Competition between cis,trans and Cyclopropane Fatty Acid Formation and its Impact on Membrane Fluidity

The impact of cis, trans and cyclopropane fatty acids on membrane fluidity was investigated using batch‐grown Pseudomonas putida P8 and Comamonas testosteroni ATCC 17454. A major difference observed between the two investigated strains is the absence of the ability to synthesize trans‐unsaturated fatty acids in Comamonas. When grown exponentially at 30 °C, a shift to 35 °C increased the trans/cis ratios of the fatty acids of P. putida P8 from 0 to 0.81 and 0 to 1.07, in lipid extracts and cell hydrolyzates, respectively. After prolonged growth followed by nutrient deprivation for 48 h, both at 30 °C, trans fatty acids were absent, but the cyclo/cis ratios rose from 0.1 to 1.55 in lipid extracts, and from 0.1 to 1.54 in cell hydrolyzates. C. testosteroni ATCC 17454 contained no cyclo fatty acids when harvested in the exponential phase after 6 h, whereas after 72 h cultivation, the cyclo/cis ratios rose to 0.49 and 0.47, in lipid extracts and cell hydrolyzates, respectively. Trans fatty acids were never observed in this strain. Increased cyclo/cis and trans/cis ratios correlated with decreased fluidity measured by the fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) intercalated in the bilayers of liposomes and by Fourier Transform Infrared (FTIR) spectroscopy of lipids prepared from the cells. The specific effect of cyclopropane fatty acids on membrane fluidity was much smaller than that of trans fatty acids. FTIR‐measurements of intact cells of P. putida P8 confirmed the high potency of trans fatty acids to decrease the fluidity. In cells with induced cyclopropane fatty acid synthesis, the membranes remained more fluidized, indicating the lower importance of these fatty acids for homeoviscosis.     

The effects of temperature on the composition and physical properties of the lipids of Pseudomonas fluorescens

1. Pseudomonas fluorescens was grown at various temperatures between 5 degrees C and 33 degrees C. The extractable lipids from organisms at various stages of growth and grown at different temperatures were examined. 2. The extractable lipids contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and an ornithine-containing lipid. The relative amounts of these lipids did not vary significantly during growth or with the changes in growth temperature. 3. The major fatty acids were hexadecanoic, hexadecenoic and octadecenoic acids and the cyclopropane acids methylene-hexadecanoic and methylene-octadecanoic acids. The relative amount of unsaturated acids (including cyclopropane acids) did not change significantly during growth, but increased with decreasing temperature. 4. Phosphatidylethanolamines with different degrees of unsaturation and containing different amounts of cyclopropane acids were isolated from organisms grown at 5 degrees C and 22 degrees C and their surface and phase behaviour in water was investigated. Thermodynamic parameters for fusion and monolayer results for cyclopropane and other fatty acids were examined. 5. The surface pressure-area isotherms of phosphatidylethanolamines containing different amounts of unsaturated fatty acids show small differences but the individual isotherms remain essentially unchanged over the temperature range 5-22 degrees C. X-ray-diffraction methods show that the structures (lamellar+hexagonal) formed in water by phosphatidylethanolamine, isolated from organisms grown at 5 degrees C and 22 degrees C, are identical when compared at the respective growth temperatures. This points to a control mechanism of the physical state of the lipids that is sensitive to the operating temperature of the organism. 6. The molecular packing of cyclopropane acids is intermediate between that of the corresponding cis- and trans-monoenoic acids. However, substitution of a cyclopropane acid for a cis-unsaturated acid has insignificant effects on the molecular packing of phospholipids containing these acids.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Effect of changes in fatty acid composition of phospholipid species on the β-galactoside transport system of Escherichia coli K-12

On lowering the growth temperature of Escherichia coli K-12 from 37 to 17 °C, the cells resumed growth after a lag period of 40 min. During the lag period, the transition points in Arrhenius plots of the preinduced β-galactoside transport system were not changed while the saturated/unsaturated fatty acids ratio decreased gradually in phosphatidylethanolamine, rapidly in phosphatidylglycerol and little in cardiolipin.     

The effects of temperature on the fatty acid and phospholipid composition of four obligately psychrophilicVibrio Spp.

The free fatty acid and phospholipid composition of 4 psychrophilic marineVibrio spp. have been determined in chemostat culture with glucose as the limiting substrate over a temperature range 0–20°C. All the isolates show maximum glucose and lactose uptake at 0°C and this correlates with maximum cell yield. None of the isolates contain fatty acids with a chain length exceeding 17 carbon atoms.Vibrio AF-1 andVibrio AM-1 respond to decreased growth temperatures by synthesizing increased proportions of unsaturated fatty acids (C15:1, C16:1 and C17:1) whereas inVibrio BM-2 the fatty acids undergo chain length shortening. The fourth isolate (Vibrio BM-4) contains high levels (60%) of hexadecenoic acid at all growth temperatures and the fatty acid composition changes little with decreasing temperature. The principal phospholipid components of the four psychrophilic vibrios were phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Lyso-phosphatidylethanolamine and 2 unknown phospholipids were additionally found inVibrio AF-1. The most profound effect of temperature on the phospholipid composition of these organisms was the marked increase in the total quantities synthesized at 0°C. At 15°C phosphatidylglycerol accumulated in the isolates as diphosphatidylglycerol levels decreased. Additionally inVibrio BM-2 andVibro BM-4 phosphatidylserine accumulates as phosphatidylethanolamine biosynthesis was similarly impaired. The observed changes in fatty acid and phospholipid composition in these organisms at 0°C may explain how solute transport is maintained at low temperature.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol - lyso PE lysophosphatidylethanolamine     

Synthesis of cyclopropane fatty acids in isolated bacterial membranes

Isolated E. coli membrane vesicles can synthesize cyclopropane fatty acids from S-adenosylmethionine using endogenous membrane phospholipid as the lipidsubstrate. The major methylated products are methylenehexadecanoic acid and methyleneoctadecenoic acid esterified to phosphatidylethanolamine. The membrane system is slightly stimulated by sodium dodecyl sulfate at low concentrations but is inhibited by neutral surfactants. The reaction is inhibited by phospholipase C and phospholipase A. The Arrhenius plot for the enzyme reaction is discontinuous over the temperature range 0–35 °C.     

Phase transition behaviour of artificial liposomes composed of phosphatidylcholines acylated with cyclopropane fatty acids

Phase transitions of liposomes composed of synthetic phosphatidylcholines acylated with the cyclopropane fatty acids, lactobacillic and dihydrosterculic acid, were studied by differential scanning calorimetry. Transition temperatures were approx. 16°C higher than for phosphatidylcholines acylated with the corresponding unsaturated fatty acids, cis-vaccenic and oleic acid. Though our transition temperatures were all several degrees lower than those determined by Silvius and McElhaney ((1979) Chem. Phys. Lipids 25, 125–134), the increase produced by replacement of the double bond with a cyclopropane ring was the same. We propose that this replacement, through its effect on membrane fluidity, may serve to regulate the activity of membrane-associated processes such as transport.     

Adaptational changes of fatty acid composition and the physical state of membrane lipids following the change of growth temperature in Yersinia enterocolitica.

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Cold adaptation of eicosapentaenoic acid-less mutant of Shewanella livingstonensis Ac10 involving uptake and remodeling of synthetic phospholipids containing various polyunsaturated fatty acids

An Antarctic psychrotrophic bacterium, Shewanella livingstonensis Ac10, produces cis-5,8,11,14,17-eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid (LPUFA), as a component of membrane phospholipids at low temperatures. The EPA-less mutant generated by disruption of the EPA synthesis gene becomes cold-sensitive. We studied whether the cold sensitivity could be suppressed by supplementation of various LPUFAs. The EPA-less mutant was cultured at 6°C in the presence of synthetic phosphatidylethanolamines (PEs) that contained oleic acid at the sn-1 position and various C20 fatty acids with different numbers of double bonds from zero to five or cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) at the sn-2 position. Mass spectrometric analyses revealed that all these fatty acids became components of various PE and phosphatidylglycerol species together with shorter partner fatty acids, indicating that large-scale remodeling followed the incorporation of synthetic PEs. As the number of double bonds in the sn-2 acyl chain decreased, the growth rate decreased and the cells became filamentous. The growth was restored to the wild-type level only when the medium was supplemented with phospholipids containing EPA or DHA. We found that about a half of DHA was converted into EPA. The results suggest that intact EPA is best required for cold adaptation of this bacterium.     

Effect of growth temperature on lipid composition of Streptococcus faecium

The effect of growth temperature on the lipid and fatty acid composition of Streptococcus faecium has been studied. No differences in the qualitative composition of S. faecium lipids were observed. In all isolated fractions (neutral lipids, glycolipids, and phospholipids plus other polar lipids), the major fatty acids were palmitic (C-16:0), palmitoleic (C-16:1), octadecenoic (C-18:1), and cyclopropane (C-19:0). Changes in the fatty acid composition of the different fractions were observed which depended on growth temperature; the most significant one was the decrease of octadecenoic acid and the increase of palmitic acid in glycolipids and polar lipids as the temperature increased. The level of cyclopropane C-19:0 was approximately eightfold lower at 8 degrees C than at the other temperatures tested (20, 30, and 45 degrees C).     

The presence of odd-chain fatty acids in Drosophila phospholipids

ABSTRACT

Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

Discontinuous thermotropic response of Tetrahymena membrane lipids correlated with specific lipid compositional changes

Steady-state fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in microsomal lipids from Tetrahymena pyriformis cells grown at 39 or 15°C revealed discrete slope discontinuities in plots of polarization vs. temperature. Two well-defined ‘break points’ were present in the 0–40°C temperature range examined and their precise location was dependent upon the growth temperature of the cells. By mixing phospholipids from cells grown at different temperatures, the break points at 17.5 and 32°C in 39°C-lipid multilayer preparations were shown to correlate with the breaks at 12 and 27°C, respectively, in similar preparations from 15°C-grown cells. The discrete break points were also present, but at slightly different characteristic temperatures, in a phosphatidylcholine fraction and a phosphatidylethanolamine plus 2-aminoethylphosphonolipid fraction purified from the phospholipids and in total microsomal lipids (phospholipids plus the sterol-like triterpenoid, tetrahymanol). However, catalytic hydrogenation of the phospholipid fatty acids or mixing the non-hydrogenated phospholipids with increasing proportions of synthetic dipalmitoyl phosphatidylcholine eliminated the break points. We interpret this discontinuous thermotropic response in microsomal lipids as signalling a lipid phase separation of importance in regulating physiological events.     

Fatty acid positional distribution in phospholipids of a psychrophilic bacterium during changes in growth temperature

The major phospholipids of the psychrophilic bacteriumMicrococcus cryophilus, phosphatidylethanolamine and phosphatidylglycerol, have similar fatty acid compositions, comprising almost entirely palmitoleic and oleic acids. We show that there is a preference for the longer chains in thesn-1 position of both phosphatidylethanolamine and phosphatidylglycerol, both during isothermal growth and after temperature shifts, despite the fact that the overall phospholipid C18/C16 acyl chain ratio decreases with a lowering of growth temperature. Although it has been shown using model systems that the isomeric configurationsn-1-long,sn-2-short lowers lipid melting temperature, this paper reports the first clear-cut demonstration of such an isomeric preference in a natural system. We discuss how this acyl chain configuration contributes to membrane fluidity inM. cryophilus, in terms of adaptation to its psychrophilic habitat.     

Studies on fluorescence polarization of 1-acyl-2-cis- or trans-parinaroyl sn-3-glycerophosphorylcholines in model systems and microsomal membranes

Fluorescent lecithin probes containing cis- or trans-parinaric acid (PnA) at the 2-position cis-parinaroylphosphatidylcholine (cis-PnPC) and trans-parinaroyl phosphatidylcholine (trans-PnPC)) showed similar behavior to that of the free cis- or trans-parinaric acids (cis-PnA or trans-PnA) in bilayer vesicles of synthetic saturated lecithins. Transition temperatures detected by cis-PnPc were about 1°C lower than those observed with trans-PnPc. In mixed lecithin vesicles, the trans-PnPc probe monitored a higher temperature melting component than did the cis-probe. Both probes were readily incorporated into microsomal membranes and into sonicated vesicles prepared from the microsomal phospholipids. With either cis- or trans-PnPc no change in polarization ratio was observed for microsomal membranes between 40°C and 0°C but this ratio increased with decreasing temperature between 0°C and ?5°C. However, vesicles of extracted phospholipids showed a continuous increase in polarization ratio with decreasing temperature between 20°C and ?15°C with trans-PnPc and bewteen 5°C and ?15°C with cis-PnPc. These results suggest that the two lecithin probes monitor different environments in the membranes and phospholipid vesicles prepared from them.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Alteration of fatty acid composition of LM cells by lipid supplementation and temperature.

Alteration of the fatty acid composition of monolayer cultures of LM cells grown in chemically defined medium was achieved by supplementation with fatty acids complexed to bovine serum albumin. Phospholipids containing up to 40% linoleate were found in cells grown in medium containing 20 mu g of linoleate/ml. Incorporation of linoleate into phospholipids reached a plateau after 12-24 hr, and cells remained viable for at least 3-4 days. Although linoleic, linolenic, and arachidonic acids were incorporated into LM cells equally well, only the latter was elongated by these cells under these experimental conditions. Nonadecanoic acid was incorporated to a lesser extent than the polyunsaturated fatty acids. Phosphatidylcholine and phosphatidylethanolamine of LM cells had different fatty acid compositions; phosphatidylethanolamine contained more longer chain and unsaturated fatty acids. Cells were also grown in the absence of choline and presence of choline analogs such as N,N-dimethylethanolamine, N-methylethanolamine, 3-amino-1-propanol, and 1-2-amino-1-butanol. The analog phospholipids in these cells had fatty acid compositions which were intermediate between those of phosphatidylethanolamine and phosphatidylcholine of control cells grown in the presence of choline. Linoleate was found in both phosphatidylcholine and phosphatidylethanolamine of cells supplemented with linoleate. The sphingolipid fraction of these cells, however, did not contain significant amounts of linoleate. When linoleate was present in the phospholipids, compensatory decreases in the oleate and palmitoleate content of phospholipids were observed. Lowering of the growth temperature to 28 degrees produced an increase in unsaturate fatty acid content of the phospholipids. When linoleate was supplied to cells grown at 28 degrees, there was no further increase in the unsaturated fatty acid composition of the phospholipids. Using both fatty acid supplementation and lowered growth temperature, LM cell membranes can be produced which have phospholipids with vastly different fatty acid compositions.