Biosynthesis of phospholipids in Clostridium butyricum: kinetics of synthesis of plasmalogens and the glycerol acetal of ethanolamine plasmalogen

The biosynthesis of the plasmalogen forms of phosphatidylethanolamine (plasmenylethanolamine) and phosphatidylglycerol (plasmenylglycerol) and of the glycerol acetal of plasmenylethanolamine has been studied in cultures of Clostridium butyricum IFO 3852. When growing cells were pulsed with [32P]orthophosphate, there was a lag of 5 to 7 min between the rapid incorporation of label into the acylphosphatides and the rapid incorporation of label into the corresponding plasmalogens. The labeling of the glycerol acetal of plasmenylethanolamine was even slower. In pulse-chase experiments with 32Pi, the kinetics of labeling indicated precursor-product relationships between phosphatidylethanolamine and plasmenylethanolamine and between the latter and its glycerol acetal. A precursor-product relationship was also seen between phosphatidylglycerol and cardiolipin, but the kinetics of labeling of the alkenyl-containing forms of these lipids were not consistent with direct precursor-product relationships with the acyl lipids. In the presence of hydroxylamine and 32Pi, both phosphatidylserine and plasmenylserine accumulated 32P in a ratio of ca. 15:1. Upon release of the inhibition of phosphatidylserine decarboxylase, label appeared in the following sequence: phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine. Acyl phosphatidylglycerol was identified as a major phospholipid (17% of lipid phosphorus) in C. butyricum grown in low-phosphate (1.13 mM) medium with 50 mM Tris buffer. Of the acyl phosphatidylglycerol, 13% was acid labile. There appear to be two plasmalogen forms of acyl phosphatidylglycerol. One of these has a single alkenyl ether group, and the other has alkenyl ether groups on both glycerols.     

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.     

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids

Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.

The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings. When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium. The acyl chains reflected the palmitic acid content of the medium more closely. Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains. The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased. However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid. In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid. These results indicate that C. acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides. These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains. Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids. The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

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.     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogen in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Taxonomy of the glycerol fermenting clostridia and description of Clostridium diolis sp. nov

Six Clostridium strains which ferment glycerol to 1,3-propanediol were tested for their taxonomic and phylogenetic relatedness. All but one were known as C butyricum. By physiological tests, 16S rDNA sequences and fatty acid composition two groups were distinguished. The first comprised the strains VPI 3266, DSM 2478 and DSM 523 (C. "kainantoi") and was consistent with the type strain of C. butyricum in almost all characters. The second group comprising the strains DSM 5430, DSM 5431 and E5 was related to C. beijerinckii. The 16S rDNAs of these strains were almost identical with that of the type strain of C. beijerinckii, DSM 791. The DNA-DNA hybridization value of DSM 5431 and ES with C. beijerinckii DSM 791 was markedly but not decisively lower (67 and 72%, respectively). However, there were significant physiological differences to C. beijerinckii which suggested to describe the strains as a separate species, Clostridium diolis with strain SH1 (= DSM 5431) as the type strain. The new species is distinguished from C. beijerinckii, which requires complex nutrients, by its ability to grow in glucose mineral medium with biotin as the only growth factor and by differences in substrate utilization. "C. kainantoi" Takeda and Matsui was recognized as a later synonym of C. butyricum.     

Lipids of isolated neurons

1. Lipids were extracted from neurons isolated from the lateral vestibular nucleus of ox (Bos taurus L.) and the ganglia of Aplysia punctata Cuvier. 2. Thin-layer chromatography of ox-neuron lipid revealed three major fractions corresponding to neutral lipid, phosphatidylethanolamine and phosphatidylserine. Part of the phosphatidylethanolamine was present as the plasmalogen. 3. Aplysia-neuron lipid contained neutral lipid, phosphatidylethanolamine and phosphatidylserine. Both phospholipids appeared to be present predominantly as the plasmalogen form. 4. The fatty acids of alkali-labile lipids of ox neurons were examined by gas–liquid chromatography. The major fatty acids were oleic acid, stearic acid and palmitic acid.     

Identification of Clostridium butyricum and Clostridium beijerinckii by gas-liquid chromatography and sugar fermentation: correlation with DNA homologies and electrophoretic patterns

Sixty-five strains of clostridia of the butyricum group were studied by DNA-DNA hybridization, electrophoresis of cell proteins, gas-liquid chromatography, and fermentation of glycerol, inositol and ribose. The DNA--DNA hybridization results confirmed that strains of this group belong to two main species, Clostridium butyricum and C. beijerinckii. Five strains did not hybridize with the reference strains of these two species. Most of the strains could be identified by quantitative gas-liquid chromatographic analysis combined with fermentation patterns. The other strains could be identified by their protein electrophoretic patterns.     

Phosphatidyltransferase of Clostridium butyricum: specificity for diacylphosphoglycerides

Phosphatidyltransferase from Clostridium butyricum, which catalyzes transfer of the phosphatidyl moiety of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) or phosphatidylserine to primary alcohols such as glycerol, serine and ethanolamine, was tested for its ability to catalyze transfer of the plasmenyl moiety from plasmalogen analogs of PE or PG to glycerol or ethanolamine, respectively. The cell membrane of C. butyricum contains high proportions of these plasmalogens. When diacyl and plasmalogen forms of PE or PG were supplied as donors in equimolar amounts to membrane particles, the diacyl forms were the preferred donors by approx. 7 and 10 to 1, respectively. When the molar ratio of PE its plasmalogen was 1:3.3, the ratio of PG formed to its plasmalogen was 1:0.66. These results show that the enzyme(s) can catalyze transfer of both the diacyl and alkenyl acyl forms of glycerophospholipids, but the diacyl forms are used preferentially.     

Reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of plasmalogens: identification of 2-chlorohexadecanal

Plasmalogens contain a vinyl ether bond linking the sn-1 aliphatic chain to the glycerol backbone of this predominant phospholipid molecular subclass, which is found in many mammalian tissues. The present study demonstrates that the vinyl ether bond of plasmalogens is a molecular target of the reactive chlorinating species produced by myeloperoxidase. Analysis by thin layer chromatography revealed that reactive chlorinating species produced by myeloperoxidase target the vinyl ether bond of the plasmalogen, lysoplasmenylcholine (1-O-hexadec-1'-enyl-sn-glycero-3-phosphorylcholine), resulting in the production of a neutral lipid. Capillary gas chromatographic analyses demonstrated that the neutral lipid generated from lysoplasmenylcholine was neither hexadecanal nor did it contain masked hexadecanal (i.e. the vinyl ether) because the dimethyl acetal of hexadecanal produced by acid methanolysis derivatization was no longer present. Electrospray ionization mass spectrometry of the myeloperoxidase-generated neutral lipid product was consistent with the production of a 16-carbon fatty aldehyde containing one chlorine atom. Furthermore, proton NMR analysis indicated that this neutral lipid product was a 2-chloro-fatty aldehyde. Additional structural analysis of this neutral lipid by gas chromatography-mass spectrometry of the underivatized product as well as its pentafluorobenzyl oxime-derivative product was consistent with the neutral lipid being 2-chlorohexadecanal. The reactive chlorinating species, hypochlorous acid and chlorine gas, both attacked the vinyl ether bond of lysoplasmenylcholine resulting in the production of 2-chlorohexadecanal. The production of 2-chlorohexadecanal was dependent on the presence of the plasmalogen masked aldehyde (i.e. the vinyl ether) in the substrate because the free fatty aldehyde, hexadecanal, was not converted to 2-chlorohexadecanal by the reactive chlorinating species generated by myeloperoxidase. Taken together, the present studies demonstrate for the first time the targeting of the vinyl ether bond of plasmalogens by the reactive chlorinating species produced by myeloperoxidase resulting in the production of novel chlorinated fatty aldehydes.     

Changes in lipid composition of the maturing rat testis

1. The lipids and fatty acids of the lipids of testes of rats aged 4 weeks to 6 months were separated and analysed. 2. A decrease in concentration of triglyceride was noted, but there was no significant change in the concentration of phospholipids, plasmalogen or cholesterol during this time. 3. There were no significant differences in the total lipid concentration of palmitic acid, stearic acid, linoleic acid, arachidonic acid and docosatetraenoic acid between the various age groups. 4. A decrease in the concentration of oleic acid in the phosphatide and triglyceride fractions and an increase in the concentration of docosapentaenoic acid (characterized as the Delta4,7,10,13,16-isomer) in phosphatides but not in triglyceride were observed during the maturation period. 5. Histological studies indicated that the lipid changes occurred at the same time as the appearance and maturation of the spermatids.     

The lipids of mammalian pancreas

1. The total lipids of ox and guinea-pig pancreas were fractionated on silicic acid columns. 2. The high lipid content of both tissues was due to triglyceride. The major fatty acids of this triglyceride were stearic acid, palmitic acid and oleic acid. 3. The pattern of individual phospholipids resembled that of liver, though pancreas contained more plasmalogen.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

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.     

Plasmenylethanolamine: growth factor for cholesterol-reducing Eubacterium.

A plasmalogen, plasmenylethanolamine, is required for in vitro growth of strains of Eubacterium which convert cholesterol to coprostanol. Plasmenylethanolamine was isolated from calf brain by selective saponification of lipid fractions separated by thin-layer or column chromatography. Cholesterol-containing thioglycolate broth plus purified plasmenylethanolamine or its 2-lyso derivative supported growth of Eubacterium ATCC 21408 and a cholesterol-reducing Eubacterium isolated from baboon feces. Plasmenylethanolamine obtained from commercial sources also supported growth of these organisms, but none of a number of other pure lipids would support growth. Metabolism of the alkenyl ether group of plasmenylethanolamine occurred during growth.     

Phosphatidylserine decarboxylase from Clostridium butyricum

Phosphatidylserine decarboxylase activity has been characterized in membrane preparations from Clostridium butyricum ATCC 19398. A particulate fraction was shown to catalyze the formation of phosphatidylethanolamine and plasmenylethanolamine when vesicles containing phosphatidylserine and plasmenylserine were used as substrate. No plasmenylethanolamine was formed when phosphatidylserine alone was used as substrate. The activity with phosphatidylserine was activated by divalent cations and was optimal under anaerobic conditions. Ionic detergents inhibited phosphatidylethanolamine formation strongly and nonionic detergents inhibited partially. In the presence of Triton X-100, phosphate from [32P]phosphatidylserine appeared in three unidentified lipid products, in addition to phosphatidylethanolamine. The formation of these products was time- and Triton X-100 concentration-dependent. Hydroxylamine inhibited phosphatidylserine decarboxylase, but did not prevent the reactions stimulated by Triton X-100.