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     

Fatty acid and phospholipid composition of five psychrotrophic Pseudomonas spp. grown at different temperatures

The free fatty acid and phospholipid composition of 5 psychrotrophic marine Pseudomonas spp. have been determined in chemostat culture with glucose as the limiting substrate over the range 0–20°C. The predominant fatty acid present in all the isolates was hexadecenoic acid (C16:1) together with lesser quantities of octadecenoic acid (C 18:1) whilst none contained acids with chain lengths exceeding 18 carbon atoms. Decreasing the growth temperature from 20°C to 0°C resulted in little significant change in fatty acid composition. The principal phospholipid components of the five psychrotrophic pseudomonads have been identified as phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Decreasing the growth temperature did not elicit significant changes either in the total quantities of phospholipid synthesized or in the concentration of individual phospholipid components in any of the isolates. All the psychrotrophs showed maximum glucose uptake between 15°C and 20°C and the rate decreased rapidly as the temperature was decreased towards 0°C.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol     

A moderate change in temperature induces changes in fatty acid composition of storage and membrane lipids in a soil arthropod

A moderate change in ambient temperature can lead to vital physiological and biochemical adjustments in ectotherms, one of which is a change in fatty acid composition. When temperature decreases, the composition of membrane lipids (phospholipid fatty acids) is expected to become more unsaturated to be able to maintain homeoviscosity. Although different in function, storage lipids (triacylglycerol fatty acids) are expected to respond to temperature changes in a similar way. Age-specific differences, however, could influence this temperature response between different life stages. Here, we investigate if fatty acid composition of membrane and storage lipids responds similarly to temperature changes for two different life stages of Orchesella cincta. Juveniles and adults were cold acclimated (15 °C → 5 °C) for 28 days and then re-acclimated (5 °C → 15 °C) for another 28 days. We found adult membranes had a more unsaturated fatty acid composition than juveniles. Membrane lipids became more unsaturated during cold acclimation, and a reversed response occurred during warm acclimation. Membrane lipids, however, showed no warm acclimation, possibly due to the moderate temperature change. The ability to adjust storage lipid composition to moderate changes in ambient temperature may be an underestimated fitness component of temperature adaptation because fluidity of storage lipids permits accessibility of enzymes to energy reserves.     

Change in chemical composition of membrane of Bacillus caldotenax after shifting the growth temperature

Membranes from Bacillus caldotenax contain neutral lipids and phospholipids such as phosphatidylethanolamine, phosphatidyl glycerol and cardiolipin. Each of the lipids has almost the same fatty acid composition. When the growth temperature decreases, not only the fatty acid composition but also the lipid composition changes such that the membrane fluidity increases, and the composition of membrane-bound proteins also changes. On shifting the growth temperature from 65° to 45°C, the bacterium grows immediately with a doubling time at 45°C, but the compositions of proteins and lipids in membranes gradually change and reach the compositions typical of cells growing at 45°C one doubling time after the temperature shift, respectively. It is concluded that the change in chemical composition of membrane of the bacterium on the temperature shift from 65° to 45°C is not prerequisite for growth at 45°C.     

Altered phospholipid metabolism in a temperature-sensitive mutant of a thermophilic bacillus

The phospholipid metabolism of a temperature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58°C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In ³²P incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 min after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative conversion of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chloramphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mechanism(s) of phospholipid metabolism in the mutant at the restrictive temperature.Nonstandard Abbreviations WT parental strain, thermophilic bacillus - TS-13 temperature-sensitive mutant of a thermophilic bacillus - CL cardiolipin - PG phosphatidylglycerol - PE phosphatidylethanolamine - l-CL lyso-cardiolipin     

Overproduction of cis-Vaccenic Acid and Altered Temperature Control of Fatty Acid Synthesis in a Mutant of Escherichia coli

A mutant of Escherichia coli has been characterized which overproduces cis-vaccenic acid in the temperature range of 30 to 42°C. The mutational lesion acts within the fatty acid biosynthetic pathway rather than at the level of fatty acid incorporation into phospholipid.     

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

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.     

A temperature sensitive nonsense mutation affecting the synthesis of a major protein of Escherichia coli K12

Summary A temperature sensitive nonsense (TSN) mutant of E. coli K12 has been isolated in which a major bacterial protein is not synthesized at 42° C. This protein is found in the parental strain at 42° C and in cells rendered temperature resistant due to the insertion of a number of different nonsense suppressors or the normal allele of the mutant locus.     

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

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     

Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria

The lipid content and fatty acid composition of a strain ofBacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio inB. caldolyticus between 60°C and 100°C and in strain NZ-2 between 45°C and 70°C were minor; inB. f avothermus the alterations in the 50°C–70°C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant inB. caldolyticus, especially between 80°C and 95°C. In contrast, the main changes inB. flavothermus and NZ-2 were found to occur between 30°C and 50°C, and between 45°C and 60°C, respectively. Based on these data, strain NZ-2 could be characterized as the least andB. flavothermus as the most versatile of the three organisms.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

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.     

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii

Summary A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii, OS15, was isolated, which required high salt or sugar concentration for growth above 30°C. Cell viability at 35°C in the presence of NaCl was higher than in the absence of NaCl, and a survival ratio of the mutant cells after incubation at 55°C was also higher in the presence of NaCl than NaCl-free condition. Furthermore, resistance to UV light, hygromycin B and geneticin was improved in the presence of NaCl. There was no difference between the parent and the mutant in fatty acid saturation and microscopic cell shape under NaCl condition.     

Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli.

Escherichia coli K-12 derivatives with a common genetic background carrying, either alone or in combination, the pss-1 allele coding for a temperature-sensitive phosphatidylserine synthase (A. Ohta and I. Shibuya, J. Bacteriol. 132:434-443, 1977) and cls- for a defective cardiolipin synthase (G. Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) were constructed. The phospholipid polar headgroup compositions of these strains were significantly different from each other depending on their genotypes and growth temperature, whereas other membrane characteristics such as the total phospholipid content, fatty acid composition, membrane protein profile, and lipopolysaccharide content were practically the same, suggesting that the phenotypes of these strains were the direct consequences of abnormalities in membrane phospholipid composition. The cls pss-1 double mutation caused an unusual accumulation of phosphatidylglycerol with an extremely low content of cardiolipin. The cls mutation alone was found to give a growth defect, and its introduction into a pss-1 mutant resulted in an enhanced temperature sensitivity of growth. Addition to a broth medium of a proper concentration of sucrose, NaCl, Mg2+, or Ca2+ allowed the growth of a pss-1 mutant at otherwise nonpermissive temperature, but a pss-1 cls double mutant required the combined addition of sucrose or NaCl and MgCl2 for full growth at 42 degrees C. The possible mechanisms for these physiological consequences of the mutations are discussed on a molecular basis. The remedial effects of culture supplements allowed the pss-1 mutants to grow at 42 degrees C resulting in enhanced abnormalities of membrane phospholipid composition.     

Effect of restrictive temperature on cell wall synthesis in a temperature-sensitive mutant of Bacillus stearothermophilus.

A temperature-sensitive mutant of Bacillus stearothermophilus, TS-13, was unable to grow above 58 degrees C, compared to 72 degrees C for the wild type. Actively growing TS-13 cells lysed within 2 h when exposed to a restrictive temperature of 65 degrees C. Peptidoglycan synthesis stopped within 10 to 15 min postshift before a shut down of other macromolecular syntheses. Composition of preexisting peptidoglycan was not altered, nor was new peptidoglycan of aberrant composition formed. No significant difference in autolysin activity was observed between the mutant and the wild type at 65 degrees C. Protoplasts of TS-13 cells were able to synthesize cell wall material at 52 degress C, but not at 65 degrees C. This wall material remained closely associated with the cell membrane at the outer surface of the protoplasts, forming small, globular, membrane-bound structures which could be visualized by electron microscopy. These structures reacted with fluorescent antibody prepared against purified cell walls. Production of this membrane-associated wall material could be blocked by bacitracin, which inhibited cell wall synthesis at the level of transport through the membrane. The data were in agreement with previous studies showing that at the restrictive temperature this mutant is unable to alter its membrane fatty acid and phospholipid composition with temperature such that it is not able to maintain a membrane lipid composition which permits normal membrane function at the restrictive temperature.     

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12

Summary A mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.