The relationship between environmental temperature,cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes.Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might be directly determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Gel to Liquid-Crystalline Phase Transitions of Lipids and Membranes Isolated from Escherichia coli Cells

Differential scanning calorimetry (DSC) was used to examine the relationship of the gel to liquid-crystalline phase transition of lipids to fatty acid composition with membrane lipids and spheroplast membranes isolated from cells of a wild strain and an unsaturated fatty acid auxotroph of Escherichia coli grown under various conditions. These lipids and membranes underwent thermotropic phase transitions at different temperatures depending on the thermal properties of their constituent fatty acids. The lipid phase transition occurred at higher temperatures in biomembranes than in extracted lipids. DSC thermograms of lipids synthesized by bacterial cells which were observed at a temperature scanning rate as slow as 0.3 K min^-1 were characterized by a distinctly plain peak summit. Endothermic peaks given by samples derived from elaidic acid-enriched cells were relatively narrow and asymmetric. The discrepancy between the transition temperatures measured with extracted lipids and with membraneous fractions, and the shape of the endothermic peaks, are discussed.     

The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus. Composition-structure-function relationships

The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var. nondiastaticus as test-organism. The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures. Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C. Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found. Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains. The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms. Unsaturated fatty acids were not produced by cells grown at 65 degrees C. In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C. The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures. Completion of the lipid melting occurred always at temperatures below those employed for growth. A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile. The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature. The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated. The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively. This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus.     

Characterization of membrane lipids of a general fatty acid auxotrophic bacterium by electron spin resonance spectroscopy and differential scanning calorimetry

Lipids in the plasma membrane of the general fatty acid auxotroph Butyrivibrio S2 pack as a bilayer that is characterized by a high order and high motional anisotropy and a low membrane fluidity compared to mammalian plasma membranes. Lipid packing as determined by the electron spin resonance (ESR) order parameter and membrane fluidity as measured by ESR correlation times are, however, comparable to those of other bacterial membranes. Membranes of the organism grown with saturated fatty acids of well-defined hydrocarbon chain length undergo a broad reversible endothermic phase transition, the peak temperature of which is well below the growth temperature; the end-point temperature of this thermal transition approximately coincides with the minimum temperature supporting significant growth of the organism. The lipid phase transition is also reflected in the temperature dependence of various ESR parameters, whereby the transition temperature thus derived is higher than the peak temperature of the endothermic transition but still lower than the growth temperature. ESR and calorimetry evidence taken together suggest that the endothermic transition is a gel to liquid-crystal transition and that, at the growth temperature, the plasma membrane of Butyrivibrio S2 is in the liquid-crystalline state. Similar values were measured for the order parameter of cell membranes of Butyrivibrio S2 regardless of whether the organism was grown on myristic, palmitic, or stearic acid. Butyrivibrio S2 has a mechanism enabling it to maintain membrane packing and fluidity at a fairly constant level.(ABSTRACT TRUNCATED AT 250 WORDS)     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

Spin-labeling studies on the lipids of psychrophilic, psychrotrophic, and mesophilic clostridia.

Spin-labeling studies were conducted to elucidate the viscosity and phase transition temperatures of lipids isolated from psychrophilic, psychrotrophic, and mesophilic clostridia. Electron spin resonance spectroscopy indicated that the lipids, for all the growth temperatures tested, were in a fluid state and from 13 to 24 C higher than the corresponding lipid transition temperatures. When the organisms were grown at different temperatures, a psychrotropic and two mesophilic clostridia were shown to be able to adjust their lipid-phase transition temperature to the growth temperature. A psychrophilic Clostridium strain, when grown at different temperatures, synthesized lipids that had the same phase transition temperature. It is suggested that this lack of growth temperature-inducible regulation of lipid-phase transition temperature may be a molecular determinant for the psychrophily of this organism. It is proposed that the growth temperature range of an organism is dependent upon the ability of the organism to regulate its lipid fluidity within a specific range.     

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane

Intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of Bacillus stearothermophilus. On the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. The excimerization rate is very sensitive to lipid phase transitions and pretransitions of synthetic pure lipid bilayers. In bacterial lipids from cultures grown at 55 and 68 degrees C, thermal profiles of excimer to monomer intensity ratios (I'/I) show a broad transition which is displaced to higher temperatures in response to the increase of the growth temperature; these results correlate well with differential scanning calorimetry data and fluorescence polarization of diphenylhexatriene. Additionally, lipid bilayers of bacteria grown at 68 degrees C exhibit a decreased membrane fluidity, as monitored by both fluorescent probes.     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

Phase properties of senescing plant membranes. Role of the neutral lipids

Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes.     

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The “PUFA index” -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.     

Regulation of fatty acid unsaturation in encystingAcanthamoeba cells

The degree of fatty acid unsaturation and average chain length are closely similar for microsomal membranes from exponential-phase trophozoites and cysts ofAcanthamoeba castellanii despite significant differences in fatty acid composition. The same trend was apparent for total fatty acids extracted from whole cells. The observations suggest that the organism regulates these lipid parameters during differentiation in order to maintain optimum membrane lipid viscosity, and are consistent with previous electron spin resonance measurements indicating that the fluidity of microsomal membranes does not change during encystment. About 75% of the microsomal fatty acids are unsaturated for both cysts and amoebae. Wide-angle X-ray diffraction of phospholipid liposomes prepared from lipid extracts of the membranes has indicted that this high level of unsaturation renders the phospholipid exclusively liquid-crystalline at temperatures as low as 9°C for rough microsomes and-1.5°C for smooth microsomes. Thus, by retaining a high proportion of unsaturated fatty acids throughout its differentiation cycle, the organism gains some protection in its natural soil habitat against lateral phase separation of membrane lipids.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Physical studies on membrane lipids of Bacillus stearothermophilus temperature and calcium effects

Bacillus stearothermophilus was grown at the optimal temperature range (center, 65 degrees C), below it (48 and 55 degrees C), and above it (68 degrees C), in a complex medium with or without 2.5 mM Ca2+. The Ca(2+)-supplement improves growth at sub- and supraoptimal temperatures and extends it to higher temperatures (Jurado et al. (1987) J. Gen. Microbiol. 133, 507-513). The phospholipid composition of cultures obtained in the different growth conditions was studied. Phosphatidylethanolamine was always the major phospholipid (40 to 50% of the total phospholipid). Diphosphatidylglycerol, phosphatidylglycerol, a phosphoglycolipid (pgl) and two minor phospholipids (not identified) were also found in the polar lipid extract. The pgl shows a threefold concentration increase as the growth temperature raises from 48 to 68 degrees C. The thermotropic behavior of membrane lipids was studied by differential scanning calorimetry (DSC) and by means of two fluorescent probes of fluidity, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(2-pyrenyl)propane (2Py(3)2Py). The results reveal similar features and clearly show a shift of the temperature range of the phase transition to higher values and an increased structural order of the bilayer, as the growth temperature rises from 55 to 68 degrees C, but an opposite effect was observed from 48 to 55 degrees C. Although the Ca(2+)-supplement to the growth medium has no detectable effect, the addition of Ca2+ to the buffer of liposomes (Ca(2+)-liposomes) has a significant ordering effect at all growth temperatures. These liposomes show a shift of the transition range to higher temperatures and the fluorescent parameters (DPH polarization and intramolecular excimerization of the 2Py(3)2Py) detected an order increase of the probes environment, along and above the main phase transition. Spectra of 31P-NMR and polarized light microscopy clearly show that the lipid extracts exhibit, in all the conditions, typical lamellar phase geometry. We concluded that B. stearothermophilus controls the membrane lipid composition to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packing of phospholipid molecules in a stable bilayer. At high temperatures, Ca(2+)-stimulatory effect on growth is presumably due to a direct Ca2+ interaction with the membrane phospholipids, inducing an increased structural order on the bilayer. The increase of the phase transition temperature in the total lipid extracts as compared with the respective polar lipid fractions probably indicates a stabilizing effect of neutral lipids on membrane bilayers.