Cold shock and adaptation

Adaptation to environmental stresses, such as temperature fluctuation, is essential for the survival of all living organisms. Cellular responses in both prokaryotes and eukaryotes to high temperature include the synthesis of a set of highly conserved proteins known as the heat shock proteins. In contrast to the heat shock response, adaptation to low temperatures has not been as extensively studied. However, a family of cold-inducible proteins is evident in prokaryotes. In addition, most organisms have developed adaptive mechanisms that alter both membrane fluidity and the protein translation machinery at low temperature. This review addresses the different adaptive mechanisms used by a variety of organisms with a focus on the molecular mechanisms of cold adaptation that have recently been identified during the cold shock response in Escherichia coli. BioEssays 20:49–57, 1998. © 1998 John Wiley & Sons, Inc.     

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.     

Cold adaptation of tropomyosin

The conformational stability of unphosphorylated and phosphorylated α,α-striated tropomyosins from rabbit and shark (95% identical sequences) has been investigated. Three additional core positions are occupied by atypical amino acids in the protein from shark: Thr179(d), Ser190(a), and Ser211(a). These changes are thought to have further destabilized most, if not all, of the carboxyl-terminal half of the molecule. Heat-induced unfolding of shark tropomyosin (2 mg/mL, 0.1 M salt, pH 7) as monitored by far-UV circular dichroism is biphasic [T(m1) ～ 33 °C (main), and T(m2) ～ 54 °C] and takes place over a wider temperature span than that of the mammalian protein. The relationship between ellipticity (and excess heat) and temperature is insensitive to the presence in either tropomyosin of covalently bound phosphate. At ～10 mg/mL, the minor endotherm of shark tropomyosin is shifted to ～60 °C and T(m2) - T(m1) is increased to 25 °C; otherwise, the results of calorimetry are in agreement with those of circular dichroism. Analyses of cyanogen bromide fragments by far-UV circular dichroism and intact protein by near-UV circular dichroism (T(m) ～ 32 °C) show that the most stable sizable portion of shark tropomyosin is located within the amino-terminal half of the molecule. These findings illuminate those regions in tropomyosin where flexibility is critical and show that substitutions predicted to be unfavorable in one temperature regime are desirable in another.     

Cold adaptation in marine organisms

Animals from polar seas exhibit numerous so called resistance adaptations that serve to maintain homeostasis at low temperature and prevent lethal freezing injury. Specialization to temperatures at or below 0 degrees C is associated with an inability to survive at temperatures above 3-8 degrees C. Polar fish synthesize various types of glycoproteins or peptides to lower the freezing point of most extracellular fluid compartments in a non-colligative manner. Antifreeze production is seasonal in boreal species and is often initiated by environmental cues other than low temperature, particularly short day lengths. Most of the adaptations that enable intertidal invertebrates to survive freezing are associated with their ability to withstand ariel exposure. Unique adaptations for freezing avoidance include the synthesis of low molecular mass ice-nucleating proteins that control and induce extracellular ice-formation. Marine poikilotherms also exhibit a range of capacity adaptations that increase the rate of some physiological processes so as to partially compensate for the effects of low temperature. However, the rate of embryonic development in a diverse range of marine organisms shows no evidence of temperature compensation. This results in a significant lengthening of the time from fertilization to hatching in polar, relative to temperate, species. Some aspects of the physiology of polar marine species, such as low metabolic and slow growth rates, probably result from a combination of low temperature and other factors such as the highly seasonal nature of food supplies. Although neuromuscular function shows a partial capacity adaptation in Antarctic fish, maximum swimming speeds are lower than for temperate and tropical species, particularly for early stages in the life history.     

Cold stress and cold adaptation

1. 1. Results from more than half a century of investigation of human adaptation to cold have been so varied that some observers have doubted whether man can adapt to cold at all.

2. 2. This paper considers what challenges to the thermoregulatory system humans experience when living and working in a cold environment (specifically the Antarctic and Subantarctic), what kinds of adaptation have been shown to develop, and what factors might have contributed to the diversity of opinion.

Cold adaptation and the human face

A framework is suggested within which the evolutionary biology of the human head and face can be explored; it includes several channels of natural and behavioral selection as well as modes of “plasticity” change. One aspect of the model is then examined by means of physiological and anthropometric experimentation. A cold room study of 33 Japanese and 25 whites, all born and raised in the tropics, was conducted at Hawaii's Pacific Biomedical Research Center. Thermal response during 70 minutes of exposure (face and hand) to moving 0°C air was electrically recorded. Assuming skin and body temperature is partially dependent upon morphology, detailed anthropometric measurements were taken and employed in thermal-morphological correlation analysis. Though results are not yet thoroughly analyzed, it appears that head surface temperatures relate to sub-cutaneous fat thickness, but not clearly to other form factors; the oriental face, supposedly a product of selection by cold, seems to respond little differently than any other.     

Cold adaptation of enzyme reaction rates

A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.     

Cold adaptation and thyroid hormone metabolism.

Resting oxygen consumption and energy expenditure is sensitive to slight alterations in thyroid function. This means that timing and magnitude of cold adaptation would to some extent depend on thyroid function. Local thyroid hormone metabolism is important for energy expenditure and dissipation of heat in special tissues. Recruitment of brown adipocytes and upregulation of uncoupling protein 1 in mitochondria depends on high tissue T3 concentrations. Most of this T3 is derived from local 5' deiodination of T4. Brown fat is vital for cold exposed mice and rats, and may be important for temperature adaptation in human neonates. The role of thyroid hormone metabolism in adult human cold adaptation has not been finally clarified. Hypothetically, cold exposure may enhance T3 production by deiodination of T4 in skeletal muscle, which may enhance heat production in muscle via a change in muscle fiber type. Another hypothetical possibility is recruitment of brown adipocytes embedded in white adipose tissue in human adults. Understanding cold adaptation in human adults may lead to development of new drugs against obesity.     

Cold adaptation in insects of Central Yakutia

The mode of cold hardening was for the first time assessed for 20 insect species living in the extremely cold climate of Yakutia. All insects tested were found to adapt through freeze tolerance, producing ice-nucleating agents that cause the hemolymph to freeze at high subzero temperatures. For the first time ice-nucleating agents were demonstrated in Lepidoptera. Pieris rapae exemplified the possibility of switchover in the survival strategy depending on the climatic conditions.     

Cold adaptation strategy in insects inhabiting central Yakutia

Cold hardiness in 20 insect species living in extremely cold climate of Yakutia has been investigated for the first time. It was shown that the Yakutian insects prefer to use the strategy of freeze tolerance according to which they produce special substances initiating the freezing of hemolymph at high subzero temperatures. The presence of ice-nucleating agents in the haemolymph of insects belonging to the phylogenetic group of Lepidopteran was shown. We postulate that Pieris rapae may shift between the different cold hardiness strategies when they move from moderately cold regions to a more severe environment.     

驯化对始红蝽(Pyrrhocoris apterus)耐寒能力的影响及越冬适应策略

为阐明越冬期间始红蝽应对低温胁迫的耐寒策略及其影响因素,从生理生化水平探讨始红蝽成虫的耐寒能力,逐月测定了12月至翌年3月始红蝽低温驯化前后的过冷却点、低温存活率、LT_(50)以及始红蝽体内耐寒物质含量。结果表明,越冬期间始红蝽自然种群过冷却点最低为(-14.01±0.53)℃,-5、-10℃驯化30min后的始红蝽过冷却点最低降至为(-19.32±0.86)℃、(-25.56±1.09)℃。0℃驯化30min后暴露于-5、-10、-15℃1h的最高存活率依次为100%、39.1%±8.6%、10%;始红蝽自然种群LT_(50)最低为-8.53℃,0℃驯化后降至-9.21℃。越冬期间雌雄始红蝽体内自由水/结合水比值和游离蛋白质含量先下降后上升,12月达到最大值,雌雄分别为144.50±26.22和140.32±21.92,(15.81±0.10)mg/g和(15.47±0.01)mg/g;脂肪、海藻糖和甘油含量先上升后下降,2月达到最大值,雌雄脂肪含量分别为(16.33±0.48)mg/g和(13.15±1.32)mg/g,海藻糖含量分别为(11.98±0.01)mg/g和(10.88±0.02)mg/g,甘油含量分别为(14.74±0.01)mg/g和(15.06±0.03)mg/g。研究证明,低温驯化后始红蝽的过冷却点和LT_(50)明显降低,低温存活率显著提高,越冬期间始红蝽可通过调整体内抗逆物质含量以增强虫体耐寒能力。     

Cold adaptation in the phytopathogenic fungi causing snow molds

Snow molds are psychrophilic or psychrotrophic fungal pathogens of forage crops, winter cereals, and conifer seedlings. These fungi can grow and attack dormant plants at low temperatures under snow cover. In this review, we describe the biodiversity and physiological and biochemical characteristics of snow molds that belong to various taxa. Cold tolerance is one of the important factors related to their geographic distribution, because snow molds develop mycelia under snow cover and because they should produce intra- and extracellular enzymes active at low temperatures for growth and infection. Basidiomycetous snow molds produce extracellular antifreeze proteins. Their physiological significance is to keep the extracellular environment unfrozen. The psychrophilic ascomycete Sclerotia borealis shows normal mycelial growth under frozen conditions, which is faster than that on unfrozen media at optimal growth temperature. This fungus does not produce extracellular antifreeze proteins, but osmotic stress tolerance enables the fungus to grow at subzero temperatures. In conclusion, different taxa of snow molds have different strategies to adapt under snow cover.     

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.     

Cold shock response and low temperature adaptation in psychrotrophic bacteria

Psychrotrophic bacteria are capable of developing over a wide temperature range and they can grow at temperatures close to or below freezing. This ability requires specific adaptative strategies in order to maintain membrane fluidity, the continuance of their metabolic activities, and protein synthesis at low temperature. A cold-shock response has been described in several psychrotrophic bacteria, which is somewhat different from that in mesophilic microorganisms: (i) the synthesis of housekeeping proteins is not repressed following temperature downshift and they are similarly expressed at optimal and low temperatures (ii) cold-shock proteins or Csps are synthesized, the number of which increases with the severity of the shock (iii) a second group of cold-induced proteins, i.e. the cold acclimation proteins or Caps, comparable with Csps are continuously synthesized during prolonged growth at low temperature. Homologues to CspA, the major cold-shock protein in E. coli, have been described in various psychrotrophs, but unlike their mesophilic counterparts, they belong to the group of Caps. Although they have been poorly studied, Caps are of particular importance since they differentiate psychrotrophs from mesophiles, and they are probably one of the key determinant that allow life at very low temperature.     

Cold adaptation of a psychrophilic chitinase: a mutagenesis study

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.     

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.