In this study, we isolated and characterized bacterial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years. The sampling site was located at Mammoth Mountain in the Aldan river valley in Central Yakutia in Eastern Siberia. Analysis of phospolipid fatty acids (PLFA) demonstrated the dominance of bacteria over fungi; the analysis of fatty acids specific for Gram-positive and Gram-negative bacteria revealed an approximately twofold higher amount of Gram-negative bacteria compared to Gram-positive bacteria. Direct microbial counts after natural permafrost enrichment showed the presence of (4.7 ± 1.5) × 10 8 cells g ？1 sediment dry mass. Viable heterotrophic bacteria were found at 0 °C, 10 °C and 25 °C, but not at 37 °C. Spore-forming bacteria were not detected. Numbers of viable fungi were low and were only detected at 0 °C and 10 °C. Selected culturable bacterial isolates were identified as representatives of Arthrobacter phenanthrenivorans, Subtercola frigoramans and Glaciimonas immobilis. Representatives of each of these species were characterized with regard to their growth temperature range, their ability to grow on different media, to produce enzymes, to grow in the presence of NaCl, antibiotics, and heavy metals, and to degrade hydrocarbons. All strains could grow at ？5 °C; the upper temperature limit for growth in liquid culture was 25 °C or 30 °C. Sensitivity to rich media, antibiotics, heavy metals, and salt increased when temperature decreased (20 °C > 10 °C > 1 °C). In spite of the ligninolytic activity of some strains, no biodegradation activity was detected.
References
[1]
Ershov, E.D. Foundations of Geocryology (in Russian); Moscow State University: Moscow, Russia, 1998; pp. 1–575.
[2]
Margesin, R. Permafrost Soils; Springer Verlag: Berlin/Heidelberg, Germany, 2009; pp. 1–348.
[3]
Johnson, S.S.; Hebsgaard, M.B.; Christensen, T.R.; Mastepanov, M.; Nielsen, R.; Munch, K.; Brand, T.B.; Gilbert, M.T.P.; Zuber, M.T.; Bunce, M.; et al. Ancient bacteria show evidence of DNA repair. Proc. Natl. Acad. Sci. USA 2007, 104, 14401–14405.
[4]
Steven, B.; Leveille, R.; Pollard, W.H.; Whyte, L.G. Microbial ecology and biodiversity in permafrost. Extremophiles 2006, 10, 259–267, doi:10.1007/s00792-006-0506-3.
[5]
Gilichinsky, D.; Vishnivetskaya, T.; Petrova, M.; Spirina, E.; Mamykin, V.; Rivkina, E. In Psychrophiles: From Biodiversity to Biotechnology; Margesin, R., Schinner, F., Marx, J.C., Gerday, C., Eds.; Springer Verlag: Berlin/Heidelberg, Germany, 2008; pp. 83–102.
[6]
Steven, B.; Niederberger, T.D.; Whyte, L.G. Permafrost Soils; Margesin, R., Ed.; Springer Verlag: Berlin/Heidelberg, Germany, 2009; pp. 59–72.
[7]
Gilichinsky, D. Encyclopedia of Environmental Microbiology; Bitton, G., Ed.; Wiley: New York, NY, USA, 2002; pp. 932–956.
[8]
Bakermans, C. Psychrophiles: From Biodiversity to Biotechnology; Margesin, R., Schinner, F., Marx, J.C., Gerday, C., Eds.; Springer Verlag: Berlin/Heidelberg, Germany, 2008; pp. 17–28.
[9]
Panikov, N.S.; Sizova, M.V. Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to ？5 degrees C. FEMS Microbiol. Ecol. 2007, 59, 500–512.
[10]
Amato, P.; Doyle, S.M.; Battista, J.R.; Christner, B.C. Implications of subzero metabolic activity on long-term microbial survival in terrestrial and extraterrestrial permafrost. Astrobiology 2010, 10, 789–798, doi:10.1089/ast.2010.0477.
[11]
Brushkov, A.V.; Melnikov, V.P.; Sukhovei, I.G.; Griva, G.I.; Repin, V.E.; Kalenova, L.F.; Brenner, E.V.; Subbotin, A.M.; Trofimova, I.B.; Tanaka, M.; et al. Relict microorganisms of cryolithozone as possible objects of gerontology (in Russian). Adv. Gerontol. 2009, 22, 253–258.
[12]
Brushkov, A.V.; Bezrukov, V.V.; Griva, G.I.; Muradyan, K.K. The effects of the relict microorganism B. sp. on development, gas exchange, spontaneous motor activity, stress resistance, and survival of Drosophila melanogaster. Adv. Gerontol. 2012, 2, 19–26, doi:10.1134/S2079057012010055.
[13]
Kalenova, L.F.; Suhovey, U.G.; Broushkov, A.V.; Melnikov, V.P.; Fisher, T.A.; Besedin, I.M.; Novikova, M.A.; Efimova, J.A.; Subbotin, A.M. Experimental study of the effects of permafrost microorganisms on the morphofunctional activity of the immune system. Bull. Exp. Biol. Med. 2011, 151, 201–204, doi:10.1007/s10517-011-1289-9.
[14]
Kalenova, L.F.; Sukhovei, U.G.; Brushkov, A.V.; Melnikov, V.P.; Fisher, T.A.; Besedin, I.M.; Novikova, M.A.; Efimova, J.A. Effects of permafrost microorganisms on the quality and duration of life of laboratory animals. Neurosci. Behav. Physiol. 2011, 41, 484–490, doi:10.1007/s11055-011-9441-6.
[15]
Fursova, O.; Potapov, V.; Brouchkov, A.; Pogorelko, G.; Griva, G.; Fursova, N.; Ignatov, S. Probiotic activity of bacterial strain isolated from ancient permafrost against Salmonella infection in mice. Probiotics Antimicrob. Proteins 2012, 403, 145–153.
[16]
Friedmann, E.I. Viable Microorganisms in Permafrost; Gilichinsky, D., Ed.; Russian Academy of Sciences: Pushchino, Russia, 1994; pp. 21–26.
[17]
Stewart, E.J.; Madden, R.; Paul, G.; Taddei, F. Ageing and death in an organism that reproduces by morphologically symmetric division. PLoS Biol. 2005, 3, e45, doi:10.1371/journal.pbio.0030045.
[18]
Johnson, L.R.; Mangel, M. Life histories and the evolution of aging in bacteria and other single-celled organisms. Mech. Ageing Dev. 2006, 127, 786–793, doi:10.1016/j.mad.2006.07.004.
[19]
Nicholson, W.L.; Munakata, N.; Horneck, G.; Melosh, H.J.; Setlow, P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. 2000, 64, 548–572, doi:10.1128/MMBR.64.3.548-572.2000.
Katayama, T.; Tanaka, M.; Moriizumi, J.; Nakamura, T.; Brouchkov, A.; Douglas, T.; Fukuda, M.; Tomita, M.; Asano, K. Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 Years. Appl. Environ. Microbiol. 2007, 73, 2360–2363, doi:10.1128/AEM.01715-06.
[22]
Clein, J.S.; Schimel, J.P. Microbial activity of tundra and taiga soils at sub-zero temperatures. Soil Biol. Biochem. 1995, 27, 1231–1234, doi:10.1016/0038-0717(95)00044-F.
[23]
Ashcroft, F. Life at the Extremes; HarperCollins: London, UK, 2000; pp. 1–326.
[24]
Jaenicke, R. Stability and folding of ultrastable proteins: Eye lens crystallins and enzymes from thermophiles. FASEB J. 1996, 10, 84–92.
[25]
Cairns, J.; Overbaugh, J.; Miller, S. The origin of mutations. Nature 1994, 335, 142–145.
[26]
Levy, M.; Miller, S.L. The stability of the RNA bases: Implications for the origin of life. Biochemistry 1998, 95, 7933–7938.
[27]
Rauser, C.L.; Mueller, L.D.; Rose, M.R. Evolution of late life. Ageing Res. Rev. 2005, 5, 14–32.
[28]
Willerslev, E.; Cooper, A. Ancient DNA. Proc. Roy. Soc. B 2005, 272, 3–16, doi:10.1098/rspb.2004.2813.
[29]
Amann, R.; Ludwig, W.; Schleifer, K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 1995, 59, 143–169.
[30]
McDougald, D.; Rice, S.A.; Kjelleberg, S. New perspectives on the viable but nonculturable response. Biologia 2009, 54, 617–623.
[31]
Spiegelman, D.; Whissell, G.; Greer, C.W. A survey of the methods for the characterization of microbial consortia and communities. Can. J. Microbiol. 2005, 51, 355–386, doi:10.1139/w05-003.
[32]
Kirk, L.J.; Beaudette, L.A.; Hart, M.; Moutoglis, P.; Klironomas, J.N.; Lee, H.; Trevors, J.T. Methods of studying soil microbiol diversity. J. Microbiol. Meth. 2004, 58, 169–188.
[33]
Baranova, U.P.; Il'inskay, I.A.; Nikitin, V.P.; Pneva, G.P.; Fradkina, A.F.; Shvareva, N.Y. Works of Geological Institute of Russian Academy of Sciences (in Russian); Nauka: Moscow, Russia, 1976; pp. 1–284.
[34]
Romanovsky, N.N. Basics of Croygenesis of Lithosphere (in Russian); Moscow State University: Moscow, Russia, 1993; pp. 1–336.
[35]
Bakulina, N.T.; Spector, V.B. Climate and Permafrost (in Russian); Maksimov, G.N., Fedorov, A.N., Eds.; Permafrost Institute: Yakutsk, Russia, 2000; pp. 21–32.
[36]
Markov, K.K. Cross-Section of the Newest Sediments; Moscow University Press: Moscow, Russia, 1973; pp. 1–198.
[37]
Vishnivetskaya, T.A.; Kathariou, S.; McGrath, J.; Gilichinsky, D.; Tiedje, J.M. Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles 2000, 4, 165–173, doi:10.1007/s007920070031.
[38]
Schlichting, E.; Blume, H.P.; Stahr, K. Bodenkundliches Praktikum, 2. Auflage (in German); Blackwell: Wissenschafts-Verlag, Berlin, Germany, 1995; pp. 1–296.
[39]
Schinner, F.; ？hlinger, R.; Kandeler, E.; Margesin, R. Methods in Soil Biology; Springer Lab Manual: Berlin/Heidelberg, Germany, 1996; pp. 1–426.
[40]
Margesin, R.; Jud, M.; Tscherko, D.; Schinner, F. Microbial communities and activities in alpine and subalpine soils. FEMS Microbiol. Ecol. 2009, 67, 208–218, doi:10.1111/j.1574-6941.2008.00620.x.
[41]
Frostegard, A.; Baath, E.; Tunlid, A. Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis. Soil Biol. Biochem. 1993, 25, 723–730, doi:10.1016/0038-0717(93)90113-P.
[42]
Bardgett, R.D.; Hobbs, P.J.; Frostegard, A. Changes in soil fungal:bacterial biomass ratios following reductions in the intensity of management of an upland grassland. Biol. Fertil. Soils 1996, 22, 261–264, doi:10.1007/BF00382522.
[43]
Federle, T.W. Perspectives in Microbial Ecology; Megusar, F., Gantar, M., Eds.; Slovene Society for Microbiology: Ljubljana, Slovenia, 1986; pp. 493–498.
[44]
Zelles, L. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: A review. Biol. Fertil. Soils 1999, 29, 111–129, doi:10.1007/s003740050533.
[45]
Haubert, D.; H？ggblom, M.M.; Langel, R.; Scheu, S.; Ruess, L. Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets. Soil Biol. Biochem. 2006, 38, 2004–2007, doi:10.1016/j.soilbio.2005.11.031.
[46]
Khotimchenko, S.V.; Vaskovsky, V.E.; Titlyanova, T.V. Fatty acids of marine algae from the pacific coast of North California. Bot. Mar. 2002, 45, 17–22.
[47]
Parkinson, D.; Gray, T.R.G.; Williams, S.T. Methods of Studying the Ecology of Soil Microorganisms; Handbooks International Biological Programme: Blackwell Sci. Publ.: Oxford/Edinburgh, UK, 1971; pp. 1–128.
[48]
Hansen, J.F.; Thingstad, T.F.; Godsoyr, J. Evaluation of fungal lengths and hyphal biomass in soil by a membrane filter technique. Oikos 1974, 25, 102–107, doi:10.2307/3543552.
[49]
Zhang, D.C.; Liu, H.C.; Xin, Y.H.; Zhou, Y.G.; Schinner, F.; Margesin, R. Sphingopyxis bauzanensis sp. nov., a novel psychrophilic bacterium isolated from soil. Int. J. Syst. Evol. Microbiol. 2010, 60, 2618–2622, doi:10.1099/ijs.0.018218-0.
[50]
Zhang, D.C.; Moertelmaier, C.; Margesin, R. Characterization of the bacterial and archaeal diversity in hydrocarbon-contaminated soil. Sci. Total Environ. 2012, 421-422, 184–196, doi:10.1016/j.scitotenv.2012.01.043.
[51]
Zhang, D.C.; Schumann, P.; Redzic, M.; Zhou, Y.G.; Liu, H.C.; Schinner, F.; Margesin, R. Nocardioides alpinus sp. nov., a psychrophilic actinomycete isolated from alpine glacier cryoconite. Int. J. Syst. Evol. Microbiol. 2012, 62, 445–450, doi:10.1099/ijs.0.031047-0.
[52]
Margesin, R.; Plaza, G.A.; Kasenbacher, S. Characterization of bacterial communities at heavy-metal-contaminated sites. Chemosphere 2011, 82, 1583–1588, doi:10.1016/j.chemosphere.2010.11.056.
[53]
Mergeay, M.; Nies, D.; Schlegel, H.G.; Gerits, J.; Charles, P.; van Gijsegem, F. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 1985, 162, 328–334.
[54]
Margesin, R.; Gander, S.; Zacke, G.; Gounot, A.M.; Schinner, F. Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 2003, 7, 451–458, doi:10.1007/s00792-003-0347-2.
[55]
Gratia, E.; Weekers, F.; Margesin, R.; D’Amico, S.; Thonart, P.; Feller, G. Selection of a cold-adapted bacterium for bioremediation of wastewater at low temperatures. Extremophiles 2009, 13, 763–768, doi:10.1007/s00792-009-0264-0.
[56]
Margesin, R.; Moertelmaier, C.; Mair, J. Low-temperature biodegradation of petroleum hydrocarbons (n-alkanes, phenol, anthracene, pyrene) by four actinobacterial strains. Int. Biodeterior. Biodegradation 2012, doi:10.1016/j.ibiod.2012.05.004.
[57]
Vorobyova, E.; Soina, V.; Gorlenko, M.; Minkovskaya, N.; Zalinova, N.; Mamukelashvili, A.; Gilichinsky, D.A.; Rivkina, E.; Vishnivetskaya, T. The deep cold biosphere: Facts and hypothesis. FEMS Microbiol. Rev. 1997, 20, 277–290, doi:10.1111/j.1574-6976.1997.tb00314.x.
[58]
Dmitriev, V.V.; Suzina, N.E.; Rusakova, T.G.; Gilichinsky, D.A.; Duda, V.I. Ultrastructural characteristics of natural forms of microorganisms isolated from permafrost grounds of Eastern Siberia by the method of low-temperature fractionation. Dokl. Biol. Sci. 2001, 378, 304–306, doi:10.1023/A:1019203800276.
[59]
Vorobyova, E.; Minkovsky, N.; Mamukelashvili, A.; Zvyagintsev, D.; Soina, V.; Polanskaya, L.; Gilichinsky, D. Permafrost Response on Economic Development, Environmental Security and Natural Resources; Paepe, R., Melnikov, V.P., Eds.; Kluwer Acedemic Publishers: Norwell, MA, USA, 2001; pp. 527–541.
[60]
Rivkina, E.; Gilichinsky, D.; Wagener, S.; Tiedje, J.; McGrath, J. Biogeochemical activity of anaerobic microorganisms from buried permafrost sediments. Geomicrobiology 1998, 15, 87–193.
[61]
Kepner, R.L.; Pratt, J.R. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: Past and present. Microbiol. Rev. 1994, 58, 603–615.
[62]
Nadeau, J.L.; Perreault, N.N.; Niederberger, T.D.; Whyte, L.G.; Sun, H.J.; Leon, R. Fluorescence microscopy as a tool for in situ life detection. Astrobiology 2008, 8, 859–874, doi:10.1089/ast.2007.0043.
[63]
Steven, B.; Briggs, G.; McKay, C.P.; Pollard, W.H.; Greer, C.W.; Whyte, L.G. Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods. FEMS Microbiol. Ecol. 2007, 59, 513–523, doi:10.1111/j.1574-6941.2006.00247.x.
[64]
Trolldenier, G. Methods in Soil Biology; Schinner, F., ？hlinger, R., Kandeler, E., Margesin, R., Eds.; Springer Lab Manual: Berlin/Heidelberg, Germany, 1996; pp. 15–19.
[65]
Gilichinsky, D.A.; Wilson, G.S.; Friedmann, E.I.; McKay, C.P.; Sletten, R.S.; Rivkina, E.M.; Vishnivetskaya, T.A.; Erokhina, L.G.; Ivanushkina, N.E.; Kochkina, G.A.; et al. Microbial populations in Antarctic permafrost: Biodiversity, state, age, and implication for astrobiology. Astrobiology 2007, 7, 275–311, doi:10.1089/ast.2006.0012.
[66]
Vishnivetskaya, T.A.; Petrova, M.A.; Urbance, J.; Ponder, M.; Moyer, C.L.; Gilichinsky, D.A.; Tiedje, J.M. Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods. Astrobiology 2006, 6, 400–414, doi:10.1089/ast.2006.6.400.
[67]
Shi, T.; Reeves, R.H.; Gilichinsky, D.A.; Friedmann, E.I. Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing. Microb. Ecol. 1997, 33, 169–179, doi:10.1007/s002489900019.
[68]
Hinsa-Leisure, S.M.; Bhavaraju, L.; Rodrigues, J.L.M.; Bakermans, C.; Gilichinsky, D.A.; Tiedje, J.A. Characterization of a bacterial community from a northeastern Siberian seacost permafrost sample. FEMS Microbiol. Ecol. 2010, 74, 103–113, doi:10.1111/j.1574-6941.2010.00945.x.
[69]
Vandera, E.; Kavakiotis, K.; Kallimanis, A.; Kyrpides, N.C.; Drainas, C.; Koukkou, A.I. Heterologous expression and characterization of two 1-hydroxy-2-naphthoic acid dioxygenases from Arthrobacter phenanthrenivorans. Appl. Environ. Microbiol. 2012, 78, 621–627, doi:10.1128/AEM.07137-11.
[70]
Mannisto, M.K.; Schumann, P.; Rainey, F.A.; Kampfer, P.; Tsitko, I.; Tiirola, M.A.; Salkinoja-Salonen, M.S. Subtercola boreus gen. nov., sp. nov. and Subtercola frigoramans sp. nov., two new psychrophilic actinobacteria isolated from boreal groundwater. Int. J. Syst. Evol. Microbiol. 2000, 50, 1731–1739.
[71]
Zhang, D.C.; Redzic, M.; Schinner, F.; Margesin, R. Glaciimonas immobilis gen. nov., sp. nov., a novel member of the family Oxalobacteraceae isolated from alpine glacier cryoconite. Int. J. Syst. Evol. Microbiol. 2011, 61, 2186–2190, doi:10.1099/ijs.0.028001-0.
[72]
Yergeau, E.; Hogues, H.; Whyte, L.G.; Greer, C.W. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses. ISME J. 2010, 4, 1206–1214, doi:10.1038/ismej.2010.41.
[73]
Ozerskaya, S.; Kochkina, G.; Ivanushhkina, N.; Gilichinsky, D.A. Permafrost Soils; Margesin, R., Ed.; Springer Verlag: Berlin/Heidelberg, Germany, 2009; pp. 85–95.
[74]
Steven, B.; Pollard, W.H.; Greer, C.W.; Whyte, L.G. Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic. Environ. Microbiol. 2008, 10, 3388–3403, doi:10.1111/j.1462-2920.2008.01746.x.
[75]
Franks, F. Nucleation of ice and its management in ecosystems. Phil. Trans. Roy. Soc. Lond. A 2003, 361, 557–574, doi:10.1098/rsta.2002.1141.
[76]
Petrova, M.; Gorlenko, Z.; Mindlin, S. Tn5045, a novel integron-containing antibiotic and chromate resistance transposon isolated from a permafrost bacterium. Res. Microbiol. 2011, 162, 337–345, doi:10.1016/j.resmic.2011.01.003.
[77]
Margesin, R.; Miteva, V. Diversity and ecology of psychrophilic microorganisms. Res. Microbiol. 2011, 162, 346–361, doi:10.1016/j.resmic.2010.12.004.
[78]
D’Costa, V.M.; King, C.E.; Kalan, L.; Morar, M.; Sung, W.W.L.; Schwarz, C.; Froese, D.; Zazula, G.; Calmels, F.; Debruyne, R.; et al. Antibiotic resistance is ancient. Nature 2011, 477, 457–461.
