bThe Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (؊5°C), hypersalinity (salinity, 24%), and reducing (Ϸ؊165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H 2 S/sulfides (up to 25 ppm), ammonia (Ϸ381 M), and methane (11.1 g day ؊1 ). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related to Cyanobacteria (19.7%), Bacteroidetes (13.3%), and Proteobacteria (6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonas spp.), sulfate reducers (Desulfobulbus spp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/ subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments.
Cryoenvironments are defined as permanently subzero or frozen environments, such as permafrost, glaciers, ice sheets, multiyear sea ice, high-elevation Antarctic dry valleys, and some cold saline springs (1-6). Microorganisms inhabiting cryoenvironments must face the challenges of subzero temperatures, low water activity, and, often, high solute concentrations to sustain their viability. The cold saline springs on Axel Heiberg Island (AHI) in the Canadian high Arctic discharge through 500 to 600 m of thick permafrost, maintain a liquid state at subzero temperatures, and offer a unique opportunity to assess microbial adaptations to extremes of both high salinity and subzero temperatures (3, 4, 7-9). These springs occur in an area with an average annual air temperature of Ϫ15°C, reaching below Ϫ40°C during the winter months, and probably originate from subpermafrost groundwater flow through carboniferous evaporites in areas of diapiric uplift on AHI (10, 11). Other Arctic cold springs, on Ellesmere Island in the Canadian high Arctic and on the Norwegian highArctic Svalbard archipelago, have been reported (12-14), although t...