A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

Eight Metagenome-Assembled Genomes Provide Evidence for Microbial Adaptation in 20,000- to 1,000,000-Year-Old Siberian Permafrost.

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as Aminicenantes, Atribacteria, Chloroflexi, and Actinobacteria within bacteria and Thermoprofundales within archaea. MAGs from these taxa have been obtained previously from nonpermafrost environments and have been suggested to show adaptations to long-term energy starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had greater proportions in the Clusters of Orthologous Groups (COGs) categories of energy production and conversion and carbohydrate transport and metabolism than did their nonpermafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost genomes. Many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of high enough quality to assemble MAGs from microorganisms with adaptations to survive long-term freezing in this extreme environment. IMPORTANCE Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. The fate of permafrost microorganisms is connected to these predicted rapid environmental changes. Studying ancient permafrost with culture-independent techniques can give a glimpse into how these microorganisms function under these extreme low-temperature and low-energy conditions. This will facilitate understanding how they will change with the environment. This study presents genomic data from this unique environment ∼20,000 to 1,000,000 years of age.

Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost.

The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d-Asp/l-Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla Actinobacteria and Proteobacteria In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within Firmicutes became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time.IMPORTANCE Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost's hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma.

In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba.

Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of "omic" approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host's ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses' diversity remains to be fully explored.

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 µm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 µm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 µm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.

Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis K5(T).

We describe cloning and expression of genes coding for lipase Lip2Pc and lipase-specific foldase LifPc from a psychrotrophic microorganism Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg (the lense of overcooled brine within permafrost). Upon expression in Escherichiacoli Lip2Pc accumulated in inclusion bodies while chaperone was synthesized in a soluble form. An efficient protocol for solubilization and subsequent refolding of the recombinant lipase in the presence of the truncated chaperone was developed. Using this procedure Lip2Pc with specific activity of 6900U/mg was obtained. Contrary to published data on other lipase-chaperone complexes, refolded Lip2Pc was mostly recovered from the complex with chaperone by metal-affinity chromatography. Recombinant Lip2Pc displayed maximum lipolytic activity at 25°C and pH 8.0 with p-nitrophenyl palmitate (C16) as a substrate. Activity assays conducted at different temperatures revealed that the recombinant Lip2Pc is a cold-adapted lipase with ability to utilize substrates with long (C10-C16) hydrocarbon chains in the temperature range from +5 to +65°C. It demonstrated relatively high stability at temperatures above 60°C and in the presence of various metal ions or organic solvents (ethanol, methanol, etc.). Non-ionic detergents, such as Triton X-100 and Tween 20 decreased Lip2Pc activity and SDS completely inhibited it.

Celerinatantimonas yamalensis sp. nov., a cold-adapted diazotrophic bacterium from a cold permafrost brine.

A facultatively anaerobic nitrogen-fixing bacterium, strain C7(T), was isolated from a permafrost cryopeg on the Yamal Peninsula, Russia. Comparative analysis of 16S rRNA gene sequences revealed that this bacterium was closely related to Celerinatantimonas diazotrophica S-G2-2(T) with a similarity of 95.5 %. Strain C7(T) differed from Celerinatantimonas diazotrophica in its ability to hydrolyse gelatin and inability to use d-mannose, melibiose, l-rhamnose, myo-inositol, lactose, lactulose, d-mannitol, trehalose, dl-lactate, glycogen or l-proline as sole carbon sources. In addition, strain C7(T) grew over a temperature range of 0-34 °C with optimum growth at 18-22 °C. The whole-cell fatty acid profile included C16 : 0, C16 : 1ω7, C18 : 1ω7, C17 cyclo and summed feature 2 [comprising C12 : 0 aldehyde and/or unknown fatty acid 10.913 (MIDI designation) and/or iso-C16 : 1/C14 : 0 3-OH]. The DNA G+C content was 44.7 mol%. Strain C7(T) is thus considered to represent a novel species, for which the name Celerinatantimonas yamalensis sp. nov. is proposed. The type strain is C7(T) ( = VKM B-2511(T) = DSM 21888(T)).

Microbial life in 25-m-deep boreholes in ancient permafrost illuminated by metagenomics.

This study describes the composition and potential metabolic adaptation of microbial communities in northeastern Siberia, a repository of the oldest permafrost in the Northern Hemisphere. Samples of contrasting depth (1.75 to 25.1 m below surface), age (from ~ 10 kyr to 1.1 Myr) and salinity (from low 0.1-0.2 ppt and brackish 0.3-1.3 ppt to saline 6.1 ppt) were collected from freshwater permafrost (FP) of borehole AL1_15 on the Alazeya River, and coastal brackish permafrost (BP) overlying marine permafrost (MP) of borehole CH1_17 on the East Siberian Sea coast. To avoid the limited view provided with culturing work, we used 16S rRNA gene sequencing to show that the biodiversity decreased dramatically with permafrost age. Nonmetric multidimensional scaling (NMDS) analysis placed the samples into three groups: FP and BP together (10-100 kyr old), MP (105-120 kyr old), and FP (> 900 kyr old). Younger FP/BP deposits were distinguished by the presence of Acidobacteriota, Bacteroidota, Chloroflexota_A, and Gemmatimonadota, older FP deposits had a higher proportion of Gammaproteobacteria, and older MP deposits had much more uncultured groups within Asgardarchaeota, Crenarchaeota, Chloroflexota, Patescibacteria, and unassigned archaea. The 60 recovered metagenome-assembled genomes and un-binned metagenomic assemblies suggested that despite the large taxonomic differences between samples, they all had a wide range of taxa capable of fermentation coupled to nitrate utilization, with the exception of sulfur reduction present only in old MP deposits.

Desulfovibrio arcticus sp. nov., a psychrotolerant sulfate-reducing bacterium from a cryopeg.

A psychrotolerant sulfate-reducing bacterium, designated B15(T), was isolated from supercooled water brine from within permafrost of the Varandey Peninsula, on the southern coast of the Barents Sea. Cells were Gram-negative, motile vibrions (3.0-4.0×0.4-0.5 µm) with a single polar flagellum. The isolate was positive for desulfoviridin as a bisulfite reductase. Strain B15(T) grew at -2 to 28 °C (optimum 24 °C) and with 0-2.0% NaCl (optimum 0.2%). The isolate used H(2) plus acetate, formate, ethanol, lactate, pyruvate and choline as electron donors and used sulfate, sulfite, thiosulfate, elemental sulfur, DMSO and Fe(3+) as electron acceptors. Pyruvate and lactate were not fermented in the absence of sulfate. The G+C content of genomic DNA was 55.2 mol%. Analysis of the 16S rRNA gene sequence showed that the isolate belonged to the genus Desulfovibrio. Its closest relatives were Desulfovibrio idahonensis CY1(T) (98.8% 16S rRNA gene sequence similarity) and Desulfovibrio mexicanus Lup1(T) (96.5%). On the basis of genotypic, phenotypic and phylogenetic characteristics, the isolate represents a novel species, for which the name Desulfovibrio arcticus sp. nov. is proposed; the type strain is B15(T) (=VKM B-2367(T)=DSM 21064(T)).

Draft Genome Sequence of a Methanogenic Archaeon from West Spitsbergen Permafrost.

Methanobacterium sp. strain VT is a psychrotolerant methanogenic archaeon that was isolated from West Spitsbergen island (Norway) permafrost. This article describes the draft genome sequence of Methanobacterium sp. strain VT.

Biogeochemical Characteristics of Earth's Volcanic Permafrost: An Analog of Extraterrestrial Environments.

This article describes a study of frozen volcanic deposits collected from volcanoes Tolbachik and Bezymianny on the Kamchatka Peninsula, Russia, and Deception Island volcano, Antarctica. In addition, we studied suprasnow ash layers deposited after the 2007 eruptions of volcanoes Shiveluch and Bezymianny on Kamchatka. The main objectives were to characterize the presence and survivability of thermophilic microorganisms in perennially frozen volcanic deposits. As opposed to permafrost from the polar regions, viable thermophiles were detected in volcanic permafrost by cultivation, microscopy, and sequencing. In the permafrost of Tolbachik volcano, we observed methane formation by both psychrophilic and thermophilic methanogenic archaea, while at 37°C, methane production was noticeably lower. Thermophilic bacteria isolated from volcanic permafrost from the Deception Island were 99.93% related to Geobacillus stearothermophilus. Our data showed biological sulfur reduction to sulfide at 85°C and even at 130°C, where hyperthermophilic archaea of the genus Thermoproteus were registered. Sequences of hyperthermophilic bacteria of the genus Caldicellulosiruptor were discovered in clone libraries from fresh volcanic ash deposited on snow. Microorganisms found in volcanic terrestrial permafrost may serve as a model for the alien inhabitants of Mars, a cryogenic planet with numerous volcanoes. Thermophiles and hyperthermophiles and their metabolic processes represent a guideline for the future exploration missions on Mars.

Methanobacterium arcticum sp. nov., a methanogenic archaeon from Holocene Arctic permafrost.

A mesophilic, non-motile, hydrogenotrophic, rod-shaped methanogen, designated M2(T), was isolated from Holocene permafrost sediments of the Kolyma lowland in the Russian Arctic. Cells were 3-6 µm long and 0.45-0.5 µm wide. Strain M2(T) grew on H(2)/CO(2) and formate. Optimum conditions for growth were 37°C, pH 6.8-7.2 and 0.1 M NaCl. The DNA G+C content was 38.1 mol%. On the basis of 16S rRNA gene sequence comparison with known methanogens, strain M2(T) was affiliated with the genus Methanobacterium and was most closely related to Methanobacterium veterum MK4(T) and Methanobacterium bryantii DSM 863(T) (both 99 % 16S rRNA gene sequence similarity). However, no significant DNA-DNA relatedness was observed between strain M2(T) and these type strains. We propose that strain M2(T) represents a novel species, with the name Methanobacterium arcticum sp. nov., with type strain M2(T) (=DSM 19844(T) =VKM B-2371(T)).

Are permafrost microorganisms as old as permafrost?

Permafrost describes the condition of earth material (sand, ground, organic matter, etc.) cemented by ice when its temperature remains at or below 0°C continuously for longer than 2 years. Evidently, permafrost is as old as the time passed from freezing of the earth material. Permafrost is a unique phenomenon and may preserve life forms it encloses. Therefore, in order to talk confidently about the preservation of paleo-objects in permafrost, knowledge about the geological age of sediments, i.e. when the sediments were formed, and permafrost age, when those sediments became permanently frozen, is essential. There are two types of permafrost-syngenetic and epigenetic. The age of syngenetic permafrost corresponds to the geological age of its sediments, whereas the age of epigenetic permafrost is less than the geological age of its sediments. Both of these formations preserve microorganisms and their metabolic products; however, the interpretations of the microbiological and molecular-biological data are inconsistent. This paper reviews the current knowledge of time-temperature history and age of permafrost in relation to available microbiological and metagenomic data.

A living bdelloid rotifer from 24,000-year-old Arctic permafrost.

In natural, permanently frozen habitats, some organisms may be preserved for hundreds to tens of thousands of years. For example, stems of Antarctic moss were successfully regrown from an over millennium-old sample covered by ice for about 400 years1. Likewise, whole campion plants were regenerated from seed tissue preserved in relict 32,000-year-old permafrost2, and nematodes were revived from the permafrost of two localities in northeastern Siberia, with source sediments dated over 30,000 years BP3. Bdelloid rotifers, microscopic multicellular animals, are known for their ability to survive extremely low temperatures4. Previous reports suggest survival after six to ten years when frozen between -20° to 0°C4-6. Here, we report the survival of an obligate parthenogenetic bdelloid rotifer, recovered from northeastern Siberian permafrost radiocarbon-dated to ∼24,000 years BP. This constitutes the longest reported case of rotifer survival in a frozen state. We confirmed the finding by identifying rotifer actin gene sequences in a metagenome obtained from the same sample. By morphological and molecular markers, the discovered rotifer belongs to the genus Adineta, and aligns with a contemporary Adineta vaga isolate collected in Belgium. Experiments demonstrated that the ancient rotifer withstands slow cooling and freezing (∼1°C min-1) for at least seven days. We also show that a clonal culture can continuously reproduce in the laboratory by parthenogenesis.

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost.

BACKGROUND: Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost. RESULTS: Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26-120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost. CONCLUSIONS: Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars. Video abstract.

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/ß hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

Frozen Zoo: a collection of permafrost samples containing viable protists and their viruses.

BACKGROUND: Permafrost, frozen ground cemented with ice, occupies about a quarter of the Earth's hard surface and reaches up to 1000 metres depth. Due to constant subzero temperatures, permafrost represents a unique record of past epochs, whenever it comes to accumulated methane, oxygen isotope ratio or stored mummies of animals. Permafrost is also a unique environment where cryptobiotic stages of different microorganisms are trapped and stored alive for up to hundreds of thousands of years. Several protist strains and two giant protist viruses isolated from permafrost cores have been already described. NEW INFORMATION: In this paper, we describe a collection of 35 amoeboid protist strains isolated from the samples of Holocene and Pleistocene permanently frozen sediments. These samples are stored at -18°C in the Soil Cryology Lab, Pushchino, Russia and may be used for further studies and isolation attempts. The collection strains are maintained in liquid media and may be available upon request. The paper also presents a dataset which consists of a table describing the samples and their properties (termed "Sampling events") and a table describing the isolated strains (termed "Occurrences"). The dataset is publicly available through the GBIF portal.

Insights into community of photosynthetic microorganisms from permafrost.

This work integrates cultivation studies of Siberian permafrost and analyses of metagenomes from different locations in the Arctic with the aim of obtaining insights into the community of photosynthetic microorganisms in perennially frozen deposits. Cyanobacteria and microalgae have been described in Arctic aquatic and surface soil environments, but their diversity and ability to withstand harsh conditions within the permafrost are still largely unknown. Community structure of photosynthetic organisms in permafrost sediments was explored using Arctic metagenomes available through the MG-RAST. Sequences affiliated with cyanobacteria represented from 0.25 to 3.03% of total sequences, followed by sequences affiliated with Streptophyta (algae and vascular plants) 0.01-0.45% and Chlorophyta (green algae) 0.01-0.1%. Enrichment and cultivation approaches revealed that cyanobacteria and green algae survive in permafrost and they could be revived during prolonged incubation at low light intensity. Among photosynthetic microorganisms isolated from permafrost, the filamentous Oscillatoria-like cyanobacteria and unicellular green algae of the genus Chlorella were dominant. Our findings suggest that permafrost cyanobacteria and green algae are expected to be effective members of the re-assembled community after permafrost thawing and soil collapse.

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.