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Seasonal cycles within the marginal ice zones in polar regions include large shifts in temperature and salinity that strongly influence microbial abundance and physiology. However, the combined effects of concurrent temperature and salinity change on microbial community structure and biochemical composition during transitions between seawater and sea ice are not well understood. Coastal marine communities along the western Antarctic Peninsula were sampled and surface seawater was incubated at combinations of temperature and salinity mimicking the formation (cold, salty) and melting (warm, fresh) of sea ice to evaluate how these factors may shape community composition and particulate metabolite pools during seasonal transitions. Bacterial and algal community structures were tightly coupled to each other and distinct across sea-ice, seawater, and sea-ice-meltwater field samples, with unique metabolite profiles in each habitat. During short-term (approximately 10-day) incubations of seawater microbial communities under different temperature and salinity conditions, community compositions changed minimally while metabolite pools shifted greatly, strongly accumulating compatible solutes like proline and glycine betaine under cold and salty conditions. Lower salinities reduced total metabolite concentrations in particulate matter, which may indicate a release of metabolites into the labile dissolved organic matter pool. Low salinity also increased acylcarnitine concentrations in particulate matter, suggesting a potential for fatty acid degradation and reduced nutritional value at the base of the food web during freshening. Our findings have consequences for food web dynamics, microbial interactions, and carbon cycling as polar regions undergo rapid climate change., (© 2023. The Author(s).)

Background: Climate change threatens Earth's ice-based ecosystems which currently offer archives and eco-evolutionary experiments in the extreme. Arctic cryopeg brine (marine-derived, within permafrost) and sea ice brine, similar in subzero temperature and high salinity but different in temporal stability, are inhabited by microbes adapted to these extreme conditions. However, little is known about their viruses (community composition, diversity, interaction with hosts, or evolution) or how they might respond to geologically stable cryopeg versus fluctuating sea ice conditions., Results: We used long- and short-read viromics and metatranscriptomics to study viruses in Arctic cryopeg brine, sea ice brine, and underlying seawater, recovering 11,088 vOTUs (~species-level taxonomic unit), a 4.4-fold increase of known viruses in these brines. More specifically, the long-read-powered viromes doubled the number of longer (≥25 kb) vOTUs generated and recovered more hypervariable regions by >5-fold compared to short-read viromes. Distribution assessment, by comparing to known viruses in public databases, supported that cryopeg brine viruses were of marine origin yet distinct from either sea ice brine or seawater viruses, while 94% of sea ice brine viruses were also present in seawater. A virus-encoded, ecologically important exopolysaccharide biosynthesis gene was identified, and many viruses (~half of metatranscriptome-inferred "active" vOTUs) were predicted as actively infecting the dominant microbial genera Marinobacter and Polaribacter in cryopeg and sea ice brines, respectively. Evolutionarily, microdiversity (intra-species genetic variations) analyses suggested that viruses within the stable cryopeg brine were under significantly lower evolutionary pressures than those in the fluctuating sea ice environment, while many sea ice brine virus-tail genes were under positive selection, indicating virus-host co-evolutionary arms races., Conclusions: Our results confirmed the benefits of long-read-powered viromics in understanding the environmental virosphere through significantly improved genomic recovery, expanding viral discovery and the potential for biological inference. Evidence of viruses actively infecting the dominant microbes in subzero brines and modulating host metabolism underscored the potential impact of viruses on these remote and underexplored extreme ecosystems. Microdiversity results shed light on different strategies viruses use to evolve and adapt when extreme conditions are stable versus fluctuating. Together, these findings verify the value of long-read-powered viromics and provide foundational data on viral evolution and virus-microbe interactions in Earth's destabilized and rapidly disappearing cryosphere. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cryopeg brines are isolated volumes of hypersaline water in subzero permafrost. The cryopeg system at Utqiaġvik, Alaska, is estimated to date back to 40 ka BP or earlier, a remnant of a late Pleistocene Ocean. Surprisingly, the cryopeg brines contain high concentrations of organic carbon, including extracellular polysaccharides, and high densities of bacteria. How can these physiologically extreme, old, and geologically isolated systems support such an ecosystem? This study addresses this question by examining the energetics of the Utqiaġvik cryopeg brine ecosystem. Using literature-derived assumptions and new measurements on archived borehole materials, we first estimated the quantity of organic carbon when the system formed. We then considered two bacterial growth trajectories to calculate the lower and upper bounds of the cell-specific metabolic rate of these communities. These bounds represent the first community estimates of metabolic rate in a subzero hypersaline environment. To assess the plausibility of the different growth trajectories, we developed a model of the organic carbon cycle and applied it to three borehole scenarios. We also used dissolved inorganic carbon and nitrogen measurements to independently estimate the metabolic rate. The model reconstructs the growth trajectory of the microbial community and predicts the present-day cell density and organic carbon content. Model input included measured rates of the in-situ enzymatic conversion of particulate to dissolved organic carbon under subzero brine conditions. A sensitivity analysis of model parameters was performed, revealing an interplay between growth rate, cell-specific metabolic rate, and extracellular enzyme activity. This approach allowed us to identify plausible growth trajectories consistent with the observed bacterial densities in the cryopeg brines. We found that the cell-specific metabolic rate in this system is relatively high compared to marine sediments. We attribute this finding to the need to invest energy in the production of extracellular enzymes, for generating bioavailable carbon from particulate organic carbon, and the production of extracellular polysaccharides for cryoprotection and osmoprotection. These results may be relevant to other isolated systems in the polar regions of Earth and to possible ice-bound brines on worlds such as Europa, Enceladus, and Mars., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kanaan, Hoehler, Iwahana and Deming.)

Marinobacter spp. are cosmopolitan in saline environments, displaying a diverse set of metabolisms that allow them to competitively occupy these environments, some of which can be extreme in both salinity and temperature. Here, we introduce a distinct cluster of Marinobacter genomes, composed of novel isolates and in silico assembled genomes obtained from subzero, hypersaline cryopeg brines, relic seawater-derived liquid habitats within permafrost sampled near Utqiaġvik, Alaska. Using these new genomes and 45 representative publicly available genomes of Marinobacter spp. from other settings, we assembled a pangenome to examine how the new extremophile members fit evolutionarily and ecologically, based on genetic potential and environmental source. This first genus-wide genomic analysis revealed that Marinobacter spp. in general encode metabolic pathways that are thermodynamically favored at low temperature, cover a broad range of organic compounds, and optimize protein usage, e.g., the Entner-Doudoroff pathway, the glyoxylate shunt, and amino acid metabolism. The new isolates contributed to a distinct clade of subzero brine-dwelling Marinobacter spp. that diverged genotypically and phylogenetically from all other Marinobacter members. The subzero brine clade displays genomic characteristics that may explain competitive adaptations to the extreme environments they inhabit, including more abundant membrane transport systems (e.g., for organic substrates, compatible solutes, and ions) and stress-induced transcriptional regulatory mechanisms (e.g., for cold and salt stress) than in the other Marinobacter clades. We also identified more abundant signatures of potential horizontal transfer of genes involved in transcription, the mobilome, and a variety of metabolite exchange systems, which led to considering the importance of this evolutionary mechanism in an extreme environment where adaptation via vertical evolution is physiologically rate limited. Assessing these new extremophile genomes in a pangenomic context has provided a unique view into the ecological and evolutionary history of the genus Marinobacter , particularly with regard to its remarkable diversity and its opportunism in extremely cold and saline environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cooper, Rapp, Shoemaker, Anderson, Zhong and Deming.)

Subzero hypersaline brines are liquid microbial habitats within otherwise frozen environments, where concentrated dissolved salts prevent freezing. Such extreme conditions presumably require unique microbial adaptations, and possibly altered ecologies, but specific strategies remain largely unknown. Here we examined prokaryotic taxonomic and functional diversity in two seawater-derived subzero hypersaline brines: first-year sea ice, subject to seasonally fluctuating conditions; and ancient cryopeg, under relatively stable conditions geophysically isolated in permafrost. Overall, both taxonomic composition and functional potential were starkly different. Taxonomically, sea-ice brine communities (∼10 5 cells mL -1 ) had greater richness, more diversity and were dominated by bacterial genera, including Polaribacter , Paraglaciecola , Colwellia , and Glaciecola , whereas the more densely inhabited cryopeg brines (∼10 8 cells mL -1 ) lacked these genera and instead were dominated by Marinobacter . Functionally, however, sea ice encoded fewer accessory traits and lower average genomic copy numbers for shared traits, though DNA replication and repair were elevated; in contrast, microbes in cryopeg brines had greater genetic versatility with elevated abundances of accessory traits involved in sensing, responding to environmental cues, transport, mobile elements (transposases and plasmids), toxin-antitoxin systems, and type VI secretion systems. Together these genomic features suggest adaptations and capabilities of sea-ice communities manifesting at the community level through seasonal ecological succession, whereas the denser cryopeg communities appear adapted to intense bacterial competition, leaving fewer genera to dominate with brine-specific adaptations and social interactions that sacrifice some members for the benefit of others. Such cryopeg genomic traits provide insight into how long-term environmental stability may enable life to survive extreme conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rapp, Sullivan and Deming.)

Arctic regions, which are changing rapidly as they warm 2 to 3 times faster than the global average, still retain microbial habitats that serve as natural laboratories for understanding mechanisms of microbial adaptation to extreme conditions. Seawater-derived brines within both sea ice (sea-ice brine) and ancient layers of permafrost (cryopeg brine) support diverse microbes adapted to subzero temperatures and high salinities, yet little is known about viruses in these extreme environments, which, if analogous to other systems, could play important evolutionary and ecosystem roles. Here, we characterized viral communities and their functions in samples of cryopeg brine, sea-ice brine, and melted sea ice. Viral abundance was high in cryopeg brine (1.2 × 10 8  ml -1 ) and much lower in sea-ice brine (1.3 × 10 5 to 2.1 × 10 5  ml -1 ), which roughly paralleled the differences in cell concentrations in these samples. Five low-input, quantitative viral metagenomes were sequenced to yield 476 viral populations (i.e., species level; ≥10 kb), only 12% of which could be assigned taxonomy by traditional database approaches, indicating a high degree of novelty. Additional analyses revealed that these viruses: (i) formed communities that differed between sample type and vertically with sea-ice depth; (ii) infected hosts that dominated these extreme ecosystems, including Marinobacter , Glaciecola , and Colwellia ; and (iii) encoded fatty acid desaturase ( FAD ) genes that likely helped their hosts overcome cold and salt stress during infection, as well as mediated horizontal gene transfer of FAD genes between microbes. Together, these findings contribute to understanding viral abundances and communities and how viruses impact their microbial hosts in subzero brines and sea ice. IMPORTANCE This study explores viral community structure and function in remote and extreme Arctic environments, including subzero brines within marine layers of permafrost and sea ice, using a modern viral ecogenomics toolkit for the first time. In addition to providing foundational data sets for these climate-threatened habitats, we found evidence that the viruses had habitat specificity, infected dominant microbial hosts, encoded host-derived metabolic genes, and mediated horizontal gene transfer among hosts. These results advance our understanding of the virosphere and how viruses influence extreme ecosystems. More broadly, the evidence that virally mediated gene transfers may be limited by host range in these extreme habitats contributes to a mechanistic understanding of genetic exchange among microbes under stressful conditions in other systems., (Copyright © 2020 Zhong et al.)

Hypersaline aqueous environments at subzero temperatures are known to be inhabited by microorganisms, yet information on community structure in subzero brines is very limited. Near Utqiaġvik, Alaska, we sampled subzero brines (-6°C, 115-140 ppt) from cryopegs, i.e. unfrozen sediments within permafrost that contain relic (late Pleistocene) seawater brine, as well as nearby sea-ice brines to examine microbial community composition and diversity using 16S rRNA gene amplicon sequencing. We also quantified the communities microscopically and assessed environmental parameters as possible determinants of community structure. The cryopeg brines harbored surprisingly dense bacterial communities (up to 108 cells mL-1) and millimolar levels of dissolved and particulate organic matter, extracellular polysaccharides and ammonia. Community composition and diversity differed between the two brine environments by alpha- and beta-diversity indices, with cryopeg brine communities appearing less diverse and dominated by one strain of the genus Marinobacter, also detected in other cold, hypersaline environments, including sea ice. The higher density and trend toward lower diversity in the cryopeg communities suggest that long-term stability and other features of a subzero brine are more important selective forces than in situ temperature or salinity, even when the latter are extreme., (© FEMS 2019.)

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 [ 14 C]-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., (© 2019 Phycological Society of America.)

Sea ice and its snow cover are critical for global processes including climate regulation and biogeochemical cycles. Despite an increase in studies focused on snow microorganisms, the ecology of snow inhabitants remains unclear. In this study, we investigated sources and selection of a snowpack-specific microbial community by comparing metagenomes from samples collected in a Greenlandic fjord within a vertical profile including atmosphere, snowpack with four distinct layers of snow, sea ice brine and seawater. Microbial communities in all snow layers derived from mixed sources, both marine and terrestrial, and were more similar to atmospheric communities than to sea ice or seawater communities. The surface snow metagenomes were characterized by the occurrence of genes involved in photochemical stress resistance, primary production and metabolism of diverse carbon sources. The basal saline snow layer that was in direct contact with the sea ice surface harbored a higher abundance of cells than the overlying snow layers, with a predominance of Alteromonadales and a higher relative abundance of marine representatives. However, the overall taxonomic structure of the saline layer was more similar to that of other snow layers and the atmosphere than to underlying sea ice and seawater. The expulsion of relatively nutrient-rich sea ice brine into basal snow might have stimulated the growth of copiotrophic psychro- and halotolerant snow members. Our study indicates that the size, composition and function of snowpack microbial communities over sea ice were influenced primarily by atmospheric deposition and inflow of sea ice brine and that they form a snow-specific assemblage reflecting the particular environmental conditions of the snowpack habitat.

Colwellia psychrerythraea 34H is a model psychrophilic bacterium found in the cold ocean-polar sediments, sea ice, and the deep sea. Although the genomes of such psychrophiles have been sequenced, their metabolic strategies at low temperature have not been quantified. We measured the metabolic fluxes and gene expression of 34H at 4 °C (the mean global-ocean temperature and a normal-growth temperature for 34H), making comparative analyses at room temperature (above its upper-growth temperature of 18 °C) and with mesophilic Escherichia coli When grown at 4 °C, 34H utilized multiple carbon substrates without catabolite repression or overflow byproducts; its anaplerotic pathways increased flux network flexibility and enabled CO 2 fixation. In glucose-only medium, the Entner-Doudoroff (ED) pathway was the primary glycolytic route; in lactate-only medium, gluconeogenesis and the glyoxylate shunt became active. In comparison, E. coli , cold stressed at 4 °C, had rapid glycolytic fluxes but no biomass synthesis. At their respective normal-growth temperatures, intracellular concentrations of TCA cycle metabolites (α-ketoglutarate, succinate, malate) were 4-17 times higher in 34H than in E. coli , while levels of energy molecules (ATP, NADH, NADPH) were 10- to 100-fold lower. Experiments with E. coli mutants supported the thermodynamic advantage of the ED pathway at cold temperature. Heat-stressed 34H at room temperature (2 hours) revealed significant down-regulation of genes associated with glycolytic enzymes and flagella, while 24 hours at room temperature caused irreversible cellular damage. We suggest that marine heterotrophic bacteria in general may rely upon simplified metabolic strategies to overcome thermodynamic constraints and thrive in the cold ocean., Competing Interests: The authors declare no conflict of interest.

A variety of ecologically important processes are driven by bacterial motility and taxis, yet these basic bacterial behaviours remain understudied in cold habitats. Here, we present a series of experiments designed to test the chemotactic ability of the model marine psychrophilic bacterium Colwellia psychrerythraea 34H, when grown at optimal temperature and salinity (8°C, 35 ppt) or its original isolation conditions (-1°C, 35 ppt), towards serine and mannose at temperatures from -8°C to 27°C (above its upper growth temperature of 18°C), and at salinities of 15, 35 and 55 ppt (at 8°C and -1°C). Results indicate that C. psychrerythraea 34H is capable of chemotaxis at all temperatures tested, with strongest chemotaxis at the temperature at which it was first grown, whether 8°C or -1°C. This model marine psychrophile also showed significant halotaxis towards 15 and 55 ppt solutions, as well as strong substrate-specific chemohalotaxis. We suggest that such patterns of taxis may enable bacteria to colonize sea ice, position themselves optimally within its extremely cold, hypersaline and temporally fluctuating microenvironments, and respond to various chemical signals therein., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and JohnWiley & Sons Ltd.)

Meaningful motion is an unambiguous biosignature, but because life in the Solar System is most likely to be microbial, the question is whether such motion may be detected effectively on the micrometer scale. Recent results on microbial motility in various Earth environments have provided insight into the physics and biology that determine whether and how microorganisms as small as bacteria and archaea swim, under which conditions, and at which speeds. These discoveries have not yet been reviewed in an astrobiological context. This paper discusses these findings in the context of Earth analog environments and environments expected to be encountered in the outer Solar System, particularly the jovian and saturnian moons. We also review the imaging technologies capable of recording motility of submicrometer-sized organisms and discuss how an instrument would interface with several types of sample-collection strategies. Key Words: In situ measurement-Biosignatures-Microbiology-Europa-Ice. Astrobiology 16, 755-774., Competing Interests: Author Disclosure Statement No competing financial interests exist.

Cryopegs are sub-surface hypersaline brines at sub-zero temperatures within permafrost; their global extent and distribution are unknown. The permafrost barrier to surface and groundwater advection maintains these brines as semi-isolated systems over geological time. A cryopeg 7 m below ground near Barrow, Alaska, was sampled for geochemical and microbiological analysis. Sub-surface brines (in situtemperature of -6 °C, salinity of 115 ppt), and an associated sediment-infused ice wedge (melt salinity of 0.04 ppt) were sampled using sterile technique. Major ionic concentrations in the brine corresponded more closely to other (Siberian) cryopegs than to Standard seawater or the ice wedge. Ionic ratios and stable isotope analysis of water conformed to a marine or brackish origin with subsequent Rayleigh fractionation. The brine contained ∼1000× more bacteria than surrounding ice, relatively high viral numbers suggestive of infection and reproduction, and an unusually high ratio of particulate to dissolved extracellular polysaccharide substances. A viral metagenome indicated a high frequency of temperate viruses and limited viral diversity compared to surface environments, with closest similarity to low water activity environments. Interpretations of the results underscore the isolation of these underexplored microbial ecosystems from past and present oceans., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sea ice is an analog environment for several of astrobiology's near-term targets: Mars, Europa, Enceladus, and perhaps other Jovian or Saturnian moons. Microorganisms, both eukaryotic and prokaryotic, remain active within brine channels inside the ice, making it unnecessary to penetrate through to liquid water below in order to detect life. We have developed a submersible digital holographic microscope (DHM) that is capable of resolving individual bacterial cells, and demonstrated its utility for immediately imaging samples taken directly from sea ice at several locations near Nuuk, Greenland. In all samples, the appearance and motility of eukaryotes were conclusive signs of life. The appearance of prokaryotic cells alone was not sufficient to confirm life, but when prokaryotic motility occurred, it was rapid and conclusive. Warming the samples to above-freezing temperatures or supplementing with serine increased the number of motile cells and the speed of motility; supplementing with serine also stimulated chemotaxis. These results show that DHM is a useful technique for detection of active organisms in extreme environments, and that motility may be used as a biosignature in the liquid brines that persist in ice. These findings have important implications for the design of missions to icy environments and suggest ways in which DHM imaging may be integrated with chemical life-detection suites in order to create more conclusive life detection packages.

The Earth's cryosphere comprises those regions that are cold enough for water to turn into ice. Recent findings show that the icy realms of polar oceans, glaciers and ice sheets are inhabited by microorganisms of all three domains of life, and that temperatures below 0 °C are an integral force in the diversification of microbial life. Cold-adapted microorganisms maintain key ecological functions in icy habitats: where sunlight penetrates the ice, photoautotrophy is the basis for complex food webs, whereas in dark subglacial habitats, chemoautotrophy reigns. This Review summarizes current knowledge of the microbial ecology of frozen waters, including the diversity of niches, the composition of microbial communities at these sites and their biogeochemical activities.

The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

Background: Psychrophiles are presumed to play a large role in the catabolism of alkanes and other components of crude oil in natural low temperature environments. In this study we analyzed the functional diversity of genes for alkane hydroxylases, the enzymes responsible for converting alkanes to more labile alcohols, as found in the genomes of nineteen psychrophiles for which alkane degradation has not been reported. To identify possible mechanisms of low temperature optimization we compared putative alkane hydroxylases from these psychrophiles with homologues from nineteen taxonomically related mesophilic strains., Results: Seven of the analyzed psychrophile genomes contained a total of 27 candidate alkane hydroxylase genes, only two of which are currently annotated as alkane hydroxylase. These candidates were mostly related to the AlkB and cytochrome p450 alkane hydroxylases, but several homologues of the LadA and AlmA enzymes, significant for their ability to degrade long-chain alkanes, were also detected. These putative alkane hydroxylases showed significant differences in primary structure from their mesophile homologues, with preferences for specific amino acids and increased flexibility on loops, bends, and α-helices., Conclusion: A focused analysis on psychrophile genomes led to discovery of numerous candidate alkane hydroxylase genes not currently annotated as alkane hydroxylase. Gene products show signs of optimization to low temperature, including regions of increased flexibility and amino acid preferences typical of psychrophilic proteins. These findings are consistent with observations of microbial degradation of crude oil in cold environments and identify proteins that can be targeted in rate studies and in the design of molecular tools for low temperature bioremediation.

Wintertime measurements near Barrow, Alaska, showed that bacteria near the surface of first-year sea ice and in overlying saline snow experience more extreme temperatures and salinities, and wider fluctuations in both parameters, than bacteria deeper in the ice. To examine impacts of such conditions on bacterial survival, two Arctic isolates with different environmental tolerances were subjected to winter-freezing conditions, with and without the presence of organic solutes involved in osmoprotection: proline, choline, or glycine betaine. Obligate psychrophile Colwellia psychrerythraea strain 34H suffered cell losses under all treatments, with maximal loss after 15-day exposure to temperatures fluctuating between -7 and -25 °C. Osmoprotectants significantly reduced the losses, implying that salinity rather than temperature extremes presents the greater stress for this organism. In contrast, psychrotolerant Psychrobacter sp. strain 7E underwent miniaturization and fragmentation under both fluctuating and stable-freezing conditions, with cell numbers increasing in most cases, implying a different survival strategy that may include enhanced dispersal. Thus, the composition and abundance of the bacterial community that survives in winter sea ice may depend on the extent to which overlying snow buffers against extreme temperature and salinity conditions and on the availability of solutes that mitigate osmotic shock, especially during melting., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Microbial enzymatic hydrolysis of marine-derived particulate organic carbon (POC) can be a dominant mechanism for attenuating carbon flux in cold Arctic waters during spring and summer. Whether this mechanism depends on composition of associated microbial communities and extends into other seasons is not known. Bacterial community composition (BCC) and extracellular enzyme activity (EEA, for leucine aminopeptidases, glucosidases and chitobiases) were measured on small suspended particles and potentially sinking aggregates collected during fall from waters of the biologically productive North Water and river-impacted Beaufort Sea. Although other environmental variables appeared influential, both BCC and EEA varied along a marine productivity gradient in the two regions. Aggregates harbored the most distinctive bacterial communities, with a small number of taxa driving differences between particle-size classes (1.0-60 and > 60 μm) and free-living bacteria (0.2-1.0 μm). Significant relationships between patterns in particle-associated BCC and EEA suggest strong links between these two variables. Calculations indicated that up to 80% of POC in the euphotic zone of the North Water, and 20% in the Beaufort Sea, may be hydrolyzed enzymatically, underscoring the importance of this mechanism in attenuating carbon fluxes in Arctic waters even as winter approaches., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Newly formed sea ice is a vast and biogeochemically active environment. Recently, we reported an unusual microbial community dominated by members of the Rhizobiales in frost flowers at the surface of Arctic young sea ice based on the presence of 16S gene sequences related to these strains. Here, we use metagenomic analysis of two samples, from a field of frost flowers and the underlying young sea ice, to explore the metabolic potential of this surface ice community. The analysis links genes for key biogeochemical processes to the Rhizobiales, including dimethylsulfide uptake, betaine glycine turnover, and halocarbon production. Nodulation and nitrogen fixation genes characteristic of terrestrial root-nodulating Rhizobiales were generally lacking from these metagenomes. Non-Rhizobiales clades at the ice surface had genes that would enable additional biogeochemical processes, including mercury reduction and dimethylsulfoniopropionate catabolism. Although the ultimate source of the observed microbial community is not known, considerations of the possible role of eolian deposition or transport with particles entrained during ice formation favor a suspended particle source for this microbial community., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Frost flowers are highly saline ice structures that grow on the surface of young sea ice, a spatially extensive environment of increasing importance in the Arctic Ocean. In a previous study, we reported organic components of frost flowers in the form of elevated levels of bacteria and exopolymers relative to underlying ice. Here, DNA was extracted from frost flowers and young sea ice, collected in springtime from a frozen lead offshore of Barrow, Alaska, to identify bacteria in these understudied environments. Evaluation of the distribution of 16S rRNA genes via four methods (microarray analysis, T-RFLP, clone library and shotgun metagenomic sequencing) indicated distinctive bacterial assemblages between the two environments, with frost flowers appearing to select for Rhizobiales. A phylogenetic placement approach, used to evaluate the distribution of similar Rhizobiales sequences in other polar marine studies, indicated that some of the observed strains represent widely distributed members of the marine rare biosphere in both the Arctic and Antarctic., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

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.

Effects of hydrostatic pressure on pure cultures of prokaryotes have been studied extensively but impacts at the community level in the ocean are less well defined. Here we consider hydrostatic pressure effects on natural communities containing both unadapted (piezosensitive) prokaryotes originating from surface water and adapted (including piezophilic) prokaryotes from the deep sea. Results from experiments mimicking pressure changes experienced by particle-associated prokaryotes during their descent through the water column show that rates of degradation of organic matter (OM) by surface-originating microorganisms decrease with sinking. Analysis of a much larger data set shows that, under stratified conditions, deep-sea communities adapt to in situ conditions of high pressure, low temperature and low OM. Measurements made using decompressed samples and atmospheric pressure thus underestimate in situ activity. Exceptions leading to overestimates can be attributed to deep mixing events, large influxes of surface particles, or provision of excessive OM during experimentation. The sediment-water interface, where sinking particles accumulate, will be populated by a mixture of piezosensitive, piezotolerant and piezophilic prokaryotes, with piezophilic activity prevailing deeper within sediment. A schematic representation of how pressure shapes prokaryotic communities in the ocean is provided, allowing a reasonably accurate interpretation of the available activity measurements., (© 2013 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Inherent to sea ice, like other high latitude environments, is the strong seasonality driven by changes in insolation throughout the year. Sea-ice organisms are exposed to shifting, sometimes limiting, conditions of temperature and salinity. An array of adaptations to survive these and other challenges has been acquired by those organisms that inhabit the ice. One key adaptive response is the production of extracellular polymeric substances (EPS), which play multiple roles in the entrapment, retention and survival of microorganisms in sea ice. In this concept paper we consider two main areas of sea-ice microbiology: the physico-chemical properties that define sea ice as a microbial habitat, imparting particular advantages and limits; and extracellular responses elicited in microbial inhabitants as they exploit or survive these conditions. Emphasis is placed on protective strategies used in the face of fluctuating and extreme environmental conditions in sea ice. Gaps in knowledge and testable hypotheses are identified for future research.

The 104 kb genome of cold-active bacteriophage 9A, which replicates in the marine psychrophilic gamma-proteobacterium Colwellia psychrerythraea strain 34H (between -12 and 8 °C), was sequenced and analyzed to investigate elements of molecular adaptation to low temperature and phage-host interactions in the cold. Most characterized ORFs indicated closest similarity to gamma-proteobacteria and their phages, though no single module provided definitive phylogenetic grouping. A subset of primary structural features linked to psychrophily suggested that the majority of annotated phage proteins were not psychrophilic; those that were, primarily serve phage-specific functions and may also contribute to 9A's restricted temperature range for replication as compared to host. Comparative analyses suggest ribonucleotide reductase genes were acquired laterally from host. Neither restriction modification nor the CRISPR-Cas system appeared to be the predominant phage defense mechanism of Cp34H or other cold-adapted bacteria; we hypothesize that psychrophilic hosts rely more on the use of extracellular polymeric material to block cell surface receptors recognized by phages. The relative dearth of evidence for genome-specific defenses, genetic transfer events or auxiliary metabolic genes suggest that the 9A-Cp34H system may be less tightly coupled than are other genomically characterized marine phage-host systems, with possible implications for phage specificity under different environmental conditions.

Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon's indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student's t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.

The physical properties of Arctic sea ice determine its habitability. Whether ice-dwelling organisms can change those properties has rarely been addressed. Following discovery that sea ice contains an abundance of gelatinous extracellular polymeric substances (EPS), we examined the effects of algal EPS on the microstructure and salt retention of ice grown from saline solutions containing EPS from a culture of the sea-ice diatom, Melosira arctica. We also experimented with xanthan gum and with EPS from a culture of the cold-adapted bacterium Colwellia psychrerythraea strain 34H. Quantitative microscopic analyses of the artificial ice containing Melosira EPS revealed convoluted ice-pore morphologies of high fractal dimension, mimicking features found in EPS-rich coastal sea ice, whereas EPS-free (control) ice featured much simpler pore geometries. A heat-sensitive glycoprotein fraction of Melosira EPS accounted for complex pore morphologies. Although all tested forms of EPS increased bulk ice salinity (by 11-59%) above the controls, ice containing native Melosira EPS retained the most salt. EPS effects on ice and pore microstructure improve sea ice habitability, survivability, and potential for increased primary productivity, even as they may alter the persistence and biogeochemical imprint of sea ice on the surface ocean in a warming climate.

A comprehensive seafloor biomass and abundance database has been constructed from 24 oceanographic institutions worldwide within the Census of Marine Life (CoML) field projects. The machine-learning algorithm, Random Forests, was employed to model and predict seafloor standing stocks from surface primary production, water-column integrated and export particulate organic matter (POM), seafloor relief, and bottom water properties. The predictive models explain 63% to 88% of stock variance among the major size groups. Individual and composite maps of predicted global seafloor biomass and abundance are generated for bacteria, meiofauna, macrofauna, and megafauna (invertebrates and fishes). Patterns of benthic standing stocks were positive functions of surface primary production and delivery of the particulate organic carbon (POC) flux to the seafloor. At a regional scale, the census maps illustrate that integrated biomass is highest at the poles, on continental margins associated with coastal upwelling and with broad zones associated with equatorial divergence. Lowest values are consistently encountered on the central abyssal plains of major ocean basins The shift of biomass dominance groups with depth is shown to be affected by the decrease in average body size rather than abundance, presumably due to decrease in quantity and quality of food supply. This biomass census and associated maps are vital components of mechanistic deep-sea food web models and global carbon cycling, and as such provide fundamental information that can be incorporated into evidence-based management.

The structure of bacterial communities in first-year spring and summer sea ice differs from that in source seawaters, suggesting selection during ice formation in autumn or taxon-specific mortality in the ice during winter. We tested these hypotheses by weekly sampling (January-March 2004) of first-year winter sea ice (Franklin Bay, Western Arctic) that experienced temperatures from -9 degrees C to -26 degrees C, generating community fingerprints and clone libraries for Bacteria and Archaea. Despite severe conditions and significant decreases in microbial abundance, no significant changes in richness or community structure were detected in the ice. Communities of Bacteria and Archaea in the ice, as in under-ice seawater, were dominated by SAR11 clade Alphaproteobacteria and Marine Group I Crenarchaeota, neither of which is known from later season sea ice. The bacterial ice library contained clones of Gammaproteobacteria from oligotrophic seawater clades (e.g. OM60, OM182) but no clones from gammaproteobacterial genera commonly detected in later season sea ice by similar methods (e.g. Colwellia, Psychrobacter). The only common sea ice bacterial genus detected in winter ice was Polaribacter. Overall, selection during ice formation and mortality during winter appear to play minor roles in the process of microbial succession that leads to distinctive spring and summer sea ice communities.

Extracellular polysaccharide substances (EPS) play critical roles in microbial ecology, including the colonization of extreme environments in the ocean, from sea ice to the deep sea. After first developing a sugar-free growth medium, we examined the relative effects of temperature, pressure, and salinity on EPS production (on a per cell basis) by the obligately marine and psychrophilic gamma-proteobacterium, Colwellia psychrerythraea strain 34H. Over growth-permissive temperatures of approximately 10 to -4 degrees C, EPS production did not change, but from -8 to -14 degrees C when samples froze, EPS production rose dramatically. Similarly, at growth-permissive hydrostatic pressures of 1-200 atm (1 atm = 101.325 kPa) (at -1 and 8 degrees C), EPS production was unchanged, but at higher pressures of 400 and 600 atm EPS production rose markedly. In salinity tests at 10-100 parts per million (and -1 and 5 degrees C), EPS production increased at the freshest salinity tested. Extreme environmental conditions thus appear to stimulate EPS production by this strain. Furthermore, strain 34H recovered best from deep-freezing to -80 degrees C (not found for Earthly environments) if first supplemented with a preparation of its own EPS, rather than other cryoprotectants like glycerol, suggesting EPS production as both a survival strategy and source of compounds with potentially novel properties for biotechnological and other applications.

A whole-core injection method was used to determine depth-related rates of microbial mineralization of (14)C-phenanthrene added to both contaminated and clean marine sediments of Puget Sound, WA. For 26-day incubations under micro-aerobic conditions, conversions of (14)C-phenanthrene to (14)CO(2) in heavily PAH-contaminated sediments from two sites in Eagle Harbor were much higher (up to 30%) than those in clean sediments from nearby Blakely Harbor (<3%). The averaged (14)C-phenanthrene degradation rates in the surface sediment horizons (0-3 cm) were more rapid (2-3 times) than in the deeper sediment horizons examined (>6 cm), especially in the most PAH polluted EH9 site. Differences in mineralization were associated with properties of the sediments as a function of sediment depth, including grain-size distribution, PAH concentration, total organic matter and total bacterial abundance. When strictly anaerobic incubations (in N(2)/H(2)/CO(2) atmosphere) were used, the phenanthrene biodegradation rates at all sediment depths were two times slower than under micro-aerobic conditions, with methanogenesis observed after 24 days. The main rate-limiting factor for phenanthrene degradation under anaerobic conditions appeared to be the availability of suitable electron acceptors. Addition of calcium sulfate enhanced the first order rate coefficient (k(1) increased from 0.003 to 0.006 day(-1)), whereas addition of soluble nitrate, even at very low concentration (<0.5 mM), inhibited mineralization. Long-term storage of heavily polluted Eagle Harbor sediment as intact cores under micro-aerobic conditions also appeared to enhance anaerobic biodegradation rates (k(1) up to 0.11 day(-1)).

Direct evidence for metabolism in a variety of frozen environments has pushed temperature limits for bacterial activity to increasingly lower temperatures, so far to -20 degrees C. To date, the metabolic activities of marine psychrophilic bacteria, important components of sea-ice communities, have not been studied in laboratory culture, not in ice and not below -12 degrees C. We measured [3H]-leucine incorporation into macromolecules (further fractionated biochemically) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H over a range of anticipated activity-permissive temperatures, from +13 to -20 degrees C, including expected negative controls at -80 and -196 degrees C. For incubation temperatures below -1 degrees C, the cell suspensions [all in artificial seawater (ASW)] were first quick-frozen in liquid nitrogen. We also examined the effect of added extracellular polymeric substances (EPS) on [3H]-leucine incorporation. Results showed that live cells of strain 34H incorporated substantial amounts of [3H]-leucine into TCA-precipitable material (primarily protein) down to -20 degrees C. At temperatures from -1 to -20 degrees C, rates were enhanced by EPS. No activity was detected in the killed controls for strain 34H (or in Escherichia coli controls), which included TCA-killed, heat-killed, and sodium azide- and chloramphenicol-treated samples. Surprisingly, evidence for low but significant rates of intracellular incorporation of [3H]-leucine into protein was observed for both ASW-only and EPS-amended (and live only) samples incubated at -80 and -196 degrees C. Mechanisms that could explain the latter results require further study, but the process of vitrification promoted by rapid freezing and the presence of salts and organic polymers may be relevant. Overall, distinguishing between intracellular and extracellular aspects of bacterial activity appears important to understanding behavior at sub-freezing temperatures.

We describe a model based on diffusion theory and the temperature-dependent mechanism of brine concentration in sea ice to argue that, if viruses partition with bacteria into sea-ice brine inclusions, contact rates between the two can be higher in winter sea ice than in seawater, increasing the probability of infection and possible virus production. To examine this hypothesis, we determined viral and bacterial concentrations in select winter sea-ice horizons using epifluorescence microscopy. Viral concentrations ranged from 1.6 to 82 x 10(6) ml(-1) of brine volume of the ice, with highest values in brines from coldest (-24 to -31 degrees C) ice horizons. Calculated virus-bacteria contact rates in underlying -1 degrees C seawater were similar to those in brines of -11 degrees C ice but up to 600 times lower than those in ice brines at or below -24 degrees C. We then incubated native bacterial and viral assemblages from winter sea ice for 8 days in brine at a temperature (-12 degrees C) and salinity ( approximately 160 psu) near expected in situ values, monitoring their concentrations microscopically. While different cores yielded different results, consistent with known spatial heterogeneity in sea ice, these experiments provided unambiguous evidence for viral persistence and production, as well as for bacterial growth, in -12 degrees C brine.

The completion of the 5,373,180-bp genome sequence of the marine psychrophilic bacterium Colwellia psychrerythraea 34H, a model for the study of life in permanently cold environments, reveals capabilities important to carbon and nutrient cycling, bioremediation, production of secondary metabolites, and cold-adapted enzymes. From a genomic perspective, cold adaptation is suggested in several broad categories involving changes to the cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and strategies to overcome temperature-dependent barriers to carbon uptake. Modeling of three-dimensional protein homology from bacteria representing a range of optimal growth temperatures suggests changes to proteome composition that may enhance enzyme effectiveness at low temperatures. Comparative genome analyses suggest that the psychrophilic lifestyle is most likely conferred not by a unique set of genes but by a collection of synergistic changes in overall genome content and amino acid composition.

The limited database on cold-active extracellular proteases from marine bacteria was expanded by successful purification and initial biochemical and structural characterization of a family M1 aminopeptidase (designated ColAP) produced by the marine psychrophile Colwellia psychrerythraea strain 34H. The 71-kDa enzyme displayed a low optimum temperature (19 degrees C) and narrow pH range (pH 6 to 8.5) for activity and greater thermolability than other extracellular proteases. Sequencing of the gene encoding ColAP revealed a predicted amino acid sequence with the highest levels of identity (45 to 55%) to M1 aminopeptidases from mesophilic members of the gamma subclass of the Proteobacteria and the next highest levels of identity (35 to 36%) to leukotriene A(4) hydrolases from mammalian sources. Compared to mesophilic homologs, ColAP had structural differences thought to increase the flexibility for activity in the cold; for example, it had fewer proline residues, fewer ion pairs, and a lower hydrophobic residue content. In addition to intrinsic properties that determine enzyme activity and stability, we also investigated effects of extracellular polymeric substances (EPS) from spent culture medium of strain 34H on ColAP activity at an environmentally relevant temperature (0 degrees C) and at 45 degrees C (the maximum temperature for activity). In both cases, ColAP stability increased significantly in the presence of EPS, indicating the importance of considering environmentally relevant extrinsic factors when enzyme structure and function are investigated.

Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.