Erik Jantzen

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.

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.

The branched-chain octose yersiniose A is a lipopolysaccharide constituent of Legionella micdadei and Legionella maceachernii

Abstract Lipopolysaccharides (LPS) were isolated from the two closely related species Legionella maceachernii and L. micdadei . The chemical composition of their polysaccharide parts have been have analysed and were found to be qualitatively similar. Hydrolysates included the sugars 2-keto-3-deoxyoctanoic acid, rhamnose, fucose, mannose, fucosaine, glucosamine, glucose and glycerol. The two latter constituents were present both phosporylated and unphosphorylated. In addition both LPS contained an uncommon sugar. Mass spectra of this compound as alditol acetate and methylglycoside (trifluoracetylated) were identical to those of yersiniose A ( 3,6- dideoxy -4-C-[1-( S )- hydroxyethyl ]- d -xylo- hexose ) isolated from Yersinia frederiksenii O:16.29. This identity was further substantiated by retention characteristics from anion-exchange chromatography, and gas chromatography both as alditol acetate and trifluoroacetylated methylglycoside. Thus, yersiniose A is a constituent of L. maceachernii and L. micdadei LPS and may prove useful as a marker in Legionella diagnostics.

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.