Norah C. Johnston

Lipid Composition in the Classification of the Butyric Acid-producing Clostridia

An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-l-enyl chains showed that the deoxyribonucleic acid homology groups I (Clostridium butyricum) and II (Clostridium beijerinckii) could be distinguished by their lipid compositions. The phospholipids of C. butyricum strains had ethanolamine as the major nitrogenous lipid polar head-group moiety, more octadecenoate plus C19-cyclopropane than hexadecenoate plus C17-cyclopropane acyl chains, and the predominant alk-l-enyl chain was C18-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C17-cyclopropane than octadecenoate plus C19-cyclopropane acyl chains, and the major alk-l-enyl chain was C16-saturated. Three species falling outside the two homology groups Clostridium fallax, Clostridium pseudofallax and Clostridium acetobutylicum had ethanolamine as the major phospholipid base, but these species could be distinguished from C. butyricum by their acyl and alk-l-enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.

Activation of Host Phospholipases C and D in Macrophages after Infection with Listeria monocytogenes

ABSTRACT Infection of the J774 murine macrophage-derived cell line withListeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770–1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC δ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC βI and βII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.

Effects of growth temperature and supplementation with exogenous fatty acids on some physical properties of Clostridium butyricum phospholipids.

Abstract The effects of the compositional alterations induced in the plasmalogen-rich phospholipids of Clostridium butyricum by growth at different temperatures with biotin or at a given temperature on supplementation with exogenous fatty acids in the absence of biotin, have been studied in lipid dispersions with the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and with the fluorescent probe diphenylhexatriene. Plots of TEMPO partitioning vs. temperature revealed deviations from linearity at 48° and 34.5°C for the phospholipids from cells grown on biotin at 37° and at 45° and 29° C for the phospholipids from cells grown on biotin at 25°C. Diphenylhexatriene fluorescence polarization showed a broad inflection beginning at 36°C for the lipids from 37°C grown cells and at 32°C for the lipids from 25°C-grown cells. Maximum polarization was reached at −5°C in each sample. Phospholipids from cells grown on oleate at 37°C had 85% 18 : 1 plus C 19 cyclopropane acyl chains and 90% 18 : 1 plus C 19 -cyclopropane alk-1-enyl chains. The phospholipids had decreased proportions of ethanolamine plus N -methylethanolamine plasmalogens (Khuller, G.K. and Goldfine, H. (1975) Biochemistry 14, 3642) and markedly increased levels of the glycerol acetal derivatives of these plasmalogens. Deviations from linearity were seen in TEMPO partitioning plots at 44° and 28°C and diphenylhexatriene polarization showed a broad transition beginning at 29°C and ending at −2°C. Polarization did not, however, reach a maximum, even at −15°C.

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.

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.

Lipid shape as a determinant of lipid composition in Clostridium butyricum. The effects of incorporation of various fatty acids on the ratios of the major ether lipids

The lipid composition of Clostridium butyricum is strongly influenced by the aliphatic chain compositions of the membrane lipids. Growth on cis-monounsaturated fatty acids in the absence of biotin was shown to affect the relative proportions of phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine most strongly, with smaller effects on the acidic lipids, phosphatidylglycerol and cardiolipin. The ratio of the glycerol acetal of plasmenylethanolamine to total phosphatidylethanolamine in cells grown on a series of fatty acids is shown to decrease in the following order; cis-vaccenic acid greater than or equal to oleic acid = C19-cyclopropane fatty acid greater than linoleic acid greater than petroselinic acid greater than elaidic acid greater than 14-methylhexadecanoic acid (anteiso-C17) greater than 12-methyltridecanoic acid (iso-C14). All fatty acids were extensively incorporated into the lipid acyl, alkenyl, and alkyl chains. There was considerable chain-elongation of the iso-C14 to iso-C16. The results are consistent with the hypothesis that the membrane lipid composition is strongly influenced by lipid shape and that the observed changes in lipid composition serve to stabilize the bilayer arrangement of the cell membrane.

Ether phospholipid asymmetry in Clostridium butyricum

Phospholipid asymmetry has been studied in the cell membrane of Clostridium butyricum IFO 3852. More than two-thirds of the phosphatidylethanolamine, which is predominantly in the plasmalogen form, was found to be in the outer monolayer by phospholipase C hydrolysis or by reaction with trinitrobenzenesulfonate. The diacyl and plasmalogen forms of phosphatidylethanolamine were similarly distributed. The glycerol acetal of phosphatidylethanolamine plasmalogen was more asymmetrically localized in the outer monolayer than was phosphatidylethanolamine. Cardiolipin and phosphatidylglycerol appeared to be largely confined to the inner monolayer.

Phospholipids ofRhizobium meliloti andAgrobacterium tumefaciens: Lack of effect of Ti plasmid

We have studied the phospholipid composition ofRhizobium meliloti strains which do or do not contain the large, tumor-inducing (Ti) plasmid ofAgrobacterium tumefaciens. The major phospholipids of stationary phase cells were phosphatidylethanolamine (PE) (22%), phosphatidyl-N-methylethanolamine (22%), phosphatidylcholine (PC) (27%), phosphatidylglycerol (11.4%), and cardiolipin (11%); as average percent of lipid phosphorus. Phosphatidyl-N,N-dimethylethanolamine (3.7%) was also present. The proportions of PE were higher, and PC lower, in logarithmic phase cells. No significant differences were seen in the proportions of phospholipids in strains with or without the Ti plasmid. Qualitative examination of the phospholipids ofA. tumefaciens with or without the Ti plasmid similarly revealed no differences.

Isolation and characterization of a novel four-chain ether lipid from Clostridium butyricum: the phosphatidylglycerol acetal of plasmenylethanolamine

We describe the isolation and characterization of a novel four-chain ether phospholipid in Clostridium butyricum grown on petroselinic acid in the absence of biotin. The results of quantitative analyses of hydrolytic products, selective hydrolysis by mild acid or phospholipase C, 1H-, 13C-, and 31P-NMR, and fast atom bombardment-mass spectroscopy (FABMS) have resulted in the determination of the structure of this lipid as a phosphatidylglycerol acetal of plasmenylethanolamine. Smaller amounts of this lipid have been found in cells grown under similar conditions in the presence of oleic, cis-vaccenic, elaidic or dihydrosterculic acids. It also appears to be present in small amounts in cells grown with biotin. This lipid is structurally related to the more plentiful glycerol acetal of plasmenylethanolamine found in these cells.

2H-NMR studies on ether lipid-rich bacterial membranes: deuterium order profile of Clostridium butyricum

Palmitic acid specifically deuterated at different carbon atoms, has been incorporated biosynthetically into the membrane lipids of Clostridium butyricum. The lipids of this organism are rich in plasmalogens and their glycerol acetals and exhibit an unusual fatty acyl and alkenyl chain distribution with saturated chains mainly at the sn-2 position and unsaturated chains at the sn-1 position. The ordering of the deuterated hydrocarbon chains in whole cells was measured with deuterium nuclear magnetic resonance and was compared to the order profiles of isolated cell membranes and membranes formed from the total phospholipid extract. The shape of the order profiles was similar for all three membranes, but the absolute values of the order profiles in whole cells and isolated membranes were lower than those of the liposomal lipids. The order profiles have the same characteristic shape as those found for the lamellar liquid-crystalline phases of synthetic diacylphospholipids.