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.     

Antibiotic AL-87 induction of changes in the fatty acid composition of phospholipids and neutral lipids in a sensitive strain of Staphylococcus aureus 209P

Under the action of the sub-bacteriostatic concentration (0.02 microgram/ml) of antibiotic AL-87 there formed in all the fractions of phospholipids and neutral lipids of S. aureus 209P unsaturated branched fatty acids not detected in the control and the content of shorter chain saturated branched fatty acids increased. This means that there were changes leading to increased lipid fluidity. The findings showed that fatty acids of the neutral lipids and phospholipids were involved in regulation of the bacterial cell functional state and participated in this case in providing increased membrane fluidity and permeability.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the n-3 and n-6 families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

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.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Adaptational changes in cellular phospholipids and fatty acid composition of the food pathogen Listeria monocytogenes as a stress response to disinfectant sanitizer benzalkonium chloride

Aims: This study provides a first approach to observing the alterations of the cell membrane lipids in the adaptation response of Listeria monocytogenes to the sanitizer benzalkonium chloride. Methods and Results: A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown when exposed to benzalkonium chloride is compared to cells optimally grown. The adaptation mechanism of L. monocytogenes in the presence of benzalkonium chloride caused (i) an increase in saturated‐chain fatty acids (mainly C_16:0 and C_18:0) and unsaturated fatty acids (mainly C_16:1 and C_18:1) at the expense of branched‐chain fatty acids (mainly C_a‐15:0 and C_a‐17:0) mainly because of neutral fatty acids; (ii) no alteration in the percentage of neutral and polar lipid content among total lipids; (iii) a decrease in lipid phosphorus and (iv) an obvious increase in the anionic phospholipids and a decrease in the amphiphilic phosphoaminolipid. Conclusions: These lipid changes could lead to decreased membrane fluidity and also to modifications of physicochemical properties of cell surface and thus changes in bacterial adhesion to abiotic surfaces. Significance and Impact of the Study: The adaptation and resistance of L. monocytogenes to disinfectants is able to change its physiology to allow growth in food‐processing plants. Understanding microbial stress response mechanisms would improve the effective use of disinfectants.     

Membrane deterioration in senescing carnation flowers : coordinated effects of phospholipid degradation and the action of membranous lipoxygenase

The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable change in fluidity was induced by in vitro aging of microsomal membranes from young flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heat-denatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated:unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both instances there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecting breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degradation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty acids from the membrane and an ensuing decrease in bulk lipid fluidity.     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Adaptation of biological membranes to temperature. The lack of homeoviscous adaptation in the sarcoplasmic reticulum

Temperature adaptation of biological membranes was examined by comparing the fragmented sarcoplasmic reticulum preparation of goldfish acclimated to different temperatures. Membrane fluidity was estimated using the fluorescence polarization technique. There was considerable variation between preparations, but no consistent differences in fluidity were observed between 5- and 25°C-acclimated goldfish, fish species adapted over an evolutionary period to arctic or desert temperatures, and rat. The fatty acid composition of the sarcoplasmic reticulum preparations of differently acclimated goldfish showed differences in the proportion of mono- and polyunsaturated fatty acids while the proportion of saturated fatty acids remained relatively constant. However, the fatty acid composition of sarcoplasmic reticulum phosphoglycerides became more unsaturated in the order rat, desert pupfish, arctic sculpin, which correlates with their respective environmental or body temperature. It is concluded that differences in membrane components other than fatty acids are important in determining membrane dynamic structure. The inability to demonstrate homeoviscous adaptation in sarcoplasmic reticulum is supported by other evidence suggesting that functions of the sarcoplasmic reticulum that are measured in vitro are not affected by such modifications of their phosphoglyceride fatty acid composition as occur during thermal acclimation.     

Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Abstract

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for β-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and α-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.     

Alteration of the phospho- or neutral lipid content and fatty acid composition in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> due to acid adaptation mechanisms for hydrochloric,acetic and lactic acids at pH 5.5 or benzoic acid at neutral pH

This study provides a first approach to observe the effects on Listeria monocytogenes of cellular exposure to acid stress at low or neutral pH, notably how phospho- or neutral lipids are involved in this mechanism, besides the fatty acid profile alteration. A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown at pH 5.5 in presence of hydrochloric, acetic and lactic acids, or at neutral pH 7.3 in presence of benzoic acid, is described relative to cells grown in acid-free medium. The results showed that only low pH values enhance the antimicrobial activity of an acid. We suggest that, irrespective of pH, the acid adaptation response will lead to a similar alteration in fatty acid composition [decreasing the ratio of branched chain/saturated straight fatty acids of total lipids], mainly originating from the neutral lipid class of adapted cultures. Acid adaptation in L. monocytogenes was correlated with a decrease in total lipid phosphorus and, with the exception of cells adapted to benzoic acid, this change in the amount of phosphorus reflected a higher content of the neutral lipid class. Upon acetic or benzoic acid stress the lipid phosphorus proportion was analysed in the main phospholipids present: cardiolipin, phosphatidylglycerol, phosphoaminolipid and phosphatidylinositol. Interestingly only benzoic acid had a dramatic effect on the relative quantities of these four phospholipids.     

Lipid Peroxides in the Free Radical Pathophysiology of Brain Diseases

1. Polyunsaturated fatty acids are essential for normal neural cell membrane functioning because many membrane properties, such as fluidity and permeability, are closely related to the presence of unsaturated and polyunsaturated side chains. Lipid peroxidation results in loss of membrane polyunsaturated fatty acids and oxidized phospholipids as polar species contributing to increased membrane rigidity.2. Polyunsaturated fatty acids are released from membrane phospholipids by a number of enzymic mechanisms involving the receptor-mediated stimulation of phospholipase A₂ and phospholipase C/diacylglycerol lipase pathways.3. The overstimulation of excitatory amino acid (EAA) receptors stimulates the activities of lipases and phospholipases, and this stimulation produces changes in membrane phospholipid composition, permeability, and fluidity, thus decreasing the integrity of plasma membranes.4. Alterations in properties of plasma membranes may be responsible for the degeneration of neurons seen in neurodegenerative diseases. Two major processes may be involved in neuronal injury caused by the overstimulation of EAA receptors. One is a large Ca²⁺ influx and the other is an accumulation of free radicals and lipid peroxides as a result of neural membrane phospholipid degradation. It is suggested that calcium and free radicals act in concert to induce neuronal injury in acute trauma (ischemia and spinal cord injury) and in neurodegenerative diseases.     

Cold hardening induces transfer of fatty acids between polar and nonpolar lipid pools in the Arctic collembollan Megaphorura arctica

Cold hardiness in the Arctic Collembola Megaphorura arctica (Tullberg), formerly Onychiurus arcticus, has been the subject of extensive studies over the last decade. This species employs an unusual strategy known as cryoprotective dehydration to survive winter temperatures as low as ?25 °C. To expand knowledge of cryoprotective dehydration in M. arctica, the present study investigates how a reduction in ambient temperature affects the fatty acid composition of the total body lipid content along with polar (mainly membrane phospholipids) and nonpolar (mainly triacylglycerols) lipids. Most ectothermic animals compensate for changes in fluidity by regulating fatty acid composition, a process often described as homeoviscous adaptation. In M. arctica, changes in the fatty acid composition of total body lipid content during cold treatment are only moderate, with no clear pattern emerging. However, the levels of unsaturated fatty acids in the polar lipids increase with cold exposure, largely attributable to 16 : 1(n? 7), 18 : 1(n? 9), 18 : 3(n? 6) and 18 : 3(n? 3), whereas unsaturated fatty acid levels in the nonpolar lipids correspondingly decrease. These results suggest a reallocation of fatty acids between the two lipid pools as a response to a temperature reduction of 6 °C. Because of hypometabolism, a characteristic of cold adaptation, such a mechanism could be less energy demanding than de novo synthesis of fatty acids and may comprise part of an adaptive homeostatic response.     

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 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 directly be 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.     

Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C_15:1 iso G, C_15:0 iso, C_15:0 anteiso, C_15:1 ω6c, C_15:0 iso 3OH, C_17:1 ω6c, C_16:0 iso 3OH and C_17:0 iso 3OH, summed features 3 (C_16:1 ω7cand/or C_16:1 ω6c) and 9 (C_16:0 10-methyl and/or C_17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.