No abstract

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.

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 × 108 ml−1) and much lower in sea-ice brine (1.3 × 105 to 2.1 × 105 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.

Cryopeg is a layer within permafrost containing a significant amount of cryotic unfrozen water due to dissolved salts. To explore the origin and development of cryopeg and associated brines found near Utqiaġvik, we sampled extensively within the Barrow Permafrost Tunnel. We found two types of cryopeg brines based on their distinctive locations: (a) intra‐ice brine (IiB), entirely bounded by massive ground ice, and not previously observed in the Northern Hemisphere; and (b) intra‐sediment brine (IsB), found as expected in unfrozen sediments within permafrost. The encountered IiBs were situated in small ellipsoidal or more complex shaped pockets within the massive ice at roughly atmospheric pressure. Radiocarbon dating suggests that the IiB segregated from IsB‐bearing cryopeg beneath the massive ice at about 11 ka BP, at the earliest. From geochemical analyses, IsB lenses were interpreted as having developed through repeated evaporation and cryoconcentration of seawater in a lagoonal environment, then isolated when the surrounding sediment froze and became covered by an upper sediment unit around 40 ka BP or earlier. The discovery of IiB and development of origin scenarios for both brine types validate the importance of high‐resolution sampling as enabled by the unique facility of a permafrost tunnel.

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.

<p>Cryopeg is a volume of permafrost with a significant amount of cryotic unfrozen water as a result of freezing-point depression by dissolved salt content. Cryopeg and saline permafrost have been reported for coastal areas of the Arctic seas, and their current distribution and future changes are a great concern for the warming Arctic, as the state of permafrost controls ground stability and the functioning of ice cellars in Arctic villages. To describe the distribution and segregation of cryopeg lenses, and to explore the origin and development of the cryopeg and associated brines found near Utqiaġvik, we conducted extensive sampling campaigns in the Barrow Permafrost Tunnel during May of 2017 and 2018.</p><p>We found two types of cryopeg brines based on their distinctive spatial occurrences: (1) intra-ice brine (IiB), entirely bounded by massive ice; and (2) intra-sediment brine (IsB), found in unfrozen sediment lenses within permafrost. While two examples of IiB have been reported previously, they were each found within ice layers below ice-sealed lakes in the McMurdo Dry Valleys of Antarctica, geological settings very different from ours. In our study, the IiBs were at roughly atmospheric pressure and situated in small pockets of ellipsoidal or more complex shape (dimensions of up to about 30 cm wide and 3 cm height) within 17–41 cm above the underlying sediment layer. Several individual IiB pockets may have been connected by porous ice of low permeability. Radiocarbon dating suggests that, at the earliest, the IiB was segregated about 11 ka BP from IsB-bearing cryopeg underneath. IsB lenses were interpreted as having developed through repeated evaporation and cryoconcentration of seawater in a lagoonal environment, then isolated at the latest when the surrounding sediment froze up and became covered by an upper sediment unit around 40 ka BP or earlier.</p><p>Considering the common characteristics among the cryopeg brines accessed from the tunnel and those found in brine-bearing marine sediment around Utqiaġvik, all occurrences of cryopeg brine in the region may have experienced analogous development despite potentially contrasting salinities and estimated ages. An increase in permafrost temperature invariably will result in expansion of cryopeg lenses and may change movement of liquids within the permafrost, which potentially become threats to Arctic coasts, infrastructure, and food security.</p>