The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus

Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol leads to (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol. Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor. Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative. In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

Phosphatidylglycerol and chilling sensitivity in plants

The hypothesis that molecular species of thylakoid phosphatidylglycerol containing two saturated fatty acids (disaturated phosphatidylglycerol) confer chilling sensitivity upon plants was tested by analyzing the fatty acid composition of phosphatidylglycerols isolated from leaves of a range of plants expected to have different sensitivities to chilling temperatures.

`Saturated' fatty acids (palmitate plus stearate plus hexadeca-trans-3-enoate) as a proportion of total phosphatidylglycerol fatty acids varied from 51 to 80 mole per cent in the plants analyzed but appeared to be rigidly fixed for a given plant species, being unaffected by leaf maturity or by environment.

Hexadeca-trans-3-enoate occurred only at the sn-2 position, whereas C-18 fatty acids occurred only at the sn-1 position of thylakoid phosphatidylglycerol. Therefore, the proportion of disaturated molecular species could be predicted accurately from the total fatty acids of phosphatidylglycerol.

Disaturated molecular species accounted for <25% of the total phosphatidylglycerol from leaves of chilling-resistant plants and for 50 to 60% of the phosphatidylglycerol in leaves from some of the most chilling-sensitive plants. However, not all chilling-sensitive plants contained high proportions of disaturated phosphatidylglycerol; solanaceous and other 16:3-plants and C₄ grasses may be important exceptions. Nonetheless, proportions of disaturated phosphatidylglycerol increased concomitantly with increasing chilling sensitivity of plants within a genus.

     

Altered phospholipid metabolism in a temperature-sensitive mutant of a thermophilic bacillus

The phospholipid metabolism of a temperature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58°C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In ³²P incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 min after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative conversion of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chloramphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mechanism(s) of phospholipid metabolism in the mutant at the restrictive temperature.Nonstandard Abbreviations WT parental strain, thermophilic bacillus - TS-13 temperature-sensitive mutant of a thermophilic bacillus - CL cardiolipin - PG phosphatidylglycerol - PE phosphatidylethanolamine - l-CL lyso-cardiolipin     

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

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

Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation,but is dispensable for mitochondrial function

Genetic dissection of the lipid bilayer composition provides essential in vivo evidence for the role of individual lipid species in membrane function. To understand the in vivo role of the anionic phospholipid, phosphatidylglycerol, the loss-of-function mutation was identified and characterized in the Arabidopsis thaliana gene coding for phosphatidylglycerophosphate synthase 1, PGP1. This mutation resulted in pigment-deficient plants of the xantha type in which the biogenesis of thylakoid membranes was severely compromised. The PGP1 gene coded for a precursor polypeptide that was targeted in vivo to both plastids and mitochondria. The activity of the plastidial PGP1 isoform was essential for the biosynthesis of phosphatidylglycerol in chloroplasts, whereas the mitochondrial PGP1 isoform was redundant for the accumulation of phosphatidylglycerol and its derivative cardiolipin in plant mitochondrial membranes. Together with findings in cyanobacteria, these data demonstrated that anionic phospholipids play an important, evolutionarily conserved role in the biogenesis and function of the photosynthetic machinery. In addition, mutant analysis suggested that in higher plants, mitochondria, unlike plastids, could import phosphatidylglycerol from the endoplasmic reticulum.     

Chlorosome lipids from Chlorobium tepidum: characterization and quantification of polar lipids and wax esters

We have extracted polar lipids and waxes from isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum and determined the fatty acid composition of each lipid class. Polar lipids amounted to 4.8 mol per 100 mol bacteriochlorophyll in the chlorosomes, while non-polar lipids (waxes) were present at a ratio of 5.9 mol per 100 mol bacteriochlorophyll. Glycolipids constitute 60 % of the polar lipids while phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and an aminoglycosphingolipid make up respectively 15, 3, 8 and 12 %. A novel glycolipid was identified as a rhamnose derivative of monogalactosyldiacylglycerol, while the other major glycolipid was monogalactosyldiacylglycerol. Tetradecanoic acid was the major fatty acid in the aminoglycosphingolipid, while the other polar lipids contained predominantly hexandecanoic acid. The chlorosome waxes are esters of unbranched fatty acids and fatty alcohols with 14 or 16 carbon atoms, joined to form molecules with between 28 and 32 carbon atoms. The stoichiometry between lipids and bacteriochlorophyll suggests that much of the chlorosome surface is covered by protein.     

Participation of the microsomal CDP-diglycerides in the mitochondrial biosynthesis of phosphatidylglycerol

Participation of microsomal CDP-diglycerides in mitochondrial biosynthesis of phosphatidylglycerol was studied by [3H]palmitoyl, [14C]linoleoyl, and [14C]arachidonoyl CDP-diglycerides and [3H]CDP-diglycerides which were bound to microsomal membranes, incubated with unlabelled mitochondrial membranes, and further incubated in the presence of radioactive sn-glycero-3-phosphate under conditions required for mitochondrial phosphatidylglycerol biosynthesis. Ten to 15% of microsomal radioactive CDP-diglycerides was transferred to mitochondrial membranes and incorporated into mitochondrial radioactive lipids identified as phosphatidylglycerol, phosphatidylglycerophosphate, and, when [14C]linoleoyl CDP-diglycerides were used, diphosphatidylglycerol (cardiolipin).     

Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS

This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion. An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8. The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol. Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Characterization of daptomycin oligomerization with perylene excimer fluorescence: stoichiometric binding of phosphatidylglycerol triggers oligomer formation

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.     

Purification and characterization of lysophospholipase L2 of Escherichia coli K-12

Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].     

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS² or MS³, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1–5 ng of injected substance.     

Formation of novel nitrofuran metabolites in xanthine oxidase-catalyzed reduction of methyl 5-nitro-2-furoate

After methyl 5-nitro-2-furoate was incubated with milk xanthine oxidase, three reduction products were isolated from the incubation mixture. Among them, two reduction products were new types of nitrofuran metabolites, i.e., metabolites 1 and 2 were identified as the dihydroxyhydrazine derivative (1,2-dihydroxy-1,2-di(5-methoxycarbonyl-2-furyl) hydrazine) and the hydroxylaminofuran derivative (methyl 5-hydroxylamino-2-furoate), respectively. Metabolite 3 was also identified as the aminofuran derivative (methyl 5-amino-2-furoate) by comparison with a synthetic sample.     

On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii.

About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A₂ from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6-trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A₂. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase A₂ from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures. Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.     

A phosphatidylinositol-containing derivative of Lactobacillus casei ATCC 7469

A derivative of Lactobaccillus casei ATCC 7469, characterized by limited growth in liquid media and an unusual phospholipid composition, has been isolated. Grown to early stationary phase on a lipid-free and inositol-free medium, the organism produces phosphatidylinositol phosphatidylglycerol, and diphosphatidylglycerol. The phosphatidylinositol was identified by thin-layer, paper, and gas chromatography, and by mass spectrometry. In agreement with published data, the conventional strain produced phosphatidylglycerol, diphosphatidylglycerol, and lysylphosphatidylglycerol, but no phosphatidylinositol. The phospholipid/glycolipid molar ratio, calculated on the basis of published glycolipid analyses, is 1.3 : 1 for the derivative and 1.5 : 1 for the conventional strain.     

The effect of the polar moiety of lipids on the ion permeability of bilayer membranes

Summary Bilayer membranes were prepared with the negatively charged lipids phosphatidylglycerol and diphosphatidylglycerol, the positively charged lipid lysyl phosphatidylglycerol, the zwitterionic lipid phosphatidylethanolamine, and an uncharged glycolipid, diglucosyldiglyceride, all isolated from gram-positive bacteria. Bilayer membranes of all these lipids manifested specific resistances of 10⁷ to 10⁹ cm² and capacitances of 0.3 to 0.4 F cm^–2. The membrane potentials of these bilayers were measured as a function of the sodium chloride, potassium chloride, and hydrogen chloride transmembrane concentration gradients (0.01 to 0.10m) and were found to be linear with the logarithm of the salt activity gradients. Membranes made from lysyl phosphatidylglycerol (one net positive charge) were almost completely chloride selective, whereas membranes from phosphatidylglycerol and diphosphatidylglycerol (one and two net negative charges, respectively) were highly cation selective. Membranes prepared with either diglucosyldiglyceride or phosphatidylethanolamine showed only slight cation selectivity. These findings indicate that the charge on the polar head group of membrane lipids plays an important role in controlling the ion-selective permeability of the bilayer.     

Modulation of Escherichia coliN-acetylmuramoyl-l-alanine amidase activity by phosphatidylglycerol

The activity of pure Escherichia coli murein (peptidoglycan) amidase ($\text{N-}\text{acetylmuramoyl-}\text{l}\text{-alanine}$ amidase, EC 3.5.1.28) was measured after preincubation with E. coli phosphatidylglycerol microdispersions in final concentration ranging over micro- and millimolarities. The enzyme activity was increased up to 160% of the control for phosphatidylglycerol concentrations increasing from 2 to 50 μM. After a plateau extending from 0.05 to 0.3 mM, higher phosphatidylglycerol concentrations inactivated the enzyme down to 15% of initial activity for concentrations of 2 mM. Positive kinetic cooperativity was observed for the activation as well as for the inactivation processes. Cardiolipin (or diphosphatidylglycerol) from the same origin and under same conditions had no significant effect. Molecular sieving experiments have shown that, when inactivated, the enzyme remained firmly bound to the phosphatidylglycerol vesicles, whereas the activated phosphatidylglycerol-enzyme complex was totally dissociable by dilution. Activated phosphatidylglycerol complexes were recovered by gel exclusion chromatography at equilibrium in 40 μM phosphatidylglycerol. Possible physiological meaning of the results is briefly discussed in the context of our work and that done previously by others.     

Synthesis of phosphatidylglycerol by chloroplasts from leaves of Spinacia oleracea L. (spinach)

Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 mM). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ ratio of 1, whereas the optimal ratio for $\text{Mg}{}^{\mathrm{2+}}\text{ATP}$ was closer to 2. The $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.     

Large decreases in membrane phosphatidylethanolamine and diphosphatidylglycerol upon mutation to duramycin resistance do not change the protonophore resistance of Bacillus subtilis

Duramycin-resistant mutant strains were selected from wild-type Bacillus subtilis (BD99) and its protonophore-resistant mutant derivative, strain AG1A3. Analyses of the membranes of the duramycin-resistant mutants showed that they had little or no phosphatidylethanolamine and diphosphatidylglycerol as determined by chemical detection after thin-layer chromatography. Small amounts of these phospholipids must remain in the mutant strains, however, because during studies of incorporation of exogenous, radioactive fatty acids, label associated with palmitoleic acid was found in chromatographic positions that corresponded to the expected positions of phosphatidylethanolamine and diphosphatidylglycerol. The duramycin-resistant strains both showed elevated levels of phosphatidylglycerol and aminoacyl(lysyl)phosphatidylglycerol. The duramycin-resistant derivative of protonophore-resistant AG1A3 (AG1A3-DR4), but not that of the wild type, also showed a decreased content of neutral relative to polar lipid in the membrane. The composition of neutral lipid in that strain was higher in free fatty acids and lower in 1,2-diacylglycerol than its parent strain. AG1A3-DR4 also contained appreciable levels of lysophosphatidylethanolamine and somewhat elevated diglycosyldiacylglycerol relative to the other strains in the study. The protonophore resistance of AG1A3 was unaltered by mutation to duramycin resistance. Nor was there any change in the efficacy of exogenous palmitoleic acid in diminishing the protonophore resistance of AG1A3-DR4. This phenomenon persists upon dramatic reduction in the content of phosphatidylethanolamine and diphosphatidylglycerol even though those phospholipids are normally the preferred sites of incorporation of the exogenous unsaturated fatty acids that mediate the effect.