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ASTROBIOLOGY: ON THE LONG-TERM PERSISTENCE OF BACTERIAL DNA

The following points are made by E. Willerslev et al (Current Biology 2004 14:R9):

1) The persistence of bacterial DNA over geological timespans remains a contentious issue. In direct contrast to in vitro based predictions, bacterial DNA and even culturable cells have been reported from various ancient specimens many million years (Ma) old [1-5]. As both ancient DNA studies and the revival of microorganisms are known to be susceptible to contamination, it is a problem that these results have not been independently replicated to confirm their authenticity. Furthermore, the results show no obvious relationship between sample age, and either bacterial composition or DNA persistence, although bacteria are known to differ markedly in hardiness and resistance to DNA degradation.

2) The authors present the first study of DNA durability and degradation of a broad variety of bacteria preserved under optimal frozen conditions using rigorous ancient DNA methods. The results demonstrate that non-spore-forming gram-positive (GP) Actinobacteria are by far the most durable, out-surviving endospore-formers such as Bacillaceae and Clostridiaceae. The observed DNA degradation rates are close to theoretical calculations, indicating a limit of approximately 400,000 years, beyond which PCR amplifications are prevented by the formation of DNA interstrand crosslinks (ICLs).

3) The twelve permafrost samples (800,000 to 1 million years old) investigated were obtained from northeast Siberia and Beacon Valley, Antarctica. DNA preservation at these sites is exceptional due to constant subzero temperatures, largely neutral pH, and anaerobic conditions. Epifluorescence microscopy revealed 10^(7) cells/gram wet-weight in the bacterial size range. The cell counts are in agreement with previous results obtained on permafrost [2,3]. 16S rDNA sequences of 120bp and 600bp could be reproducibly amplified from samples up to 400,000 to 600,000 years old, and show an inverse relationship between PCR amplification efficiency and fragment length that is typical of ancient DNA. Controls for surface contamination during sampling were negative.

4) The observed rates of DNA degradation match theory, and indicate a limit for PCR amplifiable DNA between 400,000 and 1.5 million years, beyond which DNA is either severely crosslinked or non-detectable. Thus, the results represent the oldest reproducible and authenticated bacterial DNA to date. Since cold conditions are critical for long-term DNA survival, the results strongly contradict claims of multi-Ma DNA sequences, or even putative viable cells of endospores and Proteobacteria, from non-frozen materials such as amber and halite [4,5]. None of these previous studies were confirmed by independent replication and/or measurements of DNA damage. These results also contradict previous claims of the isolation of viable bacteria from many million-year-old permafrost samples [2,3], which have also not been replicated independently.

5) The authors suggest that the superior long-term DNA persistence of non-spore-forming gram-positive bacteria has important implications for studies of cellular DNA preservation mechanisms, and for the search for extraterrestrial life in permafrost and ice deposits of Mars and Europa, where experiments have so far focused on the long-term survival of bacterial endospores under space conditions. The authors suggest their results indicate that these are probably not the most durable types of bacteria under frozen conditions.

References (abridged):

1. Kennedy, M.J., Reader, S.L., and Swierczynski, L.M. (1994). Preservation records of microorganisms: evidence of the tenacity of life. Microbiology 140, 2513-2529

2. Shi, T., Reeves, R.H., Gilichinsky, D.A., and Friedmann, E.I. (1997). Characterization of viable bacteria in Siberian permafrost by 16S rDNA sequencing. Microb. Ecol. 33, 169-179

3. Vorobyova, E., Soina, V., Gorlenko, M., Minkovskaya, N., Zalinova, N., Mamukelashvili, A., Gilichinsky, D., Rivkina, E., and Vishnivetskaya, T. (1997). The deep cold biosphere: facts and hypotheses. FEMS Microbiol. Rev. 20, 277-290

4. 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

5. Vreeland, R.H., Rosenzweig, W.D., and Powers, D.W. (2000). Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature 407, 897-900

Current Biology http://www.current-biology.com

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ASTROBIOLOGY: ON THE SEARCH FOR LIFE ON EUROPA

It is an irony that although the term "living system" is widely used in science, the term has no consensus definition. Instead, there are many definitions: physiological, biochemical, genetic, metabolic, thermodynamic, and so on. One might think the question of the definition of "life" is only of philosophical significance, but if you are designing a robot to search for "life" on another astronomical body, precisely what is the robot to search for? At the present time, the answer to this question is not at all clear.

Concerning definitions, the various definitions of "life" currently in use can in general be summarized as follows:

1) The physiological definition defines a living system as a system that performs various functions such as eating, metabolizing, excreting, breathing, moving, growing, reproducing, and being responsive to external stimuli.

2) The biochemical definition defines a living system as a system that contains reproducible hereditary information coded in nucleic acids and that metabolizes by controlling the rate of chemical reactions with protein catalysts (enzymes).

3) The genetic definition defines a living system as a system capable of evolution by natural selection.

4) The metabolic definition defines a living system as a system with a definite boundary, the system continually exchanging some of its materials with its surroundings, but the exchange not altering the general properties of the system, at least over some period of time.

5) The thermodynamic definition defines a living system as an open system manifesting a local increase in order (decrease in entropy) at the expense of a larger decrease in order outside the system.

Of course, physical scientists can immediately think of many "non-living" physical systems that can be described by one or more of the above definitions, but biologists are fully aware of such counter-examples and admit the ambiguities of all the definitions. Research on Earth-bound living systems goes on, ambiguity or no ambiguity, but when the focus is a search for living systems elsewhere, the ambiguities become critical.

Jupiter's satellite system consists of at least 16 moons, the four largest of which are called the Galilean moons, since they were discovered by Galileo Galilei (1564-1642). They are Io, Europa, Ganymede, and Callisto, in order of their orbital distance from Jupiter. Europa, which is slightly smaller than Earth's moon, has a thick icy crust, and may also have a liquid water mantle beneath this crust. Very few craters are present on Europa, which suggests an active surface that renews itself and thus erases craters as fast as they form from impacts. The surface also shows numerous lines about 30 kilometers wide and 1000 kilometers long, and these have been interpreted to be breaks in the crust where water from below has refrozen. The possible existence of a liquid water mantle beneath the ice on Europa is of great interest to planetary scientists, since such a mantle might contain life forms. [See related background material below for more information about Europa.]

The following points are made by C.F. Chyba and C.B. Phillips (Proc. Natl. Acad. Sci. 2001 98:801):

1) The authors point out that no broadly accepted definition of life exists, and that most proposed definitions face severe objections. Nevertheless, one working definition of life has become influential in the "origin-of-life" community, the definition that life is a self-sustained chemical system capable of undergoing Darwinian evolution. The idea that the origin of life is the same as the origin of evolution is a popular corollary. The authors (Chyba and Phillips) suggest, however, that such a definition is unlikely to prove useful to a remote _in situ_ search for life. In a search for extraterrestrial life in our Solar System, we instead fall back on a less ambitious notion -- "life as we know it", meaning life based on a liquid water solvent, a suite of biogenic elements (most particularly carbon), and a source of free energy. The authors state: "The availability of these on a given world would suggest life to be possible, so that further exploration may be warranted."

2) The authors point out that only once before have we conducted a robotic search for extraterrestrial life. The Viking spacecraft carried three experiments to search for life in Martian soil samples, the experiments as designed implicitly adopting a metabolic definition by searching for chemical changes associated with metabolism. But instead of finding unambiguous evidence of Martian biology, Viking appears to have encountered unanticipated non-biological oxidant chemistry. The Viking gas chromatograph mass spectrometer failed to find any organic molecules in the Martian soil at the parts-per-billion or parts-per-million level. The instrumentation provided a de facto search for life that implicitly assumed a biochemical definition: no (detected) organics, no life. In effect, a metabolic search for life that yielded some ambiguously positive results was undercut by the negative results of a search based on biochemistry.

3) The authors suggest that with the benefit of 25 years hindsight, there are a number of lessons to be learned from the Viking experience in the search for life on Europa. a) If payload limits permit, a remote search for life should use experiments that assume contrasting definitions of life. b) If only one life-detection experiment can be flown, the biochemical definition should probably be primary. c) It is crucial to establish the geological and chemical context within which biological experiments will be conducted. Had the presence of the Martian oxidants already been demonstrated, different biology experiments would have been flown on Viking. d) Life detection experiments should provide valuable information even if they fail to find life. e) Nevertheless, exploration often cannot be hypothesis-testing: much of what we do in planetary missions is simply exploration.

Proc. Nat. Acad. Sci. http://www.pnas.org

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PLANETARY SCIENCE: THE MISSING ORGANIC MOLECULES OF MARS

One of the puzzles about the planet Mars is the fact that the 1976 Viking Mars Mission found no evidence at all of any organic material on the Martian surface, including no evidence of organic material expected to result from meteorite impacts. This result has suggested that the surface layers of Mars (the Martian regolith) may contain a powerful oxidant that converts all organic molecules to carbon dioxide at a rate which is rapid relative to the rate at which they arrive, and this idea is currently influencing the planning of future Mars missions.

The following points are made by S.A. Benner et al (Proc. Natl. Acad. Sci. US 2000 97:2425):

1) The authors suggest that nonvolatile salts of benzenecarboxylic acids, and perhaps oxalic acid and acetic acid, should be metastable intermediates of meteoritic organics under oxidizing conditions. Salts of these organic acids would have been largely invisible to the gas chromatography-mass spectroscopy measurements made by the Viking Mars mission.

2) Experiments indicate that benzenehexacarbolic acid (mellitic acid) is generated by oxidation of organic matter known to come to Mars, is rather stable to further oxidation, and would not have been easily detected by the Viking experiments.

3) The authors suggest that approximately 2 kilograms of meteorite-derived mellitic acid may have been generated per square meter of Martian surface over 3 billion years. How much remains depends on decomposition rates under Martian conditions.

4) The authors suggest that as available data do not require that the surface of Mars be very strongly oxidizing, some organic molecules might be found near the surface of Mars, perhaps in amounts sufficient to be a resource. Missions should seek these organics and recognize that these complicate the search for organics from entirely hypothetical Martian life.

5) The authors state: "As in any organic reaction, the specific oxidant, specific ambient conditions, and specific catalysts determine what intermediates will accumulate in the oxidative degradation of organic compounds on Mars. Only by missions to Mars can we learn these specifics to decide what has actually happened to meteoritic organics and, by inference, to other organics that might have come to the Martian surface."

Proc. Nat. Acad. Sci. http://www.pnas.org

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