Cold adaptation in the Antarctic Archaeon Methanococcoides burtonii involves membrane lipid unsaturation

Direct analysis of membrane lipids by liquid chromatography-electrospray mass spectrometry was used to demonstrate the role of unsaturation in ether lipids in the adaptation of Methanococcoides burtonii to low temperature. A proteomics approach using two-dimensional liquid chromatography-mass spectrometry was used to identify enzymes involved in lipid biosynthesis, and a pathway for lipid biosynthesis was reconstructed from the M. burtonii draft genome sequence. The major phospholipids were archaeol phosphatidylglycerol, archaeol phosphatidylinositol, hydroxyarchaeol phosphatidylglycerol, and hydroxyarchaeol phosphatidylinositol. All phospholipid classes contained a series of unsaturated analogues, with the degree of unsaturation dependent on phospholipid class. The proportion of unsaturated lipids from cells grown at 4 degrees C was significantly higher than for cells grown at 23 degrees C. 3-Hydroxy-3-methylglutaryl coenzyme A synthase, farnesyl diphosphate synthase, and geranylgeranyl diphosphate synthase were identified in the expressed proteome, and most genes involved in the mevalonate pathway and processes leading to the formation of phosphatidylinositol and phosphatidylglycerol were identified in the genome sequence. In addition, M. burtonii encodes CDP-inositol and CDP-glycerol transferases and a number of homologs of the plant geranylgeranyl reductase. It therefore appears that the unsaturation of lipids may be due to incomplete reduction of an archaeol precursor rather than to a desaturase mechanism. This study shows that cold adaptation in M. burtonii involves specific changes in membrane lipid unsaturation. It also demonstrates that global methods of analysis for lipids and proteomics linked to a draft genome sequence can be effectively combined to infer specific mechanisms of key biological processes.     

Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii.

The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor. The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures. Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C. Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M. jannaschii's growth temperatures. Specifically, pressure shifted the temperature dependence of lipid fluidity by ca. 10 degrees C/500 atm.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.     

Effects of the temperature range and the lack of beta-ketoacyl acyl-carrier protein synthase II on fatty acid synthesis in Escherichia coli K12 after shifts in temperature

Escherichia coli K12 cells grown at higher temperatures and then subjected to lower temperatures produce fatty acids with higher unsaturated/saturated ratios than cells completely adapted to the lower temperatures (Okuyama et al. (1982) J. Biol. Chem. 257, 4812-4817). This hyper-response was not an artefact of chloramphenicol treatment and was observed when the shift-down was more than 20 degrees C in the cells grown at either 40 degrees C or 35 degrees C. In contrast, cells grown at either 25 degrees C or 30 degrees C showed no appreciable hyper-response in terms of unsaturated/saturated ratio on temperature shifts to as low as 10 degrees C. By combining shift-down and shift-up experiments, we could show the presence of different types of temperature dependency in the fatty acid-synthesizing systems of cells grown at various temperatures. Contrary to wild-type cells which synthesized mainly cis-vaccenate on down-shift to 10 degrees C, a mutant strain lacking beta-ketoacyl acyl-carrier protein synthase II synthesized more palmitoleate (16:1) and less palmitate at 10 degrees C than at 40 degrees C. The average chain lengths of saturated and unsaturated fatty acids also changed, but differently, between the mutant and wild-type cells on shifts of temperature. Thus, the mutant strain has a temperature-dependent fatty acid-synthesizing system qualitatively different from that seen in a wild-type strain.     

Positive and negative tandem mass spectrometric fingerprints of lipids from the halophilic Archaea Haloarcula marismortui

Lipids from the extremely halophilic Archaea, Haloarcula marismortui, contain abundant phytanyl diether phospholipids, namely archaetidic acid (AA), archaetidylglycerol (AG), archaetidylglycerosulfate (AGS), with mainly archaetidylglycerophosphate methyl ester (AGP-Me). These were accompanied by a triglycosyl archaeol (TGA), lacking characteristic sulfate groups. Tandem-mass spectrometry was employed to provide fingerprints for identifying these known lipids, as well as small amounts of unsaturated phospholipids. These contained 3 and 6 double bonds in their archaeol moiety, suggested by negative tandem-MS of intact phospholipids, as indicated by differences between their pseudo-molecular ion and specific fragment ions designated as π₂. The core ether lipids were confirmed by electrospray ionization mass spectrometry (ESI-MS) as 2,3-di-O-phytanyl-sn-glycerol (C20, C20), which gave rise to a precursor-ion at m/z 660 [M+Li]⁺, and its fragment ion at m/z 379 [M+Li]⁺, consistent with mono-O-phytanyl-glycerol. Furthermore, lithiated ions at m/z 654 (MS¹), 379 (MS²) and m/z 648 (MS¹), 373 (MS²), combined with ¹H/¹³C NMR chemical shifts at δ 5.31-121.6 (C2/2′-H2/2′), 5.08-124.9 (C6/6′-H6/6′) and 5.10-126.0 (10/10′-H10/10′) confirmed the presence of unsaturated homologs of archaeol. We carried out a comprehensive study on the lipids present in cells of H. marismortui. We used positive and negative ESI-MS with tandem-MS, which served as a fingerprint analysis for identifying the majority of component lipids.     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.     

Studies of the fatty acid composition and membrane microviscosity in Salmonella typhimurium TA98

When the mutagen tester bacterial strain Salmonella typhimurium TA 98 was grown at different temperatures, we found that the unsaturated fatty acid composition increased at the lower growth temperatures. Membrane microviscosity, as assessed with spin-probe fatty acids using electron spin resonance, decreased as the unsaturated fatty acid content increased. These findings are of importance in understanding our recent observation that the mutagenic response of these bacteria was increased when they were grown at 27 degrees C vs. 37 degrees C, and indicate that membrane properties may play an important role in the sequence of events leading to mutagenesis.     

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.     

Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids. Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids. In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease. Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility. Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h. The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C. No phase transition was detected between 5 and 50 degrees C as measured with both probes. In accordance with these results, no significant release of the trapped CF in this range of temperature was detected. Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane. This effect was lower with KCl, while CaCl2 did not cause any change. The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio. NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2. The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin. It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones. These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses.     

Fatty acid fingerprints of Streptococcus mutans NCTC 10832 grown at various temperatures.

Fatty acid fingerprints were determined gas chromatographically for Strepcococcus mutans NCTC 1082 which had been grown in batch brain heart infusion at a series of nine temperatures ranging from 29.0 to 40.0 degrees C. The major acids at all temperatures were n-palmitic and octadecenoic acids. Other acids detected at all temperatures included n-myristic, palmitoleic, n-stearic, and eicosenoic acids. An increase in temperature resulted in a decrease in the proportion of unsaturated to saturated fatty acids, indicating the importance of accurate temperature control in such gas-liquid chromatographic, chemotaxonomic studies.     

The tolerance of cyanobacterium Cylindrospermopsis raciborskii to low-temperature photo-inhibition affected by the induction of polyunsaturated fatty acid synthesis

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation patterns of the glycerolipids of Cylindrospermopsis raciborskii, a filamentous cyanobacterium, were determined in cells grown at 35 degrees C and 25 degrees C. The lowering of the growth temperature from 35 degrees C to 25 degrees C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. The tolerance to low-temperature photo-inhibition of the C. raciborskii cells grown at 25 degrees C and 35 degrees C was also compared. The lower growth temperature increased the tolerance of C. raciborskii cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii in algal blooming in Lake Balaton (Hungary).     

Effect of growth temperature on membrane dynamics in a thermophilic cyanobacterium: a spin label study

A strain of Synechococcus sp. was grown at its optimal growth temperature (58 degrees C) and at 38 degrees C, in order to investigate possible adaptations of membrane-related properties to growth temperature. Light-induced electron transport in thylakoid membranes from both types of cells showed linear Arrhenius plots with the same activation energy (48 kJ/mol). Membranes from cells grown at 58 degrees C had a higher temperature optimum (53 degrees C) than those from cells grown at 38 degrees C (41 degrees C). Growth at 38 degrees C caused an increase in the proportion of unsaturated fatty acids compared to growth at 58 degrees C. The fluidity of the membranes was investigated by measuring the temperature dependence of the parameters derived from electron spin resonance spectra of the spin-labels 5-doxyldecane, 5-doxylstearate and 16-doxylstearate. Only small differences between the dynamic properties of the membranes from cells grown at different temperatures could be detected. This suggests that the observed change in fatty acid composition of the membranes following the change in growth temperature does not serve to maintain a constant viscosity at the growth temperature.     

Effect of lipid composition on the stability of cellular membranes during freeze-thawing of Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at the optimal temperature of 37 degrees C (M37) appeared more sensitive to freeze-thawing than when it was grown at 25 degrees C (M25). In the first case, 87% of the cells died, in contrast to 33% for cells grown at 25 degrees C. All the surviving M37 cells showed sensitivity to NaCl. However, among the surviving M25 cells, only 85% were sensitive to NaCl. The rest of the cells were considered uninjured. Freeze-thawing in cells grown at 25 degrees C showed a liberation of nucleic acids and proteins. However, the leakage was higher in M37 cells after freeze-thawing. The greater fraction of damaged cells were observed in M25 culture after freeze-thawing. A relative increase of 81% in cardiolipid (CL), with respect to total phospholipids and 72% triglycosyldiglyceride (TGDG) with respect to the total glycolipids was observed in M37. In addition, a decrease of palmitoyl (C16:0), oleoyl (C18:0) fatty acids at CL, phosphatidylglycerol (PG), and diglicosyldiglyceride (DGDG) fractions and the increase of C19 cyc and C18:0, 10-OH fatty acids in neutral lipid, and CL fractions was also apparent. In M25 cells, the concentration of DGDG and PG was higher than in M37 cells. The difference in cryotolerance between the frozen cultures emphasizes the importance of selecting appropriate conditions of growth of microorganisms for use as dietary adjuncts.     

Involvement of membrane lipids in radiation damage to potassium ion permeability of Escherichia coli.

Radiation damage to K+ permeability of an unsaturated fatty acid auxotroph of E. coli grown with oleate or linolenate was investigated at different temperatures. A remarkable effect of radiation was observed at 0 degrees C with cells that had been grown with linolenate at 42 degrees C. This indicates that, besides protein, membrane lipids at least are involved in the radiation damage. The damage also seems to be affected by the fluidity of membrane lipids.     

Effect of Growth Temperature on the Lipid Composition of Cyanidium caldarium: II. Glycolipid and Phospholipid Components

Cyanidium caldarium was grown at 20 and 55 C and harvested during exponential growth phase. Lipids were extracted and separated by silicic acid column and thin layer chromatography. The major glycolipids were identified as mono- and digalactosyl diglyceride and sulfolipid. Major phospholipids were identified as phosphatidyl choline and phosphatidyl ethanolamine. The cells grown at 20 C contained significantly larger quantities of these glycolipids and phospholipids than cells grown at 55 C.     

Effect of temperature shifts on gliding motility, adhesion, and fatty acid composition of Cytophaga sp. strain U67.

Gliding motility and flipping of 25 degrees C-adapted Cytophaga sp. strain U67 were inhibited when the bacteria were shifted to a less than or equal to 12 degrees C environment; motility was not blocked by a shift to 13 degrees C. Bacteria adapted to 4 degrees C were motile over the entire 4 to 25 degrees C temperature range tested. U67 adhesion to the substratum appeared to be unaffected by temperature shifts. Bacteria adapted to 4 degrees C had higher proportions of unsaturated and branched-chain fatty acids than did those grown at 25 degrees C. When 25 degrees C-adapted bacteria were subjected to a gradual temperature decline, the time of reappearance of gliding competence at 4 to 5 degrees C was correlated with these changes in fatty acid composition.     

Pseudomonas putida NCTC 10936 balances membrane fluidity in response to physical and chemical stress by changing the saturation degree and the trans/cis ratio of fatty acids

This study explored the capability of Pseudomonas putida NCTC 10936 to maintain homeoviscosity after changing the growth temperature, incubating resting cells at different temperatures or at a constant temperature in the presence of 4-chlorophenol (4-CP). After raising the growth temperature from 20 to either 30 or 35 degrees C, the degree of saturation of the organism's fatty acids increased and the ratio of trans to cis unsaturated fatty acids decreased somewhat. In contrast, after the incubation temperature of resting cells was raised (grown at 30 degrees C) from 20 to 30 or 35 degrees C the degree of saturation of the fatty acids remained nearly constant, while the ratio of trans to cis unsaturated fatty acids increased. Incubating resting cells (grown at 30 degrees C) at 20 degrees C in the presence of 4-CP again caused no major changes in the degree of saturation, but cis to trans conversion of unsaturated fatty acids was induced, with a corresponding increase in the trans/cis ratios. Increases in both the saturation degree of the fatty acids and the trans/cis ratio of the unsaturated fatty acids correlated with increases in the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene intercalated in the bilayers of liposomes prepared from the cells of P. putida NCTC 10936. Electron transport phosphorylation (ETP) could be stabilized by adaptive adjustments in the fluidity of the cytoplasmic membrane mediated by changes in fatty acid composition such as those observed. Whether changes in the degree of saturation or in the trans/cis ratio are more effective can be decided by studying P. putida NCTC 10936.     

Requirement for a fluid host cell membrane in injection of coliphage T5 DNA.

Injection of T5 first-step-transfer DNA was prevented at 29 degrees C, after adsorption to an unsaturated fatty acid mutant grown on elaidate (phase transition at 35 degrees C). Local anesthetics, which increase membrane fluidity, did not inhibit injection above transition temperature and could even reverse the inhibition observed at 29 degrees C on elaidate cells.     

ESR studies on the membrane properties of a moderately halophilic bacterium. II. Effect of extreme growth conditions on liposome properties

Lipid preparations from the cells of a moderately halophilic bacterium, Pseudomonas halosaccharolytica grown under the two extreme conditions of high temperature-high NaCl concentration and low temperature-low NaCl concentration showed distinctively different profiles in phospholipid and fatty acid composition. Cells grown at 40 degrees C in medium containing 3.5 M NaCl had high concentrations of saturated and C19 cyclopropanoic fatty acids (about 50 per cent of the total), whereas cells grown at 20 degrees C in medium containing 0.5 M NaCl had decreased concentrations of these fatty acids with increased concentrations of the corresponding unsaturated fatty acids. The phospholipid composition was also affected ty the culture conditions; cells grown at 40 degrees C in 3.5 M NaCl had large amounts of acidic phospholipids, whereas those grown at 20 degrees C in 0.5 M NaCl had small amounts. ESR studies on liposomes prepared from lipids of cells grown under the two conditions showed characteristic profiles for correlation times and order parameters of three spin labels of stearic acid derivatives similar to those of membranes of whole cells of this bacterium. ESR studies showed that the physical properties of the liposomes from the total extractable lipids and isolated phosphatidylglycerol from the cells were completely different from those of synthetic dioleoylphosphatidylglycerol. Liposomes of the lipids extracted from cells grown at 40 degrees C in 3.5 M NaCl showed change in rotational viscosity on altering the NaCl concentration to 0.5M, whereas liposomes of lipids extracted from cells grown at 20 degrees C in 0.5 M NaCl did not show change in rotational viscosity on increasing the NaCl concentration to 3.5 M.