Growth of Carnobacterium spp. from permafrost under low pressure, temperature, and anoxic atmosphere has implications for Earth microbes on Mars.

The ability of terrestrial microorganisms to grow in the near-surface environment of Mars is of importance to the search for life and protection of that planet from forward contamination by human and robotic exploration. Because most water on present-day Mars is frozen in the regolith, permafrosts are considered to be terrestrial analogs of the martian subsurface environment. Six bacterial isolates were obtained from a permafrost borehole in northeastern Siberia capable of growth under conditions of low temperature (0 °C), low pressure (7 mbar), and a CO(2)-enriched anoxic atmosphere. By 16S ribosomal DNA analysis, all six permafrost isolates were identified as species of the genus Carnobacterium, most closely related to C. inhibens (five isolates) and C. viridans (one isolate). Quantitative growth assays demonstrated that the six permafrost isolates, as well as nine type species of Carnobacterium (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. pleistocenium, and C. viridans) were all capable of growth under cold, low-pressure, anoxic conditions, thus extending the low-pressure extreme at which life can function.

Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost.

Whole, fertile plants of Silene stenophylla Ledeb. (Caryophyllaceae) have been uniquely regenerated from maternal, immature fruit tissue of Late Pleistocene age using in vitro tissue culture and clonal micropropagation. The fruits were excavated in northeastern Siberia from fossil squirrel burrows buried at a depth of 38 m in undisturbed and never thawed Late Pleistocene permafrost sediments with a temperature of -7 °C. Accelerator mass spectrometry (AMS) radiocarbon dating showed fruits to be 31,800 ± 300 y old. The total γ-radiation dose accumulated by the fruits during this time was calculated as 0.07 kGy; this is the maximal reported dose after which tissues remain viable and seeds still germinate. Regenerated plants were brought to flowering and fruiting and they set viable seeds. At present, plants of S. stenophylla are the most ancient, viable, multicellular, living organisms. Morphophysiological studies comparing regenerated and extant plants obtained from modern seeds of the same species in the same region revealed that they were distinct phenotypes of S. stenophylla. The first generation cultivated from seeds obtained from regenerated plants progressed through all developmental stages and had the same morphological features as parent plants. The investigation showed high cryoresistance of plant placental tissue in permafrost. This natural cryopreservation of plant tissue over many thousands of years demonstrates a role for permafrost as a depository for an ancient gene pool, i.e., preexisting life, which hypothetically has long since vanished from the earth's surface, a potential source of ancient germplasm, and a laboratory for the study of rates of microevolution.

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)).

Structure of the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) containing a 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose moiety.

A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) and identified by 1D and 2D (1)H and (13)C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

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)).

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments.

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate approximately 100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be approximately 400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.

Biogeochemistry of methane and methanogenic archaea in permafrost.

This study summarizes the findings of our research on the genesis of methane, its content and distribution in permafrost horizons of different age and origin. Supported by reliable data from a broad geographical sweep, these findings confirm the presence of methane in permanently frozen fine-grained sediments. In contrast to the omnipresence of carbon dioxide in permafrost, methane-containing horizons (up to 40.0 mL kg(-1)) alternate with strata free of methane. Discrete methane-containing horizons representing over tens of thousands of years are indicative of the absence of methane diffusion through the frozen layers. Along with the isotopic composition of CH(4) carbon (delta(13)C -64 per thousand to -99 per thousand), this confirms its biological origin and points to in situ formation of this biogenic gas. Using (14)C-labeled substrates, the possibility of methane formation within permafrost was experimentally shown, as confirmed by delta(13)C values. Extremely low values (near -99 per thousand) indicate that the process of CH(4) formation is accompanied by the substantial fractionation of carbon isotopes. For the first time, cultures of methane-forming archaea, Methanosarcina mazei strain JL01 VKM B-2370, Methanobacterium sp. strain M2 VKM B-2371 and Methanobacterium sp. strain MK4 VKM B-2440 from permafrost, were isolated and described.

Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods.

The microbial composition of ancient permafrost sediments from the Kolyma lowland of Northeast Eurasia was examined through culture and culture-independent approaches. These sediments have been continuously frozen for 5,000 to 2-3 million years. A total of 265 Bacteria 16S rRNA gene sequences were amplified from the permafrost total-community genomic DNA and screened by amplified ribosomal 16S rRNA restriction analysis. Members of three major lineages were found: gamma-Proteobacteria (mostly Xanthomonadaceae), Actinobacteria, and Firmicutes. We also determined partial 16S rRNA gene sequences of 49 isolates from a collection of 462 aerobes isolated from these sediments. The bacteria included Actinomycetales (Arthrobacter and Microbacteriaceae); followed by the Firmicutes (Exiguobacterium and Planomicrobium); the Bacteroidetes (Flavobacterium); the gamma-Proteobacteria (Psychrobacter); and the alpha-Proteobacteria (Sphingomonas). Both culture and culture-independent approaches showed the presence of high and low G+C Gram-positive bacteria and gamma-Proteobacteria. Some of the 16S rRNA gene sequences of environmental clones matched those of Arthrobacter isolates. Two-thirds of the isolates grew at -2.5 degrees C, indicating that they are psychroactive, and all are closely related to phylogenetic groups with strains from other cold environments, mostly commonly from Antarctica. The culturable and non-culturable microorganisms found in the terrestrial permafrost provide a prototype for possible life on the cryogenic planets of the Solar System.

Biodiversity of cryopegs in permafrost.

This study describes the biodiversity of the indigenous microbial community in the sodium-chloride water brines (cryopegs) derived from ancient marine sediments and sandwiched within permafrost 100-120,000 years ago after the Arctic Ocean regression. Cryopegs remain liquid at the in situ temperature of -9 to -11 degrees C and make up the only habitat on the Earth that is characterized by permanently subzero temperatures, high salinity, and the absence of external influence during geological time. From these cryopegs, anaerobic and aerobic, spore-less and spore-forming, halotolerant and halophilic, psychrophilic and psychrotrophic bacteria, mycelial fungi and yeast were isolated and their activity was detected below 0 degrees C.

Aspartic acid racemization and age-depth relationships for organic carbon in Siberian permafrost.

We have analyzed the degree of racemization of aspartic acid in permafrost samples from Northern Siberia, an area from which microorganisms of apparent ages up to a few million years have previously been isolated and cultured. We find that the extent of aspartic acid racemization in permafrost cores increases very slowly up to an age of approximately 25,000 years (around 5 m in depth). The apparent temperature of racemization over the age range of 0-25,000 years, determined using measured aspartic acid racemization rate constants, is -19 degrees C. This apparent racemization temperature is significantly lower than the measured environmental temperature (-11 to -13 degrees C) and suggests active recycling of D-aspartic acid in Siberian permafrost up to an age of around 25,000 years. This indicates that permafrost organisms are capable of repairing some molecular damage incurred while in a "dormant" state over geologic time.

Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg.

A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5(T) was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by P. cryohalolentis K5(T) , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0-5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca(2+) , Mn(2+) , and EDTA whereas Zn(+2) , Cu(+2) , Co(+2) , Ni(+2) , and Mg(+2) inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.

Cryptic diversity within the choanoflagellate morphospecies complex Codosiga botrytis - phylogeny and morphology of ancient and modern isolates.

Choanoflagellates are closely related to metazoans and fungi according to recent phylogenetic studies; therefore the systematics of these organisms is of particular interest. The choanoflagellate morphospecies Codosiga botrytis is the first described choanoflagellate, and is one of the most frequently reported choanoflagellate species. In this study we present phylogenetic and morphological data on eight different strains of Codosiga botrytis. Among these there are five ancient strains; these cultures have been established from up to 43,000 years old cysts from Siberian permafrost. We found that based on the variable V4 region of the small subunit (SSU) of the rDNA, all the investigated freshwater isolates of Codosiga botrytis, together with Sphaeroeca volvox, form a cluster at the base of all other choanoflagellate species. Moreover, the morphospecies described classically as Codosiga botrytis contains at least four different genotypes separated by considerably high genetic distance. All these 'cryptic species' have identical general morphology and cell structure. Strains have a similar life cycle with several different life forms and large morphological plasticity. For the first time we were able to establish cultures from cryo-conserved cysts of choanoflagellates. The ancient strains did not differ significantly in partial SSU rDNA from the modern ones. Besides, no biogeographically pattern could be established. This fact and the low genetic distances of some strains from remote locations support the distribution of dormant stages via air.

Ancient fungi in Antarctic permafrost environments.

Filamentous fungi in 36 samples of Antarctic permafrost sediments were studied. The samples collected during the Russian Antarctic expedition of 2007-2009 within the framework of the Antarctic Permafrost Age Project (ANTPAGE) were recovered from different depths in ice-free oases located along the perimeter of the continent. Fungal diversity was determined by conventional microbiological techniques combined with a culture-independent method based on the analysis of internal transcribed spacer (ITS2) sequences in total DNA of the samples. The study revealed a rather low fungal population density in permafrost, although the diversity found was appreciable, representing more than 26 genera. Comparison of the data obtained by different techniques showed that the culture-independent method enabled the detection of ascomycetous and basidiomycetous fungi not found by culturing. The molecular method failed to detect members of the genera Penicillium and Cladosporium that possess small-sized spores known to have a high resistance to environmental changes.

Structure of the O-polysaccharide chain of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled water brines within permafrost.

Psychrotrophic bacteria of the genus Psychrobacter have not been studied in respect to lipopolysaccharide structure. In this work, we determined the structure of the O-specific polysaccharide of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled (-9°C) water brines within permafrost. The polysaccharide was found to be acidic due to the presence of an amide of 2-acetamido-2-deoxy-l-guluronic acid with glycine (l-GulNAcA6Gly), which has not been hitherto found in nature. The following structure of the disaccharide repeating unit of the polysaccharide was established using composition analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: →4)-α-l-GulpNAcA6Gly-(1→3)-ß-d-GlcpNAc-(1→

Structure of an acidic polysaccharide isolated from Psychrobacter maritimus 3pS containing a bacillosamine derivative.

An acidic polysaccharide was obtained from Psychrobacter maritimus 3pS isolated from a Siberian cryopeg sample (Kolyma lowland). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar analysis along with (1)H and (13)C NMR spectroscopy: →2)-α-L-Rhap-(1→4)-α-D-GalpNAcA-(1→3)-α-D-QuipNAc4NHb-(1→3)-ß-D-QuipNAc4NHb-(1→ where D-GalNAcA indicates 2-acetamido-2-deoxy-D-galacturonic acid and d-QuiNAc4NHb indicates 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutanoyl]amino-D-glucose.

Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample.

A combination of culture-dependent and -independent techniques was used to characterize a bacterial community, examine cold adaptation of isocitrate lyase (icl) genes, and detect genes with important ecological functions in a permafrost sample from the Bykovsky Peninsula on the Laptev Sea coast of northeast Siberia. According to the 16S rRNA gene sequence, 47 of the cultured isolates were members of the phyla Firmicutes, Proteobacteria, and Actinobacteria, with 85% of the isolates belonging to the genera Arthrobacter and Planococcus. The 16S rRNA gene clone library derived from DNA from the same permafrost sample contained sequences from the same phyla plus a few from Acidobacteria, but favored the Firmicutes at the cost of the Actinobacteria. A partial sequence of the icl gene, a proposed marker for cold adaptation, was determined for 25 isolates that grew at 0 °C. Two Psychrobacter isolates contained two of the four residues shown to be important for low-temperature activity in Colwellia maris or Colwellia psychrerythreaea. The presence in the permafrost DNA of genes with ecosystem functions was determined using geochip 2.0. The highest number of genes identified was from the categories of aromatic and natural polymer degradation genes, perhaps reflecting selection for the use of tundra vegetation-produced carbon.

Biogeography of two cold-adapted genera: Psychrobacter and Exiguobacterium.

The genera Exiguobacterium and Psychrobacter have been frequently detected in and isolated from polar permafrost and ice. These two genera have members that can grow at temperatures as low as -5 and -10 degrees C, respectively. We used quantitative PCR (Q-PCR) to quantify members of these genera in 54 soil or sediment samples from polar, temperate and tropical environments to determine to what extent they are selected by cold environments. These results were further analyzed by multiple linear regression to identify the most relevant environmental factors corresponding to their distribution. Exiguobacterium was detected in all three climatic zones at similar densities, but was patchier in the temperate and tropical samples. Psychrobacter was present in almost all polar samples, was at highest densities in Antarctica sediment samples, but was in very low densities and infrequently detected in temperate and tropical soils. Clone libraries, specific for the 16S rRNA gene for each genus, were constructed from a sample from each climatic region. The clone libraries were analyzed for alpha and beta diversities, as well as for variation in population structure by using analysis of molecular variance. Results confirm that both genera were found in all three climatic zones; however, Psychrobacter populations seemed to be much more diverse than Exiguobacterium in all three climatic zones. Furthermore, Psychrobacter populations from Antarctica are different from those in Michigan and Puerto Rico, which are similar to each other.

Clostridium tagluense sp. nov., a psychrotolerant, anaerobic, spore-forming bacterium from permafrost.

A strictly anaerobic, Gram-positive, psychrotolerant, endospore-forming bacterium (strain A121(T)) was isolated from a permafrost sample collected in the Canadian High Arctic. Phylogenetic analysis of the 16S rRNA gene sequence of strain A121(T) showed its affiliation with the group of psychrophilic and psychrotolerant members of cluster I of the genus Clostridium, Clostridium bowmanii DSM 14206(T) being the closest relative (sequence similarity 98.5 %). Levels of DNA-DNA relatedness between strain A121(T) and the type strains of phylogenetically related species ranged from 33 to 52 %. Strain A121(T) grew in PY broth at temperatures between 4 and 28 degrees C (optimum 15-20 degrees C), at pH 6.0-8.0 (optimum pH 6.5-7.2) and in NaCl concentrations of 0-10.0 % (optimum 0-2.0 %). The strain utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose, fructose, trehalose, maltose and starch; it also hydrolysed gelatin. Predominant fatty acids were C(16 : 1) cis9, C(16 : 1) cis9 DMA, C(16 : 0) and C(14 : 0). The DNA G+C content was 31.5 mol%. On the basis of its overall genotypic and phenotypic characteristics, strain A121(T) is classified within a novel species of the genus Clostridium, Clostridium tagluense sp. nov. The type strain is A121(T) (=VKM B-2369(T) =DSM 17763(T)).

Ancient bacteria show evidence of DNA repair.

Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability.