Metabolic responses to the reduction in palmitate caused by disruption of the FATB gene in Arabidopsis

Disruption of the FATB gene in Arabidopsis results in a two-thirds reduction in saturated fatty acids, largely palmitate, in the leaf extra-plastidic phospholipids and a reduction in the growth rate of the mutant compared to wild type (Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB [2003] Plant Cell 15: 1020-1033). In this study, we report that although fatb-ko seedlings grow more slowly than wild type, the rate of fatty acid synthesis in leaves of the mutant increases by 40%. This results in approximately the same amount of palmitate exported from the plastid as in wild type but an increase in oleate export of about 55%. To maintain constant amounts of fatty acids in leaves, thereby counterbalancing their higher rate of production, the mutant also increases its rate of fatty acid degradation. Although fatb-ko leaves have higher rates of fatty acid synthesis and turnover, the relative proportions of membrane lipids are similar to wild type. Thus, homeostatic mechanisms to preserve membrane compositions compensate for substantial changes in rates of fatty acid and glycerolipid metabolism in the mutant. Pulse-chase labeling studies show that in fatb-ko leaves there is a net increase in the synthesis of both prokaryotic and eukaryotic lipids and consequently of their turnover. The net loss of palmitate from phosphatidylcholine plus phosphatidylethanolamine is similar for wild type and mutant, suggesting that mechanisms are not present that can preferentially preserve the saturated fatty acids. In summary, the leaf cell responds to the loss of saturated fatty acid production in the fatb-ko mutant by increasing both fatty acid synthesis and degradation, but in doing so the mechanisms for increased fatty acid turnover contribute to the lowering of the percentage of saturated fatty acids found in eukaryotic lipids.     

Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis.

Attempts to manipulate the level of C16:1 fatty acids in membrane phospholipids were made by using Bacillus subtilis and its protonophore-resistant mutants to test the hypothesis that C16:1 fatty acid levels relate to the bioenergetic properties of the mutant strains. Growth of the three mutants in the presence of palmitoleic acid restored the level of C16:1 fatty acids in the membrane lipids to somewhat above those found in the wild type. The palmitoleic acid was preferentially incorporated into diphosphatidylglycerol (cardiolipin) and phosphatidylethanolamine and was associated with increased levels of these phospholipids. These membrane preparations showed no increase in the levels of free fatty acids. The increase in C16:1 fatty acids achieved by growth in the presence of palmitoleic acid was accompanied by secondary changes in membrane lipids as well as a pronounced diminution in the protonophore resistance of growth and ATP synthesis. Other membrane-associated properties that had been observed in these mutants, e.g., elevated ATPase levels, were not altered coordinately with protonophore resistance and C16:1 fatty acid levels. Growth of the wild type in the presence of palmitic acid caused a modest elevation of the C16:0 of the membrane lipids and a modest increase in the protonophore resistance of growth and ATP synthesis. Growth of the wild type at elevated temperatures, in the absence of fatty acid supplementation, also enhanced its resistance to protonophores. The results support the hypothesis that specific changes in membrane lipid composition underlie the bioenergetic changes associated with protonophore resistance.     

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extremely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Chlolesterol was shown to suppress the polymyxin-induced response in liposomes.     

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids.

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extemely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Cholesterol was shown to suppress the polymyxin-induced response in liposomes.     

The protonophore resistance of Bacillus megaterium is correlated with elevated ratios of saturated to unsaturated fatty acids in membrane phospholipids

Growth of the protonophore-resistant strain of Bacillus megaterium, strain C8, in the presence of oleic acid markedly reduced its resistance to low concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth of the CCCP-sensitive wild-type strain in the presence of stearic acid increased the resistance of that strain to growth inhibition by protonophore. Studies of the membrane lipids indicated that in the absence of additions to the medium, membranes from C8 contained greatly reduced levels of monounsaturated fatty acids relative to the wild type; wild-type levels were restored by growth of C8 in the presence of oleic acid, concomitant with the loss of resistance. Conversely, growth of the wild type on stearic acid increased the ratio of saturated/unsaturated fatty acids in the membrane, concomitant with a modest increase in the resistance of the wild-type strain to CCCP. The exogenous oleic acid was preferentially incorporated into phosphatidylethanolamine, diphosphatidylglycerol, and 1,2-diacylglycerol, whereas stearic acid was incorporated preferentially into phosphatidylglycerol, and into the small component of free fatty acids. Depending upon the growth conditions, changes in membrane lipid-to-membrane protein ratio and in the ratios of polar lipid components were observed, but none of those changes correlated as did the changes in saturated fatty-acid-to-unsaturated fatty-acid ratio with protonophore resistance. This latter correlation was further suggested by experiments in which the protonophore resistance of wild type B. megaterium was shown to increase with increasing growth temperature without any temperature-dependent loss of protonophore efficacy. The experiments here support the hypothesis developed from work with Bacillus subtilis that changes in the fatty acid composition of the membrane phospholipids affect energy coupling, and make it clear that simple increases or decreases in the hydrolytic activity of ATPase in the uncoupler-resistant mutants of bacilli are not correlated with resistance in some direct way.     

Lipid alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

The lipid composition of cells of Pseudomonas aeruginosa strains resistant to polymyxin was compared with the lipid composition of cells of polymyxin-sensitive strains as to their content of readily extractable lipids (RELs), acid-extractable lipids, the fatty acid composition of RELs, and the contents of various phospholipids in the REL fraction. The polymyxin-resistant strains had an increased content of RELs, but a decreased phospholipid content. The REL fraction from the polymyxin-resistant strains had an increased content of unsaturated fatty acids accompanied by a decreased content of cyclopropane fatty acids as compared with the fatty acid composition of RELs from polymyxin-sensitive strains. The phosphatidylethanolamine content was greatly reduced in the polymyxin-resistant strains, whereas the content of an unidentified lipid, thought to be a neutral lipid lacking either a phosphate, free amino, or choline moiety, was greatly increased. Cell envelopes of the polymyxin-resistant strains contained reduced concentrations of Mg2+ and Ca2+ as compared with the cell envelopes of polymyxin-sensitive strains. It appears that polymyxin resistance in these strains is associated with a significant alteration in the lipid composition and divalent cation content of the cell envelope.     

Lipids and fatty acids of a moderately halophilic bacterium, No. 101.

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.     

Differences in tissue-specific lipid composition between embryos of wild and captive breeding alligators (Alligator mississippiensis)

Fertile eggs obtained from alligators reared in captivity typically exhibit high rates of embryonic mortality. Also, the fatty acid composition of the yolk lipid of the captive eggs is markedly different from that observed in eggs from wild alligators, possibly as a result of differences in maternal diet in the two situations. The fatty acid compositions of tissue lipids during the embryonic development of wild and captive alligators were compared. The lipids of liver, adipose tissue and heart of the two types of embryo displayed fatty acid profiles which generally reflected the acyl compositions of the respective yolks. Thus the lipids from these tissues of the captive embryos contained markedly higher proportionate levels of linoleic and linolenic acids, lower levels of palmitoleic acid, and, in general, lower levels of docosahexaenoic acid and other C20 and C22 polyunsaturates, in comparison to the values for the wild embryos. In contrast, the fatty acid composition of the brain phosphoglycerides was very similar in the two types of embryo. Thus, at least in those embryos which had survived during the developmental period studied, the brain was able to maintain a relatively constant fatty acid composition, in spite of major differences between the wild and captive eggs in the proportions of the various fatty acids supplied from the yolk. It is suggested that a major cause of embryonic mortality in the captive embryos could be a failure to maintain an adequate level of docosahexaenoic acid in the developing brain.     

Characteristic lipids of Bordetella pertussis: simple fatty acid composition, hydroxy fatty acids, and an ornithine-containing lipid.

The lipids and fatty acids of Bordetella pertussis (phases I to IV) were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry and compared with those of B. parapertussis and B. bronchiseptica. The major lipid components of the three species were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, lysophosphatidylethanolamine, and an ornithine-containing lipid. The ornithine-containing lipid was characteristic of the genus Bordetella. The fatty acid composition of the total extractable cellular lipids of B. pertussis was mostly hexadecanoic and hexadecenoic acids (90%) in a ratio of about 1:1. The hexadecenoic acid of B. pertussis was in the cis-9 form. The fatty acid composition of the residual bound lipids was distinctly different from that of the extractable lipids, and residual bound lipids being mainly 3-hydroxytetradecanoic, tetradecanoic, and 3-hydroxydecanoic acids, with 3-hydroxydodecanoic acid occurring in some strains. It was determined that the 3-hydroxy fatty acids were derived from lipid A. The fatty acid composition of the total extractable cellular lipids of B. parapertussis and B. bronchiseptica, mainly composed of hexadecanoic and heptadecacyclopropanoic acid, differed from that of B. pertussis. Although the fatty acid composition of the residual bound lipids of B. parapertussis was similar to that of the residual bound lipids of B. pertussis, 2-hydroxydodecanoic acid was detected only in the bound lipids of B. bronchiseptica.     

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

Lipids and lipid metabolism in the brown alga, Fucus serratus

The lipids of the brown alga Fucus serratus were isolated, identified and quantified. The major acyl lipids were the three glycosylglycerides, diacylgalactosylglycerol, diacyldigalactosylglycerol and diacylsulphoquinovosylglycerol. These represent over 70% of the total acyl lipids. The fatty acid compositions of the major lipids were examined and most showed rather distinctive fatty acid contents. For example, diacylgalactosylglycerol was enriched in n-3 polyunsaturated fatty acids while phosphatidylcholine and phosphatidylethanolamine had very high levels of arachidonate. Phosphatidylglycerol contained the unusual trans-Δ3-hexadecenoic acid. The labelling of lipids and fatty acids from [¹⁴C]acetate was examined and the distribution of label between individual components as a function of the incubation period and in algae collected at different times of the year is reported. Algae collected in the winter incorporated much more radioactivity into non-esterified fatty acids when compared to algae collected in the summer. All algae could label myristate, palmitate, stearate and oleate at high rates. Longer incubation times allowed the labelling of polyunsaturated fatty acids such as linoleic acid.     

Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Whole-cell and membrane lipids of the methylotrophic bacterium Methylosinus trichosporium.

The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed.     

Physiology of Sporeforming Bacteria Associated with Insects II. Lipids of Vegetative Cells

Lipid composition was studied in two strains each of mid-log phase cells of Bacillus thuringiensis, B. larvae, B. popilliae, B. alvei, and B. lentimorbus. Total lipids varied from 2.5 to 3.5% of the cell dry weight of B. thuringiensis to 4.3 to 5.0% of B. popilliae. Phospholipids in the organisms examined ranged from 55 to 79% of total lipids; neutral lipids averaged from 13 to 45%. Common phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and lysophosphatidylethanolamine. 1,2-Diglycerides, methyl esters, free fatty acids, and hydrocarbons were found in all the organisms studied. Branched-chain fatty acids constituted more than 50% of the total fatty acids in B. thuringiensis, B. larvae, B. popilliae, and B. alvei, whereas, in B. lentimorbus, normal-chain acids constituted more than 50%. Anteiso-C₁₅ (12-methyltetradeconoate) was the most abundant acid (30 to 50%) in B. alvei, B. larvae, B. popilliae, and B. lentimorbus. In contrast, B. thuringiensis contained more iso-C₁₃ (7%), iso-C₁₅ (17%), normal-C₁₆ (24%), and iso-C₁₇ (18%) than anteiso-C₁₅ (6%). The distribution of individual fatty acids was similar in the phospholipids and neutral lipids of each organism. However, the total amount of iso, anteiso, and normal isomers differed.     

Polar glycerolipids of Chlamydomonas moewusii

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate: phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [32P]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

Correlation of Bacterial Lipid Composition with Antibiotic Resistance

The correlation of bacterial lipid composition with antibiotic resistance was investigated with particular emphasis on those organisms in which resistance may be related to membrane or envelope structure or function, as in resistance to tetracyclines and polymyxin. Chloroform-methanol-extractable lipids, phosphatidyl ethanolamine fractions, and both fatty acids of these lipid fractions and total fatty acids were studied by using thin-layer chromatography, gas chromatography, and infrared spectroscopy. Consistent quantitative differences were found between the fatty acid compositions of sensitive and resistant strains. Most notable was the fact that, in gram-negative organisms, resistant strains showed decreases in cyclopropane acids as compared with sensitive strains. These changes were found to be inherent in the strains and not due to growth stage or culture age. No significant qualitative differences were noted. In contrast, no such variation in fatty acid content was observed in penicillin-sensitive and resistant strains of gram-positive cocci. As significant alterations of fatty acid composition were noted in gram-negative strains resistant to antibiotics, we suggest that resistance is correlated to membrane or envelope lipid composition.