Cold-adapted Organisms Research Articles

Bioinformatics survey of the metal usage by psychrophilic yeast Glaciozyma antarctica PI12.

Metal ions are one of the essential elements which are extensively involved in many cellular activities. With rapid advancements in genome sequencing techniques, bioinformatics approaches have provided a promising way to extract functional information of a protein directly from its primary structure. Recent findings have suggested that the metal content of an organism can be predicted from its complete genome sequences. Characterizing the biological metal usage of cold-adapted organisms may help to outline a comprehensive understanding of the metal-partnerships between the psychrophile and its adjacent environment. The focus of this study is targeted towards the analysis of the metal composition of a psychrophilic yeast Glaciozyma antarctica PI12 isolated from sea ice of Antarctica. Since the cellular metal content of an organism is usually reflected in the expressed metal-binding proteins, the putative metal-binding sequences from G. antarctica PI12 were identified with respect to their sequence homologies, domain compositions, protein families and cellular distribution. Most of the analyses revealed that the proteome was enriched with zinc, and the content of metal decreased in the order of Zn > Fe > Mg > Mn, Ca > Cu. Upon comparison, it was found that the metal compositions among yeasts were almost identical. These observations suggested that G. antarctica PI12 could have inherited a conserved trend of metal usage similar to modern eukaryotes, despite its geographically isolated habitat.

A FRET-based, continuous assay for the helicase activity of DEAD-box proteins.

Enzymes from cold-adapted organisms are generally endowed with lower activation enthalpies than their counterparts from organisms growing at higher temperatures, making them better catalysts in the cold. However, the enzymes of RNA metabolism have not been examined in this respect. A challenge for studying cold adaptation of DEAD-box RNA helicases is the low precision of the classical, discontinuous helicase assay based on electrophoretic separation of duplexes and isolated strands. Here, we describe a continuous, FRET-based assay that allows the measurement of the helicase activities of DEAD-box proteins with a precision high enough to detect changes in activation enthalpies associated with cold adaptation.

C-2030 : Investigation of anti-freeze proteins effect on vitrification–devitrificartion processes in a micro-scale view

Ice crystals can greatly damage biological matter which is kept at low temperature for preservation. This problem can be avoided using vitrification, a process by which a liquid is solidified into a non-crystalline (glassy) state by lowering the temperature in the presence of solutes and greatly increasing the viscosity. This process is also beneficiary since it avoids the formation of concentration gradients introduced during crystallization and thereby avoids osmotic injuries resulting in loss of sample viability. Since the late thirties, when Luyet raised the idea of cells cryopreservation by vitrification, a lot of progress has been made in understanding this process and developing this technique to suit biological needs. However, there is still the main difficulty which has not come into a successful solution – the phenomena called ’devitrification’, when ice crystals form during the rewarming step of partially vitrified biological material. We suggest a natural alternative to face this challenge by using antifreeze proteins (AFPs) found in cold-adapted organisms. These proteins are known to inhibit ice growth in slightly supercooled conditions, and inhibit recrystalization of ice in frozen tissues. In this study we investigate the influence of AFPs on the vitrification and de-vitrification processes under a microscope equipped with controlled cold stage and by differential scanning calorimetry (DSC). Observation on vitrification and de-vitrification of Me2SO solutions at different volumes, temperatures and cooling and warming rates have demonstrated these processes in a micro scale. Preliminary results show that addition of the hyperactive AFP fromTenibrio molitor in various concentrations appear to have less ice nucleation centers during warming. Moreover, ice crystals, at post vitrification temperatures (i.e ∼−105 °C), display slower growth in solutions containing AFPs. These novel effects of TmAFP will be further characterized and developed in a course of utilizing these findings into biological matter cryopreservation applications.

β-Amylase1 and β-amylase3 are plastidic starch hydrolases in Arabidopsis That Seem to Be Adapted for Different Thermal, pH, and stress conditions.

Starch degradation in chloroplasts requires β-amylase (BAM) activity, which is encoded by a multigene family. Of nine Arabidopsis (Arabidopsis thaliana) BAM genes, six encode plastidic enzymes, but only four of these are catalytically active. In vegetative plants, BAM1 acts during the day in guard cells, whereas BAM3 is the dominant activity in mesophyll cells at night. Plastidic BAMs have been difficult to assay in leaf extracts, in part because of a cytosolic activity encoded by BAM5. We generated a series of double mutants lacking BAM5 and each of the active plastidic enzymes (BAM1, BAM2, BAM3, and BAM6) and found that most of the plastidic activity in 5-week-old plants was encoded by BAM1 and BAM3. Both of these activities were relatively constant during the day and the night. Analysis of leaf extracts from double mutants and purified BAM1 and BAM3 proteins revealed that these proteins have distinct properties. Using soluble starch as the substrate, BAM1 and BAM3 had optimum activity at pH 6.0 to 6.5, but at high pH, BAM1 was more active than BAM3, consistent with its known daytime role in the guard cell stroma. The optimum temperature for BAM1, which is transcriptionally induced by heat stress, was about 10°C higher than that of BAM3, which is transcriptionally induced by cold stress. The amino acid composition of BAM1 and BAM3 orthologs reflected differences that are consistent with known adaptations of proteins from heat- and cold-adapted organisms, suggesting that these day- and night-active enzymes have undergone thermal adaptation.

Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

Role of the occluded conformation in bacterial dihydrofolate reductases.

Dihydrofolate reductase (DHFR) from Escherichia coli (EcDHFR) adopts two major conformations, closed and occluded, and movement between these two conformations is important for progression through the catalytic cycle. DHFR from the cold-adapted organism Moritella profunda (MpDHFR) on the other hand is unable to form the two hydrogen bonds that stabilize the occluded conformation in EcDHFR and so remains in a closed conformation during catalysis. EcDHFR-S148P and MpDHFR-P150S were examined to explore the influence of the occluded conformation on catalysis by DHFR. Destabilization of the occluded conformation did not affect hydride transfer but altered the affinity for the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP(+)) and changed the rate-determining step of the catalytic cycle for EcDHFR-S148P. Even in the absence of an occluded conformation, MpDHFR follows a kinetic pathway similar to that of EcDHFR with product release being the rate-limiting step in the steady state at pH 7, suggesting that MpDHFR uses a different strategy to modify its affinity for NADP(+). DHFRs from many organisms lack a hydrogen bond donor in the appropriate position and hence most likely do not form an occluded conformation. The link between conformational cycling between closed and occluded forms and progression through the catalytic cycle is specific to EcDHFR and not a general characteristic of prokaryotic DHFR catalysis.

Computational study on ice growth inhibition of Antarctic bacterium antifreeze protein using coarse grained simulation.

Antarctic bacterium antifreeze proteins (AFPs) protect and support the survival of cold-adapted organisms by binding and inhibiting the growth of ice crystals. The mechanism of the anti-freezing process in a water environment at low temperature of Antarctic bacterium AFPs remains unclear. In this research, we study the effects of Antarctic bacterium AFPs by coarse grained simulations solution at a temperature range from 262 to 273 K. The results indicated that Antarctic bacterium AFPs were fully active in temperatures greater than 265 K. Additionally, the specific temperature ranges at which the water molecules become completely frozen, partially frozen, and not frozen were identified.

Psychrophilic endophytic fungi with biological activity inhabit Cupressaceae plant family

Psychrophilic microorganisms are cold-adapted organisms that have an optimum growth temperature below 15 °C, and often below 5 °C. Endophytic microorganisms live inside healthy plants and biosynthesize an array of secondary metabolites which confer major ecological benefits to their host. We provide information, for the first time, on an endophytic association between bioactive psychrophilic fungi and trees in Cupressaceae plant family living in temperate to cold, semi-arid habitats. We have recovered psychrophilic endophytic fungi (PEF) from healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens and Thuja orientalis (Cupressaceae, Coniferales). In total, 23 such fungi were found out of 110 endophytic fungal isolates. They were identified as ascomycetous fungi, more specifically Phoma herbarum, Phoma sp. and Dothideomycetes spp., all from Dothideomycetes. The optimal growth temperature for all these 23 fungal isolates was 4 °C, and the PEF isolates were able to biosynthesize secondary metabolite at this temperature. Extracted metabolites from PEF showed significant antiproliferative/cytotoxic, antifungal and antibacterial effects against phytopathogenic fungi and bacteria. Of special interest was their antibacterial activity against the ice-nucleation active bacterium Pseudomonas syringae. Accordingly, we suggest that evergreen Cupressaceae plants may benefit from their psychrophilic endophytic fungi during cold stress. Whether such endosymbionts confer any ecological and evolutionary benefits to their host plants remains to be investigated in vivo.

Inferring the past and present connectivity across the range of a North American leaf beetle: combining ecological niche modeling and a geographically explicit model of coalescence.

The leaf beetle Chrysomela aeneicollis occurs across Western North America, either at high elevation or in small, isolated populations along the coast, and thus has a highly fragmented distribution. DNA sequence data (three loci) were collected from five regions across the species range. Population connectivity was examined using traditional ecological niche modeling, which suggested that gene flow could occur among regions now and in the past. We developed geographically explicit coalescence models of sequence evolution that incorporated a two-dimensional representation of the hypothesized ranges suggested by the niche-modeling estimates. We simulated sequence data according to these models and compared them to observed sequences to identify most probable scenarios regarding the migration history of C. aeneicollis. Our results disagreed with initial niche-modeling estimates by clearly rejecting recent connectivity among regions, and were instead most consistent with a long period of range fragmentation, extending well beyond the last glacial maximum. This application of geographically explicit models of coalescence has highlighted some limitations of the use of climatic variables for predicting the present and past range of a species and has explained aspects of the Pleistocene evolutionary history of a cold-adapted organism in Western North America.

New Insights on the Mechanism of Action of Ice-Binding Proteins

Ice-binding proteins (IBPs) aid the survival of cold-adapted organisms by inhibiting the growth of endogenous ice crystals. The binding of IBPs to ice causes a separation between the melting point and the freezing point of the ice crystal (Thermal hysteresis, TH). Although the discovery of IBPs was more than 45 years ago, the mechanism of action is still unclear. For moderately active IBPs it is known that thermal hysteresis values increase with annealing time between ice and IBP before cooling is started. We have extended this observation to hyperactive IBPs. Using a custom-made nanoliter osmometer and a novel microfluidics system [1], we show that the exposure time of crystals to IBPs is a crucial factor and that their activity could be increased up to 40-fold over long periods of time (13 h). We found that each IBP has a different time-dependent behavior thus the structure of the IBP molecule brings about a distinct kinetic behavior. Using our cooled microfluidic system, we show that hyperactive IBPs accumulate progressively on the basal plane, and rapidly on the prism plane. Fluorescence intensity analysis showed that the distance between IBPs on the ice surface is 7-20 nm (the ice-binding site of the protein is 3 nm long), and a correlation between the surface density to the measured TH activity was obtained. Microfluidic solution exchange experiments show that both moderate and hyperactive IBPs prevent the growth of ice crystals after the removal of the protein solution. These results have a significant contribution to understanding the IBP mechanism and can be helpful in applying these proteins in different fields.Funded by ISF, CIHR and ERC.1) Celik, Y., Drori, R., et al., Proc Natl Acad Sci U S A, 2013. 110(4): p. 1309-14.

136 The effect of antifreeze proteins on vitrification–devitrificartion processes in a micro-scale view

Vitrification, an “ice-free” cryopreservation method, is an effective way to preserve biological matter, as it avoids the damages of ice growth. This method involves solidification of a liquid into a glass state without crystallization due to high values of viscosity at low temperatures. In the recent years it became widely used in preserving oocytes, embryos, organs and seeds. However this method encounters some difficulties in achieving successful preservation. The main difficulty is avoiding ice crystal formation during rewarming, a phenomena called de-vitrification. So far the solutions found to face this problem are: to rewarm at extremely high rates in a homogeneously manner (Mazur, 1992); to use synthetic anti-nucleators, such as polyvinyl alcohol (PVA) (Wowk, 2000); or to use cryoprotectant mixtures at high concentration. The later may be too toxic to maintain sample viability. Solutions with less toxicity that avoid devitrification are desirable. Natural antifreeze proteins (AFPs), found in cold-adapted organisms, might be an alternative for the aforementioned solutions. While interacting with ice crystals, AFPs arrest ice growth at temperatures below the equilibrium melting temperature and also inhibit damaging recrystalization processes. We investigate the influence of AFPs on the vitrification and de-vitrification processes using optical cryo-microscopy. We use the Linkam MDBCS196 controlled cold stage, which can get to liquid nitrogen temperatures with relatively high cooling rates (5000 °C/min). The microscopy includes bright field and fluorescence microscopy with labeled AFPs. Vitrification and de-vitrification of various cryoprotectant solutions as Me 2 SO and Glycerol at different volumes, temperatures and cooling and warming rates were examined. At slow cooling rates in which crystallization occurs, results show that addition of hyperactive AFP from Tenibrio molitor drops the nucleation temperature of the solution and thus extends its suppercooling range. Also, initial results show that while Me 2 SO solution at 30% v/v concentration facilitate vitrification when the sample was cooled at the high rate of the stage, at 20%, addition of AFP at tens of micromolar concentrations were required to achieve vitrification. Devitrification has been observed in both cases and will be further investigated. These results indicate that the AFPs are a promising candidate to reduce the toxicity of cryoprotectant solutions for vitrification. Source of funding: The European Research Council (ERC). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

142 Ice growth in the presence of an antifreeze protein

Antifreeze proteins (AFPs) have evolved in cold-adapted organisms to control ice crystal growth when exposed to sub-zero temperature conditions. It has been suggested that the effect of the proteins results in small ice crystal size, thus avoiding the mechanical damage to frozen tissues and cells caused by large ice grains. The polar diatom Fragilariopsis cylindrus , a dominant species within sea-ice assemblages, produces AFPs. We expressed in E. coli a recombinant form of this protein and isolated it by affinity chromatography. We studied its effect on ice grain size after shock-freezing and subsequent annealing, and under slow freezing conditions. Shock-freezing (−40 °C) produced small sized crystals, and during annealing at −4 °C AFPs successfully inhibited recrystallization already at low concentrations (1.2 μM), as observed at light microscopy and using the Otago optical recrystallometer. However, slow ice growth at −5 °C, more likely to resemble natural freezing conditions, surprisingly resulted in the formation of larger crystals in the presence of AFPs than in the negative controls. Further characteristic microstructural features, such as gradual c -axis transition within individual grains and sublimation etching patterns, were observed under crossed polarizers and at light microscopy. These features are possibly due to the incorporation of proteins into the ice lattice during growth, causing local defects. Our observations remain to be clarified, but should be taken into account when considering the biological role of AFPs as well as potential industrial applications of the proteins. Source of funding: Public funding by German AiF/IGF. Conflict of interest: None declared. maddalena.bayer@awi.de

144 Kinetics of hyperactive and moderate antifreeze proteins

Ice binding proteins (IBPs) protect cold-adapted organisms from freeze injuries by inhibiting the growth of endogenous ice crystals. IBPs have been found in fish, insects, plants, fungi and bacteria, and hold great potential in different fields such as the food industry, medicine and agriculture. IBPs include antifreeze proteins (AFPs), which adsorb to the surface of ice crystals and lower the temperature at which ice crystals grow, thereby creating a gap (thermal hysteresis [TH]) between the melting point and the non-equilibrium freezing point, within which ice growth is arrested. The accepted model explaining the mechanism of action of AFPs is the adsorption–inhibition model (Raymond and DeVries, 1977). Although this model requires irreversible binding of AFP to ice, there is still an ongoing debate over whether AFPs adsorb to ice irreversibly; furthermore, the time-dependence of this process is still unclear. Our results on the kinetics of AFP binding show that for a hyperactive AFP from Tenebrio molitor (TmAFP), TH activity increased with time by 3- to 10-fold for all of the concentrations measured (1–40 μM). In an AFP from spruce budworm ( Choristoneura fumiferana ) (sbwAFP) the increase of TH activity with time was the highest, reaching a 43-fold increase. Using a novel microfluidic system (Celik, Drori et al., 2013) to investigate the binding mechanism of AFPs to ice, we exposed an ice crystal to AFP molecules, and then exchanged the bathing solution with AFP-free buffer. Assuming irreversible binding, upon exchange of the AFP solution, the crystal must remain covered with AFPs. Our results with a TmAFP-GFP solution show that after the removal of the AFP solution, TH activity was similar to that before the exchange, and the crystal remains covered with TmAFP-GFP (Celik, Drori et al., 2013). Similar to the exchange of TmAFP-GFP solution, after the removal of an AFPIII-GFP solution, bound AFP were sufficient to inhibit ice growth in a supercooled solution (0.11–0.23 °C). However, the ice growth inhibition of AFPIII (which do not bind to the basal plane of the crystal) is dependent on the crystal shape; if the bipyramidal shape is complete, the basal plane is minimal and ice growth is suppressed. Our microfluidic system also enables us to measure and calculate the spacing between AFP molecules on the ice surface. We found that depending on solution concentration and exposure time of the crystal, the distance between AFPs is 5–30 nm (note that the dimensions of AFPs are about 3 nm 2 ), and this value has a direct correlation to the TH activity. Our results suggest that AFPs accumulate on the ice surface, and that any desorption rate must be much slower than the adsorption rate. These findings reveal new insights about the mechanism of action of AFPs, and assist in better understanding how to manipulate these proteins for our benefit. Source of funding: This research was funded by Israel Science Foundation, The European Research Council, and the Canadian Institutes of Health Research. Conflict of interest: None declared. drori.ran@gmail.com

Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities

BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments.We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.The Cold Stage SystemThe cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments. We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details. To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16. The Cold Stage System The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Antarctic DNA moving forward: genomic plasticity and biotechnological potential

Antarctica is the coldest, driest, and windiest continent, where only cold-adapted organisms survive. It has been frequently cited as a pristine place, but it has a highly diverse microbial community that is continually seeded by nonindigenous microorganisms. In addition to the intromission of 'alien' microorganisms, global warming strongly affects microbial Antarctic communities, changing the genes (qualitatively and quantitatively) potentially available for horizontal gene transfer. Several mobile genetic elements have been described in Antarctic bacteria (including plasmids, transposons, integrons, and genomic islands), and the data support that they are actively involved in bacterial evolution in the Antarctic environment. In addition, this environment is a genomic source for the identification of novel molecules, and many investigators have used culture-dependent and culture-independent approaches to identify cold-adapted proteins. Some of them are described in this review. We also describe studies for the design of new recombinant technologies for the production of 'difficult' proteins.

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

BackgroundPsychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.ResultsWe examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.ConclusionsThese findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Antarctic yeastLeucosporidiumsp. AY30

Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a=b=98.05, c=106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.