Klaus Bryn

Analysis of lipopolysaccharides by methanolysis, trifluoroacetylation, and gas chromatography on a fused-silica capillary column

A gas chromatographic method for simultaneous analysis of fatty acids and sugars of lipopolysaccharides (LPS) has been developed. The sample (1 mg or less) is methanolyzed at 85°C overnight in 2 M HCl in methanol. The released methyl esters and methyl glycosides are trifluoroacetylated and chromatographed on a methylsilicone-impregnated fused-silica capillary column. This column resolves all ordinary LPS sugars and fatty acids, and quantitative analysis is possible, including 2-deto-3-deoxyoctanoic acid (KDO), glucosamine and heptoses. 3,6-Dideoxyhexoses show some thermal degradation at 85°C during methanolysis, but this can be overcome by lowering the temperature to 37°C. For KDO the higher temperature similarly causes some degradation, but a reproducile response factor was found. The method appears to be useful for analysis of purified LPS as well as a means for monitoring for LPS content during purification of bacterial antigents of different kinds.

Quantitative and structural characteristics of lipids in Chlorobium and Chloroflexus

The lipid compositions of Chlorobium limicola (4 strains) and Chloroflexus aurantiacus (2 strains) have been compared. Both species contained straight-chain, saturated and monosaturated fatty acids as their main fatty acid constituents but the patterns were distinctly different. Chlorobium contained acids of chain-length essentally in the range C12−C18 with n-tetradecanoate, hexadecenoate and n-hexadecanoate predominating. Chloroflexus was characterized by the presence of significant amounts of C17 and C18−C20 fatty acids not detected in Chlorobium. The latter, on the other hand, contained hydroxylated and cyclopropane-substituted acids not detected in Chloroflexus. Simple wax esters (C28−C38) were found solely in Chloroflexus and accounted for 2.5–3.0% of the cell dry weight. Their fatty acid constituents ranged from C12−C19 (both saturated and monounsaturated isomers) whereas the alcohols were generally saturated and of chain-length C16−C19. Waxes in the range C34−C36 accounted for more than 60% of the total.The polar lipid patterns of the two genera also showed marked differences. All strains contained phosphatidyl-glycerol, monogalactosyl diglyceride and sulfoquinovosyldiglyceride. Chlorobium contained in addition cardiolipin, phosphatidylethanolamine, the unidentified “glycolipid II” and several other unidentified glycolipids, whereas phosphatidyl inositol and a diglycosyl diglyceride were specific for Chloroflexus. The latter lipid contained equimolar amounts of glucose and galactose.Phenol-water extraction yielded material comprising 14% of the dry cell weight for Chlorobium but only 2.5% for Chloroflexus. The Chlorobium material contained two 3-hydroxy fatty acids and several uncommon sugars (not identified). The analytical results were inconclusive regarding occurrence of 2-keto-3-deoxyoctonate. No typical lipopoly-saccharide constituents were found in Chloroflexus.

Chemical composition of a lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.

Composition of 2,3-dihydroxy fatty acid-containing lipopolysaccharides from Legionella israelensis, Legionella maceachernii and Legionella micdadei

Lipopolysaccharides (LPS) from Legionella israelensis, L. maceachernii and L. micdadei were analysed for chemical composition. The main sugar of the lipid A fractions was in each case 2,3-diamino-2,3-dideoxy-D-glucose. Lipid A of L. israelensis also contained a substantial amount of D-glucosamine. In each lipid A fraction a complex fatty acid pattern was detected. This comprised at least 19 different 3-hydroxy fatty acids (amide-linked), three 2,3-dihydroxy fatty acids (amide-linked), non-hydroxy fatty acids (ester-linked) as well as long-chain (omega-1)-oxo, (omega-1)-hydroxy and (1,omega)-dioic fatty acids (ester-linked). In addition, L. maceachernii and L. micdadei contained alpha-hydroxylated long-chain (omega-1)-oxo and (1,omega)-dioic fatty acids. The polysaccharide parts of L. maceachernii and L. micdadei LPS were similar and contained mainly L-rhamnose, L-fucose, D-mannose, D-glucose, L-fucosamine, D-glucosamine, 2-keto-3-deoxy-octonic acid (Kdo) as well as the rare octose yersiniose A. The corresponding composition of L. israelensis LPS was simpler and consisted mainly of L-rhamnose and 3-amino-3,6-dideoxy-D-mannose. LPS of L. israelensis and L. micdadei contained, in addition, 2-keto-octonic acid linked to Kdo. Phosphorylated sugar constituents were detected in all three LPS, whereas ethanolamine was found only in LPS from L. maceachernii. The SDS-PAGE band pattern of L. micdadei differed from the two others in a higher proportion of the low molecular mass constituents.

Quantification of 2-keto-3-deoxyoctonate in (lipo)polysaccharides by methanolytic release, trifluoroacetylation and capillary gas chromatography

Several conditions of acidic anhydrous methanolysis were examined to optimize the release and minimize the degradation of unphosphorylated 2-keto-3-deoxy-D-manno-octonic acid (KDO) from bacterial lipopolysaccharides and polysaccharides. The reaction was monitored by capillary gas chromatography after derivatization by trifluoroacetic anhydride. The best results were obtained by use of 2 M hydrochloric acid at 60 degrees C for 2 h. Under these conditions a single KDO component appeared, and KDO was quantitatively released from all model compounds except when glycosidically linked to hexosamines. For quantitative cleavage of this linkage a reaction time of 6 h was required at 60 degrees C, giving rise to 5-10% of secondary KDO products. The KDO detection limit was about 250 pmol (50 ng) and the molar response was the same as for glucose. The KDO derivative gave a mass spectrometric fragmentation pattern consistent with a pyranosidic methylketoside methyl ester structure. Differentiation of KDO linkage types could be obtained by determination of the rates of KDO release by mild methanolysis.

Heterogeneity of lipopolysaccharides of Neisseria meningitidis revealed by thin-layer chromatography combined with monoclonal antibodies

Abstract Thin-layer chromatography (TLC) was used for revealing the heterogeneity of lipopolysaccharides (LPS) from Neisseria meningitidis. A complex pattern consisting of at least 4 bands was achieved for the vaccine strain N. meningitidis 44 76 . The complexity remained unchanged after dephosphorylation, but removal of both phosphate and O-acyl groups simplified the pattern into two bands. The band patterns of LPS of strain 44 76 after growth in two different media were substantially different, but became identical upon dephosphorylation. Staining of LPS on TLC plates by use of immunotype specific monoclonal antibodies provided specific band patterns at high sensitivity. In addition to revealing structural heterogeneity, the TLC system may thus be used for characterisation and differentiation of LPS-specific antibodies.

Gas chromatographic determination of (phosphorylated) 2-keto-3-deoxyoctonic acid, heptoses and glucosamine in bacterial lipopolysaccharides after treatment with hydrofluoric acid, methanolysis and trifluoroacetylation

Quantification of phosphorylated sugar constituents of lipopolysaccharides has been performed by the following sequence: dephosphorylation by treatment with hydrofluoric acid, cleavage to monomeric constituents by methanolysis and analysis of the released sugars by capillary gas chromatography. Lipopolysaccharides of Salmonella minnesota Rd1P+, Bordetella pertussis NIH 114 and Vibrio cholerae, NAG and 95R strains, were used as model substances. Comparison of the chromatographic data obtained from hydrofluoric acid-treated and untreated lipopolysaccharide preparations indicated that all lipopolysaccharides examined contained one moiety of glucosamine bound to phosphate in a stable linkage. 2-Keto-3-deoxyoctonic acid appeared phosphorylated to a variable extent. Lipopolysaccharides of the two V. cholerae strains contained one moiety of fully phosphorylated 2-keto-3-deoxyoctonic acid, whereas in that of S. minnesota Rd1P+ only one of the three moieties was phosphorylated. Lipopolysaccharide of B. pertussis had one moiety of 2-keto-3-deoxyoctonic acid, ca. 70% phosphorylated. All four of the preparations examined contained L-glycero-D-manno-heptose in amounts varying from 2.6 to 5.2 moieties. In the lipopolysaccharides of B. pertussis and strain 95R of V. cholerae this sugar was unphosphorylated, whereas the two remaining strains contained one phosphorylated moiety of this sugar. Phosphorylated lipopolysaccharide constituents can be analysed by this approach on a 50-100 micrograms scale.

Lipids of heliobacteria are characterised by a high proportion of monoenoic fatty acids with variable double bond positions.

The fatty acid composition and lipid pattern of six strains of heliobacteria have been analysed. The results were fairly uniform for all strains. Phosphatidyl ethanolamine and phosphatidyl glycerol were the dominating lipids found, with the former as the major one. No glycolipids were detected. The general fatty acid pattern was dominated by acids of chain length C16 to C18. An unusually large proportion of monoenoic acids was seen, with up to four positional isomers for each chain length. Methyl branched (iso) fatty acids were present, but not cyclopropyl or hydroxy fatty acids nor fatty alcohols.

A polysaccharide produced by a mucoid strain of Moraxella nonliquefaciens with a 2-acetamido-2-deoxy-5-O-(3-deoxy-β-d-manno-octulopyranosyl)-β-d-galactopyranosyl repeating unit

A capsular polysaccharide, isolated from the mucoid Moraxella nonliquefaciens strain 3828/60, has been investigated by component analyses, periodate oxidation, methylation analyses, mass spectrometry, 1H and 13C NMR spectroscopy, and hydrolysis to give a disaccharide that was isolated and characterised. The results showed that the polysaccharide has the repeating unit-->3)-beta-D- GalpNAc-(1-->5)-beta-Kdo p-(2-->, with approximately 40% of O-8 of Kdo being acetylated.