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A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

There are a large number of active cold-adapted microorganisms in the perennial cold environment. Due to their high-efficiency and energy-saving catalytic properties, cold-adapted microorganisms have become valuable natural resources with potential in various biological fields. In this study, a series of cold response strategies for microorganisms were summarized. This mainly involves the regulation of cell membrane fluidity, synthesis of cold adaptation proteins, regulators and metabolic changes, energy supply, and reactive oxygen species. Also, the potential of biocatalysts produced by cold-adapted microorganisms including cold-active enzymes, ice-binding proteins, polyhydroxyalkanoates, and surfactants was introduced, which provided a guidance for expanding its application values. Overall, new insights were obtained on response strategies of microorganisms to cold environments in this review. This will deepen the understanding of the cold tolerance mechanism of cold-adapted microorganisms, thus promoting the establishment and application of low-temperature biotechnology.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at low temperatures down to − 0.4 °C. Maintaining cytoplasmic membrane fluidity by changing the lipid membrane composition is important during growth at low temperatures. In Listeria monocytogenes, the dominant adaptation effect is the fluidization of the membrane by shortening of fatty acid chain length. In some strains, however, an additional response is the increase in menaquinone content during growth at low temperatures. The increase of this neutral lipid leads to fluidization of the membrane and thus represents a mechanism that is complementary to the fatty acid-mediated modification of membrane fluidity. This study demonstrated that the reduction of menaquinone content for Listeria monocytogenes strains resulted in significantly lower resistance to temperature stress and lower growth rates compared to unaffected control cultures after growth at 6 °C. Menaquinone content was reduced by supplementation with aromatic amino acids, which led to a feedback inhibition of the menaquinone synthesis. Menaquinone-reduced Listeria monocytogenes strains showed reduced bacterial cell fitness. This confirmed the adaptive function of menaquinones for growth at low temperatures of this pathogen.

Atriplex halimus grows in humid and saline warm regions of Mediterranean area showing high adaptability but less information is available on resistance to chilling temperatures. Potted plants of fi...

In recent years, the prevalence of obesity has increased dramatically and has become a 21st century epidemic. Obesity is associated with the development of many diseases, and therefore treatments that can reduce body mass are actively sought. The aim of this study was to examine the effect of 20 cryostimulation sessions on body composition in obese/high body mass (HBM, n = 12) males and normal body mass (NBM, n = 9) controls. The HBM group had a mean age = 29.08 ± 4.19 years, body fat percentage = 32.08 ± 6.16%, body mass index = 36.23 ± 8.13 kg/m2) and NBM group had a mean age = 22.00 ± 2.45 years, body fat percentage = 12.14 ± 4.93%, body mass index = 23.58 ± 2.00 kg/m2. Kilocalorie intake was similar for both groups. All participants received 20 sessions of systemic cryostimulation at −120°C for 2–3 min in a cryochamber. Blood samples were collected before the first session, 1 h after the 10th session, and 1 h after the 20th cryostimulation session. C-reactive protein (CRP) plasma concentrations, and expression of the heat shock protein genes (HSPA1A, HSPB1) and CRP mRNA in leukocytes were evaluated after 10 and 20 cryostimulation sessions. In both groups, 20 sessions were associated with a significant decrease in body mass, fat mass and the percentage of body fat. CRP concentrations were significantly higher in obese people before the first session and after 10 treatments, but not at the end of study. Expression of HSPA1A and HSPB1 mRNA gradually decreased with the number of cryostimulation sessions. A significant difference in HSPA1A expression was found after 20 sessions (NBM > HBM) and for HSPB1 at baseline and after 20 sessions (HBM > NBM). Our results show that cryostimulation influences body composition and that cryostimulation-induced HSP genes expression depends on the number of cryosessions and baseline body mass, and is differentially altered in HBM individuals. Further research on the interaction between body mass and cold adaptation is warranted.

Introduction: Extreme-altitudes (5500 m/18045 ft and higher) pose environmental, psychophysiological, infrastructural, logistic, and ergonomic challenges that question explorer’s adaptability and mission-efficiency due to isolation, monotony, intimidating environment and terse health conditions. The assessment of an explorer’s comprehensive adaptability in extreme-altitudes is of paramount importance in ensuring mission-preparedness. Knowledge, attitude, and practices (KAP) of explorers staying on extreme-altitudes was assessed through personal interview technique. Methods: 125 healthy, acclimatized, mountain-trained explorers staying above 4570 m/15000 ft in winter-season for at least 30 days on extreme-altitudes were assessed by a single cross-sectional study through personal interviews on KAP related to extreme-altitude and cold-adaptation. Results: Mean duration of stay on extreme-altitude was 55.7 days. All explorers knew about difficulties and health-problems at extremealtitude. All explorers felt that mountain-training and acclimatization were beneficial. 92% felt that mission tenure of 90 days on the extreme-altitude was adequate. 92.8% felt they were adequately trained for the extreme-altitude; however, only 52% felt confident about health-training. 66.4% did preventive rewarming of extremities. 66.4% regularly smoked/chewed tobacco. 57.6% had sleep problems and 64% had altered appetite. 26.4% felt difficulties related to living conditions, infrastructure, and logistics. Conclusion: Explorers at extreme-altitude exhibited adequate knowledge and performed activities with progressive attitude and healthy practices. Explorers endured altered psychophysiology at extreme-altitude and regarded the utility of training and acclimatization programs. Knowledge-practice gap existed towards tobacco consumption. There was a felt need for the improvement of living conditions, infrastructure, logistics, and health-related training.

[cat] Els fruits climatèrics es caracteritzen per un procés de maduració comercial estretament vinculat a la producció d’etilè, que comporta la maduració i sobre-maduració del fruit al llarg de la cadena de producció, tot portant a pèrdues que afecten a la qualitat i a la seva apreciació pels consumidors. Diverses estratègies com l’emmagatzematge en fred s’implementen actualment per tal de frenar la maduració comercial i allargar la vida útil del fruit i, així, millorar-ne la comercialització. Per aquesta raó, l’estudi de processos fisiològics desencadenats en resposta a l’exposició a temperatures fredes i l’impacte en la qualitat és essencial per comprendre els avantatges d’aquesta tècnica i les seves limitacions com, per exemple, els danys per fred que tant afecten a la vida post-collita. En aquest estudi, es van seleccionar fruits d’alvocat de la varietat Bacon (Persea americana Mill. cv. Bacon) amb l’objectiu d’aprofundir en el coneixement sobre el procés de maduració comercial, determinar l’impacte de l’emmagatzematge en fred sobre el contingut d’antioxidants en el fruit i estudiar tècniques innovadores per augmentar la qualitat del fruit en la post- collita. En base als resultats, la maduració comercial dels alvocats està regulada, a part de l’etilè, per altres fitohormones estretament relacionades entre si i l’emmagatzematge en fred altera la resposta hormonal tot retardant aquest procés. Ara bé, les baixes temperatures indueixen una resposta d’aclimatació regulada principalment per jasmonats, els quals tenen un rol crucial en la tolerància al fred i en evitar el desordre de danys per fred en el fruit. Per una banda, la resposta dels jasmonats contra els danys per fred pot potenciar-se mitjançant l’aplicació directa en el fruit de metil jasmonat (100µM) en esprai en la post-collita sense afectar les propietats antioxidants del fruit i, per altra banda, el contingut antioxidant, principalment en vitamines E i B6, es pot incrementar notablement durant un llarg termini de temps d’emmagatzematge en fred (30d) amb tractaments post-collita de piridoxal 5’-fosfat (9mM) per immersió, els quals tenen a més un impacte positiu en fruits exposats a temperatura ambient. En conclusió, cal destacar la varietat ‘Bacon’ pel seu elevat contingut en vitamina E. A més, s’ha establert que les baixes temperatures són eficients per retardar la resposta hormonal implicada en la senyalització de maduració de l’alvocat. No obstant, el fred pot arribar a ocasionar danys al fruit que afecten negativament en el mercat. Així mateix, aplicacions de metil jasmonat en esprai i tractaments de piridoxal 5’-fosfat per immersió poden afavorir l’emmagatzematge i qualitat del fruit al llarg de la cadena de producció., [eng] Climacteric fruit are characterized by a ripening process tightly related to ethylene production, thus inducing ripening and overripening along the supply chain causing quality losses that affect consumers acceptance. Some strategies are implemented currently aiming to delay or inhibit ripening to prolong shelf life and increase avocado marketability. For this reason, the knowledge in physiological events triggered by cold exposure and its impact on fruit quality is of paramount importance to understand the advantages and disadvantages of the technique, for instance, chilling injury disorder that highly affects postharvest quality in avocados. Here, ‘Bacon’ avocados (Persea americana Mill. cv. Bacon) were selected aiming to deepen into our understanding of hormonal response regulating ripening, to determine the impact of low temperature exposure on antioxidants and to study some innovative techniques to increase postharvest quality on the fruit. According to the results, avocado ripening is regulated, rather than by ethylene alone, by orchestrated phytohormonal interactions and, cold storage alters these hormonal modulations, therefore delaying ripening. Differently, cold storage induced an acclimation response modulated by jasmonates participating in cold tolerance events for preventing chilling injury. In this sense, jasmonates response against chilling injury can be enhanced by spraying harvested fruit with methyl jasmonate (100µM) solutions, which resulted in a reduced number of damaged fruits without negatively affecting antioxidant properties due to vitamin E. Moreover, antioxidant content related to vitamins E and B6 can be triggered in long-term cold-stored fruit (30d) by submersing avocados in pyridoxal 5’-phosphate (9mM) solutions which, at room temperature, slightly delayed early ripening of fruit. In conclusion, ‘Bacon’ variety has higher amounts of vitamin E than the widely commercialised ‘Hass’ or ‘Fuerte’ avocados. Moreover, cold temperatures prevent hormonal responses related to ripening and activates cold tolerance mechanisms but, long term exposure can induce chilling injury in ‘Bacon’ avocados, thus affecting its marketability. Noteworthy, methyl jasmonate and pyridoxal 5’-phosphate treatments can improve ‘Bacon’ avocado storage and quality along the supply chain.

Carotenoids have several crucial biological functions and are part of the cold adaptation mechanism of some bacteria. Some pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, whose function in these bacteria is unclear and is found mainly in halophilic archaea. Strains Arthrobacter agilis DSM 20550T and Arthrobacter bussei DSM 109896T show an increased bacterioruberin content if growth temperature is reduced from 30 down to 10 °C. In vivo anisotropy measurements with trimethylammonium-diphenylhexatriene showed increased membrane fluidity and a broadening phase transition with increased bacterioruberin content in the membrane at low-temperature growth. Suppression of bacterioruberin synthesis at 10 °C using sodium chloride confirmed the function of bacterioruberin in modulating membrane fluidity. Increased bacterioruberin content also correlated with increased cell resistance to freeze–thaw stress. These findings confirmed the adaptive function of bacterioruberin for growth at low temperatures for pink-pigmented Arthrobacter species.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni–NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0–50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20–30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

It is known that adaptive evolution in permanently cold environments drives cold adaptation in enzymes. However, how the relatively high enzyme activities were achieved in cold environments prior to cold adaptation of enzymes is unclear. Here we report that an Antarctic strain of Chlorella vulgaris, called NJ-7, acquired the capability to grow at near 0 °C temperatures and greatly enhanced freezing tolerance after systematic increases in abundance of enzymes/proteins and positive selection of certain genes. Having diverged from the temperate strain UTEX259 of the same species 2.5 (1.1–4.1) to 2.6 (1.0–4.5) Ma, NJ-7 retained the basic mesophilic characteristics and genome structures. Nitrate reductases in the two strains are highly similar in amino acid sequence and optimal temperature, but the NJ-7 one showed significantly higher abundance and activity. Quantitative proteomic analyses indicated that several cryoprotective proteins (LEA), many enzymes involved in carbon metabolism and a large number of other enzymes/proteins, were more abundant in NJ-7 than in UTEX259. Like nitrate reductase, most of these enzymes were not upregulated in response to cold stress. Thus, compensation of low specific activities by increased enzyme abundance appears to be an important strategy for early stage cold adaptation to Antarctica, but such enzymes are mostly not involved in cold acclimation upon transfer from favorable temperatures to near 0 °C temperatures.

Purpose We summarized the evidence on the effects of heat and cold exposures on mortality in China. We included studies published on this topic in both Chinese and English, thereby filling a gap in knowledge using data from a country that consists of one-fifth of the world's population. Methods We conducted a systematic search of peer-reviewed studies on the association between daily mean temperature and mortality published from 2001 up to July 2018. We searched one Chinese database (China National Knowledge infrastructure, http://www.cnki.net ) and three English databases (PubMed, Scopus, Web of Science). We converted the effect estimates of heat/cold to rate ratios (RRs) associated with 1° increase/decrease beyond the heat/cold reference temperatures. For studies that provided lag-specific estimates, we used both the maximum and minimum of RR estimates. We calculated summary effect estimates for all-cause and cause-specific mortalities, as well as RRs stratified by sex, age, and socioeconomic status. We also investigated patterns of heat and cold adaptation at different latitudes, and at different reference temperatures. Results In total, 45 articles were included in this systematic review. For every 1° temperature increase/decrease beyond reference points, the rate of non-accidental mortality increased by 2% (RR, 1.02; 95% confidence interval (95% CI [1.01–1.02]) for heat and 4% (RR, 1.04; 95% CI [1.03–1.04]) for cold, respectively; the rate of cardiovascular mortality increased 3% (RR, 1.03; 95% CI [1.03–1.04]) for heat and 6% (RR, 1.06; 95% CI [1.04–1.07]) for cold; the rate of respiratory mortality increased 2% (RR, 1.02; 95% CI [1.01–1.03]) for heat and 2% (RR, 1.02; 95% CI [1.00–1.04]) for cold; the rate of cerebrovascular mortality increased 2% (RR, 1.02; 95% CI [1.02–1.03]) for heat and 3% (RR, 1.03; 95% CI [1.02–1.04]) for cold. We identified a variation in optimal temperature range related to latitude of the residential area, and differences in people's capability to adapt to heat versus cold. Conclusion We found consistent evidence of the association between temperature and mortality, as well as evidence of patterns in human adaptation, and we discussed the implications of our findings.

As global temperatures rise, the mechanistic links between temperature, physiology and behaviour will increasingly define predictions of ecological change. However, for many taxa, we currently lack consensus about how thermal performance traits vary within and across populations, and whether and how locally adaptive trait plasticity can buffer warming effects. The metabolic cold adaptation hypothesis posits that cold environments (e.g. high elevations and latitudes) select for high metabolic rates (MR), even after controlling for body size differences, and that this enables high activity levels when an organism is near its cold lower thermal limits. Steep MR reaction norms are further predicted at cold temperatures to enable rapid behavioural activation with rising temperatures needed to exploit brief thermal windows suitable for performing eco-evolutionary tasks. We tested these predictions by performing common garden experiments comparing thermal reaction norms of MR (from 15 to 32°C) and behaviour (from 10 to 40°C) across populations of the ant Aphaenogaster iberica sampled from a 2 km elevation gradient in the Sierra Nevada Mountains of southern Spain. As predicted, high-elevation ants had higher MR and steeper MR-temperature reaction norms. However, higher rates of energy use did not yield the predicted benefits of steeper activity-level reaction norms. The evidence for locally adaptive metabolic physiology only became apparent at intermediate temperatures, highlighting the importance of testing thermal performance hypotheses across thermal gradients, rather than focusing only on performance at thermal limits (i.e. critical thermal values). The partial support for the metabolic cold adaptation hypothesis highlights that while organisms likely show a wealth of unexplored metabolic temperature plasticity, the physiological mechanisms and eco-evolutionary trade-offs underlying such local adaptation remain obscure., J.Z.S. was supported by a postdoctoral fellowship from the Smithsonian Institution Competitive Grants Program, by a Marie Curie International Incoming Fellowship (327940 INSEAME) and by the Centre for Social Evolution at the University of Copenhagen. X.A. was supported by a Ramón y Cajal research contract by the Spanish Ministry of Economy and Competitiveness (RYC-2015-18448). X.C. and R.B. were funded by the Spanish Ministry of Economy and Competitiveness and FEDER (CGL2015-65807-P).

We present here a review of the literature analyzing the link between the physiological mechanisms of cold adaptation and cold-induced prooxidant and compensatory antioxidant processes. Cold adaptation has been shown to increase the number and activity of mitochondria to support increases in ATP consumption; this leads to increased generation of reactive oxygen species (ROS), as mitochondria are among the main sources of ROS in physiological conditions. Studies in recent decades have shown that protein PGC-1α – one of the main regulators of mitochondrial biogenesis – also affects the synthesis of antioxidant enzymes, thus controlling the mitochondrial generation of reactive oxygen species. At the whole-body level, systematic cold stimulates intrinsic protective resources by enhancing oxidative processes, which in turn initiates activation of the antioxidant systems and increases the overall resistance of the body to stress factors of different types.

Pedobacter cryoconitis BG5 is an obligate psychrophilic bacterium that was first isolated on King George Island, Antarctica. Over the last 50 years, the West Antarctic, including King George Island, has been one of the most rapidly warming places on Earth, hence making it an excellent area to measure the resilience of living species in warmed areas exposed to the constantly changing environment due to climate change. This bacterium encodes a genome of approximately 5694 protein-coding genes. However, 35% of the gene models for this species are found to be hypothetical proteins (HP). In this study, three conserved HP genes of P. cryoconitis, designated pcbg5hp1, pcbg5hp2 and pcbg5hp12, were cloned and the proteins were expressed, purified and their functions and structures were evaluated. Real-time quantitative PCR analysis revealed that these genes were expressed constitutively, suggesting a potentially important role where the expression of these genes under an almost constant demand might have some regulatory functions in thermal stress tolerance. Functional analysis showed that these proteins maintained their activities at low and moderate temperatures. Meanwhile, a low citrate synthase aggregation at 43 °C in the presence of PCBG5HP1 suggested the characteristics of chaperone activity. Furthermore, our comparative structural analysis demonstrated that the HPs exhibited cold-adapted traits, most notably increased flexibility in their 3D structures compared to their counterparts. Concurrently, the presence of a disulphide bridge and aromatic clusters was attributed to PCBG5HP1’s unusual protein stability and chaperone activity. Thus, this suggested that the HPs examined in this study acquired strategies to maintain a balance between molecular stability and structural flexibility. Conclusively, this study has established the structure–function relationships of the HPs produced by P. cryoconitis and provided crucial experimental evidence indicating their importance in thermal stress response.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at −1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

In order to investigate the effect of intermittent mild cold stimulation (IMCS) on immune function of spleens and adaptability to cold stress in broilers, 400 healthy 1-day-old Ross-308 chickens were divided into 5 groups: CC (control) reared in normal thermal environment from 1 to 49 d; CS3, CS4, CS5, and CS6 (treatments) raised at 3°C below the temperature of CC for 3, 4, 5, or 6 h at 1-d intervals from 15 to 35 d, respectively. Subsequently, CS3-6 was raised at 20°C from 36 to 49 d. At 50 d, all groups were exposed to acute cold stress (ACS) for 12 h. The spleen immunity index at 22, 29, 36, 43, and 49 d, expression levels of toll-like receptors (TLRs), cytokines and immunoglobulins at 22, 43, and 49 d and heat shock proteins (HSPs) before and after ACS at 50 d were examined. The spleen index of broilers aged 22 to 49 d did not differ between CS and CC (P > 0.05), and the spleen index of CS5 was higher than that of CS3 at 49 d (P < 0.05). The mRNA levels of TLR5, TLR15, TLR21, and IL-2 in CS3, TLR3, TLR4, TLR15, TLR21, IL-2, IL-6, and IFN-ϒ in CS4, TLR1, TLR3, TLR4, TLR21, IL-2, IFN-a, IFN-ϒ, IgA, and IgG in CS6, but all TLRs, immunoglobulins and cytokines except IFN-ϒ in CS5 differential expressed stably compared with CC at 43 and 49 d (P < 0.05). Compared with Pre-ACS, the mRNA levels of HSP60, HSP70, and HSP90 were upregulated in CS after ACS (P < 0.05). Except for HSP90 mRNA and HSP70 protein in CS6, and HSP90 protein in CS3, the levels of HSPs after ACS in all treatment groups were higher than those in CC (P < 0.05), and the highest HSPs levels after ACS were found in CS5. We concluded that IMCS could enhance immunity of spleens and adaptability to ACS in broilers, besides CS5 was the optimal program.

The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that Ubqln1, 2 and 4 are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. Inconclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.

Graphical abstract, Highlights • A high-resolution structure of a psychrophilic RNA polymerase contributes to our knowledge of cold adaptation. • While catalytic core motifs are conserved, at least one shows cold adaptation. • Loss of helix-capping increases structural flexibility in a catalytic core motif. • Overall reduction of alpha-helical elements appears as a strategy for cold adaptation., CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence C-C-A at tRNA 3‘-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to −15 °C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation - a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6–7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Native cattle breeds represent an important cultural heritage. They are a reservoir of genetic variation useful for properly responding to agriculture needs in the light of ongoing climate changes. Evolutionary processes that occur in response to extreme environmental conditions could also be better understood using adapted local populations. Herein, different evolutionary histories of the world northernmost native cattle breeds from Russia were investigated. They highlighted Kholmogory as a typical taurine cattle, whereas Yakut cattle separated from European taurines approximately 5,000 years ago and contain numerous ancestral and some novel genetic variants allowing their adaptation to harsh conditions of living above the Polar Circle. Scans for selection signatures pointed to several common gene pathways related to adaptation to harsh climates in both breeds. But genes affected by selection from these pathways were mostly different. A Yakut cattle breed-specific missense mutation in a highly conserved NRAP gene represents a unique example of a young amino acid residue convergent change shared with at least 16 species of hibernating/cold-adapted mammals from six distinct phylogenetic orders. This suggests a convergent evolution event along the mammalian phylogenetic tree and fast fixation in a single isolated cattle population exposed to a harsh climate.

Background Asian tiger mosquito Aedes albopictus is an arbovirus vector that has spread from its native habitation areal in Southeast Asia throughout North and South Americas, Europe, and Africa. Ae. albopictus was first detected in the Southern Federal District of the Russian Federation in the subtropical town of Sochi in 2011. In subsequent years, this species has been described in the continental areas with more severe climate and lower winter temperatures. Methods Genomic analysis of pooled Ae. albopictus samples collected in the mosquito populations in the coastal and continental regions of the Krasnodar Krai was conducted to look for the genetic changes associated with the spread and potential cold adaptation in Ae. albopictus. Results The results of the phylogenetic analysis based on mitochondrial genomes corresponded well with the hypothesis that Ae. albopictus haplotype A1a2a1 was introduced into the region from a single source. Population analysis revealed the role of dispersal and genetic drift in the local adaptation of the Asian tiger mosquito. The absence of shared haplotypes between the samples and high fixation indices suggest that gene flow between samples was heavily restricted. Mitochondrial and genomic differentiation together with different distances between dispersal routes, natural and anthropogenic barriers and local effective population size reduction could lead to difficulties in local climatic adaptations due to reduced selection effectiveness. We have found genomic regions with selective sweep patterns which can be considered as having been affected by recent selection events. The genes located in these regions participate in neural protection, lipid conservation, and cuticle formation during diapause. These processes were shown to be important for cold adaptation in the previous transcriptomic and proteomic studies. However, the population history and relatively low coverage obtained in the present article could have negatively affect sweep detection.

We here report the de novo transcriptome assembly and functional annotation of Eusirus cf. giganteus clade g3, providing the first database of expressed sequences from this giant Antarctic amphipod. RNA-sequencing, carried out on the whole-body of a single juvenile individual likely undergoing molting, revealed the dominant expression of hemocyanins. The mRNAs encoding these oxygen-binding proteins cumulatively accounted for about 40% of the total transcriptional effort, highlighting the key biological importance of high hemocyanin production in this Antarctic amphipod species. We speculate that this observation may mirror a strategy previously described in Antarctic cephalopods, which compensate the decreased ability to release oxygen to peripheral tissues at sub-zero temperatures by massively increasing total blood hemocyanin content compared with temperate species. These preliminary results will undoubtedly require confirmation through proteomic and biochemical analyses aimed at characterizing the oxygen-binding properties of E. cf. giganteus clade g3 hemocyanins, and at investigating whether other Antarctic arthropod species exploit similar adaptations to cope with the challenges posed by the extreme conditions of the polar environment.

Synechococcus are picocyanobacteria with a cosmopolitan distribution. They are capable of surviving in a wide variety of environmental conditions. Synechococcus have been isolated from the Chesapeake Bay during winter months, and they show an impressive tolerance to cold temperatures. Cold-adapted Synechococcus are unique and diverse, as they have complex phylogenetic lineages closely related to subalpine cluster II, Bornholm Sea cluster, CB7 cluster, and some novel lineages which are independent from summer estuarine strains in subcluster 5.2. CBW1002 and CBW1006 are the first complete genomes to represent Bornholm Sea cluster Synechococcus strains. They have some of the largest genomes among the Synechococcus (3.8 Mb) and share many unique and cryptic homologs which could give insight into their ability to tolerate such cold and dynamic conditions in the Chesapeake Bay estuary.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.

The study of enzymes from extremophiles arouses interest in Protein Science because of the amazing solutions these proteins adopt to cope with extreme conditions. Recently solved, the structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) pinpoints a mechanism of dimerization unusual for this class of enzymes. The quaternary structure of SpAAP relies on a domain-swapping mechanism involving the N-terminal A1 helix. The A1 helix is conserved among homologous mesophilic and psychrophilic proteins and its deletion causes the formation of a monomeric enzyme, which is inactive and prone to aggregate. Here, we investigate the dimerization mechanism of SpAAP through the analysis of chimeric heterodimers where a protomer lacking the A1 helix combines with a protomer carrying the inactivated catalytic site. Our results indicate that the two active sites are independent, and that a single A1 helix is sufficient to partially recover the quaternary structure and the activity of chimeric heterodimers. Since catalytically competent protomers are unstable and inactive unless they dimerize, SpAAP reveals as an “obligomer” for both structural and functional reasons.

Thyroid hormone (TH) is an important regulator of mammalian metabolism and facilitates cold induced thermogenesis (CIT) in brown adipose tissue (BAT). Profound hypothyroidism or hyperthyroidism lead to alterations in BAT function and CIT. In euthyroid humans the inter-individual variation of thyroid hormones is relatively large. Therefore, we investigated whether levels of free thyroxine (T4) or free triiodothyronine (T3) are positively associated with CIT in euthyroid individuals. We performed an observational study in 79 healthy, euthyroid volunteers (mean age 25.6 years, mean BMI 23.0 kg · m-2). Resting energy expenditure (REE) was measured by indirect calorimetry during warm conditions (EEwarm) and after a mild cold stimulus of two hours (EEcold). CIT was calculated as the difference between EEcold and EEwarm. BAT activity was assessed by 18F-FDG-PET after a mild cold stimulus in a subset of 26 participants. EEcold and CIT were significantly related to levels of free T4 (R2 = 0.11, p=0.0025 and R2 = 0.13, p=0.0011, respectively) but not to free T3 and TSH. Cold induced BAT activity was also associated with levels of free T4 (R2 = 0.21, p=0.018). CIT was approximately fourfold higher in participants in the highest tertile of free T4 as compared to the lowest tertile. Additionally, free T4 was weakly, albeit significantly associated with outdoor temperature seven days prior to the respective study visit (R2 = 0.06, p=0.037). These finding suggests that variations in thyroid hormone levels within the euthyroid range are related to the capability to adapt to cool temperatures and affect energy balance., Frontiers in Endocrinology, 12, ISSN:1664-2392

With accelerating global warming, understanding the evolutionary dynamics of plant adaptation to environmental change is increasingly urgent. Here we reveal the enigmatic history of the genus Cochlearia (Brassicaceae), a Pleistocene relic that originated from a drought-adapted Mediterranean sister genus during the Miocene. Cochlearia rapidly diversified and adapted to circum-Arctic regions and other cold-characterized habitat types during the Pleistocene. This rapid change in ecological preferences was accompanied by a highly complex, reticulate polyploid evolution, which was apparently triggered by the impact of repeated Pleistocene glaciation cycles. Our results illustrate that two early diversified arctic-alpine diploid gene pools contributed differently to the evolution of this young polyploid genus now captured in a cold-adapted niche. Metabolomics revealed ancestral central carbon metabolism responses to cold in diverse ecotypes, likely due to continuous connections to cold habitats that we hypothesize facilitated widespread parallel adaptation to alpine and subalpine habitats, and which we speculate were coopted from existing drought adaptations. Given the growing scientific interest in adaptive evolution of temperature-related traits, our results provide much-needed taxonomic and phylogenomic resolution of a model system as well as first insights into the origins of its adaptation to cold.

Cold climates represent one of the major environmental challenges that anatomically modern humans faced during their dispersal out of Africa. The related adaptive traits have been achieved by modulation of thermogenesis and thermoregulation processes where nuclear (nuc) and mitochondrial (mt) genes play a major role. In human populations, mitonuclear genetic interactions are the result of both the peculiar genetic history of each human group and the different environments they have long occupied. This study aims to investigate mitonuclear genetic interactions by considering all the mitochondrial genes and 28 nuclear genes involved in brown adipose tissue metabolism, which have been previously hypothesized to be crucial for cold adaptation. For this purpose, we focused on three human populations (i.e., Finnish, British, and Central Italian people) of European ancestry from different biogeographical and climatic areas, and we used a machine learning approach to identify relevant nucDNA–mtDNA interactions that characterized each population. The obtained results are twofold: (i) at the methodological level, we demonstrated that a machine learning approach is able to detect patterns of genetic structure among human groups from different latitudes both at single genes and by considering combinations of mtDNA and nucDNA loci; (ii) at the biological level, the analysis identified population-specific nuclear genes and variants that likely play a relevant biological role in association with a mitochondrial gene (such as the “obesity gene” FTO in Finnish people). Further studies are needed to fully elucidate the evolutionary dynamics (e.g., migration, admixture, and/or local adaptation) that shaped these nucDNA–mtDNA interactions and their functional role.

Cold environments, such as glaciers and alpine regions, constitute unique habitats for organisms living on Earth. In these harsh ecosystems, snow algae survive, florish, and even become primary producers for microbial communities. How the snow algae maintain physiological activity during violent ambient temperature changes remains unsolved. To explore the cold adaptation mechanisms of the unicellular snow alga Chlamydomonas nivalis, we compared its physiological responses to a model organism from the same genus, Chlamydomonas reinhardtii. When both cell types were exposed to a shift from 22°C to 4°C, C. nivalis exhibited an apparent advantage in cold tolerance over C. reinhardtii, as C. nivalis had both a higher growth rate and photosynthetic efficiency. To determine the cold tolerance mechanisms of C. nivalis, RNA sequencing was used to compare transcriptomes of both species after 1 h of cold treatment, mimicking temperature fluctuations in the polar region. Differential expression analysis showed that C. nivalis had fewer transcriptomic changes and was more stable during rapid temperature decrease relative to C. reinhardtii, especially for the expression of photosynthesis related genes. Additionally, we found that transcription in C. nivalis was precisely regulated by the cold response network, consisting of at least 12 transcription factors and 3 RNA-binding proteins. Moreover, genes participating in nitrogen metabolism, the pentose phosphate pathway, and polysaccharide biosynthesis were upregulated, indicating that increasing resource assimilation and remodeling of metabolisms were critical for cold adaptation in C. nivalis. Furthermore, we identified horizontally transferred genes differentially expressed in C. nivalis, which are critical for cold adaptation in other psychrophiles. Our results reveal that C. nivalis adapts rapid temperature decrease by efficiently regulating transcription of specific genes to optimize resource assimilation and metabolic pathways, providing critical insights into how snow algae survive and propagate in cold environments.

A large body of work focuses on the unique aspects of Neanderthal anatomy, inferred physiology, and behavior to test the assumption that Neanderthals were hyper-adapted to living in cold environments. This research has expanded over the years to include previously unexplored and potentially adaptive features such as brown adipose tissue and fire-usage. Here we review the current state of knowledge of Neanderthal cold adaptations along morphological, physiological, and behavioral lines. While highlighting foundational as well as recent work, we also emphasize key areas for future research. Despite thriving in a variety of climates, it is well-accepted that Neanderthals appear to be the most cold-adapted of known fossil hominin groups; however, there are still many unknowns. There is a great deal yet to be uncovered about the nature and manifestation of Neanderthal adaptation and how the synergy of biology and culture helped buffer them against extreme and variable environments.

Antarctic regions involve a great variety of habitats characterized by environmental stressors and life forms of autotrophic organisms with unique survival and functioning mechanisms. Lichens and mosses from these regions, similarly to high altitude alpine locations, have evolved physiological adaptations to perform photosynthesis at subzero temperatures. In this study we applied linear cooling technique in order to analyze interspecific differences in primary photosynthetic processes in Antarctic species affected by low and subzero temperature stress. We exposed Sanionia uncinata, Rhizoplaca aspidophora, Ochrolechia frigida, Cladonia sp., Himantormia lugubris and Umbilicaria decussata to the cooling from 20 to -35°C at a constant rate of 2°C min-1. Fluorometric parameters were measured during the cooling experiments: FV/FM - potential yield of photosynthetic processes in photosystem II, and F0 - minimal chlorophyll fluorescence. All the species showed S-curves for FV/FM in response to decreasing temperature and interspecific differences in the parameters of S-curve equation. Critical temperature for FV/FM was found -35°C for U. decussata, while the other species ranged between -16 to -20°C. The changes of F0 with thallus temperature decrease were species-specific. F0 decrease followed by an increase was found with cooling from 20 to -20°C, and from -20 to -35°C, respectively, in the majority of cases. These results suggest that the experimental moss and lichen species from Antarctica have a high resistance to freezing temperatures. The underlying physiological mechanisms are constitutive features of Antarctic lichens and mosses. They are a crucial part of the adaptation and short-term acclimatory changes in ecophysiological performance of the organisms in harsh polar environments.

A novel psychrotroph Cryobacterium sp. GCJ02 was isolated and characterized, which showed gorwth well at 4 ºC. The assembled whole genome of strain GCJ02 is 4.39 Mb, including 4,139 protein coding genes with G+C content of 68.41mol%. In this study, we report the complete genome sequence of a novel strain of the genus Cryobacterium, affording feasibility to elucidate the molecular mechanism of cold adaptation, and facilitate genetic manipulation of this bacterium.

In the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Chionodraco hamatus, an Antarctic teleost widely distributed in many areas of the Ross Sea that plays a pivotal role in the Antarctic food chain. The primary sequence was obtained using biochemical and molecular biology approaches and compared with Cu,Zn SODs from other organisms. Multiple sequence alignment using the amino acid sequence revealed that Cu,Zn SOD showed considerable sequence similarity with its orthologues from various vertebrate species, but also some specific substitutions directly linked to cold adaptation. Phylogenetic analyses presented the monophyletic status of Antartic Teleostei among the Perciformes, confirming the erratic differentiation of these proteins and concurring with the theory of the “unclock-like” behavior of Cu,Zn SOD evolution. Expression of C. hamatus Cu,Zn SOD at both the mRNA and protein levels were analyzed in various tissues, highlighting the regulation of gene expression related to environmental stress conditions and also animal physiology. The data presented are the first on the antioxidant enzymes of a fish belonging to the Channichthyidae family and represent an important starting point in understanding the antioxidant systems of these organisms that are subject to constant risk of oxidative stress.

It is well known that the chicken gene pools have high adaptive abilities, including adaptation to cold environments. This research aimed to study the genomic distribution of runs of homozygosity (ROH) in a population of Russian White (RW) chickens as a result of selection for adaptation to cold environments in the early postnatal period, to perform a structural annotation of the discovered breed-specific regions of the genome (compared to chickens of the Amroks breed) and to suggest key candidate genes associated with the adaptation of RW chickens to cold environments. Genotyping of individual samples was performed using Illumina Chicken 60K SNP BeadChip® chips. The search for homozygous regions by individual chromosomes was carried out using the PLINK 1.9 program and the detectRuns R package. Twelve key genes on breed-specific ROH islands were identified. They may be considered as potential candidate genes associated with the high adaptive ability of chickens in cold environments in the early postnatal period. Genes associated with lipid metabolism (SOCS3, NDUFA4, TXNRD2, IGFBP 1, IGFBP 3), maintaining body temperature in cold environments (ADIPOQ, GCGR, TRPM2), non-shivering thermogenesis (RYR2, CAMK2G, STK25) and muscle development (METTL21C) are perspectives for further research. This study contributes to our understanding of the mechanisms of adaptation to cold environments in chickens and provides a molecular basis for selection work.

Basal metabolic activity and freezing of body fluids create reactive oxygen species (ROS) in freeze-tolerant organisms. These sources of ROS can have an additive negative effect via oxidative stress. In cells, antioxidant systems are responsible for removing ROS in order to avoid damage due to oxidative stress. Relatively little is known about the importance of metabolic rate for the survival of freezing, despite a good understanding of several cold tolerance related physiological mechanisms. We hypothesized that low basal metabolism would be selected for in freeze-tolerant organisms where winter survival is important for fitness for two reasons. First, avoidance of the additive effect of ROS production from metabolism and freezing, and second, as an energy saving mechanism under extended periods of freezing where the animal is metabolically active, but unable to feed. We used the terrestrial oligochaete, Enchytraeus albidus, which is widely distributed from Spain to the high Arctic and compared eight populations originating across a broad geographical and climatic gradient after they had been cold acclimated at 5 degrees C in a common garden experiment. Cold tolerance (lower lethal temperature: LT50) and the potential metabolic activity (PMA, an estimator of the maximal enzymatic potential of the mitochondria) respiration chain) of eight populations were positively correlated amongst each other and correlated negatively with latitude and positively with average yearly temperature and the average temperature of the coldest month. These results indicate that low PMA in cold tolerant populations is important for survival in extremely cold environments.

Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on truncated hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of the unusual geminate rebinding phase. Water molecules are present in the distal pocket, but their stabilization by TrpG8, TyrB10, and HisCD1 is much weaker than in thermophilic Thermobifida fusca truncated hemoglobin, resulting in a faster geminate rebinding. Our results support the hypothesis that weaker water-molecule interactions at the reaction site are associated with cold adaptation. Fil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Boechi, Leonardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Patrizi, Barbara. Università di Firenze. Lens European Laboratory For Non-linear Spectroscopy; Italia. Istituto Nazionale di Ottica; Italia Fil: Di Donato, Mariangela. Università di Firenze. Lens European Laboratory For Non-linear Spectroscopy; Italia. Istituto Nazionale di Ottica; Italia Fil: Iagatti, Alessandro. Università di Firenze. Lens European Laboratory For Non-linear Spectroscopy; Italia Fil: Giordano, Daniela. Consiglio Nazionale delle Ricerche; Italia. Stazione Zoologica Anton Dohrn; Italia Fil: Verde, Cinzia. Consiglio Nazionale delle Ricerche; Italia. Stazione Zoologica Anton Dohrn; Italia Fil: Bruno, Stefano. Università di Parma; Italia Fil: Abbruzzetti, Stefania. Università di Parma; Italia Fil: Viappiani, Cristiano. Università di Parma; Italia

Cold-active cellulases have received great attention for both industrial applications and fundamental research because of their high activity at low temperatures and their unique structural characters. In this study, the cold-active endoglucanase CelX from psychrotrophic Pseudoalteromonas sp. DY3 was successfully overexpressed in E. coli, partly purified and characterized in detail. CelX showed the highest activity at pH 5.5, and exhibited moderate activity and superior pH stability over a wide pH range (pH 5.0–pH 9.0). It displayed the highest activity at 45 °C, and kept 34.7% residual activity even at 5 °C. It was stable below 35 °C and lost activity very quickly above 45 °C, which is consistent with its cold adaptability. The apparent kinetic parameters CelX against CMC (carboxymethyl cellulose) were determined, with the Km and kcat values of 6.4 mg/ml and 4.2 s−1 respectively. Mn2+ and Co2+ enhanced the cellulolytic activity of CelX by 28.8% and 20.6% respectively, whereas Pb2+ and Cu2+ inhibited its activity by 14.9% and 6.5% separately. The cold adaptation of CelX is possibly due to the presence of the unusually long linker between the catalytic module and the cellulose-binding domain.

Protein evolution is potentially governed by protein stability. Here, we investigated the relationship between protein evolution and stability through the random mutational drift of a thermophilic bacterial protein, an esterase of Alicyclobacillus acidocaldarius (Aac-Est), at high and low temperatures. In the first random mutation of Aac-Est, few proteins exhibit increased activity at 65 °C, indicating that the wild-type (WT) Aac-Est is located on the peak of a mountain in a fitness landscape for activity at high temperature. To obtain higher active variants than those of WT, it must go down the mountain once and climb another, higher mountain. In the second and third generations from lower active templates, the evolvability (the proportion of variants with higher activity in all the variants obtained in a given generation than a parent protein) depended on the stability of the template proteins. Compared to WT, the stability-maintaining template could recover the activity more. Thus, a low-activity variant with high stability is able to drift vastly in sequence space and reach the foot of a higher mountain. Meanwhile, random mutations in stability-loss templates produced several variants with higher activity at 40 °C than those produced by WT, via cold adaptation. Our results indicate that maintaining protein stability enables the protein to search sequence space and evolve in the original environment, and proteins with lost stability use a cold adaptation path.