A diphytanyl ether analog of phosphatidylserine from a methanogenic bacterium, Methanobrevibacter arboriphilus

Several ninhydrin-positive lipids were found in methanogenic bacteria and the structure of one of them, designated as PNL2 from Methanobrevibacter arboriphilus, was identified as a diphytanyl ether analog of phosphatidylserine. The chromatographic behavior of the lipid on thin-layer plates and on a DEAE-cellulose column was identical to the ester form of phosphatidylserine. The infrared spectra showed the presence of amino, carboxyl, ether, and phosphate groups, and the absence of an ester linkage. The hydrophobic portion of the lipid was identified as diphytanyl glycerol diether on the basis of the mass spectrum of the acetolysis product and gas-liquid chromatography of the iodinated alkyl chain prepared by hydroiodic acid cleavage of PNL2. The fast atom bombardment-ionization and field desorption mass spectrum provided a molecular weight of 819 and several fragment ions consistent with the proposed structure. Hydrofluoric acid hydrolysis resulted in water-soluble products including serine, phosphoserine, and ammonia, which accounted for 95% of hydrolyzed PNL2. The lipid product of the hydrolysis was mainly the diether form of phosphatidic acid. This is the first report on the structural characterization of an amino-containing phospholipid in archaebacteria. Amino lipids have been found in many other methanogenic bacteria.     

Lipid and lipopolysaccharide composition of Acholeplasma oculi.

The total lipid content of Acholeplasma oculi comprises 13.3% of the dry weight of the organism and is about equally distributed between the neutral lipids plus glycolipids and the phospholipids. The phospholipids were identified as phosphatidyl glycerol and diphosphatidyl glycerol. The glycolipid fraction contained O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol and O-alpha-D-glucopyranosyl-(1 leads to 2)-O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol. The neutral lipid contained pigmented carotenoids. Hot aqueous phenol extraction of lipid-extracted whole cells yielded a polymeric carbohydrate comprising 2.3% of the dry weight of the organism. The A. oculi lipopolysaccharide was found to contain only neutral sugars and no amino sugar, in contrast to other acholeplasmas. The neutral sugars consisted of fucose, galactose, and glucose in a ratio of 2:19:3.     

Lipid composition of Mycoplasma neurolyticum

The total lipid content of Mycoplasma neurolyticum comprises about 14% of the dry weight of the organisms and is about equally distributed between the phospholipid and the neutral-glycolipid fractions. The neutral lipids were identified as triglycerides, diglycerides, and cholesterol. The glycolipid fraction contained 1-O-beta-glucopyranosyl-d-2,3-diglyceride and 1-[O-beta-d-glycopyranosyl-(1-->6)-O-beta-d-glucopyranosyl]-d-2,3-diglyceride. The latter lipid is structurally identical to the diglucosyl diglyceride which occurs in Staphylococcus aureus. The phospholipids of the organism consist of a fully acylated glycerophosphoryl-glycerophosphoryl glycerol, phosphatidic acid, diphosphatidyl glycerol, phosphatidyl glycerol, and amino acyl esters of phosphatidyl glycerol. Phosphatidic acid and phosphatidyl glycerol account for greater than 90% of the phospholipids of organisms in the exponential phase of growth. The predominant fatty acids found in all of the acyl lipids were palmitic, stearic, and oleic acids.     

Archaea contain a novel diether phosphoglycolipid with a polar head group identical to the conserved core of eucaryal glycosyl phosphatidylinositol.

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-GlcN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be 1-D-myo-inositol 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 1H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an alpha-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycero-1-phospho- 1'[6'-O-(2"-amino-2"-deoxy-alpha-D-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(alpha 1-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.     

Osmometric and microscopic studies on bilayers of polar lipids from the extreme halophile, halobacterium cutirubrum

The major membrane polar lipid components in Halobacterium cutirubrum are the diphytanyl ether analogues of phosphatidylglycerol phosphate, glycolipid sulfate, phosphatidylglycerol and phosphatidylglycerol sulfate. Dispersions of total polar lipids in water formed large birefringent liposomes showing concentric lipid bilayers in the elctron microscope; they behaved as ideal osmometers in KCl or NaCl solutions in the concentration range 0.005–0.2 M. At concentrations above 0.2 M KCl the liposomes shrank to spherical particles which were much less birefringent, showed no distinct bilayer structures by electron microscopy, and no longer behaved as ideal osmometers. Dispersions of phosphatidylglycerol phosphate, phosphatidylglycerol or phosphatidylglycerol sulfate alone did not behave as osmometers at any concentration of KCl or NaCl, but glycolipid sulfate alone or mixed with phosphatidylglycerol phosphate or phosphatidylglycerol phosphate + phosphatidylglycerol sulfate showed ideal osmometer behavior in 0.005–0.2 M KCl or NaCl. The highly negatively charged total polar lipids of H. cutirubrum thus can form stable lipid bilayers only at low ionic concentrations (0.005–0.2 M), much lower than the salt concentration (4 M) of the growth medium, and the presence of glycolipid sulfate is essential. Stability of the membrane in 4 M salt appears to require direct participation of the protein components.     

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.     

Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960.

The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy. The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments. The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot. The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds. Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid.     

Extraction and composition of polar lipids from the archaebacterium, Methanobacterium thermoautotrophicum: effective extraction of tetraether lipids by an acidified solvent

The usual Bligh and Dyer method could extract only a small part of the lipids of Methanobacterium thermoautotrophicum. When the water in the solvent was replaced by 5% trichloroacetic acid, the lipid recovery reached the maximum level, which was 6 times higher than that by the former method. The use of HCl (2 M) or disruption of cells was also effective but prolonged extraction with the HCl-containing solvent caused degradation of some phosphoglycolipids. Twenty-three spots of polar lipids were detected on a thin-layer chromatogram of the total lipid. These were 10 phospholipids (18%), 6 aminophospholipids (17%), 3 aminophosphoglycolipids (15%), 2 phosphoglycolipids (31%), and 2 glycolipids (19%). The predominant polar lipids were a highly polar phosphoglycolipid (PGL1, 30%) and a glycolipid (GL1a, 16%). The other major lipids included an aminophospholipid (PNL1a, 9%), and an aminophosphoglycolipid (PNGL1, 7%). The complete structure determination of PNL1a, GL1a, and PNGL1 is described in the accompanying paper. Acetolysis of the total lipids followed by acid methanolysis was required for the complete cleavage of polar head groups, releasing core residues of diphytanyl glycerol diether (C20 diether) and dibiphytanyl diglycerol tetraether (C40 tetraether). A densitometric assay of a thin-layer chromatogram showed that the ratio of C20 diether and C40 tetraether was 1:14. GLC analysis of alkyl chlorides prepared from the total lipid by BCl3 treatment showed that phytanyl (C20), biphytanyl (C40), and unidentified alkyl chains accounted for 10, 83, and 7 mol% of the total alkyl chains, respectively. Strong acid hydrolysis of the macromolecular residue obtained after lipid extraction gave a significant amount of C40 tetraether, which had probably been bound covalently to other substances in the cells.     

Novel, acid-labile, hydroxydiether lipid cores in methanogenic bacteria

Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties. Major amounts of hydroxydiether core lipid were confirmed for Methanosaeta concilii and discovered in two Methanosarcina species, Methanococcus voltae, and tentatively in several Methanobacterium species. Signals at 1.24 and 1.8-1.9 ppm in 1H NMR spectra are characteristic of Methanosaeta concilii lipids hydroxylated on carbon-3 (sn-3 chain). Related signals, which were shifted slightly, appeared in spectra of the polar lipids extracted from both Methanosarcina species. Following mild hydrolysis to remove the polar head groups, only two chromatographically distinct core lipids were found in significant amounts in Methanosarcina barkeri (and Methanosarcina mazei) consisting of 43% 2,3-di-O-phytanyl-sn-glycerol (C20,20-diether) and 57% C20,20-hydroxydiether. This latter core lipid differed from the hydroxydiether from M. concilii by hydroxylation, on carbon-3, of the phytanyl chain in ether linkage to the sn-2 carbon of glycerol. The structural assignment was based on identification of the novel hydroxydiether core and its methylation products by 1H NMR, 13C NMR, and mass spectroscopy. The hydroxy core lipid degraded to various products during standard methanolic HCl and sulfuric acid procedures, including a methoxy derivative (methanolic HCl) and the 3-mono-O-phytanyl-sn-glycerol.     

Synthesis and characterization of deoxy analogues of diphytanylglycerol phospholipids

Novel analogues of diphytanyl phospholipids, 2,3-diphytanyl sn-1-glycerol-1-phosphoryl-1'-(1',3'-propanediol) (dPG), 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-propanol (ddPG) and 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-(1',3'-propanediol-3'-p hosphate) (dPGP), were synthesized according to modifications of previously published procedures. The samples were TLC and analytically pure and were characterized by 13C- and 1H-NMR and negative FAB/MS. The pK values of dPGP in aqueous dispersions or in methanol/water (1:1, v/v) were determined by potentiometric titration and compared with those of 2,3-diphytanyl-sn-glycerol-1-phosphoryl-3'-sn-glycerol-1'-phosphat e (PGP). The dissociation constant of the third ionizable POH group of dPGP was more than 2 pK units higher than that of PGP, indicating that the free glycerol hydroxyl group plays an important role in headgroup conformation and stabilization, perhaps through hydrogen bonding with the phosphate group(s).     

The glycolipids from the non-capsulated strain of Pneumococcus I-192R, A.T.C.C. 12213.

1. The total lipid was extracted from the non-capsulated strain of Pneumococcus I-192R, A.T.C.C. 12213, with chloroform-methanol mixtures. Two glycolipids were isolated by chromatography on silicic acid and DEAE-cellulose (acetate form). 2. The major glycolipid was obtained pure in a yield of 640mg./34g. dry wt. of cells and represents about 34% of the total lipid. It contained galactose, glucose, glycerol and fatty acid ester residues in the proportions 1:1:1:2, and yielded on saponification a crystalline non-reducing glycoside. 3. The structure of the glycoside was shown to be O-alpha-d-galactopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol. The fatty acids obtained on saponification were identified by gas-liquid partition chromatography of their methyl esters. 4. The minor glycolipid was obtained as a 1:1 (w/w) mixture with the major component, but after saponification the two glycosides were separated by paper chromatography. Evidence was obtained for the structure of the glycoside derived from the minor glycolipid as 1-O-alpha-d-glucosylglycerol. 5. A general method is described for determining the stereochemistry of the glycerol moiety in 1-linked glycerol glycosides.     

Synthesis of sulfate esters of phosphatidylglycerol (diphytanyl ether analog)

Synthesis of 1-sn-phosphatidyl-3'-sn-glycero-1'-sulfate (phosphatidylglycero-1-sulfate) was achieved by monosulfation of 1-sn-phosphatidyl-3'-sn-glycerol (diphytanyl ether analog) with an equimolar amount of SO(3)-pyridine complex at room temperature; with excess sulfation reagent at 60 degrees C, the 1',2'-disulfate ester was obtained. The phosphatidyl-glycero-2-sulfate isomer was synthesized by an unambiguous route starting from the bacterial 2,3-di-O-phytanyl-sn-glycerol. The synthetic phosphatidylglycerosulfates were characterized by analytical, chromatographic, optical rotatory, and spectral (infrared and NMR) data and compared with the phosphatidylglycerosulfate isolated from Halobacterium cutirubrum.     

Characterization and in vivo production of three glycolipids from Candida Bogoriensis: 13-glucopyranosylglucopyranosyloxydocosanoic acid and its mono- and diacetylated derivatives

Three glycosides of 13-hydroxydocosanoic acid isolated from Candida bogoriensis were characterized by quantitating the amount of carbohydrate, acetate, and hydroxy acid in each, and by gas-liquid chromatography and mass spectrometry of their methyl ester, trimethylsilyl ether derivatives. One of the glycosides was the diacetylated derivative of 13-glucosylglucosyloxydocosanoic acid previously characterized by Tulloch, Spencer, and Deinema (Can. J. Chem., 46: 345 [1968]), in which the disaccharide had the beta(1 --> 2) sophorose linkage and the acetyl groups were attached to the 6' and 6" positions of the glucose residues. The other two glycosides were 13-glucosylglucosyloxydocosanoic acid and its monoacetylated derivative. A comparison of the mass spectra of derivatives indicates that the acetyl group of the monoacetyl lipid is on the internal glucose. Methyl 13-glucosyloxydocosanoate was produced by acid hydrolysis of the methyl ester of the unacetylated glycolipid and was characterized by the same techniques as the other glycolipids. Time course of production of the three glycolipids is consistent with the diacetylated derivative being the first extra-cellular product and the other two glycolipids being formed by deacetylation. 13-Hydroxy[13-(3)H]docosanoic acid, methyl 13-hydroxy[13-(3)H]docosanoate, and 9-hydroxy[11,12-(3)H]-stearic acid were each incorporated into the glycolipid fraction.     

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.     

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.     

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.     

1-(O-β-Glucosaminyl)-2,3-diglyceride in Bacillus megaterium

1. A lipid that contains glucosamine but not phosphorus has been isolated from Bacillus megaterium. It constitutes about 5% of the total lipid glucosaminide in this organism and can be distinguished chromatographically from 2'-(O-beta-glucosaminyl)phosphatidylglycerol and 3'-(O-beta-glucosaminyl)phosphatidylglycerol, which are also present. 2. The lipid contains glycerol, fatty acids and glucosamine in the molar proportion 1:2:1. The fatty acids are bound by an ester linkage and are similar to those found in other lipids of this organism. Partial acid hydrolysis or alkaline hydrolysis of the lipid yields 1-(O-beta-glucosaminyl)glycerol and degradation with nitrite yields 2,5-anhydromannose and diglyceride. 3. The lipid has been identified as 1-(O-beta-glucosaminyl)-2,3-diglyceride.     

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.     

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.     

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.