Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum

Abstract

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.