Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli

Abstract

The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-- The gram-negative bacteria of the Rhizobiaceae family include the nitrogen-fixing symbionts of legumes (Rhizobium,Azorhizobium, and Bradyrhizobium spp.) and the pathogenic Agrobacterium genus. These species produce various types of extracellular polysaccharides (EPSs) and cell surface polysaccharides that appear to be important factors in symbiotic and pathogenic effectiveness.Lipopolysaccharides (LPSs), unique to gram-negative bacteria, are outer membrane inclusions that consist of (i) a glycolipid membrane anchor, which contains characteristic Il-hydroxy fatty acids on a glucosamine backbone (lipid A);(ii) a core oligosaccharide that is linked to the lipid A via 3-deoxy-D-manno-2-octulosonic acid (Kdo) and is highly conserved within a species; and (iii) a highly variable 0 antigen. Polyacrylamide gel electrophoresis (PAGE)-silver stain analyses of rhizobial LPSs frequently result in banding regions (5, 9), indicating that the 0 antigens of these LPSs may commonly consist of oligosaccharides of specific lengths.Rhizobia also produce acidic EPSs and cyclic P-glucans.EPSs are secreted compounds and are isolated from the growth media of cultured cells. The EPSs produced by rhizobia are high-molecular-weight heteropolysaccharides composed of small repeating units that contain either uronic acids or noncarbohydrate substituents, such as succinate and pyruvate, as the acidic functions (4, 22, 29). The cyclic 3-glucans are commonly harbored within the periplasmic space but may also be secreted; consequently, they are found in both the growth media and the bacterial pellet of cultured cells (42). The role of the 13-glucans in symbiosis remains a moot subject. * Corresponding author.Numerous studies have shown that bacterial LPS and EPS are important in the infection and nodulation processes; however, the symbiotic phenotype of LPS-and EPS-defective microsymbionts appears to depend on the type of nodule formed by the host.(i) Inoculation of determinate nodule-forming host plants with rhizobial LPS mutants that are affected ...

Characterization of Galacturonosyl Transferase Genes rgtA, rgtB, rgtC, rgtD, and rgtE Responsible for Lipopolysaccharide Synthesis in Nitrogen-fixing Endosymbiont Rhizobium leguminosarum

“…DOC-PAGE Analysis-Gel electrophoresis in the presence of DOC was performed as previously described (13). Briefly, 1 g of extracted LPS material was loaded onto an 18% acrylamide gel, and current was applied (30 mA (constant), 400 V) in the presence of DOC buffer.…”

The Neisseria meningitidis Serogroup A Capsular Polysaccharide O-3 and O-4 Acetyltransferase

“…Column fractions were tested for neutral sugar estimation by phenol sulfuric acid assay (20). Void volume fractions were pooled and concentrated by speed vacuuming and analyzed by deoxycholate-PAGE and Alcian blue staining (21).…”

Immunochemical characterization of an Ogawa-Inaba common antigenic determinant of Vibrio cholerae O1

“…Fractions corresponding to bands I and II were pooled (pool I) and fractions corresponding to band III were pooled (pool II). These pools were separately dialysed against 10 % ethanol at room temperature to remove buffer salts and DOC as previously described (Reuhs et al, 1993), and freezedried.…”