Enhanced thermotolerance and temperature-induced changes in protein composition in the hyperthermophilic archaeon ES4.

The hyperthermophilic archaeon ES4, a heterotrophic sulfur reducer isolated from a deep-sea hydrothermal vent, is capable of protecting itself from thermal stress at temperatures above its optimum for growth. The thermotolerance of ES4 was determined by exposing log-phase cells to various lethal high temperatures. When ES4 was shifted from 95 to 102 degrees C, it displayed recovery from an exponential rate of death, followed by transient thermotolerance. When ES4 was shifted directly from 95 to either 105 or 108 degrees C, only exponential death occurred. However, a shift from 95 to 105 degrees C with an intermediate incubation at 102 degrees C also gave ES4 transient thermotolerance to 105 degrees C. The protein composition of ES4 was examined at temperatures ranging from 75 to 102 degrees C by one-dimensional electrophoresis. Two proteins with molecular masses of approximately 90 and 150 kDa significantly decreased in abundance with increasing growth temperature, while a 98-kDa protein, present at very low levels at normal growth temperatures (76 to 99 degrees C), was more abundant at higher temperatures. The enhanced tolerance to hyperthermal conditions after a mild hyperthermal exposure and the increased abundance of the 98-kDa protein at above-optimal temperatures imply that ES4 is capable of a heat shock-like response previously unseen in hyperthermophilic archaea.     

Cold shock induction of thermal sensitivity in Listeria monocytogenes

Cold shock at 0 to 15 degrees C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60 degrees C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8 degrees C for controls and 7.7 degrees C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28 degrees C followed by heating at 60 degrees C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D(60) values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Kinetics of petite mutation and thermal death in Saccharomyces cerevisiae growing at superoptimal temperatures.

Mass formation of petite mutants took place in a strain of Saccharomyces cerevisiae when grown at superoptimal temperatures. After an initial period of exponential growth, a second period followed during which exponential death and net exponential petite mutation concurred with exponential growth. The specific rates of the three exponential processes were of the same order of magnitude and varied with the temperature. Net exponential petite mutation did not occur during the deathless first period of growth at superoptimal temperatures nor at any time during growth at suboptimal temperatures. Mitochondria are discussed as possible targets of thermal death in mesophilic yeasts.     

Effects of cycloheximide on the temperature profile of Sacharomyces cerevisiae

Tmax, the maximum temperature for growth of Saccharomyces cerevisiae, decreased linearly with increasing concentrations of cycloheximide added to the medium, to about 20 degrees C at 2.5 microgram ml-1. In this concentration range thermal death was not enhanced. The Arrhenius plot of growth was shifted to lower temperatures as a function of the cycloheximide concentration and became dissociated from the Arrhenius plot of thermal death. It was concluded that the target site of cycloheximide, the cytoplasmic ribosome, is not identical with the physiological Tmax site of S. cerevisiae and that the binding of cycloheximide to its target sites is strongly enhanced by the temperature.     

Temperature-induced protein synthesis in Bacillus stearothermophilus NUB36.

Cultures of Bacillus stearothermophilus subjected to a temperature shift-up or shift-down of 15 degrees C within the normal temperature range of growth (45 to 65 degrees C) enter a transient adaptation period before exponential growth at the new temperature. The de novo synthesis of some proteins coincides with the adaptation period.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Cultures and thermal analysis of the marine microalga Tetraselmis suecica Kylin (Butch).

In this paper, the growth of the marine microalga Tetraselmis suecica was investigated using thermogravimetry (TG) and differential thermal analysis (DTA) to determine suitable diets for larval and juvenile development in aquaculture systems. Microalgae were maintained in synthetic sea water (19@1000 salinity, 18 degrees C constant temperature) and the algal growth was evaluated by cell abundance. Exponential, stationary and senescence cells were analyzed by TG and DTA. The results of thermal analysis pointed out marked differences between exponential, stationary and senescence phases and showed that exponentially growing microalgae could represent a suitable food in aquaculture systems.     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.     

Thermal death of temperature-sensitive lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis: effect of culture medium and developmental stage

The growth of thermosensitive Bacillus subtilis lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants (lysS1 and trypS1) (l-lysine:transfer ribonucleic acid [tRNA] ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) was terminated when exponential phase cells were shifted from 30 to 43 C in a rich medium. Under these conditions, the temperature-inhibited cells undergo thermal death; they rapidly lose their ability to form colonies at 30 C. Another lysyl-tRNA synthetase mutant (lysS2) is refractory to thermal death even though its growth is inhibited at 43 C. The thermal death response of the lysS1 mutant is affected by the stage of cell development. At periods in spore outgrowth and sporogenesis these cells become refractory to thermal death. The refractory state does not result from the production of an inhibitor, or from the degradation of an activator of thermal death. However, culture medium composition does modify the thermal death response. Rich media enhance the effect, and no thermal death occurs in the lysS1 strain grown in a minimal medium. Temperature-sensitive cells can grow in a lysine- (0.25 mM) or tryptophan- (0.25 mM) supplemented minimal medium at 43 C, but amino acid concentrations of 25 mM only transiently protect trypS1 and lysS1 cells from thermal death in a rich medium. Osmotic agents such as sucrose (0.5 M) and NaCl (0.34 M) completely prevent thermal death in the lysS1 strain, although growth is still arrested. On solid media, sucrose stabilized lysS1 cells can form colonies at the restrictive temperature, but neither sucrose (0.5 M) nor NaCl (0.34 M) stabilized the lysS1 enzyme in vitro. Chloramiphenicol increased the rate of thermal death of the lysS1 strain but decreased the thermal death response of the trypS1 mutant. Considering the nature of the enzyme defect in the lysS1 strain, the common genetic origin of the spore and vegetative lysyl-tRNA synthetase, and the protective effects exerted by lysine and osmotic agents, it is tentatively concluded that thermal death results from irreversible inactivation of the mutant gene product. According to this hypothesis, either the lysS1 enzyme is altered during sporogenesis or some physiological or structural aspect of this developmental phase can stabilize the mutant phenotype and thereby rescue cells from thermal death.     

Effect of Several Environmental Conditions on the “Thermal Death Rate” of Endospores of Aerobic, Thermophilic Bacteria

The composition of the recovery medium affected the apparent heat resistance of Bacillus stearothermophilus when the pH of the medium was 7.0 but not when the pH was 6.5. The rate of thermal death at 110 C was exponential. Deviations from exponential rates of thermal death during the initial phases of heating at 96 C were observed with endospores of B. coagulans under different conditions of sporulation. Additionally, the apparent heat resistance was influenced by the composition of the media used for sporulation and recovery and by the composition of the suspending menstruum. The presence of 0.001 m sorbic acid in the suspending menstruum at pH 7.0 and the temperature of incubation of the cultures after heating did not affect the apparent heat resistance of B. coagulans. Several explanations are discussed for the observed deviations from exponential thermal death rates and the effect of the environment on the apparent heat resistance of B. coagulans.     

Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions

Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 degrees C) with or without NaCl (7%), and their fatty acid compositions were analysed. The results showed that low thermal adaptation response of L. monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids. However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0. In hyperosmotic medium and over the entire temperature range (4 degrees C, 20 degrees C and 37 degrees C) the two L. monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0. In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain. These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Changes in membrane fatty acid composition of Pediococcus sp. strain NRRL B-2354 in response to growth conditions and its effect on thermal resistance.

Membrane fatty acid composition and thermal resistance (D value) of Pediococcus sp. were determined for mid-exponential-phase (ME) and stationary-phase (ST) cells grown in tryptic soy broth (TSB) and tryptone-glucose-yeast extract (TGY) at 28 and 37 degrees C. As the cells entered the stationary phase of growth, the unsaturated fatty acid, C18:1 n11c, produced during the exponential phase of growth was converted to its cyclic form, C19:0 Delta9c. This shift in membrane fatty acid composition was accompanied by an increase in the D values of this bacterium. Data from this study suggest that the membrane fatty acid composition of Pediococcus sp. is dependent on the growth conditions and that membrane fatty acid composition plays a critical role in thermal resistance. Thermal inactivation curves of Pediococcus sp. cells grown in TGY at 28 degrees C indicated the presence of a cell population that is heterogeneous in thermal resistance. The growth of this bacterium in TGY at 37 degrees C and in TSB at 28 and 37 degrees C resulted in cell populations that were uniform in thermal resistance with a lag time for thermal inactivation. Thermal inactivation curves of ME and ST cultures were similar. The data presented here suggest that the cell population's uniformity of thermal inactivation is independent of the growth phase of the culture.     

Thermal dosimetry of normal porcine tissue

The response of normal porcine fat and muscle to graduated doses of hyperthermia provided by an annularly focused acoustic source was measured. Temperatures and exposure times were varied between 43 degrees C (20-90 min), 45 and 47 degrees C (20-60 min), and 49 degrees C (20 min). Response, based on histologic grading of the treated sites 30 days after exposure, was found to correlate well when mapped against several methods of estimating thermal energy deposition. The threshold for damage production was at or near 43 degrees C. For a given temperature, a nearly exponential increase in relative tissue damage as a function of increased exposure time was found. A twofold increase in tissue damage was produced in fat relative to muscle at any given thermal dose.     

Death and injury of Staphytococcus aureus during thermal treatment of milk

Staphylococcus aureus isolated from milk and grown in milk was heated in milk. The phenomena of death as well as injury was investigated in the range of 50 to 75 degrees C. The D60 value (decimal reduction time on salt-free medium) was 0.87 min, the D'60 value (decimal reduction time in salt-containing medium) was 0.62 min. Cultures were injured as soon as heating started. This initial thermal shock increased with increasing temperature. At 50-60 degrees C injury was more rapid than death. At greater than 60 degrees C death became faster than injury and the two processes coincided at 70 degrees C. The Z value was 9.46 degrees C and the Z' value was 9.93 degrees C.     

Effects of temperature on the yeast cell cycle analyzed by flow cytometry

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila.

The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48 degrees-65 degrees C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7 degrees C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30 degrees C) were twice as resistant as those in the early stage (5 h at 30 degrees C), and the maximum D-value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z-values significantly. The heat resistance of cells incubated for 48 h at 30 degrees C increased (twice) after holding at 7 degrees C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30 degrees C) did not influence their D-values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 to 4.0 reduced D-values by a factor of 5. Although the strain studied was heat-sensitive (D55 degrees C = 0.17 min; z = 5.11 degrees C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Oxygen limitation of thermal tolerance in cod, Gadus morhua L., studied by magnetic resonance imaging and on-line venous oxygen monitoring

The hypothesis of an oxygen-limited thermal tolerance due to restrictions in cardiovascular performance at extreme temperatures was tested in Atlantic cod, Gadus morhua (North Sea). Heart rate, changes in arterial and venous blood flow, and venous oxygen tensions were determined during an acute temperature change to define pejus ("getting worse") temperatures that border the thermal optimum range. An exponential increase in heart rate occurred between 2 and 16 degrees C (Q(10) = 2.38 +/- 0.35). Thermal sensitivity was reduced beyond 16 degrees C when cardiac arrhythmia became visible. Flow-weighted magnetic resonance imaging (MRI) measurements of temperature-dependent blood flow revealed no exponential but a hyperbolic increase of blood flow with a moderate linear increase at temperatures >4 degrees C. Therefore, temperature-dependent heart rate increments are not mirrored by similar increments in blood flow. Venous Po(2) (Pv(O(2))), which reflects the quality of oxygen supply to the heart of cod (no coronary circulation present), followed an inverse U-shaped curve with highest Pv(O(2)) levels at 5.0 +/- 0.2 degrees C. Thermal limitation of circulatory performance in cod set in below 2 degrees C and beyond 7 degrees C, respectively, characterized by decreased Pv(O(2)). Further warming led to a sharp drop in Pv(O(2)) beyond 16.1 +/- 1.2 degrees C in accordance with the onset of cardiac arrhythmia and, likely, the critical temperature. In conclusion, progressive cooling or warming brings cod from a temperature range of optimum cardiac performance into a pejus range, when aerobic scope falls before critical temperatures are reached. These patterns might cause a shift in the geographical distribution of cod with global warming.     

Individual leaf development in Arabidopsis thaliana: a stable thermal-time-based programme

In crop species, the impact of temperature on plant development is classically modelled using thermal time. We examined whether this method could be used in a non-crop species, Arabidopsis thaliana, to analyse the response to temperature of leaf initiation rate and of the development of two leaves of the rosette. The results confirmed the large plant-to-plant variability in the studied isogenic line of the Columbia ecotype: 100-fold differences in leaf area among plants sown on the same date were commonly observed at a given date. These differences disappeared in mature leaves, suggesting that they were due to a variability in plant developmental stage. The whole population could therefore be represented by any group of synchronous plants labelled at the two-leaf stage and followed during their development. Leaf initiation rate, duration of leaf expansion and maximal relative leaf expansion rate varied considerably among experiments performed at different temperatures (from 6 to 26 degrees C) but they were linearly related to temperature in the range 6-26 degrees C, with a common x-intercept of 3 degrees C. Expressing time in thermal time with a threshold temperature of 3 degrees C unified the time courses of leaf initiation and of individual leaf development for plants grown at different temperatures and experimental conditions. The two leaves studied (leaf 2 and leaf 6) had a two-phase development, with an exponential phase followed by a phase with decreasing relative elongation rate. Both phases had constant durations for a given leaf position if expressed in thermal time. Changes in temperature caused changes in both the rate of development and in the expansion rate which mutually compensated such that they had no consequence on leaf area at a given thermal time. The resulting model of leaf development was applied to ten experiments carried out in a glasshouse or in a growth chamber, with plants grown in soil or hydroponically. Because it predicts accurately the stage of development and the relative expansion rate of any leaf of the rosette, this model facilitates precise planning of sampling procedures and the comparison of treatments in growth analyses.     

Induction of proteins in response to low temperature in Escherichia coli.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.