Trypanosoma cruzi: growth requirements at different temperature in fetal bovine serum or peptide supplemented media

An enriched synthetic medium with low molecular weight peptides allows Trypanosoma cruzi epimastigotes to grow at 26-37 C. Using this medium, the growth requirements of T. cruzi were compared at different temperatures. When supplemented with fetal bovine serum or serum peptides, nine amino acids were absolutely required from the first passage, while additional amino acids and amino acid precursors were needed to support growth during a second passage. Five amino acids (beta-alanine, glutamine, cysteine, ornithine, and threonine) were also required absolutely at temperatures ranging between 30 and 37 C. Nine vitamins were needed at all temperatures, while ascorbic acid and ergocalciferol were not necessary at any temperature. The remaining amino acids and vitamins showed a variable role as growth factors depending on the temperature increase. In peptide supplemented media, requirements for amino acids and their precursors, as well as vitamins and nucleotides, increased markedly when compared with the protein supplemented medium. A peptide composed of one glutamic acid, two alanines, and one lysine can substitute for serum for trypanosomal growth at all temperatures. Several minimum media have been prepared in which epimastigote forms of T. cruzi can grow at 26-37 C for more than 10 passages.     

Impact of concentration, temperature, and pH on inactivation of Salmonella spp. by volatile fatty acids in anaerobic digestion

It is known that the presence of volatile fatty acids may play a role in the inactivation of pathogens for systems that employ an acid phase reactor. This study was conducted to investigate the influence of volatile fatty acids on the inactivation of Salmonella spp. over a range of digestion temperatures. In this study, digesters that were treating municipal wastewater treatment plant sludges were operated at temperatures that ranged from 35 to 49 degrees C and had a solids residence time of 15 days. Samples collected from the effluent of the digesters were dosed with solutions containing acetic, propionic, and butyric acids alone and in mixtures, and the dosed effluents were analyzed for Salmonella spp. over time. In the first round of testing, the digester effluents were dosed with individual organic acids and also a mixture containing all three volatile fatty acids over a range of concentrations from 750 to 6000 mg/L, and the pH of the samples was fixed at a value of 5.5. In the second round of testing, the sample sludges were spiked with a fixed amount of organic acid mixture, and the pH was varied from 4.5 to 7.5. The reduction of Salmonella spp. in digester effluents, when dosed with volatile organic acids, was found to depend on pH, temperature, the chain length of the acids, and the concentration and composition of the acids present. Increases in temperature appeared to increase the inhibitory effects of the volatile organic acids. At mesophilic temperatures, acidic pHs resulted in a greater inhibition of Salmonella spp.; whereas at higher temperatures neutral pHs were found to be more inhibitory. The results suggest that acid phase digesters that operate at elevated temperatures and low pH can achieve substantial reduction of Salmonella spp.     

Effect of Temperature on the Fatty Acid Composition of the Extreme Thermophiles, Bacillus caldolyticus and Bacillus caldotenax

Gas chromatographic analysis of extracts from Bacillus caldolyticus and Bacillus caldotenax grown at 60, 70, or 80 C showed that both contain branched-chain fatty acids as major constituents at all temperatures tested. With increasing temperature, a decrease of i-C15 and an increase of i-C17 fatty acids were observed in both strains, as well as a decrease of i-C16 fatty acids corresponding to an increase of n-C16 fatty acids. The most obvious difference was that the shifts observed with B. caldolyticus occurred mainly upon raising the temperature to 10 C above, and in B. caldotenax upon lowering the temperature to 20 C below, the optimal growth temperature.     

Perkinsus marinus, a protozoan parasite of the Eastern oyster (Crassostrea virginica): effects of temperature on the uptake and metabolism of fluorescent lipid analogs and lipase activities

The effects of temperature on the uptake and metabolism of fluorescent labeled palmitic acid (FLC16) and phosphatidylcholine (FLPC) and lipase activities in the oyster protozoan parasite, Perkinsus marinus, meront stage were tested at 10, 18, and 28 degrees C. Temperature significantly affected not only the uptake, assimilation, and metabolism of both FLC16 and FLPC in P. marinus, but also its triacylglycerol (TAG) lipase activities. The incorporation of both FLC16 and FLPC increased with temperature and paralleled the increase in the amount of total fatty acids in P. marinus meront cultures. The incorporation of FLC16 was higher than FLPC at all temperatures. The percentage of FLC16 metabolized to TAG was significantly higher at higher temperatures. Trace amounts of incorporated FLC16 were detected in monoacylglycerol (MAG) and PC at 18 and 28 degrees C. P. marinus meronts metabolized FLPC to TAG, diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids (FFA), phosphatidylethanolamine (PE), and cardiolipin (CL). The conversion of FLPC to TAG and PE was highest at 28 degrees C. The relative proportions of individual fatty acids and total saturated, monounsaturated and polyunsaturated fatty acids changed with temperatures. While total saturated fatty acids (SAFAs) increased with temperature, total monounsaturated fatty acids (MUFAs) decreased with temperature. Total polyunsaturated fatty acids (PUFAs) increased from 28 to 18 degrees C. The findings of increase of total SAFAs and decrease of total MUFAs with the increase of temperatures and upward shift of total PUFAs from 28 to 18 degrees C suggest that, as in other organisms, P. marinus is capable of adapting to changes in environmental temperatures by modifying its lipid metabolism. Generally, higher lipase activities were noted at higher cultivation temperatures. Both TAG lipase and phospholipase activities were detected in P. marinus cells and their extra cellular products (ECP), but phospholipase activities in both the cell pellets and ECP were very low. Also, lipase activities were much lower in ECP than in the cells. The observations of low metabolism, bioconversion of incorporated fluorescent lipid analogs and lipase activities at low temperatures are consistent with the low in vitro growth rate and low infectivity of P. marinus at low temperatures.     

The Effect of Temperature on the Level and Biosynthesis of Unsaturated Fatty Acids in Diacylglycerols of Brassica napus Leaves

Experiments on the effects of temperature on the levels of unsaturated fatty acids and their rates of desaturation in Brassica napus leaf lipids have shown that significant differences occur in the composition of all diacylglycerols in the leaf between plants grown at high and low temperatures. In the major thylakoid diacylglycerols, monogalactosyl-diacylglycerol and digalactosyldiacylglycerol, not only is there an increase in the level of unsaturation at low temperatures, but there is a change in the balance between molecular species of chloroplastic origin (16/18C) and cytosolic origin (18/18C). Radioactivity tracer data indicate that at low temperatures there are two distinct phases of desaturation in the fatty acids of the major diacylglycerols of these leaves. A rapid phase, which appears in plants grown at low temperatures and results in the desaturation of palmitic acid to hexadecadienoic acid and oleic acid to linoleic acid may explain the high levels of unsaturated fatty acids found in the leaf diacylglycerols from plants grown at low temperatures. The appearance of this rapid phase is controlled by the temperature at which the plant is grown and is not subject to rapid variations in environmental temperature.     

Emergence timing and fitness consequences of variation in seed oil composition in Arabidopsis thaliana

Early seedling emergence can increase plant fitness under competition. Seed oil composition (the types and relative amounts of fatty acids in the oils) may play an important role in determining emergence timing and early growth rate in oilseeds. Saturated fatty acids provide more energy per carbon atom than unsaturated fatty acids but have substantially higher melting points (when chain length is held constant). This characteristic forms the basis of an adaptive hypothesis that lower melting point seeds (lower proportion of saturated fatty acids) should be favored under colder germination temperatures due to earlier germination and faster growth before photosynthesis, while at warmer germination temperatures, seeds with a higher amount of energy (higher proportion of saturated fatty acids) should be favored. To assess the effects of seed oil melting point on timing of seedling emergence and fitness, high‐ and low‐melting point lines from a recombinant inbred cross of Arabidopsis thaliana were competed in a fully factorial experiment at warm and cold temperatures with two different density treatments. Emergence timing between these lines was not significantly different at either temperature, which aligned with warm temperature predictions, but not cold temperature predictions. Under all conditions, plants competing against high‐melting point lines had lower fitness relative to those against low‐melting point lines, which matched expectations for undifferentiated emergence times.     

INFLUENCE OF TEMPERATURE ON THE FATTY ACID PROFILE OF LISTERIA MONOCYTOGENES

Variation in the fatty acid profile of two Listeria monocytogenes strains grown at varying temperatures was determined. The fatty acid profiles varied greatly at different temperatures. General decreases in relative percentages of branched and medium chain (up to C16:0) fatty acids and variable changes in long chain fatty acids were found with increasing growth temperature. Individual fatty acid percentages between strains were variable. The relative percentages of unknown long chain fatty acids, detected in both strains at various temperatures, were greatest in Scott A (7.07%) and ATCC 19114 (13.15%) at 35C. Results demonstrated that L. monocytogenes had altered fatty acid profile in response to changes in growth temperature.     

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.     

Arabidopsis mutants reveal that short- and long-term thermotolerance have different requirements for trienoic fatty acids

The photosynthetic thylakoid has the highest level of lipid unsaturation of any membrane. In Arabidopsis thaliana plants grown at 22°C, approximately 70% of the thylakoid fatty acids are trienoic - they have three double bonds. In Arabidopsis, and other species, the levels of trienoic fatty acids decline substantially at higher temperatures. Several genetic studies indicate that reduced unsaturation improves photosynthetic function and plant survival at high temperatures. Here, these studies are extended using the Arabidopsis triple mutant, fad3-2 fad7-2 fad8 that contains no detectable trienoic fatty acids. In the short-term, fluorescence analyses and electron-transport assays indicated that photosynthetic functions in this mutant are more thermotolerant than the wild type. However, long-term photosynthesis, growth, and survival of plants were all compromised in the triple mutant at high temperature. The fad3-2 fad7-2 fad8 mutant is deficient in jasmonate synthesis and this hormone has been shown to mediate some aspects of thermotolerance; however, additional experiments demonstrated that a lack of jasmonate was not a major factor in the death of triple-mutant plants at high temperature. The results indicate that long-term thermotolerance requires a basal level of trienoic fatty acids. Thus, the success of genetic and molecular approaches to increase thermotolerance by reducing membrane unsaturation will be limited by countervailing effects that compromise essential plant functions at elevated temperatures.     

The Hydrophobic Temperature Dependence of Amino Acids Directly Calculated from Protein Structures

The hydrophobic effect is the main driving force in protein folding. One can estimate the relative strength of this hydrophobic effect for each amino acid by mining a large set of experimentally determined protein structures. However, the hydrophobic force is known to be strongly temperature dependent. This temperature dependence is thought to explain the denaturation of proteins at low temperatures. Here we investigate if it is possible to extract this temperature dependence directly from a large set of protein structures determined at different temperatures. Using NMR structures filtered for sequence identity, we were able to extract hydrophobicity propensities for all amino acids at five different temperature ranges (spanning 265-340 K). These propensities show that the hydrophobicity becomes weaker at lower temperatures, in line with current theory. Alternatively, one can conclude that the temperature dependence of the hydrophobic effect has a measurable influence on protein structures. Moreover, this work provides a method for probing the individual temperature dependence of the different amino acid types, which is difficult to obtain by direct experiment.     

Influence of rearing temperature on triacylglycerol storage in the pea aphid,Acyrthosiphon pisum

Total fatty acids in the pea aphid reared at low temperatures increased significantly compared to that at high rearing temperatures. This change is reflected in a large increase of myristic acid, which occurs exclusively in triacylglycerols. When aphids were moved from 25°C to a lower rearing temperature at 10°C, saturated fatty acids accumulated over time, reaching a maximum at 16th day. When aphids were moved to 4°C, a temperature below the developmental threshold, those aphids did not accumulate saturated fatty acids. Similar results were observed when aphids were exposed to sequential decrease in rearing temperature. However, both total fatty acids and myristic acid in the aphids from the treatments of sequential decreasing rearing temperature were significantly higher compared to those in the aphids from the treatments of sudden decreasing rearing temperature. This result, therefore, supports the hypothesis that cold‐adapted aphids can survive under threshold temperature for a longer period of time than noncold‐adapted aphids. Acetyl‐CoA carboxylase activity in the aphids at 25°C was twofold higher than that in the aphids at 10°C, whereas fatty acid synthase activities in the aphids reared at 25 and 10°C are similar. Aphids reared at 10°C showed a threefold reduction in reproduction rates. This reduced production of new nymphs reduces energy demand and would allow for accumulation of energy in the form of triacylglycerols. Therefore, the increased level of saturated fatty acids in aphids reared at low temperature is probably related to lower utilization of fatty acids rather than increased rates of biosynthesis.     

Influence of anaerobiosis and low temperature on Bacillus cereus growth, metabolism, and membrane properties

The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD(+). Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.     

Solubility of saturated fatty acids in water at elevated temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230 degrees C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160 degrees C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens.

We investigated the fatty acid composition of the membrane of Bacillus amyloliquefaciens grown at different temperatures. A decrease in growth temperature was accompanied by an increase in the ratio of branched- to straight-chain fatty acids and a marked increase in the level of unsaturation of branched-chain fatty acids. When cells of this organism grown at 30 degrees C were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween 80 to cells caused the critical temperature zone for cold shocking to be lowered significantly. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.     

Solubility of Saturated Fatty Acids in Water at Elevated Temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230°C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160°C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Factors affecting carbohydrate and free amino acid content in overwintering larvae of Enosima leucotaeniella

Amounts of several metabolites were measured in overwintering larvae of Enosima leucotaeniella acclimated to temperatures between -5 and 15 degrees C for 30days. In the diapausing stage, cold hardiness, as shown by the survival rate, began rising below 15 degrees C. Glycogen content decreased as the temperature decreased from 10 to 0 degrees C. Trehalose content rose as the temperature decreased from 15 to 5 degrees C, but remained unchanged as the temperature decreased from 5 and 0 degrees C. Twenty-eight free amino acids were detected in the haemolymph; levels of proline, glutamine and glutamic acid increased at high temperatures, but alanine increased at low temperatures, especially as temperature decreased from 5 to 0 degrees C. Lipid content was unchanged by the different acclimation temperatures. The effects of temperature, diapause and aerobic conditions on the levels of carbohydrates and amino acids in overwintering larvae were analyzed. Alanine levels rose at low temperature only when the larvae were in the diapausing stage. The level of trehalose rose at low temperature in both the diapausing and post-diapausing stages, although it was higher at aerobic conditions in the post-diapausing stage. These results suggest that efficient trehalose synthesis occurs under the combination of low temperature and aerobic conditions of the post-diapausing stage, so that cold hardiness in overwintering E. leucotaeniella larvae may rise to a high level in winter.     

Effects of Hydrostatic Pressure and Temperature on the Uptake and Respiration of Amino Acids by a Facultatively Psychrophilic Marine Bacterium

Studies of pressure and temperature effects on glutamic acid transport and utilization indicated that hydrostatic pressure and low temperature inhibit glutamate transport more than glutamate respiration. The effects of pressure on transport were reduced at temperatures near the optimum. Similar results were obtained for glycine, phenylalanine, and proline. Pressure effects on the transport systems of all four amino acids were reversible to some degree. Both proline and glutamic acid were able to protect their transport proteins against pressure damage. The data presented indicate that the uptake of amino acids by cells under pressure is inhibited, which is the cause of their inability to grow under pressure.     

Acholeplasma laidlawii membranes: an electron spin resonance study of the influence on molecular order of fatty acid composition and cholesterol.

The effects on membrane structure of including various fatty acids and cholesterol in the growth medium of Acholeplasma laidlawii were investigated by the use of spin-labeled fatty acids. Although the order-mobility parameters varied significantly at some temperatures with the nature of the fatty acid incorporated, the value measured at the growth temperature was only slightly affected by changes in the fatty acid composition of the membranes. The data confirm previous assertions that despite a high level of incorporation of fatty acids of various chain lengths or degree of unsaturation, A. laidlawii regulates its overall membrane fluidity within close limits at the growth temperature. Incorporation of cholesterol increased the degree of order at all temperatures. The coexistence of two lipid phases, one protein-dependent, could be observed in membranes. The order-mobility parameter of spin probes proved less satisfactory for the observation of a gel to liquid crystal transition of the membrane lipid than the partition parameter of a fatty acid spin probe. Order parameters measured by fatty acid spin probes were somewhat higher than those measured by the analogous ²H nmr probes.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.