Bioavailability of Mineral-Bound Iron to a Snow Algal-Bacterial Coculture and Implications for Albedo-Altering Snow Algal Blooms.

Snow algae can form large-scale blooms across the snowpack surface and near-surface environments. These pigmented blooms can decrease snow albedo and increase local melt rates, and they may impact the global heat budget and water cycle. Yet, the underlying causes for the geospatial occurrence of these blooms remain unconstrained. One possible factor contributing to snow algal blooms is the presence of mineral dust as a micronutrient source. We investigated the bioavailability of iron (Fe)-bearing minerals, including forsterite (Fo90, Mg1.8Fe0.2SiO4), goethite, smectite, and pyrite as Fe sources for a Chloromonas brevispina-bacterial coculture through laboratory-based experimentation. Fo90 was capable of stimulating snow algal growth and increased the algal growth rate in otherwise Fe-depleted cocultures. Fo90-bearing systems also exhibited a decrease in the ratio of bacteria to algae compared to those of Fe-depleted conditions, suggesting a shift in microbial community structure. The C. brevispina coculture also increased the rate of Fo90 dissolution relative to that of an abiotic control. Analysis of 16S rRNA genes in the coculture identified Gammaproteobacteria, Betaproteobacteria, and Sphingobacteria, all of which are commonly found in snow and ice environments. Archaea were not detected. Collimonas and Pseudomonas, which are known to enhance mineral weathering rates, comprised two of the top eight (>1%) operational taxonomic units (OTUs). These data provide unequivocal evidence that mineral dust can support elevated snow algal growth under otherwise Fe-depleted growth conditions and that snow algal microbial communities can enhance mineral dissolution under these conditions.IMPORTANCE Fe, a key micronutrient for photosynthetic growth, is necessary to support the formation of high-density snow algal blooms. The laboratory experiments described herein allow for a systematic investigation of the interactions of snow algae, bacteria, and minerals and their ability to mobilize and uptake mineral-bound Fe. Results provide unequivocal and comprehensive evidence that mineral-bound Fe in Fe-bearing Fo90 was bioavailable to Chloromonas brevispina snow algae within an algal-bacterial coculture. This evidence includes (i) an observed increase in snow algal density and growth rate, (ii) decreased ratios of bacteria to algae in Fo90-containing cultures relative to those of cultures grown under similarly Fe-depleted conditions with no mineral-bound Fe present, and (iii) increased Fo90 dissolution rates in the presence of algal-bacterial cocultures relative to those of abiotic mineral controls. These results have important implications for the role of mineral dust in supplying micronutrients to the snow microbiome, which may help support dense snow algal blooms capable of lowering snow albedo and increasing snow melt rates on regional, and possibly global, scales.

Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.

Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src.

The function of autophosphorylation in Src family protein tyrosine kinases is not fully understood. In this paper we compared the catalytic and ligand-binding properties of autophosphorylated and nonautophosphorylated (control) Src. The following are the main differences we found. First, while both forms had the same K(m) for ATP-Mg, autophosphorylated Src had significantly higher K(m) values for the phosphate-accepting substrates, polyE(4)Y, and RCM-lysozyme. The autophosphorylated form also had significantly higher V(max) values than the control. The substrate specificity, as measured by V(max)/K(m) ratio, was altered by autophosphorylation and was dependent on the phosphate-accepting substrate. Second, while autophosphorylation did not affect Src activation by free Mg(2+), Zn(2+), which inhibited Src by competing against an essential Mg(2+) activator, inhibited the control threefold more potently than the autophosphorylated form. Third, autophosphorylation significantly reduced the ability of its SH2 domain to bind phosphotyrosine. Fourth, a Pro-rich Src SH3 domain binding peptide activated the control, but not the autophosphorylated Src even though the apparent binding affinity was not significantly affected by autophosphorylation. These differences indicated that autophosphorylation induced significant and widespread changes in the catalytic and regulatory properties of Src. The implications of these findings relative to Src biological regulation are discussed.

Semipurification and ice recrystallization inhibition activity of ice-active substances associated with Antarctic photosynthetic organisms.

Ice-active substances (IASs), i.e., macromolecular substances that modify the shape of growing ice crystals, were previously found to be associated with various terrestrial and aquatic photosynthetic organisms from Antarctica, but their chemical nature and function are unknown. In this study, we used the ice-binding properties of the IASs to semipurify IASs from a cyanobacterial mat, a eukaryotic green alga (Prasiola sp.), and a moss (Bryum sp.) and examined the ice recrystallization inhibition (RI) activities of the semipure materials. The semipure materials contain both protein and carbohydrate in which the carbohydrate accounted for 73, 52, and 37%, respectively, of the total carbohydrate + protein. The IASs had RI activity at concentrations of 1.4, 0.05, and 0.01 microg ml-1, respectively. RI activity was greatly reduced by heat treatment, suggesting that the IASs inhibit recrystallization through a specific interaction with ice. These results raise the possibility that the IASs increase freezing tolerance of their respective organisms by preventing the recrystallization of ice.

Trimethylamine oxide and urea synthesis in rainbow smelt and some other northern fishes.

Trimethylamine oxide (TMAO) and urea levels in the blood of rainbow smelt, Osmerus mordax, were previously shown to increase dramatically in winter, but the means by which these osmolytes are acquired has remained unclear. In this study, I show that the smelt can synthesize TMAO via liver trimethylamine oxidase activity and thus are not completely dependent on a dietary source of TMAO. Cold-acclimatized Pacific herring, Clupea harengus, were also found to have high levels of TMAO in the blood, while individuals from a temperate-water population of herring did not. Herring also had liver TMA oxidase activity, which appeared to be due to a flavin-containing monooxygenase. In both species, TMA oxidase activity did not appear to be strongly affected by temperature. TMAO data were obtained for three other northern species (Macrozoarces americanus, Eleginus gracilis, and Platichthys stellatus), and these results, together with previously reported data, suggest that TMA oxidase activity is a necessary condition for high levels of TMAO in the blood. In the smelt, urea appears to be synthesized via uricolysis and also through the action of arginase on dietary arginine, while the ornithine urea cycle appears to be nonfunctional. There was no relation among several species of northern fishes between levels of urea in the blood and levels of uricase or arginase activity. Together, these results provide further evidence of the importance of TMAO and urea in some cold water fishes, demonstrate that the synthetic machinery for these osmolytes is present in the liver, and suggest that the elevated levels in response to cold may be due to conservation rather than to increased production.

Seasonal variations of trimethylamine oxide and urea in the blood of a cold-adapted marine teleost, the rainbow smelt.

Trimethylamine oxide (TMAO), which has previously not been known to occur in significant amounts in the blood of marine teleosts, rose to concentrations of approximately 50 mM in the blood of winter-acclimatized rainbow smelt, Osmerus mordax. Urea also increased in the blood of cold-acclimatized smelt, and, with TMAO, contributed significantly to the winter freezing point depression. TMAO and urea also varied seasonally in muscle and liver tissues. TMAO and urea appeared to be reabsorbed from the urine. Losses of TMAO and urea from the head region of the fish, where most of the losses appeared to occur, were approximately 9 µmol and 8 µmol 100 g(-1) h(-1), respectively. Despite the effluxes, TMAO and urea levels in both the blood and muscle either increased or were maintained in starved, cold-acclimated fish, suggesting that they were synthesized in response to cold temperature. TMAO was also found in the blood of some other cold-hardy teleosts.

Inhibition of growth of nonbasal planes in ice by fish antifreezes.

Peptide and glycopeptide antifreezes from a variety of cold-water fishes cause ice single crystals grown from the melt to assume unusual and strikingly similar habits. The antifreezes inhibit growth on the prism faces but allow limited growth on the basal plane. As new layers are deposited on the basal plane, pyramidal surfaces develop on the outside of the crystal, and large hexagonal pits form within the basal plane. The pits are rotated 30 degrees with respect to the normal orientation of hexagonal ice crystals. Growth inhibition on the prism, pyramidal, and pit faces indicates that these faces contain sites of adsorption of the antifreeze molecules. Several properties of the antifreeze pits are consistent with (but do not prove) an origin of the pits at dislocations. The similarity of crystal habit imposed on ice by antifreezes with wide differences in composition and structure indicates a common mechanism.

Fish, crustaceans, and the sea floor under the ross ice shelf.

Baited traps and a camera lowered through the Ross Ice Shelf, Antarctica, at a point 475 kilometers from the open Ross Sea and to 597 meters below sea level revealed the presence of fish, many amphipods, and one isopod. Biological or current markings were not evident on a soft bottom littered with subangular lumps. A fish was caught through a crevasse 80 kilometers from the shelf edge.

Adsorption inhibition as a mechanism of freezing resistance in polar fishes.

Polar fishes are known to have serum proteins and glycoproteins that protect them from freezing, by a noncolligative process. Measurements of antifreeze concentrations in ice and scanning electron micrographs of freeze-dried antifreeze solutions indicate that the antifreezes are incorporated in ice during freezing. The antifreezes also have a pronounced effect on the crystal habit of ice grown in their presence. Each of four antifreezes investigated caused ice to grow in long needles whose axes were parallel to the ice c axis. Together these results indicate the antifreezes adsorb to ice surfaces and inhibit their growth. A model in which adsorbed antifreezes raise the curvature of growth steps on the ice surface is proposed to account for the observed depression of the temperature at which freezing occurs and agrees well with experimental observations. The model is similar to one previously proposed for other cases of crystal growth inhibition.

Some respiratory characteristics of the blood of four antarctic fishes.

Oxygen equilibrium curves of blood from four antarctic fishes were obtained at subzero temperatures. Oxygen contents were measured directly and the results on two of these fishes agreed favorably with previous studies in which oxygen electrodes were used.

Prediction of inspired oxygen concentration within a circle anaesthetic system.

Fresh gas flows of nitrous oxide, oxygen and halothane at 6, 3, 2 and 1 litre/min were introduced into a circle absorber system. Spontaneous respiration and IPPV were studied and a regression of inspired on delivered oxygen concentration % was calculated. The difference between delivered and inspired oxygen concentration % was increased by decreasing the fresh gas flow and by decreasiing the proportion of oxygen in that flow, especially during IPPV. Circuits designed to allow a maximum overflow of alveolar gas provided a greater inspired oxygen concentration. The patients' height and weight were related to the scatter of inspired values observed at 1 litre/min of fresh gas flow with IPPV.

Glycoprotein and protein antifreezes in two Alaskan fishes.

Two macromolecular freezing point depressants have been isolated from the serums of two species of Alaskan fishes. A common property of the four antifreezes thus far isolated is a high alanine content which suggests that the mechanism of these antifreezes may be related to a repeating tripeptide unit.