Intact DNA in ancient permafrost

Update Deutsche Forschungsgemeinschaft (SFB576, project B1 to R.H.) and by the Federal Ministry of Education and Research (Nationales Genom Forschungsnetz, NGFN-2; project NIE-S31T13) to R.H.

References 1 Leunk, R.D. et al. (1988) Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. J. Med. Microbiol. 26, 93–99 2 Cover, T.L. and Blanke, S.R. (2005) Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat. Rev. Microbiol. 3, 320– 332 3 McClain, M.S. et al. (2003) Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin. J. Biol. Chem. 278, 12101–12108 4 Reyrat, J.M. et al. (1999) 3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin. J. Mol. Biol. 290, 459–470 5 Gangwer, K.A. et al. (2007) Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc. Natl. Acad. Sci. U. S. A. 104, 16293–16298 6 Roche, N. et al. (2007) Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA. Microbes Infect. 9, 605– 614 7 Yahiro, K. et al. (1999) Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta. J. Biol. Chem. 274, 36693–36699 8 Yahiro, K. et al. (2003) Protein-tyrosine Phosphatase a, RPTPa, is a Helicobacter pylori VacA Receptor. J. Biol. Chem. 278, 19183–19189 9 Yahiro, K. et al. (2004) Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin. J. Biol. Chem. 279, 51013–51021 10 Sewald, X. et al. (2008) Integrin subunit CD18 is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin. Cell Host Microbe 3, 20–29 11 Pagliaccia, C. et al. (1998) The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity. Proc. Natl. Acad. Sci. U. S. A. 95, 10212–10217 12 Ji, X. et al. (2000) Cell specificity of Helicobacter pylori cytotoxin is determined by a short region in the polymorphic midregion. Infect. Immun. 68, 3754–3757 13 de Guzman, B.B. et al. (2005) Cytotoxicity and recognition of receptorlike protein tyrosine phosphatases, RPTPalpha and RPTPbeta, by Helicobacter pylori m2VacA. Cell. Microbiol. 7, 1285–1293

Trends in Microbiology Vol.16 No.3 14 Letley, D.P. et al. (2006) Paired cysteine residues are required for high levels of the Helicobacter pylori autotransporter VacA. Microbiology 152, 1319–1325 15 Ye, D. et al. (1999) Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin. J. Biol. Chem. 274, 9277–9282 16 de Bernard, M. et al. (1998) Identification of the Helicobacter pylori VacA toxin domain active in the cell cytosol. Infect. Immun. 66, 6014– 6016 17 Torres, V.J. et al. (2006) Mapping of a domain required for protein– protein interactions and inhibitory activity of a Helicobacter pylori dominant-negative VacA mutant protein. Infect. Immun. 74, 2093–2101 18 El Bez, C. et al. (2005) High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy. J. Struct. Biol. 151, 215–228 19 Czajkowsky, D.M. et al. (1999) The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH. Proc. Natl. Acad. Sci. U. S. A. 96, 2001–2006 20 Kim, S. et al. (2004) Membrane channel structure of Helicobacter pylori vacuolating toxin: role of multiple GXXXG motifs in cylindrical channels. Proc. Natl. Acad. Sci. U. S. A. 101, 5988–5991 21 Molinari, M. et al. (1998) The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies. Biochem. Biophys. Res. Commun. 248, 334–340 22 Nakayama, M. et al. (2006) Clustering of Helicobacter pylori VacA in lipid rafts, mediated by its receptor, receptor-like protein tyrosine phosphatase beta, is required for intoxication in AZ-521 Cells. Infect. Immun. 74, 6571–6580 23 Ricci, V. et al. (2000) High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis. Mol. Biol. Cell 11, 3897–3909 24 Rhead, J.L. et al. (2007) A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 133, 926–936 25 Skibinski, D.A. et al. (2006) The cell-specific phenotype of the polymorphic vacA midregion is independent of the appearance of the cell surface receptor protein tyrosine phosphatase beta. Infect. Immun. 74, 49–55 0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.01.001 Available online 14 February 2008

Research Focus

Intact DNA in ancient permafrost Kim Lewis, Slava Epstein, Veronica G. Godoy and Sun-Hee Hong Department of Biology, Northeastern University, Boston, MA 02115, USA

Contrary to the generally held notions about microbial survival, the recently published paper by Johnson et al., ‘Ancient bacteria show evidence of DNA repair’, presents evidence suggesting that non-spore-forming bacteria in ancient samples are apparently alive, as judged by intact DNA, and fare better than spores. The data presented in this work raise intriguing questions about the nature of bacteria in many of the ancient samples reported to date: are they spores, persisters, sessile vegetative cells or do they make up a slow-growing population? Microbes are highly adaptable organisms capable of withstanding the harshest conditions on our planet [1,2]. Corresponding author: Lewis, K. ([email protected]).

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Spores are generally thought of as unmatched champions of survival. A multilayered envelope, together with desiccation, which slows down chemical reactions, and coating of DNA with protective proteins enable spores to survive not only harsh conditions but also over long periods of time. In 1995, a striking report of reviving a 40-million year old bacterial spore [3] began to fuel the interest in studying ancient cells and their DNA. The isolation of a previously unrecognized spore-forming bacterium (Bacillus species) from approximately 250 million-year-old brine samples [4] was reported but remains controversial. Recently, it has been suggested that bacteria from old samples are different from their modern counterparts [5], indicating that careful isolation might give us access to live organisms from ancient samples [6].

Update Spores versus persisters The recent paper by Johnson and coauthors [7] presents evidence of DNA repair in ancient bacteria from 500 000year-old permafrost samples. Provocatively, the authors report that spore-forming species decline over time and it is actually the non-spore formers that retain the ability to repair their DNA and, by extension, are expected to show better recovery from ancient samples. Perhaps these bacterial cells are persisters, which is another important form of cell survival. Like spores, persisters are specialized survivor cells that are produced stochastically by bacterial populations [8]. Persisters are an intermediate form between a growing cell and a spore – they have diminished metabolism and are tolerant to a variety of stresses but are unremarkable morphologically and do not exhibit the typical spore-style desiccation. Persisters are expected to maintain cell and DNA integrity and might actually represent the surviving form of cells, which are capable of repairing their DNA actively over prolonged periods of time. There is indeed an apparently strong argument against the prolonged survival of spores – DNA will degrade over geological periods of time owing to hydrolysis and crosslinking and there is no active DNA-repair machinery within spores to stop the decay process [9]. Only small, approximately 200 bp, fragments of DNA have been recovered from ancient plant and animal samples [10,11]. Spontaneous DNA degradation at the freezing temperatures of permafrost is supposed to occur within 100–1000 kyrs, according to Johnson and coauthors [7]. At the same time, it is not clear how this estimate will change in the desiccated environment of a spore. Evidence and approaches Johnson and coworkers used an elegant approach to detect possible ancient DNA that is under constant repair by live bacterial cells. The approach is based on well known spontaneous hydrolytic deamination of DNA cytosine to uracil. Treating DNA with uracil-N-glycosylase (UNG) would then fragment damaged DNA in the sample but leave DNA in metabolically active cells intact. Long (4 kb) amplicons would only be obtained from DNA of live cells. Armed with this approach, the authors attempted to amplify 4 kb fragments that included portions of 16S and 23S ribosomal RNA genes as well as the intervening ITS region genes (after UNG digestion) from permafrost and Antarctic ice samples of varying age, from modern to up to 1 Myr old. The recovered and sequenced fragments suggest the presence of numerous bacterial phylotypes in less than 3  104 yr-old samples, less diverse – yet detectable – bacterial assemblages in 4–6  105-yr-old samples and a complete lack of detection in older samples. Importantly, live spore-forming species present in the 5–30 kyr samples apparently disappear in the older samples and only non-spore formers have been detected by this method in samples of more than 600 kyrs. In an independent approach, the authors show that CO2 evolution from samples, measured under anaerobic conditions after a 9–month long incubation at in situ temperature, can be detected from 4–6  105- but not 7  105-yr old samples [7]. The authors’ interpretation of these obser-

Trends in Microbiology

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vations is that cellular metabolic activities and DNA repair are a prerequisite for long-term survival and that spore dormancy is an inferior strategy for such survival. They conclude that the amplicons from UNG-treated DNA represent ‘. . .the oldest independently authenticated DNA to date obtained from viable cells’. Bacterial abundance and survival But what about claims of reviving considerably older spores? It is important to note that the oldest examples of revived spore formers might not have come from spores but rather from vegetative, DNA-repairing cells of sporeforming species. At the same time, it is important to note that the disappearance of spore formers from older samples does not necessarily mean that spores to do not survive well. The disappearance of spore-forming species in the Johnson et al. study [7] in the very old samples could be owing to their low abundance in that environment. Indeed, according to recent metagenomics studies, sporeforming bacteria might be fairly rare in the biosphere [12] and the relative abundance of spore formers in the modern sample was reported as negligible [7]. When specific organisms make up only a small minority and cells begin to die, further decreasing their count, only repair-competent cells of the more abundant or enriched species will be detected. A note of caution is that DNA extraction from spores might be inefficient and this might also skew results. A more sensitive nested PCR or the use of DNA polymerases especially designed to amplify ancient DNA [13] might have indicated the presence of intact spore DNA. Moreover, a possibility for incomplete digestion of damaged DNA by UNG might also complicate the picture, in this case over-reporting the degree of DNA repair. Long-term survival? There is an interesting alternative to the long-term survival of both spores and metabolizing vegetative cells (i.e. active growth). Arctic ice and permafrost (as well as seawater ice and glaciers) have interconnected channels filled with aqueous solutions of varying osmolarity [14–17]. This provides cells with liquid water, at least down to 208C. Microbial cells in the liquid-filled channels remain viable and fairly active. For example, the Arctic bacterium Colwellia psychroerythraea maintains motility at 108C, with swimming speeds comparable with that of enteric bacteria at room temperature [18]. Respiratory activity of bacteria in ice was detected using fluorescent dyes at all temperatures tested to date, down to 208C [15]. This qualitative observation of activity was confirmed using C14-labeled substrates by measuring CO2 evolution from Antarctic ice collected above Lake Vostok in Antarctica [19]. There is strong evidence that this activity is not a reflection of basic metabolic maintenance. Rather, the cells in, for example, permafrost seem to grow in the laboratory at reasonable rates, with minimum doubling time of approximately 20 days at 108C and 160 days at 208C [20]. Soil in frozen permafrost could conceivably contain sufficient nutrients for growth. Glacial ice contains enough carbon to sustain activity of at least several cells per cm3 for approximately half a million years [21,22]. This might be a conservative estimate, considering the 93

Update surprising number and diversity of viable and culturable cells discovered recently from 120 000-yr-old ice from Greenland [23]. Conclusions and future perspectives It is possible that, in at least some of the cases where metabolic activity or growth was detected in old samples, the organisms might not have been so ancient after all. Thus, it is possible that permafrost and ice might contain growing – albeit slowly – communities of microorganisms ([24] and references therein). One of the exciting questions to address is whether the surviving bacteria in permafrost are some form of persister cells or simply vegetative and slowly growing bacteria. Research is starting to unravel the diversity of bacteria in permafrost [25,26], which will give insights to the metabolic pathways used by bacteria under these extreme conditions. It is interesting to note that studies of bacterial communities in permafrost are also being used as a model system for astrobiology [27,28], considering that the conditions in permafrost are similar to those on Mars. Like most studies that break new ground, this paper raises more questions than it provides answers to. This work is sufficiently captivating to inspire a search for answers to these intriguing questions. References 1 Gold, T. (1992) The deep, hot biosphere. Proc. Natl. Acad. Sci. U. S. A. 89, 6045–6049 2 Moyer, C.L. and Morita, R.Y. (1989) Effect of growth rate and starvation-survival on the viability and stability of a psychrophilic marine bacterium. Appl. Environ. Microbiol. 55, 1122–1127 3 Cano, R.J. and Borucki, M.K. (1995) Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. Science 268, 1060–1064 4 Vreeland, R.H. et al. (2000) Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature 407, 897–900 5 Vreeland, R.H. et al. (2006) Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives. Extremophiles 10, 71–78 6 Hebsgaard, M.B. et al. (2005) Geologically ancient DNA: fact or artefact? Trends Microbiol. 13, 212–220 7 Johnson, S.S. et al. (2007) Ancient bacteria show evidence of DNA repair. Proc. Natl. Acad. Sci. U. S. A. 104, 14401–14405

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Trends in Microbiology Vol.16 No.3 8 Lewis, K. (2007) Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5, 48–56 9 Lindahl, T. (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715 10 Woodward, S.R. et al. (1994) DNA sequence from Cretaceous period bone fragments. Science 266, 1229–1232 11 DeSalle, R. and Grimaldi, D. (1994) Very old DNA. Curr. Opin. Genet. Dev. 4, 810–815 12 von Mering, C. et al. (2007) Quantitative phylogenetic assessment of microbial communities in diverse environments. Science 315, 1126–1130 13 d’Abbadie, M. et al. (2007) Molecular breeding of polymerases for amplification of ancient DNA. Nat. Biotechnol. 25, 939–943 14 Ershov, E.D. (1998) General geocryology. Cambridge University Press 15 Junge, K. et al. (2004) Bacterial activity at -2 to -20 degrees C in Arctic wintertime sea ice. Appl. Environ. Microbiol. 70, 550–557 16 Mulvaney, R. et al. (1988) Sulphuric acid at grain boundaries in Antarctic ice. Nature 331, 247–249 17 Thomas, D.N. and Dieckmann, G.S. (2002) Antarctic Sea ice – a habitat for extremophiles. Science 295, 641–644 18 Junge, K. et al. (2003) Motility of Colwellia psychrerythraea strain 34H at subzero temperatures. Appl. Environ. Microbiol. 69, 4282–4284 19 Karl, D.M. et al. (1999) Microorganisms in the accreted ice of Lake Vostok, Antarctica. Science 286, 2144–2147 20 Rivkina, E.M. et al. (2000) Metabolic activity of permafrost bacteria below the freezing point. Appl. Environ. Microbiol. 66, 3230–3233 21 Price, P.B. (2000) A habitat for psychrophiles in deep Antarctic ice. Proc. Natl. Acad. Sci. U. S. A. 97, 1247–1251 22 Price, P.B. and Sowers, T. (2004) Temperature dependence of metabolic rates for microbial growth, maintenance, and survival. Proc. Natl. Acad. Sci. U. S. A. 101, 4631–4636 23 Miteva, V.I. and Brenchley, J.E. (2005) Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core. Appl. Environ. Microbiol. 71, 7806–7818 24 Steven, B. et al. (2006) Microbial ecology and biodiversity in permafrost. Extremophiles 10, 259–267 25 Steven, B. et al. (2007) Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culturedependent and culture-independent methods. FEMS Microbiol. Ecol. 59, 513–523 26 Gilichinsky, D. et al. (2008) Psychrophiles: from Biodiversity to Biotechnology. Springer-Verlag 27 Soina, V.S. and Vorobyova, E.A. (2004) Adaptation of bacteria to the terrestrial permafrost environment: a biomodel for astrobiology. In Origins (Seckbach, J., ed.), Kluwer Academic Publishers 28 Morozova, D. et al. (2007) Survival of methanogenic Archaea from Siberian permafrost under simulated martian thermal conditions. Orig. Life Evol. Biosph. 37, 189–200 0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.01.002