Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein.

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

Protein engineering of antifreeze proteins reveals that their activity scales with the area of the ice-binding site.

Antifreeze proteins (AFPs) protect organisms from freezing by binding to ice crystals to prevent their growth. Here, we have investigated how the area of an AFP's ice-binding site (IBS) changes its antifreeze activity. The polyproline type II helical bundle fold of the 9.6-kDa springtail (Collembola) AFP from Granisotoma rainieri (a primitive arthropod) facilitates changes to both IBS length and width. A one quarter decrease in area reduced activity to less than 10%. A one quarter increase in IBS width, through the addition of a single helix, tripled antifreeze activity. However, increasing IBS length by a similar amount actually reduced activity. Expanding the IBS area can greatly increase antifreeze activity but needs to be evaluated by experimentation on a case-by-case basis.

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Toxicity profiles and protective effects of antifreeze proteins from insect in mammalian models.

Antifreeze proteins (AFPs), found in many cold-adapted organisms, can protect them from cold and freezing damages and have thus been considered as additional protectants in current cold tissue preservation solutions that generally include electrolytes, osmotic agents, colloids and antioxidants, to reduce the loss of tissue viability associated with cold-preservation. Due to the lack of toxicity profile studies on AFPs, their inclusion in cold preservation solutions has been a trial-and-error process limiting the development of AFPs' application in cold preservation. To assess the feasibility of translating the technology of AFPs for mammalian cell cold or cryopreservation, we determined the toxicity profile of two highly active beetle AFPs, DAFP1 and TmAFP, from Dendroides canadensis and Tenebrio molitor in this study. Toxicity was examined on a panel of representative mammalian cell lines including testicular spermatogonial stem cells and Leydig cells, macrophages, and hepatocytes. Treatments with DAFP1 and TmAFP at up to 500 µg/mL for 48 and 72 h were safe in three of the cell lines, except for a 20% decrease in spermatogonia treated with TmAFP. However, both AFPs at 500 µg/mL or below reduced hepatocyte viability by 20-40% at 48 and 72 h. At 1000 µg/mL, DAFP1 and TmAFP reduced viability in most cell lines. While spermatogonia and Leydig cell functions were not affected by 1000 µg/mL DAFP1, this treatment induced inflammatory responses in macrophages. Adding 1000 µg/mL DAFP1 to rat kidneys stored at 4 °C for 48 h protected the tissues from cold-related damage, based on tissue morphology and gene and protein expression of two markers of kidney function. However, DAFP1 and TmAFP did not prevent the adverse effects of cold on kidneys over 72 h. Overall, DAFP1 is less toxic at high dose than TmAFP, and has potential for use in tissue preservation at doses up to 500 µg/mL. However, careful consideration must be taken due to the proinflammatory potential of DAFP1 on macrophages at higher doses and the heighten susceptibility of hepatocytes to both AFPs.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.

[Physical Basis of Functioning of Antifreeze Protein].

Antifreeze proteins, expressed in cold-blooded organisms, prevent ice formation in their bodies, and thus help them to survive in extremely cold winter temperatures. However, the mechanism of action of these proteins is still not clear. In any case, it is not simply a decrease in the temperature of normal ice formation. In this work, investigating the ice-binding protein (a mutant form of the antifreeze protein cfAFP from the spruce budworm Choristoneura fumiferana, which overwinters in needles), we showed that this antifreeze protein does not at all lower the freezing point of water and, paradoxically, increases the melting point of ice. On the other hand, calculations based on the theory of crystallization show that at temperatures of 0° to -30°C ice can only appear on surfaces that contact water, but not in the body of water. These facts suggest a new perspective on the role of antifreeze proteins: their task is not (as it is commonly believed) to bind with nascent ice crystals already formed in the organism and stop their growth, but to bind to those surfaces, on which ice nuclei can appear, and thus completely inhibit the ice formation in supercooled water or biological fluid.

Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates.

Ice-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival at sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more IBPs than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an IBP was significantly greater in intertidal species than in those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk and that there is an array of uncharacterized IBPs, and highlight the need for broader laboratory-based surveys of the diversity of ice-binding activity across diverse taxonomic and ecological groups.

Identification and Functional Characterization of Antifreeze Protein and Its Mutants in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) Larvae Under Cold Stress.

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is considered to be the most destructive forest pest in the Qinling and Bashan Mountains of China. Low winter temperatures limit insect's populations, distribution, activity, and development. Insects have developed different strategies such as freeze-tolerance and freeze-avoidance to survive in low temperature conditions. In the present study, we used gene cloning, real-time polymerase chain reaction (PCR), RNA interference (RNAi), and heterologous expression to study the function of the D. armandi antifreeze protein gene (DaAFP). We cloned the 800 bp full-length cDNA encoding 228 amino acids of DaAFP and analyzed its structure using bioinformatics analysis. The DaAFP amino acid sequence exhibited 24-86% similarity with other insect species. The expression of DaAFP was high in January and in the larvae, head, and midgut of D. armandi. In addition, the expression of DaAFP increased with decreasing temperature and increasing exposure time. RNAi analysis also demonstrated that AFP plays an important role in the cold tolerance of overwintering larvae. The thermal hysteresis and antifreeze activity assay of DaAFP and its mutants indicated that the more regular the DaAFP threonine-cystine-threonine (TXT) motif, the stronger the antifreeze activity. These results suggest that DaAFP plays an essential role as a biological cryoprotectant in overwintering D. armandi larvae and provides a theoretical basis for new pest control methods.

Beetle and mussel-inspired chimeric protein for fabricating anti-icing coating.

Ice accretion on surfaces can cause serious damages and economic losses in industries and civilian facilities. Antifreeze proteins (AFPs) as evolutionary adaptation products of organisms to cold climates, provide solutions for alleviating icing problems. In this work, a chimeric protein Mfp-AFP was rationally designed combining mussel-inspired adhesive domain with Tenebrio molitor-derived antifreeze protein domain. Expectedly, the multifunctional Mfp-AFP can lower the freezing point of water and inhibit ice recrystallization. The chimeric protein could also readily modify diverse solid surfaces due to the adhesive domain containing Dopa, and resist frosting and delay ice formation due to the beetle-derived antifreeze fragment. Moreover, Mfp-AFP coatings display excellent biocompatibility proved by cytocompatibility and hemolysis assays. Here, the designed multifunctional protein coatings provide an alternative strategy for fabricating anti-icing surfaces.

AFP-CMBPred: Computational identification of antifreeze proteins by extending consensus sequences into multi-blocks evolutionary information.

In extremely cold environments, living organisms like plants, animals, fishes, and microbes can die due to the intracellular ice formation in their bodies. To sustain life in such cold environments, some cold-blooded species produced Antifreeze proteins (AFPs), also called ice-binding proteins. AFPs are not only limited to the medical field but also have diverse significance in the area of biotechnology, agriculture, and the food industry. Different AFPs exhibit high heterogeneity in their structures and sequences. Keeping the significance of AFPs, several machine-learning-based models have been developed by scientists for the prediction of AFPs. However, due to the complex and diverse nature of AFPs, the prediction performance of the existing methods is limited. Therefore, it is highly indispensable for researchers to develop a reliable computational model that can accurately predict AFPs. In this connection, this study presents a novel predictor for AFPs, named AFP-CMBPred. The sequences of AFPs are formulated via four different feature representation methods, such as Amphiphilic pseudo amino acid composition (Amp-PseAAC), Dipeptide Deviation from Expected Mean (DDE), Multi-Blocks Position Specific Scoring Matrix (MB-PSSM), and Consensus Sequence-based on Multi-Blocks Position Specific Scoring Matrix (CS-MB-PSSM) to collect local and global descriptors. In the next step, the extracted feature vectors are evaluated via Support Vector Machine (SVM) and Random Forest (RF) based classification learners. The prediction performance of both classifiers is further assessed using three validation methods i.e., jackknife test, 10-fold cross-validation test, and independent test. After examining the prediction rates of all validation tests, it was found that our proposed model achieved the higher prediction accuracies of ∼2.65%, ∼2.84%, and ∼3.37% using jackknife, K-fold, and independent test, respectively. The experimental outcomes validate that our proposed "AFP-CMBPred" predictor secured the highest prediction results than the existing models for the identification of AFPs. It is further anticipated that our proposed AFP-CMBPred model will be considered a valuable tool in the research academia and drug development.

Propagation of a De Novo Gene under Natural Selection: Antifreeze Glycoprotein Genes and Their Evolutionary History in Codfishes.

The de novo birth of functional genes from non-coding DNA as an important contributor to new gene formation is increasingly supported by evidence from diverse eukaryotic lineages. However, many uncertainties remain, including how the incipient de novo genes would continue to evolve and the molecular mechanisms underlying their evolutionary trajectory. Here we address these questions by investigating evolutionary history of the de novo antifreeze glycoprotein (AFGP) gene and gene family in gadid (codfish) lineages. We examined AFGP phenotype on a phylogenetic framework encompassing a broad sampling of gadids from freezing and non-freezing habitats. In three select species representing different AFGP-bearing clades, we analyzed all AFGP gene family members and the broader scale AFGP genomic regions in detail. Codon usage analyses suggest that motif duplication produced the intragenic AFGP tripeptide coding repeats, and rapid sequence divergence post-duplication stabilized the recombination-prone long repetitive coding region. Genomic loci analyses support AFGP originated once from a single ancestral genomic origin, and shed light on how the de novo gene proliferated into a gene family. Results also show the processes of gene duplication and gene loss are distinctive in separate clades, and both genotype and phenotype are commensurate with differential local selective pressures.

Identification of Biomolecules Involved in the Adaptation to the Environment of Cold-Loving Microorganisms and Metabolic Pathways for Their Production.

Cold-loving microorganisms of all three domains of life have unique and special abilities that allow them to live in harsh environments. They have acquired structural and molecular mechanisms of adaptation to the cold that include the production of anti-freeze proteins, carbohydrate-based extracellular polymeric substances and lipids which serve as cryo- and osmoprotectants by maintaining the fluidity of their membranes. They also produce a wide diversity of pigmented molecules to obtain energy, carry out photosynthesis, increase their resistance to stress and provide them with ultraviolet light protection. Recently developed analytical techniques have been applied as high-throughoutput technologies for function discovery and for reconstructing functional networks in psychrophiles. Among them, omics deserve special mention, such as genomics, transcriptomics, proteomics, glycomics, lipidomics and metabolomics. These techniques have allowed the identification of microorganisms and the study of their biogeochemical activities. They have also made it possible to infer their metabolic capacities and identify the biomolecules that are parts of their structures or that they secrete into the environment, which can be useful in various fields of biotechnology. This Review summarizes current knowledge on psychrophiles as sources of biomolecules and the metabolic pathways for their production. New strategies and next-generation approaches are needed to increase the chances of discovering new biomolecules.

Predicting antifreeze proteins with weighted generalized dipeptide composition and multi-regression feature selection ensemble.

BACKGROUND: Antifreeze proteins (AFPs) are a group of proteins that inhibit body fluids from growing to ice crystals and thus improve biological antifreeze ability. It is vital to the survival of living organisms in extremely cold environments. However, little research is performed on sequences feature extraction and selection for antifreeze proteins classification in the structure and function prediction, which is of great significance. RESULTS: In this paper, to predict the antifreeze proteins, a feature representation of weighted generalized dipeptide composition (W-GDipC) and an ensemble feature selection based on two-stage and multi-regression method (LRMR-Ri) are proposed. Specifically, four feature selection algorithms: Lasso regression, Ridge regression, Maximal information coefficient and Relief are used to select the feature sets, respectively, which is the first stage of LRMR-Ri method. If there exists a common feature subset among the above four sets, it is the optimal subset; otherwise we use Ridge regression to select the optimal subset from the public set pooled by the four sets, which is the second stage of LRMR-Ri. The LRMR-Ri method combined with W-GDipC was performed both on the antifreeze proteins dataset (binary classification), and on the membrane protein dataset (multiple classification). Experimental results show that this method has good performance in support vector machine (SVM), decision tree (DT) and stochastic gradient descent (SGD). The values of ACC, RE and MCC of LRMR-Ri and W-GDipC with antifreeze proteins dataset and SVM classifier have reached as high as 95.56%, 97.06% and 0.9105, respectively, much higher than those of each single method: Lasso, Ridge, Mic and Relief, nearly 13% higher than single Lasso for ACC. CONCLUSION: The experimental results show that the proposed LRMR-Ri and W-GDipC method can significantly improve the accuracy of antifreeze proteins prediction compared with other similar single feature methods. In addition, our method has also achieved good results in the classification and prediction of membrane proteins, which verifies its widely reliability to a certain extent.

A minimalistic cyclic ice-binding peptide from phage display.

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.

Antifreeze protein from Ammopiptanthus nanus functions in temperature-stress through domain A.

Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In our previous study, the AnAFP gene cloned from Ammopiptanthus nanus was confirmed to be an excellent candidate enhancing plant cold resistance. But, AnAFP protein shared similar structures with KnS type dehydrins including K, N and S domains except ice crystal binding domain A. Here, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. Expression of AnAFPΔA decreases cold and heat tolerance in E. coli, meanwhile, AnAFP enhances heat tolerance in Arabidopsis, suggesting that domain A is a thermal stable functional domain. AnAFP, AnAFPΔA and AnAFPΔS localize in whole cell, but AnAFPΔK and AnAFPΔN only localizes in nucleus and cytoplasm, respectively, exhibiting that K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The result of RT-qPCR showed that expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway. Taken together, the study provides insights into understanding the mechanism of AnAFP in response to temperature stress and gene resource to improve heat or cold tolerance of plants in transgenic engineering.

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai.

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

Complete genome sequence of Marinomonas arctica BSI20414, a giant antifreeze protein-producing bacterium isolated from Arctic sea ice.

Sea ice in the polar oceans is a dynamic and challenging environment for life to survive, with extreme gradients of temperature, salinity and nutrients etc., as well as formation of ice crystals. Bacteria surviving in sea ice attract broad attention from academia and industry, due to fascinating mechanisms for adaptation. Here we described the complete genome sequence of Marinomonas arctica BSI20414, isolated from Arctic sea ice. The strain tolerated high salinity and low temperature. Genetic features commonly related to adaptation to oxidative stress, osmotic stress and cold stress were detected in the genome. In addition, a large adhesion protein containing a putative antifreeze protein (AFP) domain was detected in the genome, similar with the giant AFP MpIBP from M. primoryensis. The presence of the putative AFP could facilitate M. arctica BSI20414 to bind to sea ice for favorable conditions and protect it from freezing. The genome sequence and the AFP reported here can provide insights into adaptation to sea ice and can be explored further for biotechnological applications.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6-8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.