James A. Raymond

Ice-binding proteins from sea ice diatoms (Bacillariophyceae)

Sea ice diatoms thrive under conditions of low temperature and high salinity, and as a result are responsible for a significant fraction of polar photosynthesis. Their success may be owing in part to secretion of macromolecules that have previously been shown to interfere with the growth of ice and to have the ability to act as cryoprotectants. Here we show that one of these molecules, produced by the sea ice diatom Navicula glaciei Vanheurk, is a ∼25 kDa ice‐binding protein (IBP). A cDNA obtained from another sea ice diatom, Fragilariopsis cylindrus Grunow, was found to encode a protein that closely matched the partially sequenced N. glaciei IBP, and enabled the amplification and sequencing of an N. glaciei IBP cDNA. Similar proteins are not present in the genome of the mesophilic diatom Thalassiosira pseudonana. Both proteins closely resemble antifreeze proteins from psychrophilic snow molds, and as a group represent a new class of IBPs that is distinct from other IBPs found in fish, insects and plants, and bacteria. The diatom IBPs also have striking similarities to three prokaryotic hypothetical proteins. Relatives of both snow molds and two of the prokaryotes have been found in sea ice, raising the possibility of a fungal or bacterial origin of diatom IBPs.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Possible Role of Horizontal Gene Transfer in the Colonization of Sea Ice by Algae

Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Proteins with similar sequences and ice-binding activities are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 47% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes.

NOVEL ICE-BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE, CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.

Ice-binding proteins from enoki and shiitake mushrooms.

Fungi have developed a variety of mechanisms for tolerating cold, including production of proteins that bind to ice, as shown by their ability to slightly lower the freezing point. At present, only one of these proteins, from the snow mold Typhula ishikariensis, and partial transcripts of a similar protein from shiitake mushroom, Lentinula edodes, have been identified. Here, we report the full sequences of ice-binding proteins from shiitake and another mushroom, the cold-adapted Flammulina populicola (enoki mushroom), and show that the recombinant proteins have ice-binding activity. The three proteins share 50-55% identities and are similar to other ice-binding proteins recently identified in ice bacteria and sea ice diatoms. The possibility that ice-binding protein genes have spread among these phyla by horizontal transfer is discussed.

Distribution and partial characterization of ice-active molecules associated with sea-ice diatoms

Abstract Macromolecular ice-active substances (IASs) that cause pitting of ice-crystal surfaces were previously shown to be associated with two species of Antarctic sea-ice diatom. Here it is shown that IASs are associated with many, if not all, sea-ice diatoms of McMurdo Sound, Antarctica, are present in the bottom layer of sea ice in rough proportion to the density of cells, and exist in winter-grown, algae-containing sea ice in different parts of the Southern Ocean and in at least one location in the Arctic. The IASs are retained to varying degrees by dialysis tubing with 50-, 100- and 300-kDa molecular weight cut-offs, suggesting a large molecular weight range, and have a solute requirement (∼≥200 mOsm/kg) for activity. The IASs occur as smears on both native and denaturing electrophoretic gels, which further suggests a heterogeneous nature. One apparently pure sample contained carbohydrate and protein (at an approximate 3:2 ratio on a weight basis), suggesting that the IASs are glycoproteins. The IASs preferentially bind to ice crystals, as they are concentrated in the ice phase of partially frozen solutions.

Ice-active substances associated with Antarctic freshwater and terrestrial photosynthetic organisms

Macromolecular substances that cause pitting and other modifications of growing ice crystals were found to be associated with cyanobacterial mats, eukaryotic algae and mosses from Ross Island and the McMurdo Dry Valleys, Antarctica. Ice-pitting activities were largely retained by dialysis membranes with molecular weight cut-offs of up to 300 kDa. Unlike most aqueous solutes, the ice-active molecules were not excluded from the ice phase during freezing. The ice-pitting activities of each of the samples tested was destroyed by exposure to temperatures between 45 and 65°C, suggesting that they have a protein component. Ice-active substances were not found in cyanobacteria or mosses from temperate climates, but ice-activity was found to be associated with mosses from cold habitats in North America. Although the function of the ice-active substances is not known, their apparent confinement to cold environments suggests that they have a cryoprotective role.

Amino Acids Are a Source of Glycerol in Cold-Acclimatized Rainbow Smelt

Abstract Rainbow smelt ( Osmerus mordax ) produce high concentrations of glycerol in winter to protect against freezing. The glycerol is lost to the environment and so must be continually replaced. Glycogen has previously been shown to be a source of glycerol in this species, but glycerol levels are maintained even when glycogen levels have been depleted. In the present study, using radiolabeled substrates, it is qualitatively shown that the carbon skeletons of alanine and glutamate are largely converted to glycerol and glucose in −1°C-acclimatized smelt, suggesting that protein is another important source of glycerol. Bicarbonate carbon was also largely converted to glycerol and glucose, apparently through its involvement in the conversion of amino acids to oxaloacetate. Together, these results indicate an active gluconeogenesis in winter-acclimatized rainbow smelt.

Enzyme activity levels associated with the production of glycerol as an antifreeze in liver of rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) tolerate temperatures close to the freezing point of sea water, in part, through the use of glycerol as an antifreeze. Potential mechanisms for glycerol production by liver were assessed by comparing activities of key enzym es of carbohydrate and amino acid metabolism in rainbow smelt to those in Atlantic tomcod (Microgadus tomcod) and smooth flounder (Liopsetta putmani). The latter two species inhabit the same environment but do not maintain high levels of blood glycerol. The enzyme profile of liver from rainbow smelt is substantially different from those of the other species and is poised for glycerol production. With respect to carbohydrate metabolism, glycerol-3-phosphate dehydrogenase activity in rainbow smelt liver was 156 µmoles min-1 g-1, a level which was 28 and 12-fold higher than activities in tomcod and flounder liver, respectively. Glycerol-3-phosphatase activity in smelt liver was 1.95 µmol min-1 g-1. This activity was 2.7 and 5.4-fold higher than those in tomcod and flounder liver, respectively. As such, the production of glycerol appears to be dependent upon the concerted action of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. The enzyme profile also suggests that amino acids are a potential source of carbon for glycerol. Aspartate aminotransferase activity in rainbow smelt was 7 to 14-fold higher in comparison to tomcod and flounder liver, respectively. Activities of alanine aminotransferase and glutamate dehydrogenase in liver were between 2 and 3-fold higher in rainbow smelt than in the other two species. Finally, it is shown that in vitro preparations of smelt liver sections produce glycerol at 0°C lending support to the concept that liver is a site of glycerol synthesis in vivo.

Separate origins of ice-binding proteins in antarctic chlamydomonas species.

The green alga Chlamydomonas raudensis is an important primary producer in a number of ice-covered lakes and ponds in Antarctica. A C. raudensis isolate (UWO241) from Lake Bonney in the McMurdo Dry Valleys, like many other Antarctic algae, was found to secrete ice-binding proteins (IBPs), which appear to be essential for survival in icy environments. The IBPs of several Antarctic algae (diatoms, a prymesiophyte, and a prasinophyte) are similar to each other (here designated as type I IBPs) and have been proposed to have bacterial origins. Other IBPs (type II IBPs) that bear no resemblance to type I IBPs, have been found in the Antarctic Chlamydomonas sp. CCMP681, a putative snow alga, raising the possibility that chlamydomonad IBPs developed separately from the IBPs of other algae. To test this idea, we obtained the IBP sequences of C. raudensis UWO241 by sequencing the transcriptome. A large number of transcripts revealed no sequences resembling type II IBPs. Instead, many isoforms resembling type I IBPs were found, and these most closely matched a hypothetical protein from the bacterium Stigmatella aurantiaca. The sequences were confirmed to encode IBPs by the activity of a recombinant protein and by the matching of predicted and observed isoelectric points and molecular weights. Furthermore, a mesophilic sister species, C. raudensis SAG49.72, showed no ice-binding activity or PCR products from UWO241 IBP primers. These results confirm that algal IBPs are required for survival in icy habitats and demonstrate that they have diverse origins that are unrelated to the taxonomic positions of the algae. Last, we show that the C. raudensis UWO241 IBPs can change the structure of ice in a way that could increase the survivability of cells trapped in the ice.