A phosphoethanolamine-modified glycosyl diradylglycerol in the polar lipids of Clostridium tetani

The polar lipids of the anaerobic bacterium Clostridium tetani, the causative agent of tetanus, have been examined by two-dimensional thin layer chromatography, ESI mass spectrometry, and NMR spectroscopy. Plasmalogen and di- and tetra-acylated species of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and N-acetylglucosaminyl diradylglycerol were the major lipids present in most strains examined except for strain ATCC 10779, the parent of strain E88, the first C. tetani strain to have its genome sequenced. This strain contained the same di- and tetra-acylated species but did not contain plasmalogens. All strains contained a novel derivative of N-acetylglucosaminyl diradylglycerol in which a phosphoethanolamine unit is attached to the 6’-position of the sugar, as judged by selective ³¹P-decoupled, ¹H-detected NMR difference spectroscopy. The N-acetylglucosamine (GlcNAc) residue is presumably linked to the 3-positon of the diradylglycerol moiety, and it has the β-anomeric configuration. Very little plasmalogen component was detected by mass spectrometry in the precursors phosphatidic acid and phosphatidylserine, consistent with the idea that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid assembly in anaerobic clostridia.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Δ9cis and C18:1 Δ11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

The polar lipids of Clostridium psychrophilum, an anaerobic psychrophile

We have examined the polar lipids of Clostridium psychrophilum, a recently characterized psychrophilic Clostridium isolated from an Antarctic microbial mat. Lipids were extracted from cells grown near the optimal growth temperature (+ 5 °C) and at − 5 °C, and analyzed by two-dimensional thin layer chromatography and liquid chromatography coupled with mass spectrometry. The major phospholipids of this species are: cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol. Phosphatidylserine and lyso-phosphatidylethanolamine were found as minor components. The most abundant glycolipids are a monoglycosyldiradylglycerol (MGDRG) and a diglycosyldiradylglycerol (DGDRG). The latter was only seen in cells grown at − 5 °C. An ethanolamine-phosphate derivative of N-acetylglucosaminyldiradylglycerol was seen in cells grown at − 5 °C and an ethanolamine-phosphate derivative of MGDRG was found in cells grown at + 5 °C. All lipids were present in both the all acyl and plasmalogen (alk-1′-enyl acyl) forms with the exception of PS and MGDRG, which were predominantly in the diacyl form. The significance of lipid changes at the two growth temperatures is discussed.     

Lipid diversity in clostridia

Studies of the lipidomes of twenty-one species of clostridia have revealed considerable diversity. Even among those species now defined as Clostridium sensu stricto, which are related to Clostridium butyricum, the type species, lipid analysis has shown that a number of distinct clades have characteristic polar lipids. All species of Clostridium sensu stricto have phosphatidylethanolamine, phosphatidylglycerol and cardiolipin which are present as all acyl or alk-1′-enyl acyl (plasmalogen) species. In addition, almost every clade has specialized polar lipids. For example, the group closely related to Clostridium beijerinckii and several other solventogenic species has glycerol acetals of plasmenylethanolamine, which protects the membrane bilayer arrangement when the lipids are highly unsaturated or in the presence of solvents. The group related to Clostridium novyi has aminoacyl-phosphatidylglycerol, which protects these pathogens from cationic antimicrobial peptides (CAMPs) of innate immunity. Clostridium botulinum species, which fall into several groups, align with these clades, and have the same specific lipids. This review will present the current state of knowledge on clostridial lipids.     

Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Delta9cis and C18:1 Delta11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

Polar lipids and fatty acids of three wild cyanobacterial strains of the genus<Emphasis Type="Italic">Chroococcidiopsis</Emphasis>

The occurrence of n-saturated, branched, and unsaturated fatty acids of 3 wild terrestrial strains of the genus Chroococcidiopsis (Order Chroococcales): C. supralittoralis, C. umbratilis, and C. versatilis collected from Lake Kinneret, Dead Sea, and Ein Kerem (Jerusalem) was investigated and individual compounds identified by gas chromatography-mass spectrometry. Polar lipids also were examined. Among polar lipids (studied using two-dimensional thin-layer chromatography) were as major glycolipids isolated: monogalactosyl-diacylglycerols, digalactosyl-diacylglycerols, 6-sulfoquinovosyl-diacylglycerols and phosphatidylglycerol. Nonphosphorus betaine-containing lipid, viz. N,N,N-trimethylhomoserin-4-O-yl-diacylglycerol, was found for the first time in cyanobacterial species.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Separation and Identification of the Polar Lipids of Chromatium Strain D

The polar lipids of the autotrophically grown, obligately anaerobic, photosynthetic bacterium Chromatium strain D were separated by paper chromatography. Four major phospholipids were identified: lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In addition, three glycolipids were observed and characterized, namely, monoglucosyldiglyceride, which is found in other biological systems, and (mannosyl, glucosyl)-diglyceride and (dimannosyl, glucosyl)-diglyceride, which heretofore have not been observed in nature.     

Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii

As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8. H. volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.7% of archaeal cardiolipin, 2.5% archaetidic acid, and 14% sulfated glycolipids 1 and 2. Nuclear magnetic resonance (NMR) and Fast atom bombardment mass spectrometry (FAB MS) data determined the glycolipids to be 6-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol] and a novel glycocardiolipin 6'-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol]. The polar lipids of Planococcus H8 consisted of 49% saturated phosphatidylglycerol and cardiolipin (9:1, w/w), and surprisingly 51% of the photosynthetic membrane lipid sulfoquinovosyldiacylglycerol (SQDG). This study documents archaeal cardiolipin and a novel glycocardiolipin in H. volcanii (lacking purple membrane), and is the first report of SQDG in a non-photosynthetic, halophilic bacterium.     

Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry

By use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, phosphatidic acid was found to be the main product of γ radiolysis of cardiolipin, phosphatidylinositol, and phosphatidylglycerol. It has been shown that γ irradiation of such glycolipids as cerebroside and galactosyl diglyceride leads to formation of ceramide and diglyceride, respectively. These findings, combined with those obtained earlier, allowed an assumption to be made that, owing to radiation-induced free radical fragmentation of lipids in their polar moiety, formation of signaling molecules can occur.     

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.     

Structural identities of four glycosylated lipids in the oral bacterium Streptococcus mutans UA159

The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have α-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.     

Lipid composition of aminopterin-resistant and sensitive strains of Streptococcus pneumoniae. Effect of aminopterin inhibition.

The polar lipids of Streptococcus pneumoniae wild type and aminopterin-resistant strains were analysed. The membrane contained only two acid phospholipids, phosphatidylglycerol and cardiolipin, and a large amount of two glycolipids, glucosyldiglyceride and galactosylglucosyldiglyceride. The unsaturated acyl chains ranged from 58 to 87% of total fatty acids, depending on the strain and on growth conditions. No relation could be established between aminopterin resistance and polar lipid or fatty acid compositions. However, in the presence of bacteriostatic concentrations of aminopterin, the wild type and the resistant mutant did not have the same behavior. The resistant strain maintained its fatty acid composition and a normal [32P]phosphate distribution among phospholipids while the wild type shifted to a higher content in unsaturated fatty acids and to a high relative cardiolipin labelling. Such a differencein [32P] distribution was not observed when bacteriostatic concentrations of chloramphenicol were used, or when growth was stopped after amino acid deprivation induced by high concentrations of isoleucine. The biochemical basis of the aminopterin resistant character of the amiA mutants are not yet well understood but the present study establishes that the mutation confers a certain insensitivity of the lipid metabolism to aminopterin.     

Halobellus rarus sp. nov., a halophilic archaeon from an inland salt lake of China

Two halophilic archaeal strains, YC21^T and YC77, were isolated from an inland salt lake of China. Both have pleomorphic rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require at least 2.1 M NaCl for growth under the optimum growth temperature of 37 °C. The major polar lipids of the two strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), two major glycolipids (GL1 and GL2) chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Trace amounts of two unidentified lipids (GL0-1 and GL0-2) were also detected. The 16S rRNA gene sequences of the two strains are 99.9 % identical, show 94.0–98.9 % similarity to the closest relative members of Halobellus of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC21^T and YC77 is 99.8 % and show 90.3–95.3 % similarity to the closest relative members of Halobellus. The DNA G+C content of strains YC21^T and YC77 were 66.1 and 66.2 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 89 %, and the two strains showed low DNA–DNA relatedness with Halobellus limi TBN53^T, the most related member of Halobellus. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC21^T and YC77 represent a novel species of the genus Halobellus, for which the name Halobellus rarus sp. nov. is proposed. The type strain is YC21^T (=CGMCC 1.12121^T = JCM 18362^T).     

Preliminary Analysis of Lipids and Fatty Acids of Green Bacteria and Chloroflexus aurantiacus

The complex lipids and fatty acids of the seven type species of green bacteria and three strains of Chloroflexus aurantiacus were analyzed. The green bacteria contained lipids that behaved as cardiolipin and phosphatidylglycerol on thin-layer chromatography. They did not contain phosphatidylethanolamine or phosphatidylserine. Similarly, Chloroflexus contained lipids that behaved as phosphatidylglycerol and phosphatidylinositol on thin-layer chromatography and did not contain phosphatidylethanolamine or phosphatidylserine. The green bacteria contained glycolipids I and II of Constantopoulos and Bloch (monogalactosyldiglyceride and a galactose- and rhamnose-containing diglyceride). Chloroflexus exhibited galactose-containing glycolipids that behaved identically with the mono- and digalactosyldiglycerides of spinach on thin-layer chromatography, and each contained galactose as well as at least one other sugar. The fatty acids of both groups of bacteria consisted entirely of saturated and monounsaturated fatty acids. In the green bacteria, myristic, palmitic, and hexadecenoic acids predominated. In Chloroflexus, palmitic, stearic, and oleic acids predominated. The positions of the double bonds in the monounsaturated fatty acids of Chloroflexus indicated synthesis by the anaerobic pathway. The lipid analyses suggest a close relationship between the green bacteria and Chloroflexus and further suggest that these groups of photosynthetic bacteria are more closely related to the blue-green algae than are the purple bacteria.     

Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modern procedures for identifying Bifidobacterium species.     

Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi

The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea.     

Novel ether lipid cardiolipins in archaeal membranes of extreme haloalkaliphiles

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyticus, has been examined by means of combined thin-layer chromatography and MALDI-TOF/MS analyses. The detailed investigation of lipid profiles has confirmed the presence of i) ether lipid phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components, ii) both C(20) and C(25) isopranoid chains in the lipid core and yielded new findings on membrane lipids of these unusual organisms. Besides some novel minor or trace phospholipids and glycolipids, data indicate the presence of ether lipid cardiolipin variants constituted by different combinations of C(20) and C(25) isopranoid chains, never before described in archaea. The role of C(25) isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.     

Lipid Composition of Some Species of Arthrobacter

The lipids from Arthrobacter crystallopoietes, A. pascens, and A. globiformis were investigated. Each strain contained three glycolipids, a monogalactosyl diglyceride, a digalactosyl diglyceride, and a dimannosyl diglyceride, and traces of triand tetraglycosyl diglycerides. The phospholipids in all three strains consisted of bisphosphatidylglycerol, phosphatidylglycerol, and phosphatidylmyoinositol. No evidence could be obtained for the occurrence of mannophosphoinositides. Analysis of the fatty acids by gas-liquid chromatography showed that they are predominantly C_15:0anteiso and C_17:0anteiso compounds. No significant differences were observed in the composition of lipids extracted from homogeneous cell preparations of the rod and sphere forms of A. crystallopoietes.