The glycolipids of Lactobacillus casei A.T.C.C. 7469

1. The lipids were extracted from Lactobacillus casei A.T.C.C. 7469 with chloroform-methanol mixtures. The glycolipids were obtained by chromatography on silicic acid and DEAE-cellulose (acetate form). 2. Hydrolysis of the glycolipids with alkali gave two glycerol glycosides and a mixture of fatty acids. 3. The glycosides were separated and their structures elucidated. The major component was O-alpha-d-galactopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-glycerol and the minor component O-alpha-d-glucopyranosyl-(1-->6)-O-alpha-d-galactopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-glycerol. 4. Analysis of the fatty acids by gas-liquid chromatography showed that they were predominantly palmitic acid, octadecenoic acid and lactobacillic acid.     

Bacterial glycolipids. Glycosyl diglycerides in Gram-positive bacteria

1. The lipids of ten Gram-positive bacteria have been isolated and the presence in each of a glycosyl diglyceride was established. 2. The glycolipid fractions were isolated and deacylated to give water-soluble glycosides which were purified by paper chromatography. Partial structures for the glycosides have been deduced from chemical and enzymic studies. 3. Nine of the glycosides were disaccharides glycosidically linked to the 1-position of glycerol: the remaining glycoside contained a trisaccharide similarly linked to glycerol.     

Cell wall teichoic acids of Actinomadura viridis VKM Ac-1315T.

The cell walls of Actinomadura viridis contain poly(glycosylglycerol phosphate) chains of complex structure. On the basis of NMR spectroscopy of the polymer and glycosides thereof the following structural units were found: beta-D-Galp3Me-(1-->4)[beta-D-Glcp-(1-->6)]-beta-D-Galp-(1-->1)-++ +snGro (G1); beta-D-Galp-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2); beta-D-Galp3Me-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2a); beta-D-Galp-(1-->1)-snGro (G3); beta-D-Galp-(1-->1)[beta-D-Galp-(1-->2)]-snGro (G4); beta-D-Glcp-(1-->2)-snGro (G5). Glycosides G1, G2 and G3 were the predominant components of the teichoic acid: they formed the polymer chain via phosphodiester bonds involving C-3 of the glycerol residue and C-3 of the galactosyl residue which in turn glycosylates C-1 of the glycerol residue. Whether the different glycosides make up the one chain or whether there are several poly(glycosylglycerol phosphate) chains in the cell wall remains to be determined. It was suggested that the minor component G5 is located at the nonterminal end of the chains. Compound G4 which contains disubstituted glycerol residues (unusual for the teichoic acid) was also found as a minor component; this may be the glycoside of a new type of teichoic acid, or a glycoside on the terminal end of the above mentioned chains. In addition, small amounts of 1,3-poly(glycerol phosphate) chains were found in the cell wall.     

Characterization of the Lipids of Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens strain D-1 was grown on a lipid-free chemically defined medium. The lipids were extracted with chloroform-methanol and separated into nonpolar and polar fractions by silicic acid column chromatography. Further separations were made by preparative thin-layer chromatography. The lipid fractions were identified by specific staining reactions and R(F) values, by phosphorus and nitrogen determinations, by chromatography of hydrolysis products, and by the use of infrared spectroscopy. The major nonpolar lipid was free fatty acid. Four major polar lipids were identified: phosphatidylethanolamine, phosphatidyl glycerol, lipoaminoacid, and glycolipid. The lipoaminoacid contained alanine, leucine, and isoleucine. The glycolipid contained galactose. The major fatty acids identified were C16:0 and C18:1. The significance of the presence of lipoaminoacid is discussed.     

Structure of a major glycolipid from Thermus oshimai NTU-063

The structure of a major glycolipid isolated from the thermophilic bacteria Thermus oshimai NTU-063 was elucidated. The sugar and fatty acid compositions were determined by GC-MS and HPLC analysis on their methanolysis and methylation derivatives, respectively. After removal of both O- and N-acyl groups by alkaline treatment, the glycolipid was converted to a fully acetylated tetraglycosyl glycerol derivative, the structure of which was then determined by NMR spectroscopy (TOCSY, HSQC, HMBC). Thus, the complete structure of the major glycolipid from T. oshimai NTU-063 was established as beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-GlcpNAcyl-(1-->2)-alpha-Glcp-(1-->1)-glycerol diester. The N-acyl groups on the 2-amino-2-deoxy-glucopyranose residue are C15:0 and C17:0 fatty acids, whereas the fatty acids of glycerol diester are more heterogeneous including both straight and branched fatty acids from C15:0 to C18:0.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

The lipid composition of Mycoplasma laidlawii strain B

1. Total lipid was extracted from Mycoplasma laidlawii strain B with chloroform-methanol mixtures and fractionated into neutral lipid, glycolipid and phospholipid components by chromatography on silicic acid. 2. Saponification of the glycolipid fraction, which represented nearly half of the total lipid, yielded two glycosides for which the structures O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol were established. 3. The ratio of monoglucosyl diglyceride to diglucosyl diglyceride increased with the age of the culture, though the total glycolipid concentration remained virtually constant. The glycolipid concentration was unaffected by the addition of cholesterol to the culture medium. 4. The phospholipid fraction consisted of two components, phosphatidylglucose and phosphatidylglycerol. Organisms harvested at acidic pH also contained O-amino acyl esters of phosphatidylglycerol. No lipids containing inositol could be detected.     

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.     

The structure and possible function of the glycolipid from Staphylococcus lactis I3

1. The total lipid was extracted from Staphylococcus lactis I3 with chloroform-methanol mixtures and the glycolipid component was isolated by chromatography on silicic acid. 2. Saponification yielded a non-crystalline glycoside for which the structure O-beta-d-glucopyranosyl-(1-->6)-O-beta-d-glucopyranosyl-(1-->1)-d-glycerol has been established by chemical degradations and by comparison with synthetic material. 3. The role of the glycosyl diglycerides in bacterial membranes is discussed.     

Isolation and characterization of a glycolipid from Dictyostelium discoideum

A glycolipid was isolated from a lipid extract of the cellular slime mold Dictyostelium discoideum and characterized. From the results of analyses by thin-layer chromatography and infrared spectrometry, it was identified as a steryl glycoside. The steryl glycoside was further analyzed by gas-liquid chromatography/mass spectrometry as a trimethylsilyl ether derivative, and its quantitative and qualitative changes during the development of D. discoideum were examined. Δ²²-Stigmastenyl-d-glucoside was the major constituent of the steryl glycoside and comprised more than 90% of the total steryl glycoside fraction in cells at all stages of development. The content of the steryl glycoside was higher in vegetative-stage cells, late aggregation-stage cells, and 1-day sorocarps than in cells of other stages. The glycolipid fraction was often contaminated by a lipid which was also isolated and identified as a ceramide containing 2-hydroxy fatty acids and 4D-hydroxysphinganine.     

Structure of the major glycolipid from Rothia dentocariosa

Structural studies of the major glycolipid isolated from Rothia dentocariosa were carried out by specific chemical degradation and nuclear magnetic resonance spectroscopy. The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-sn-Gro, and the internal mannose was esterified at C-6 by fatty acid residue. The other fatty acyl chain substituted the primary methylene position of glycerol. The occurrence of this glycolipid is limited to the related microorganisms. The structural characteristics can facilitate the differentiation of some genera.     

Characterization of lyso-galactolipids, C-2 and C-3 O-acyl trigalactosylglycerol isomers, obtained from the lichenized fungus Dictyonema glabratum

A mixture of two lyso isomers of a galactolipid was obtained from Dictyonema glabratum. Aqueous hydrolysis gave rise to galactose and glycerol in a 3:1 molar ratio. ESI-MS spectroscopy gave, in the positive-ion mode, a pseudomolecular ion at m/z 839 and daughter ions with m/z 677, 600, 515 and 353, suggesting three galactosyl units linked to a glycerol moiety, substituted by one O-acyl group. 1D and 2D NMR experiments were used to characterize the glycolipid, and HMQC examination showed three anomeric signals, corresponding to two alpha-Galp and one beta-Galp residue liked to glycerol. The glycolipid structure was shown to be O-alpha-D-Galp-(1-->6)-O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1<-->1)-2- and -3-monoacyl-D-glycerol, the latter structures not having been previously found in nature. The fatty acid composition was determined by GC-MS of derived methyl esters: that of palmitic acid C(16:0) was the most abundant, although the presence of C(12:0), C(14:0), C(16:1) and C(18:0) esters was observed.     

Lipids of Thermoplasma acidophilum

Cells of Thermoplasma acidophilum contain about 3% total lipid on a dry weight basis. Total lipid was found to contain 17.5% neutral lipid, 25.1% glycolipid, and 56.6% phospholipid by chromatography on silicic acid. The lipids contain almost no fatty acid ester groups but appear to have long-chain alkyl groups in ether linkages to glycerol. The phospholipid fraction includes a major component which represents about 80% of the lipid phosphorus and 46% of the total lipids. We believe this component to be a long-chain isopranol glycerol diether analogue of glycerolphosphoryl monoglycosyl diglyceride. The glycolipids appear to contain isopranol diether analogues. Several components of the complex, neutral lipid fraction have been identified as hydrocarbons, vitamin K(2)-7, and isopranol glycerol diether analogues. Sterols are present in the neutral lipids but do not appear to be synthesized by the organism.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Isolation and characterization of a sulfated glyceroglucolipid from alveolar lavage of rabbit.

A major acidic glycolipid of the rabbit alveolar lavage has been isolated and characterized. The isolation procedure involved extraction of lipids, column fractionation on DEAE-Sephadex and silicic acid, and thin-layer chromatography. Chemical analysis of the glycolipid revealed the presence of glucose, fatty acids, glycerol mono-ethers and sulfate. By partial acid and alkaline methanolysis, oxidation with periodate and chromium trioxide, and methylation analysis of the native and desulfated compound, the structure of this glycolipid is proposed to be: SO3H-6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 3-1,(3)-O-alkyl-2-0-acylglycerol.     

Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).     

Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae.

The Streptococcus sp. studied here is closely related to Streptococcus pneumoniae with 98.6% 16S rRNA similarity and 65% DNA/DNA homology. We isolated the lipoteichoic acid and the membrane glycolipids whose structures were established using conventional procedures and NMR spectroscopy. The lipoteichoic acid contains a linear 1,3-linked poly(glycerophosphate) chain which is partly substituted with D-alanine ester and is phosphodiester-linked to O6 of beta-D-Galf(1-->3)acyl2Gro. This lipoteichoic acid is the first example in which a monohexosylglycerol serves as the glycolipid anchor; and with an average chain length of 10 glycerophosphate residues it is the shortest known to date. MS analysis, applied for the first time to a native acylated lipoteichoic acid, revealed a continuous increase in chain length from seven to 17 glycerophosphate residues with a maximum at 10, and allowed identification of the fatty acid combinations. Membrane glycolipids consisted of beta-D-Galf(1-->3)acyl2Gro (9%), alpha-D-Glcp(1-->3)acyl2Gro (22%), alpha-D-Galp(1-->2)-alpha-D-Glcp(1-->3)acyl2Gro (64%) and alpha-D-Galp(1-->2)-(6-O-acyl)-alpha-D-Glcp(1-->3)acyl2Gro (5%). It is noteworthy that in lipoteichoic acid biosynthesis, Galfacyl2Gro, a less abundant membrane glycolipid, is selected as the lipid anchor. Despite the genetic relatedness to Streptococcus pneumoniae, the lipoteichoic acid structure is quite different to the complex structure of pneumococcal lipoteichoic acid [T. Behr et al. (1992) Eur. J. Biochem. 207, 1063-1075], thus providing an example that minor differences in DNA sequence exert major changes in macromolecular structure.     

Isolation of a novel sphingoglycolipid containing glucuronic acid and 2-hydroxy fatty acid from Flavobacterium devorans ATCC 10829

A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).