Proteomics of Colwellia psychrerythraea at subzero temperatures – a life with limited movement,flexible membranes and vital DNA repair

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (?1, and ?10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]‐leucine and [3H]–thymidine incubations indicated active protein and DNA synthesis to ?10°C. Mass spectrometry‐based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo‐taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold‐adapted marine organisms to sustain cellular function in their habitat.     

Potential effect of ice formation on Antarctic pelagic copepods: salinity induced mortality of Calanus propinquus and Metridia gerlachei in comparison to sympagic acoel turbellarians

The salinity tolerance of two dominant Antarctic planktic copepods (Calanus propinquus and Metridia gerlachei) was tested over a range from 34 to 85 PSU and compared with that of sympagic turbellarians. The copepods survived only at a salinity of 34, higher salinities causing death within days. The turbellarians survived at salinities up to 75. The data imply that C. propinquus and M. gerlachei will not survive incorporation into newly forming sea ice because of the increasing brine salinity in new ice. Received: 27 January 1998 / Accepted: 11 April 1998     

Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis

Fragilariopsis is a dominating psychrophilic diatom genus in polar sea ice. The two species Fragilariopsis cylindrus and Fragilariopsis curta are able to grow and divide below freezing temperature of sea water and above average sea water salinity. Here we show that antifreeze proteins (AFPs), involved in cold adaptation in several psychrophilic organisms, are widespread in the two polar species. The presence of AFP genes (afps) as a multigene family indicated the importance of this group of genes for the genus Fragilariopsis, possibly contributing to its success in sea ice. Protein phylogeny showed the potential mobility of afps, which appear to have crossed kingdom and domain borders, occurring in Bacteria, diatoms, crustaceans and fungi. Our results revealed a broad distribution of AFPs not only in polar organisms but also in taxa apparently not related to cold environments, suggesting that these proteins may be multifunctional. The relevance of AFPs to Fragilariopsis was also shown by gene expression analysis. Under stress conditions typical for sea ice, with subzero temperatures and high salinities, F. cylindrus and F. curta strongly expressed selected afps. An E/G point mutation in the Fragilariopsis AFPs may play a role in gene expression activity and protein function.     

Cold resistance and metabolic responses to salinity variations in the amphipod Eusirus antarcticus and the krill Euphausia superba

Summary The krill Euphausia superba, unlike the amphipod, Eusirus antarcticus, tolerates being frozen into solid sea-ice at temperatures down to about-4°C. Cooled in air, the amphipod and the krill freeze and will die at temperatures of-11° and-9°C respectively, representing the supercooling points of the animals. The krill is an osmoconformer in the salinity range of 25 to 45 ppt, while the amphipod conforms in the salinity range of 26 to 40 ppt. The animals thereby lower the melting point of their body fluids in the vicinity of the freezing sea ice, preventing internal ice formation at low temperatures. The mean oxygen consumption rates, at raised and lowered salinities, were not significantly different from rates obtained in normal (35 ppt.) seawater, indicating that salinity has little effect on the metabolism of either species.     

GROWTH RESPONSES TO SALINITY VARIATION IN FOUR ARCTIC ICE DIATOMS1,2

During spring, extensive blooms of microalgae grow on the underside of arctic sea ice. The brownish, algal layer penetrates ca. 2 cm into the bottom surface of the ice and the algae are potentially exposed to very high salinities. Four diatom species, Melosira juergensii Ag., Porosira glacialis (Grun.) Jørg., Navicula transitans var. derasa (Grun.) Cleve, and Coscinodiscus lacustris Grun., isolated from, sea ice samples taken from the Beaufort and Chukchi seas near Barrow, Alaska, were grown at 11 salinities ranging from 5 to 70‰ at 5 C under constant illumination. All of the species grew at 5‰ except N. transitans whose lower growth limit was 15‰. Growth was high over a broad range of salinities, but none of the species grew at salinities above 60‰. These diatom species appear to be well suited to tolerate the salinities in the brine pockets near the bottom of annual arctic sea ice where they are found. High brine-cell salinity, however, may limit the upward, penetration of ice algae into the bottom of sea ice.     

An inter-order horizontal gene transfer event enables the catabolism of compatible solutes by Colwellia psychrerythraea 34H

Colwellia is a genus of mostly psychrophilic halophilic Gammaproteobacteria frequently isolated from polar marine sediments and sea ice. In exploring the capacity of Colwellia psychrerythraea 34H to survive and grow in the liquid brines of sea ice, we detected a duplicated 37 kbp genomic island in its genome based on the abnormally high G + C content. This island contains an operon encoding for heterotetrameric sarcosine oxidase and is located adjacent to several genes used in the serial demethylation of glycine betaine, a compatible solute commonly used for osmoregulation, to dimethylglycine, sarcosine, and glycine. Molecular clock inferences of important events in the adaptation of C. psychrerythraea 34H to compatible solute utilization reflect the geological evolution of the polar regions. Validating genomic predictions, C. psychrerythraea 34H was shown to grow on defined media containing either choline or glycine betaine, and on a medium with sarcosine as the sole organic source of carbon and nitrogen. Growth by 8 of 9 tested Colwellia species on a newly developed sarcosine-based defined medium suggested that the ability to catabolize glycine betaine (the catabolic precursor of sarcosine) is likely widespread in the genus Colwellia. This capacity likely provides a selective advantage to Colwellia species in cold, salty environments like sea ice, and may have contributed to the ability of Colwellia to invade these extreme niches.     

POPULATION DYNAMICS OF MICROALGAE IN THE UPPER LAND-FAST SEA ICE AT A SNOW-FREE LOCATION

The population dynamics of interior ice microalgae were investigated at a snow-free site on annual land-fast sea ice in McMurdo Sound, Antarctica, during the austral spring and summer of 1995 – 96. A dynamic successional sequence was observed with life history transformations playing an important role. During late November and early December (austral spring), cryo- and halotolerant dinoflagellates and chrysophytes bloomed in brine channels within the upper ice. At this time, competition and grazing pressure are low because of the inability of most marine species to grow under the extreme environmental conditions found in the upper ice during the austral spring. In November and December, dinoflagellates, chrysophytes, and prasinophytes contributed an average of 66%, 44%, and < 1% of the phytoflagellate biomass, respectively. Both the dinoflagellates and the chrysophytes encysted in December, with cyst formation most intense just prior to surface melt and flushing of the ice. The cysts appear to be an adaptation for survival and dispersal in the plankton during ice decay and/or overwintering in the sea ice. In January (austral summer), when ice temperatures were similar to those in the water column, pennate diatoms replaced flagellates as the photosynthetic dominants in the upper sea ice. The upper land-fast sea ice undergoes dramatic seasonal changes in light availability, temperature, brine salinity, and inorganic nutrient availability. Ephemeral blooms of cyst-forming phytoflagellates exploit this habitat in the austral spring, when both inorganic nutrients and light are available but temperatures <− 2° C and brine salinities elevated.     

The nutrient status in sea ice of the Weddell Sea during winter: effects of sea ice texture and algae

Summary Cores and brine samples from sea ice of the Weddell Sea were analyzed for nutrients (phosphate, nitrate and silicate), salinity and chlorophyll a during winter. Stratigraphic analyses of the cores were also carried out. Bulk nutrient concentrations in the sea ice fluctuated widely and did not correlate with salinity. Nutrient concentrations in cores were normalized to sea-water salinity to facilitate comparison. They varied between zero and two or three times those measured in the water column. Differentiation into young and old sea ice, however, revealed that nutrient concentrations in the young ice in many cases corresponded to those in surface seawater. In older ice, nutrients showed signs of increase as well as depletion or exhaustion relative to the water column. Differentiation of core sections according to ice textural classes and analyses of brine samples clarified some relationships between nutrients, salinity and algal biomass. Most of the changes in the nutrient concentrations are attributed to an increase in biological activity as the seasons progress. Silicate is expected to become the first nutrient likely to limit growth of diatoms in the ice which is ascribed to slower regeneration or dissolution of this nutrient relative to phosphate and nitrate. A consequence of silicate exhaustion may be the succession of different algal assemblages, from a diatom dominated community to one in which autotrophic flagellates form the largest component.     

THE INFLUENCE OF SALINITY AND TEMPERATURE COVARIATION ON THE PHOTOPHYSIOLOGICAL CHARACTERISTICS OF ANTARCTIC SEA ICE MICROALGAE1

The responses of sea ice microalgae to variation in ambient irradiance (0 to 150 μE · m^?2· s^?1), temperature (–6° to + 6° C), and salinity (0 to 100 ppt) were tested to determine whether these variables act independently or in concert to influence rates of microalgal photosynthesis. The photosynthetic efficiency and maximum photosynthetic rate for sea ice microalgae increased as a function of incubation temperature between -6° and + 6° C. Furthermore, photosynthetic efficiency, maximum photosynthetic rate, and quantum yield were greatest at salinities between SO and 50 ppt. In contrast, the mean specific absorption coefficients were lowest near seawater salinities, and the saturating irradiance, I_s, appeared to be inversely proportional to salinity. Results also suggest that the effects of salinity on the growth of sea ice microalgae are independent of those elicited by temperature or light, and that the functional relationship between salinity and light or temperature is multiplicative. This information is essential to the proper formulation of algorithms used to describe algal growth in environments where light, temperature, and salinity are changing simultaneously, such as within sea ice or within the water column at the marginal ice edge zone.     

Physiological adaptations to low temperature and brine exposure in the circumpolar amphipod Gammarus wilkitzkii

Summary The amphipod Gammarus wilkitzkii does not survive being frozen totally into solid sea ice. When the animals are cooled in air or freezing seawater, they will freeze and die at a temperature of about-4° C. However, during sea ice growth, the amphipods may tolerate to stay in the vicinity of the ice by conforming to the ambient brine in a salinity range of 34 ppt to about 60 ppt. A passive relationship between the concentrations of the haemolymph and seawater Na⁺ and Cl^-, lowers the melting point of the body fluids of the animals, thus preventing internal ice formation at low temperatures.     

Use of exogenous glycine betaine and its precursor choline as osmoprotectants in Antarctic sea‐ice diatoms1

Wide salinity ranges experienced during the seasonal freeze and melt of sea ice likely constrain many biological processes. Microorganisms generally protect against fluctuating salinities through the uptake, production, and release of compatible solutes. Little is known, however, about the use or fate of glycine betaine (GBT hereafter), one of the most common compatible solutes, in sea‐ice diatoms confronted with shifts in salinity. We quantified intracellular concentrations and used [¹⁴C]‐labeled compounds to track the uptake and fate of the nitrogen‐containing osmolyte GBT and its precursor choline in three Antarctic sea‐ice diatoms Nitzschia lecointei, Navicula cf. perminuta, and Fragilariopsis cylindrus at ?1°C. Experiments show that these diatoms have effective transporters for GBT, but take up lesser amounts of choline. Neither compound was respired. Uptake of GBT protected cells against hyperosmotic shock and corresponded with reduced production of extracellular polysaccharides in N. lecointei cells, which released 85% of the retained GBT following hypoosmotic shock. The ability of sea‐ice diatoms to rapidly scavenge and release compatible solutes is likely an important strategy for survival during steep fluctuations in salinity. The release and recycling of compatible solutes may play an important role in algal–bacterial interactions and nitrogen cycling within the semi‐enclosed brines of sea ice.     

Osmotic and ionic regulation in response to salinity variations and cold resistance in the Arctic under-ice amphipod <Emphasis Type="Italic">Apherusa glacialis</Emphasis>

Amphipods living at the underside of Arctic sea ice are exposed to varying salinities due to freezing and melting, and have to cope with the resulting osmotic stress. Extracellular osmotic and ionic regulation at different salinities, thermal hysteresis, and supercooling points (SCPs) were studied in the under-ice amphipod Apherusa glacialis. The species is euryhaline, capable to regulate hyperosmotically at salinities S _R < 30 g/kg, and osmoconforms at salinities S _R ≥ 30 g/kg. Hyperosmotic regulation is an adaptation to thrive in low-salinity meltwater below the ice. Conforming to the ambient salinity during freezing reduces the risk of internal ice formation. Thermal hysteresis was not observed in the haemolymph of A. glacialis. The SCP of the species was −7.8 ± 1.9°C. Several ions were specifically downregulated ([Mg²⁺], [SO₄ ²⁻]), or upregulated ([K⁺], [Ca²⁺]) in comparison to the medium. Strong downregulation of [Mg²⁺], is probably necessary to avoid an anaesthetic effect at low temperatures.     

INFECTION POTENTIAL AND GROWTH OF LAGENIDIUM CHTHAMALOPHILUM

Johnson , T. W., Jr . (Duke U., Durham, N. C.) Infection potential and growth of Lagenidium chthamalophilum. Amer. Jour. Bot. 47(5): 383—385. Illus. 1960.–Temperature and salinity influence infection of Chthamalus fragilis and Balanus amphitrite denticulata ova by Lagenidium chthamalophilum. Optimum conditions for infection are 20—22°C. in raw (as opposed to aged) sea water of 34 0/00 salinity. With increases in incubation temperature, the percentage of infection occurring in egg lamellae submerged in hypersaline waters also increases. Nutrition has essentially the same effect on salinity tolerance as water temperature: only on enriched agar media will L. chthamalophilum grow vegetatively at salinities well above that of normal sea water. Ova of B. amphitrite denticulata become infected by L. chthamalophilum only at a temperature of 15°C. in hypersaline water, or if the Balanus ova are subjected to below-freezing temperatures before exposure to the fungus.     

Composition and succession of dinoflagellates and chrysophytes in the upper fast ice of Davis Station, East Antarctica

Little is known of the wider Antarctic distribution of the upper fast ice community now comprehensively described from McMurdo Sound. We determined the fast ice protist community at Davis Station, East Antarctica and compared it with that of McMurdo Sound. As at McMurdo Sound, Davis fast ice is characterised by extreme and transitory salinities (96–2.5 psu) and temperatures (−4.5 to −0.1°C) during the spring/summer transition. Both communities are dominated by Polarella glacialis (an autotrophic dinoflagellate), chrysophytes and their life cycle stages. Furthermore, the physical parameters of brine temperature and salinity at which these successions occurred approximated those of McMurdo Sound. The high degree of similarity between the communities from the geographically disparate locations indicates that this community type has a circum-Antarctic distribution. Confirming the areal extent and seasonality of this community type will assist in future predictions of sea ice productivity.     

Fast-start escape performance across temperature and salinity gradients in mummichog Fundulus heteroclitus

Fast-start predator-escape performance of mummichogs Fundulus heteroclitus was tested across field-informed variation in temperature (24, 30 and 36°C) and salinity (2, 12 and 32 ppt). Performance was similar across temperatures and salinities when fish were allowed to acclimate to these conditions. However, when mummichogs experienced acute temperature changes, performance exhibited thermal dependence in two contrasting ways. Fast-start turning rates and linear speeds varied directly with the temperature at which the manoeuvre was executed, but these aspects of performance varied inversely with acclimation temperature, with cool-acclimated fish exhibiting faster starts across test temperatures. Temperature effects were consistent across salinities. These results suggest that while mummichogs increase performance with acute temperature increases, long-term rises in sea temperature may cause these fish to become more susceptible to predation during abrupt cooling events, such as when storm events flood shallow water estuaries with cool rainwater.     

Organism losses during ice melting: A serious bias in sea ice community studies

Summary When ice samples are melted, microorganisms living within the brine inclusions are subjected to rapid and extreme changes in salinities. This procedure results in substantial losses of flagellates and ciliates. Most of these losses can be prevented if ice samples are melted in larger volumes of sterile sea water to buffer salinity and osmotic changes. Since most studies on the ice biota have ignored, or have been unable to avoid this bias, current views of the composition and activity of sea ice communities are based on assemblages over-representing organisms with rigid cell material.     

Evidence for active microbial nitrogen transformations in sea ice (Gulf of Bothnia, Baltic Sea) in midwinter

Nutrient concentrations, chlorophyll-a, bacterial biomass and relative activity of denitrifying organisms were investigated from ice-core, brine and underlying water samples in February 1998 in the Gulf of Bothnia, Baltic Sea. Examined sea ice was typical for the Baltic Sea; ice bulk salinity varied from 0.1 to 1.6 psu, and in underlying water salinity was from 4.2 to 4.7 psu. In 2- to 3-months-old sea ice (thickness 0.4–0.6 m), sea-ice communities were at the winter stage; chl-a concentrations were generally below 1 mg m⁻³ and heterotrophic organisms composed 7–20% of organism assemblage. In 1-month-old ice (thickness 0.2–0.25 m), an ice spring bloom was already developing and chl-a concentrations were up to 5.6 mg m⁻³. In relation to low salinity, high concentrations of NH⁺ ₄, NO⁻ ₂, PO³⁺ ₄ and SiOH₄ were found in the ice column. The results suggest that the upper part of ice accumulates atmospheric nutrient load during the ice season, and nutrients in the upper 10–20 cm of ice are mainly of atmospheric origin. The most important biological processes controlling the sea-ice nutrient status are nutrient regeneration, nutrient uptake and nitrogen transformations. Nutrient regeneration is specially active in the middle parts of the 50- to 60-cm-thick ice and subsequent accumulation of nutrients probably enhances the ice spring bloom. Nitrite accumulation and denitrifying activity were located in the same ice layers with nutrient regeneration, which together with the observed significant correlation between the concentrations of nitrogenous nutrients points to active nitrogen transformations occurring in the interior layers of sea ice in the Baltic Sea. Accepted: 12 June 2000     

Sea ice microbial communities (SIMCO)

Summary Sea ice microbial communities (SIMCO) grow luxuriantly within several microhabitats of sea ice, indicating that the microorganisms comprising these communities are well adapted to the physicochemical gradients which characterize sea ice. We used SIMCO obtained from the bottom of congelation ice in McMurdo Sound, Antarctica, to test the hypothesis that low temperature limits microbial productivity in polar oceans and also to investigate the effect of salinity on rates of autotrophic and heterotrophic metablism. Substantial rates of carbon fixation, incorporation of thymidine, and uptake of glutamate occurred at the in situ temperatures of-1.9°C, with maximum rates at temperatures considerably warmer but below 15°C. Microalgae and bacteria of SIMCO are thus indicated to be psychrophiles. The relative rates of autotrophic and heterotrophic microbial growth (based on rates of fixation of ¹⁴CO₂ by microalgae and incorporation of ³H-thymidine by bacteria, respectively) were similar and overlapped from 4° and 7°C. These data suggest that a recent hypothesis proposing the uncoupling of primary production and bacterial production in cold water, due to differential growth of phytoplankton and bacterioplankton at low temperatures, is refuted with respect to SIMCO. Maximum rates of carbon fixation by autotrophs of SIMCO occurred at salinities which characterized the ice from which the SIMCO were collected. In contrast, heterotrophs of SIMCO exhibited a more stenohaline response to variable salinity, with maximum incorporation of thymidine and uridine from 20 to 30. Adaptations by autotrophs and heterotrophs of SIMCO that permit substantial metabolism and growth at very low temperatures and variable salinities are significant when considering production and trophodynamics in polar oceans. Actively growing microorganisms in these unique communities contribute to overall production in polar oceans, provide carbon for food webs associated with sea ice, and upon release from melting ice may contribute to microbial blooms in marginal ice edge zones, which in turn support cryopelagic food webs.     

Acquisition of freeze protection in a sea-ice crustacean through horizontal gene transfer?

Sea ice is permeated by small brine channels, which are characterised by sub-zero temperatures and varying salinities. Despite sometimes extreme conditions a diverse fauna and flora thrives within the brine channels. The dominant calanoid copepods of Antarctic sea ice are Stephos longipes and Paralabidocera antarctica. Here, I report for the first time thermal hysteresis (TH) in the haemolymph of a crustacean, S. longipes, whereas P. antarctica has no such activity. TH, the non-colligative prevention of ice growth, seems to enable S. longipes to exploit all available microhabitats within sea ice, especially the surface layer, in which strong temperature fluctuations can occur. In contrast, P. antarctica only thrives within the lowermost centimetres of sea ice, where temperature fluctuations are moderate. S. longipes possesses two isoforms of a protein with TH activity. A high homology to a group of (putative) antifreeze proteins from diatoms, bacteria and a snow mold and, in contrast, no homologs in any metazoan lineage suggest that this protein was obtained through horizontal gene transfer (HGT). Further analysis of available sequence data from sea-ice organisms indicates that these antifreeze proteins were probably transferred horizontally several times. Temperature and salinity fluctuations within the brine channel system are proposed to provide “natural transformation” conditions enabling HGT and thus making this habitat a potential “hot spot” for HGT.     

Salinity affects growth but not thermal preference of adult mummichogs Fundulus heteroclitus

The effects of an ecologically relevant range of salinities (2, 12, 22, 32) on thermal preferences and growth of adult mummichogs Fundulus heteroclitus were determined for fish from a southern Chesapeake Bay population. Salinity did not affect the mean temperature selected by F. heteroclitus in a thermal gradient, which was identified as 26.6°C based on observations of 240 individuals. Salinity and temperature had significant and interacting effects on growth rates of F. heteroclitus measured over 12 weeks. Growth rates were highest overall and remained high over a broader range of temperatures at moderate salinities (12 and 22), while high growth rates were shifted toward lower temperatures for fish grown at a salinity of 2 and higher temperatures at a salinity of 32. Significant reductions in growth relative to the optimal conditions (28.6°C, salinity of 22) were observed at the coolest (19.6°C) and warmest (33.6°C) temperature tested at all salinities, as well as temperatures ≥ 26.6°C at a salinity of 2, ≥ 28.6°C at a salinity of 12 and ≤ 26.6°C at a salinity of 32. Growth rates provide a long-term, organismal measure of performance and results of this study indicate that performance may be reduced under conditions that the highly euryhaline F. heteroclitus can otherwise easily tolerate. The combination of reduced salinity and increased temperature that is predicted for temperate estuaries as a result of climate change may have negative effects on growth of this ecologically important species.