Cold-Adapted Enzymes from Marine Antarctic Microorganisms

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology.     

Mechanism of bacterial adaptation to low temperature

Survival of bacteria at low temperatures provokes scientific interest because of several reasons. Investigations in this area promise insight into one of the mysteries of life science —namely, how the machinery of life operates at extreme environments. Knowledge obtained from these studies is likely to be useful in controlling pathogenic bacteria, which survive and thrive in cold-stored food materials. The outcome of these studies may also help us to explore the possibilities of existence of life in distant frozen planets and their satellites.     

Absence of metabolic cold adaptation and compensatory acclimation in the Antarctic fly, Belgica antarctica

The respiratory metabolism in larvae of the Antarctic fly, Belgica antarctica Jacobs (Diptera: Chironomidae) was investigated at Palmer Station, Anvers Island (64°46′S, 64°03′W). Oxygen consumption was linearly related to temperature from 0 to 20°C, respectively, 49 and 338 nl/mg live wt/hr. Maintenance at 0 and 10°C for 8 days had no differential effect on the metabolic rate, suggesting that larvae lack the ability for compensatory acclimation. A comparison of standard metabolism for polar and temperate chironomids revealed no elevation of metabolic rate in polar forms. However, polar species exhibited lower activation energies than temperate forms indicating that the respiratory metabolism of polar chironomids is relatively temperature independent.     

Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.     

Energy partitioning in the Antarctic collembolan Cryptopygus antarcticus

Abstract. 1. Consumption, production and assimilation rates were determined for two age groups of Crypropygus antarcticus to give an estimate of energy utilization, and to investigate low temperature adaptation in its energy partitioning.
2. Feeding selectivity shown in laboratory preference tests was supported by gut analysis of field animals from contrasting sites. Although moulting rate was not significantly affected by food type, rates of growth were slowest and mortality highest when fed on a non-preferred substrate.
3. Both a radio labelling and a more direct method for measuring dry weight consumed gave similar results for Cvpropygus feeding on algae. The consuniption rate for animals when feeding on algae was lower than that on moss peat. The assimilation efficiency for immature animals feeding on algae was 46% and for mature animals was 19%; the values when feeding on moss peat were 7% and lo%, respectively, The net production efficiency ranged from 35%(inimatures) to 13% (matures) and was similar on both substrates.
4. Food consumption exceeded assimilation over the range 2.5–10°C, but the two converged from 2.5 to 0°C. Immature Cryptopygus maintained a net positive energy balance over 0–10°C, whilst below 1S°C respiration exceeded assimilation for mature individuals.
5. An estimate of the annual dry matter consumption (7 g m^-1 y^-1) by Ctypropygus in a moss turf at Signy Island agrees with one based on respiration data alone (Davis, 1981). The consumption at an alga-dominated site was c . 26 g m^-2 y^-l, and Crypropygus may have a locally limiting effect on net priniary production at such sites.     

微生物产生的冷休克蛋白研究进展

冷休克蛋白(cold shock protein,Csp)首先在大肠杆菌中发现,它与微生物对冷环境的适应及多种细胞功能有关。冷休克蛋白基因是一段编码70个左右氨基酸的DNA序列,在这段序列中有5′非翻译区(5′UTR)、冷盒及下游盒等特征。冷休克蛋白作为DNA或RNA结合蛋白在基因表达调控过程中起重要作用。冷休克蛋白在转录、mRNA稳定性及翻译等几个水平上被严格调控。     

Temperature preferences of the mite, Alaskozetes antarcticus, and the collembolan, Cryptopygus antarcticus from the maritime Antarctic

Abstract. The thermal preferences of Alaskozetes antarcticus (Acari, Cryptostigmata) and Cryptopygus antarcticus (Collembola, Isotomidae) were investigated over 6 h within a temperature gradient (?3 to +13 °C), under 100% relative humidity (RH) conditions. After 10 days of acclimation at ?2 or +11 °C, individual supercooling points (SCP) and thermopreferences were assessed, and compared with animals maintained for 10 days under fluctuating field conditions (?6 to +7 °C). Acclimation at ?2 °C lowered the mean SCP of both A. antarcticus (?24.2 ± 9.1) and C. antarcticus (?14.7 ± 7.7) compared to field samples (?19.0 ± 9.0 and ?10.7 ± 5.2, respectively). Acclimation at +11 °C increased A. antarcticus mean SCP values (?13.0 ± 8.5) relative to field samples, whereas those of C. antarcticus again decreased (?16.7 ± 9.1). Mites acclimated under field conditions or at +11 °C selected temperatures between ?3 and +1 °C. After acclimation at ?2 °C, both species preferred +1 to +5 °C. Cryptopygus antarcticus maintained under field conditions preferred +5 to +9 °C, whereas individuals acclimated at +11 °C selected +9 to +13 °C. For A. antarcticus, thermopreference was not influenced by its cold hardened state. The distribution of field specimens was further assessed within two combined temperature and humidity gradient systems: (i) 0–3 °C/12% RH, 3–6 °C/33% RH, 6–9 °C/75% RH and 9–12 °C/100% RH and (ii) 0–3 °C/100% RH, 3–6 °C/75% RH, 6–9 °C/33% RH and 9–12 °C/12% RH. In gradient (i), C. antarcticus distributed homogeneously, but, in gradient (ii), C. antarcticus preferred 0–3 °C/100% RH. Alaskozetes antarcticus selected temperatures between 0 and +6 °C regardless of RH conditions. Cryptopygus antarcticus appears better able than A. antarcticus to opportunistically utilize developmentally favourable thermal microclimates, when moisture availability is not restricted. The distribution of A. antarcticus appears more influenced by temperature, especially during regular freeze‐thaw transitions, when this species may select low temperature microhabitats to maintain a cold‐hardened state.     

Experimental studies on the cold tolerance of Alaskozetes antarcticus

The cold tolerance mechanism of the Antarctic terrestrial mite Alaskozetes antarcticus (Michael) was investigated in cultured animals. Freezing is fatal in this species and winter survival occurs by means of supercooling, which is enhanced by the presence of glycerol in the body. There is an inverse, linear relationship between the concentration of glycerol and the supercooling point, which may be as low as ?30°C. Feeding detracts from supercooling ability by providing ice nucleators in the gut which initiate freezing at relatively high sub-zero temperatures. Experiments on the effects of various environmental factors showed that low temperature acclimation gave rise to increased glycerol concentrations and suppressed feeding, while desiccation also stimulated glycerol production. Photoperiod had no effect on cold tolerance in this species. The juvenile instars of A. antarcticus were found to possess a greater degree of low temperature tolerance than adults.     

Protein adaptation to low temperatures: a comparative study of α-tubulin sequences in mesophilic and psychrophilic algae

The α-tubulin genes from two psychrophilic algae belonging to the genus Chloromonas (here named ANT1 and ANT3) have been isolated and sequenced. The genes ant1 and ant3 contain 4 and 2 introns, respectively. The coding DNA sequences are 90% identical but the degree of isology is very high at the polypeptide level (more than 97% strict identities). The ANT1 and ANT3 α-tubulin amino acid sequences were compared to the corresponding sequence of the mesophilic alga Chlamydomonas reinhardtii. Of the 15 substitutions detected in ANT1 and/or ANT3, 5 are common to both psychrophilic algae. The recorded substitutions have been analyzed in terms of cold adaptation on the basis of the available three-dimensional structure of the α,β-tubulin heterodimer from pig brain. Most of these are subtle changes, but two substitutions, M268V and A295V occurring in the region of interdimer contacts, could be of great significance for the cold stability of Antarctic algae microtubules due to the fact that the entropic control of microtubule assembly is particularly high in cold adaptes species. Received: December 24, 1998 / Accepted: April 2, 1999     

The effect of thermal adaptation on the level of nitrogenous excretion of Channa punctatus (bloch)

The rates of ammonia-N and urea-N excretion have been measured in summer- winter-adapted Channa punctatus, a representative of an Indian freshwater air-breathing teleost. The season-specific difference in these rates have been analyzed with respect to thermal adaptation during the variation of seasonal temperature of summer and winter months. Due to cold adaptation (either in nature's laboratory or a man made laboratory) the rates of ammonia-N and urea-N excretion are decreased, suggesting a lesser mobilization-N excretion during warm adaptation (either summer acclimation or 32°C acclimation) are noticed, suggesting a greater mobilization of protein and ureogenesis.     

Rapamycin inhibits aldolase A expression during human lymphocyte activation

Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.