Enhanced protein synthetic capacity in Atlantic cod (Gadus morhua) is associated with temperature-induced compensatory growth

Atlantic cod (Gadus morhua) were held either at seasonal ambient temperatures (-0.3 to 11 degrees C) or at a relatively constant control temperature (8-11 degrees C) to investigate aspects of protein synthesis during a period of compensatory growth. Protein synthesis rate, total RNA, and RNA-specific protein synthesis rate were determined in white muscle and liver when ambient temperatures were -0.3, 4.5, and 11 degrees C in February, June, and July, respectively. To allow for comparisons between treatment temperatures, fish were also acutely transferred to a comparable assay temperature in February and June. Over the transition from 4.5 to 11 degrees C (June to July), the ambient-held cod had a significant increase in size and a substantially higher growth rate relative to control-held fish over the same period, consistent with cold-induced compensatory growth. During the onset of this enhanced growth, in June when ambient temperature was approximately 4.5 degrees C, ambient-held fish elevated their capacity for protein synthesis in the white muscle and liver via elevation of the RNA content. When ambient temperature reached the same point as for the control fish (11 degrees C), the rate of white muscle protein synthesis remained higher in the ambient-held vs. that in the control-held fish, a process facilitated by elevated RNA content and greater RNA-specific rate of protein synthesis. In the liver, all measured characteristics of protein synthesis were the same for ambient and control fish in July. The latter suggests that compensatory growth may be in part explained by improved efficiency of protein synthesis.     

Cold adaptation of microorganisms

Psychrophilic and psychrotrophic microorganisms are important in global ecology as a large proportion of our planet is cold (below 5 degrees C); they are responsible for the spoilage of chilled food and they also have potential uses in low-temperature biotechnological processes. Psychrophiles and psychrotrophs are both capable of growing at or close to zero, but the optimum and upper temperature limits for growth are lower for psychrophiles compared with psychrotrophs. Psychrophiles are more often isolated from permanently cold habitats, whereas psychrotrophs tend to dominate those environments that undergo thermal fluctuations. The molecular basis of psychrophily is reviewed in terms of biochemical mechanisms. The lower growth temperature limit is fixed by the freezing properties of dilute aqueous solutions inside and outside the cell. In contrast, the ability of psychrophiles and psychrotrophs to grow at low, but not moderate, temperatures depends on adaptive changes in cellular proteins and lipids. Changes in proteins are genotypic, and are related to the properties of enzymes and translation systems, whereas changes in lipids are genotypic or phenotypic and are important in regulating membrane fluidity and permeability. The ability to adapt their solute uptake systems through membrane lipid modulation may distinguish psychrophiles from psychrotrophs. The upper growth temperature limit can result from the inactivation of a single enzyme type or system, including protein synthesis or energy generation.     

Effect of temperature on RNA synthesis in Escherichia coli CRT 266 (dna Bts) following infection with bacteriophage T3]

Infection of the temperature-sensitive E. coli CRT 266 (dnaBts) with T3-phages at the temperature of 30 degrees C and 35 degrees C, respectively, induced T3-specific RNA synthesis with a maximum rate at 7 min (30 degrees C) and 4.5 min (35 degrees C) after infection. At temperatures above 40 degrees C no T3-induced RNA synthesis could be observed. Infection of E. coli CR 34--45 (dnaB+) with T3 phages at 30 degrees C, 35 degrees C and at temperatures above 40 degrees C, however, produced T3-specific RNA synthesis. The maximum of T3-induced RNA synthesis could be observed between 7 min and 3 min depending on the temperature during infection. The inability to form T3-specific RNA after infection of E. coli CRT 266 at nonpermissive temperatures may be a cause for the absence of the formation of T3 phages and lysis of the host cells.     

High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature.

Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

Effect of cultivation temperature on nucleic acid level in bacteria Listeria monocytogenes and Yersinia pseudotuberculosis

The relationship between the multiplication of bacteria, the content of nucleic acid and the specific rate of their growth during their batch cultivation in nutrient broth and mineral medium at temperatures of 37 degrees C and 4-6 degrees C was studied in the causative agents of saprozoonotic infections with L. monocytogenes and Y. pseudotuberculosis used as typical representatives of such bacteria. The content of DNA was shown to remain practically unchanged after the alteration of cultivation temperature and the conditions of nutrition. The linear relationship between the content of RNA and specific growth rate was registered both at 37 degrees C and 4-6 degrees C. However a higher content of RNA at low temperatures was found to correspond to one and the same specific growth rate, which was linked with the additional synthesis of this nucleic acid.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Maximizing growth rate at low temperatures: RNA:DNA allocation strategies and life history traits of Arctic and temperate Daphnia

Many short-lived or univoltine organisms at high latitudes and altitudes face the challenge to complete their life-cycle within a brief growing season. This means that they need to maintain a high growth rate at low temperatures, and one way of doing this is to allocate limiting resources like phosphorus to RNA in order to maximize protein synthesis. We here explore the allocations of phosphorus to RNA relative to DNA, and the potential bearings on growth rate and life history traits of polyploid (high-Arctic) and diploid (temperate) Daphnia pulex. The polyploid clone matured earlier at low temperature (8°C) but later than the diploid clone at high temperature (18°C). Juveniles of Arctic Daphnia had both higher specific levels of RNA and higher growth rates at low temperature compared with the temperate clone of Daphnia. We hypothesize that Arctic Daphnia may overcome growth constraints posed by low temperature and polyploidy by increasing their allocation of resources to RNA. The prevalence of polyploidy in Arctic populations strongly suggests that the potential drawbacks of polyploidy are counteracted by an increased allocation of resources to RNA to keep a high rate of protein synthesis even under low temperatures.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Molecular basis for the maximum growth temperature of an obligately psychrophilic yeast, Leucosporidium stokesii.

Cells of the obligately psychrophilic yeast Leucosporidium stokesii were subjected to permissive (15 and 20 degrees C) and restrictive (23 and 25 degrees C) temperatures to determine the event(s) responsible for the low maximum growth temperature of this organism. An investigation of subcellular morphology by nuclear staining revealed that buds formed at 20 degrees C were anucleate but showed nuclear migration within the parent cell. Cells incubated initially at 23 degrees C and then shifted down to a permissive growth temperature of 15 degrees C in the presence of a deoxyribonucleic acid (DNA) synthesis inhibitor, hydroxyurea, confirmed the observation that the anucleate condition of atypical buds was the result of temperature-sensitive DNA synthesis. Concomitantly, the incorporation of labeled adenine into DNA was inhibited at 23 and 25 degrees C. The synthesis of ribonucleic acid, however, was enhanced at 23 degrees C but impaired at 25 degres C. Similarly, protein synthesis was unaffected at either restrictive temperature.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

Psychrophilic pseudomonas from soil

A psychrophilic culture of Pseudomonas inhabiting soil was cultivated at a temperature close to the minimum one (0 degrees C) and at a temperature close to a maximum one 28 degrees C) for growth on the original and diluted MPB. No changes were found in the formation of biomass, RNA and DNA. The maximum possible biomass yield was attained sooner at 0 degrees C on a diluted medium than on an original medium. At a low temperature, the specific growth rate remained the same, but the content of RNA increased.     

Compositional changes in RNA,DNA and proteins for bacterial adaptation to higher and lower temperatures

It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed.     

Phenethyl Alcohol Resistance in ESCHERICHIA COLI. III. a Temperature-Sensitive Mutation (dnaP) Affecting DNA Replication

A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.     

Effect of increased temperatures on RNA and protein synthesis in the cells of a synchronous Candida utilis culture

The rate of incorporation of labeled precursors for RNA ([14C]uracil) and protein ([14C]DL-leucine) into the cells of the synchronous culture of Candida utilis VKMY-1668 (the optimum temperature of growth, 31--32 degrees C) was studied as a function of different temperatures (28, 31, 32, 34, 36, 38, and 41 decrees C). The yeast was grown on a simple mineral medium containing glycerol. RNA synthesis was found to be more susceptible to elevated temperature than protein synthesis: the maximum rate of incorporation was registered at 32--34 degrees C for [14C]DL-leucine and only at 32 degrees C for [14C]uracil (the rate of its incorporation at 34 degrees C decreased by 50% as compared to that at 32 degrees C). The rate of incorporation of [14C]uracil at 34 degrees C reached 100% (the rate at 32 degrees C) when yeast autolysate was added to the medium, and 75 and 70%, respectively, upon the addition of DL-methionine or Mg2+ (as compared to 50% without them).     

Increased risk of phosphorus limitation at higher temperatures for Daphnia magna

Invertebrate herbivores frequently face growth rate constraints due to their high demands for phosphorus (P) and nitrogen (N). Temperature is a key modulator of growth rate, yet the interaction between temperature and P limitation on somatic growth rate is scarcely known. To investigate this interaction, we conducted a study on the somatic growth rate (SGR) of the cladoceran Daphnia magna, known to be susceptible to P-limitation. We determined the SGR across a broad range of dietary P content of algae (carbon (C):P ratios (125?C790), and at different temperatures (10?C25°C). There was a strong impact of both temperature and C:P ratio on the SGR of D. magna, and also a significant interaction between both factors was revealed. The negative effect of dietary C:P on growth rate was reduced with decreased temperature. We found no evidence of P limitation at lowest temperature, suggesting that enzyme kinetics or other measures of food quality overrides the demands for P to RNA and protein synthesis at low temperatures. These findings also indicate an increased risk of P limitation and thus reduced growth efficiency at high temperatures.     

A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.     

Comparison of temperature effects on soil respiration and bacterial and fungal growth rates

Temperature is an important factor regulating microbial activity and shaping the soil microbial community. Little is known, however, on how temperature affects the most important groups of the soil microorganisms, the bacteria and the fungi, in situ. We have therefore measured the instantaneous total activity (respiration rate), bacterial activity (growth rate as thymidine incorporation rate) and fungal activity (growth rate as acetate-in-ergosterol incorporation rate) in soil at different temperatures (0-45 degrees C). Two soils were compared: one was an agricultural soil low in organic matter and with high pH, and the other was a forest humus soil with high organic matter content and low pH. Fungal and bacterial growth rates had optimum temperatures around 25-30 degrees C, while at higher temperatures lower values were found. This decrease was more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. A tendency towards the opposite effect was observed at low temperatures, indicating that fungi were more adapted to low-temperature conditions than bacteria. The temperature dependence of all three activities was well modelled by the square root (Ratkowsky) model below the optimum temperature for fungal and bacterial growth. The respiration rate increased over almost the whole temperature range, showing the highest value at around 45 degrees C. Thus, at temperatures above 30 degrees C there was an uncoupling between the instantaneous respiration rate and bacterial and fungal activity. At these high temperatures, the respiration rate closely followed the Arrhenius temperature relationship.