E. V. Spirina

Survival of Micromycetes and Actinobacteria under Conditions of Long-Term Natural Cryopreservation

Almost all of the investigated samples of the Arctic and Antarctic permafrost sediments of different genesis with ages from 5–10 thousand to 2–3 million years were found to contain viable micromycete and bacterial cells. The maximum amounts of viable cells of fungi (up to 104CFU/g air-dried sample) and bacteria (up to 107–109CFU/g air-dried sample) were present in fine peaty sediment samples taken from different depths. The identified micromycetes belonged to more than 20 genera of the divisions Basidiomycota, Ascomycota, and Zygomycota, and some represented mitosporic fungi. Thawing the samples at 35 and 52°C allowed the number of detected fungal genera to be increased by more than 30%. Aerobic heterotrophic prokaryotes were dominated by coryneform, nocardioform, and spore-forming microorganisms of the order Actinomycetales.Analysis of the isolated fungi and actinomycetes showed that most of them originated from the microbial communities of ancient terrestrial biocenoses.

Bacteria in Permafrost

Significant numbers of viable ancient microorganisms are known to be present within the permafrost. They have been isolated in both polar regions from the cores up to 400 m deep and ground temperatures of -27 C. The age of the cells corresponds to the longevity of the permanently frozen state of the soils, with the oldest cells dating back to {approx}3 million years in the Arctic, and {approx}5 million years in the Antarctic. They are the only life forms known to have retained viability over geological time. Thawing of the permafrost renews their physiological activity and exposes ancient life to modern ecosystems. Thus, the permafrost represents a stable and unique physicochemical complex, which maintains life incomparably longer than any other known habitats. If we take into account the depth of the permafrost layers, it is easy to conclude that they contain a total microbial biomass many times higher than that of the soil cover. This great mass of viable matter is peculiar to permafrost only.

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.

The resistance of viable permafrost algae to simulated environmental stresses: implications for astrobiology

54 strains of viable green algae and 26 strains of viable cyanobacteria were recovered from 128 and 56 samples collected from Siberian and Antarctic permafrost, respectively, with ages from modern to a few million years old. Although species of unicellular green algae belonged to Chlorococcales were subdominant inside permafrost, green algae Pedinomonas sp . were observed in Antarctic permafrost. Filamentous cyanobacteria of Oscillatoriales , Nostocales were just found in Siberian permafrost. Algal biomass in the permanently frozen sediments, expressed as concentration of chlorophyll a , was 0.06–0.46 μg g −1 . The number of viable algal cells varied between 2 and 9×10 3 cfu g −1 , but the number of viable bacterial cells was usually higher from 10 2 to 9.2×10 5 cfu g −1 . Frozen but viable permafrost algae have preserved their morphological characteristics and photosynthetic apparatus in the dark permafrost. In the laboratory, they restored their photosynthetic activity, growth and development in favourable conditions at positive temperatures and with the availability of water and light. The discovery of ancient viable algae within permafrost reflects their ability to tolerate long-term freezing. In this study, the tolerance of algae and cyanobacteria to freezing, thawing and freezing–drying stresses was evaluated by short-term (days to months) low-temperature experiments. Results indicate that viable permafrost microorganisms demonstrate resistance to such stresses. Apart from their ecological importance, the bacterial and algal species found in permafrost have become the focus for novel biotechnology, as well as being considered proxies for possible life forms on cryogenic extraterrestrial bodies.

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

Fungal diversity in the Antarctic active layer

Taxonomic diversity of fungi in the samples of the active layer of Antarctica was investigated using conventional microbiological techniques and metagenomic analysis of total DNA extracted from environmental samples. The list of Antarctic microscopic fungi was expanded, including detection of the species representing a portion of the fungal complex which is nonculturable or sterile on conventional nutrient media.

Halophilic-psychrotrophic bacteria of an Alaskan cryopeg—a model for astrobiology

Cryopegs, lenses of hypersaline unfrozen soil or water within permafrost, are a model for astrobiology, since free water can only be present on cryogenic bodies and planets in the form of brine. In this paper the diversity of aerobic halophilic-psychrotrophic microorganisms from an Alaskan cryopeg (Barrow Cape) were studied and described for the first time. This cryopeg is characterized by a constant subzero temperature (–7°C), high salinity (total mineralization is about 120 g/L) and isolation from external influences for a geologically significant period of time. Our study has revealed a large number of microorganisms capable of growth at low temperature (4°C) in a wide range of salinities from 5 to 250 g/L of NaCl, the latter being 3 times higher than the natural salt concentration of the Alaskan cryopeg. The microorganisms identified are comprised of four major phyla: Actinobacteria (genera Brevibacterium, Citricoccus, Microbacterium), Firmicutes (genus Paenibacillus), Bacteroidetes (genus Sphingobacterium), and Proteobacteria (genus Ochrobactrum).

Lipolytic enzymes of microorganisms from permafrost cryopegs

279 Permafrost, which occupies 26% of the earths surr face and 50% of Russia, is a unique habitat simultaa neously exposed to constant subzero temperature, background ionizing radiation, low water activity, and nutrient shortage over a geologically significant duraa tion of time. Many researchers have demonstrated the ability of proo and eukaryotic microorganisms to remain viable in permafrost [1], and specifically in cryopegs, unfrozen lens of highly mineralized water existing at subzero temperatures in the bulk of permaa frost sedimentary deposits of marine origin [2]. Study of the physiological mechanisms of microorr ganism survival under the conditions characteristic of permafrost contributes to the broadening of the views on the spatial and temporal extent of the biosphere, as well as to the detection of novel biologically active compounds potentially useful for biotechnology, such as fatty acids, pigments, antimicrobial compounds and proteins, including enzymes [3]. Colddactive enzymes display increased activities at low temperatures and high affinities for their subb strates, thus providing for efficient catalysis and high reaction rates. The use of such enzymes in biotechnoll ogy is of special interest. For example, colddactive lipases and esterases, which catalyze the hydrolysis of triglycerides into glycerin and fatty acids, can be used in food industry, as well as for fine organic synthesis of thermolabile compounds, and in bioremediation of polluted northern territories. To detect microorganisms producing colddactive lipolytic enzymes in cryopeg samples from the Kolyma Lowland, Yamal Peninsula, and Point Barrow (Alaska) (Table 1), we used both the traditional microo biological approach involving the isolation of pure cultures of microbial community members displaying the desired activity, and the upptoodate methods of genetic engineering and bioinformatics. The use of these approaches allowed the isolation and partial characterization of aerobic halophilic microorganisms producing lipolytic enzymes from cryopeg samples. The genes of two enzymes of especial interest, namely, colddactive lipases from Psychroo bacter cryohalolentis K5 T were cloned, proteins were produced in milligram quantities, and the physicoo chemical properties of the proteins were analyzed. Microorganisms were isolated from cryopegs by enrichment cultivation at 4 and 20°C on the standard nutrient media R2A and 1/2 TSB (Difco) containing 100–250 g/L NaCl. As a result, pure cultures of 51 bacterial strains were obtained: most of the bacteria were either pigmented Grammpositive cocci or Grammpositive rods. Partial sequences of 16S DNA were obtained for 36 strains by direct sequencing of DNA fragments obtained in a PCR with universal bacterial …

Chromatic Adaptation of Viable Ancient Cyanobacteria from Arctic Permafrost

Chromatic adaptation of cyanobacteria involves the regulation of the synthesis of the main cellular phycobilins, C-phycoerythrin and phycocyanin, by red and green light [1, 2]. The chromatic adaptation of modern nitrogen-fixing cyanobacteria was most pronounced when they were grown on nitrogen-free media. The addition of nitrogen sources to the cultivation media reduces the effect of green light and increases the effect of red light on the accumulation of C-phycoerythrin and phycocyanin [3]. The organisms studied in this work was the ancient and viable heterocystous cyanobacterium Nostoc sp., taken from a depth of 10 m in 5000-year old Holocene lake sediments [4]. In this study of the ancient bacterium, as in modern nitrogenfixing bacteria, the amount of C-phycoerythrin is the greatest with growth on a nitrogen-free medium and decreases in the presence of various nitrogen sources [5]. To discern the capacity of this cyanobacterium for chromatic adaptation, we studied the effect of various nitrogen sources on the levels of C-phycoerythrin and phycocyanin in cells grown under red and green light. The amount of pigments in the cells was estimated from the absorption and fluorescence excitation spectra.

Methanogens in the Antarctic Dry Valley permafrost

Polar permafrost is at the forefront of climate change, yet only a few studies have enriched the native methane-producing microbes that might provide positive feedbacks to climate change. Samples Ant1 and Ant2, collected in Antarctic Miers Valley from permafrost sediments, with and without biogenic methane, respectively, were evaluated for methanogenic activity and presence of methanogens. After a one-year incubation of both samples under anaerobic conditions, methane production was observed only at room temperature in microcosm Ant1 with CO2/H2 (20/80) as carbon and energy sources and was monitored during the subsequent 10 years. The concentration of methane in the headspace of microcosm Ant1 changed from 0.8% to a maximum of 45%. Archaeal 16S rRNA genes from microcosm Ant1 were related to psychrotolerant Methanosarcina lacustris. Repeated efforts at achieving a pure culture of this organism were unsuccessful. Metagenomic reads obtained for the methane-producing microcosm Ant1 were assembled and resulted in a 99.84% complete genome affiliated with the genus Methanosarcina. The metagenome assembled genome contained cold-adapted enzymes and pathways suggesting that the novel uncultured Methanosarcina sp. Ant1 is adapted to sub-freezing conditions in permafrost. This is the first methanogen genome reported from the 15 000 years old permafrost of the Antarctic Dry Valleys.