly, synthesis of the LPS O-chain in Sinorhizobium sp. NGR234 is regulated differently in the free-living versus endosymbiotic state (31), suggesting that the relative proportion of R-form to S-form LPS species may be a critical factor for the facultative intracellular life style of this plant symbiont. In future it will be interesting to recognize whether the facultative intracellular pathogen B. henselae can regulate synthesis of different amounts of R-form versus S-form LPS species in response to environmental signals as a specific adaptation to the host.…”

“…However, be- cause of a limited number of detailed structural studies on lipid A's from these organisms, and because of difficulties associated with analyzing the long-chain fatty acids, the location, stoichiometry, and type of attachment of these substituents is known only for a few species, including Sinorhizobium sp. NGR234 and R. etli-R. leguminosarum (30,31). Interestingly, LPS of the unrelated intracellular pathogen L. pneumophila appears to have a related structure too (32) with similar long-chain fatty acids.…”

“…Heterogeneity of LPS in regard to the presence of an O-chain has previously been reported for the closely related species B. bacilliformis (14) and B. quintana, 2 as well as for some other rhizobacteria, including the plant symbionts Bradyrhizobium japonicum (28) and Sinorhizobium sp. NGR234 (31), and may be a typical feature of the Rhizobiales. Interestingly, synthesis of the LPS O-chain in Sinorhizobium sp.…”

Lipopolysaccharides as a communication signal for progression of legume endosymbiosis

“…LPSs of A. caulinodans were prepared with the hot-phenol water method, purified by size exclusion chromatography, and analyzed by deoxycholate PAGE combined with oxidation and silver staining to visualize the LPS, and with Alcian blue followed by silver staining, but without the oxidation step to address purity as described (27). For coinoculations, 10 9 bacteria of either ORS571 or ORS571-oac2 were resuspended in LPS solutions of 100 or 10 g͞ml in 2.5 mg͞ml deoxycholic acid.…”