Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

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

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

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Effects of growth temperature on transport and membrane viscosity in Streptococcus faecalis

A shift in the growth temperature of Streptococcus faecalis from 37 to 10°C resulted in an 18% increase in the proportion of unsaturated fatty acids. Electron spin resonance spectra of spin-labeled membranes and extracted phospholipids indicated viscosity changes consistent with the alterations in fatty acid composition. Growth temperature had no significant effect on the active transport of leucine and alanine; uptake rates assayed at 10 or 35°C were essentially the same in cells grown at either 10 or 37°C. The relative rapidity of amino acid transport, which presumably contributes to the ability of S. faecalis to thrive in cold environments, is evidently unrelated to adaptive changes in the viscosity of membrane lipids.Abbreviations doxyl 4-4-dimethyloxazolidine-N-oxyl - proxyl 2,2-disubstituted 5,5-dimethylpyrrolidine-N-oxyl     

Dietary fats,selected body temperature and tissue fatty acid composition of agamid lizards (Amphibolurus nuchalis)

The composition of tissue and membrane fatty acids in ectothermic vertebrates is influenced by both temperature acclimation and diets. If such change in body lipid composition and thermal physiology were linked, a diet-induced change in body lipid composition should result in a change in thermal physiology. We therefore investigated whether the selected body temperature of the agamid lizardAmphibolurus nuchalis (body mass 20 g) is influenced by the lipid composition of dietary fatty acids and whether diet-induced changes in thermal physiology are correlated with changes in body lipid composition. The selected body temperature in two groups of lizards was indistinguishable before dietary treatments. The selected body temperature in lizards after 3 weeks on a diet rich in saturated fatty acids rose by 2.1 °C (photophase) and 3.3 °C (scotophase), whereas the body temperature of lizards on a diet rich in unsaturated fatty acids fell by 1.5 °C (photophase) and 2.0 °C (scotophase). Significant diet-induced differences were observed in the fatty acid composition of depot fat, liver and muscle. These observations suggest that dietary lipids may influence selection of body temperature in ectotherms via alterations of body lipid composition.Abbreviations bm body mass - FA fatty acid(s) - MUFA monounsaturated fatty acids - PUFA polyunsaturated fatty acids - SFA saturated fatty acids - T _a air temperature - T _b body temperature - UFA unsaturated fatty acids     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.     

Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli

In contrast to stringent (relA⁺) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (-lactamase, interferon 1) into the culture medium during amino acid limitation. Comparative analyses of overall fatty acid composition in relA⁺ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response. The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17_cyc and C19_cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased. In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset amino acid limitation. These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.     

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.     

Biochimica et Biophysica Acta: Vol. 307 (1973)

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ($\text{12-Me-14:0}$) or $\text{cis-Δ}{}^9\text{-}\text{octadecenoate}$ ($\text{cis-δ}{}^9\text{-18:1}$). When this strain is grown for many generations on $\text{12-Me-14:0}$, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of $\text{12:0}$ (8%) and $\text{14:0}$ (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.     

The degree of dietary fatty acid unsaturation affects torpor patterns and lipid composition of a hibernator

Diets rich in unsaturated and polyunsaturated fatty acids have a positive effect on mammalian torpor, whereas diets rich in saturated fatty acids have a negative effect. To determine whether the number of double bonds in dietary fatty acids are responsible for these alterations in torpor patterns, we investigated the effect of adding to the normal diet 5% pure fatty acids of identical chain length (C18) but a different number of double bonds (0, 1, or 2) on the pattern of hibernation of the yellow-pine chipmunk, Eutamias amoenus. The response of torpor bouts to a lowering of air temperature and the mean duration of torpor bouts at an air temperature of 0.5°C (stearic acid C18:0, 4.5±0.8 days, oleic acid C18:1, 8.6±0.5 days; linoleic acid C18:2, 8.5±0.7 days) differed among animals that were maintained on the three experimental diets. The mean minimum body temperatures (C18:0, +2.3±0.3°C; C18:1, +0.3±0.2°C; C18:2,-0.2±0.2°C), which torpid individuals defended by an increase in metabolic rate, and the metabolic rate of torpid animals also differed among diet groups. Moreover, diet-induced differences were observed in the composition of total lipid fatty acids from depot fat and the phospholipid fatty acids of cardiac mitochondria. For depot fat 7 of 13 and for heart mitochondria 7 of 14 of the identified fatty acids differed significantly among the three diet groups. Significant differences among diet groups were also observed for the sum of saturated, unsaturated and polyunsaturated fatty acids. These diet-induced alterations of body fatty acids were correlated with some of the diet-induced differences in variables of torpor. The results suggest that the degree of unsaturation of dietary fatty acids influences the composition of tissues and membranes which in turn may influence torpor patterns and thus survival of hibernation.Abbreviations bm body mass - T _a air temperature - T _b body temperature - FA fatty acid - MR metabolic rate - MUFA monounsaturated fatty acids - PUFA polyunsaturated fatty acids - VO₂ rate of oxygen consumption - SFA saturated fatty acids - UFA unsaturated fatty acids - UI unsaturation index - SNK Student-Newman-Keuls test     

Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum

Summary In order to improve the economic value of lipids produced by the oleaginous yeast strain Apiotrichum curvatum ATCC 20509, a search was made for mutants defective in the conversion of stearic acid to oleic acid. Mutants could be selected as unsaturated fatty acid auxotrophs, since unsaturated fatty acids are essential componenets in membrane lipids. After treatment of A. curvatum wild-type with N-methyl-N-nitro-N-nitrosoguanidine, 58 fatty-acid-requiring mutants were isolated. On the basis of (1) the growth response to saturated and unsaturated fatty acids and (2) the fatty acid composition of lipids produced by these mutants, it was concluded that only 18 of them were real unsaturated fatty acid (Ufa) mutants, while the other 40 were designated as fatty acid synthetase (Fas) mutants. It is further shown that Ufa mutants of A. curvatum are able to produce high amounts of lipids consisting of more than 90% triacylglycerols with a percentage of saturated fatty acids resembling that of cocoa butter, when grown in the presence of relatively small amounts of oleic acid in the growth medium. This may offer an economically favourable alternative in comparison with other methods that have been developed for the production of cocoa butter equivalents by microorganisms.Offprint requests to: H. Smit     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

Summary The effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.     

Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Physical properties of murine fibroblasts with altered acyl chain unsaturation.

Murine fibroblasts, LM cells, were cultured in suspension with laurate (12:0), myristate (14:0), palmitate (16:0), palmitoleate (16:1), or palmitate + palmitoleate (16:0 + 16:1) bound to fatty acid-free bovine serum albumin. Supplementation with saturated fatty acids decreased the ratio of unsaturated/saturated fatty acids in membrane phospholipids as much as 3.4-fold (palmitate-enriched cells). Concomitantly fluorescence polarization, absorption-corrected fluorescence, and relative fluorescence efficiency of the fluorescence probe molecule, β-parinaric acid, increased 1.5-, 2.9-, and 1.8-fold, respectively, in the membrane phospholipids. Unsaturated fatty acid (palmitoleate) increased the unsaturated/saturated fatty acid ratio by 20% but did not significantly alter the fluorescence parameters. When the cells were fed mixtures of palmitate and palmitoleate, the unsaturated/saturated fatty acid ratio of the membrane phospholipids and the above fluorescence parameters had values intermediate between those if each fatty acid had been fed separately. All fatty acid supplements caused a loss of two characteristic temperatures in Arrhenius plots of relative fluorescence efficiency. However, no shifts or appearance of new characteristic temperatures occurred. The break points at approximately 42, 37, and 22 °C were essentially un-altered. The data were consistent with the possibility that LM cells were unable to maintain constant fluidity, as indicated by fluorescence polarization, when supplemented with different fatty acids. A good correlation could be made between the phospholipid unsaturated/ saturated fatty ratio, the fluorescence polarization, and the toxicity elicited by different fatty acid supplements.     

Properties of Bacillus acidocaldarius mutants deficient in ω-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in -cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing -cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.     

Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol