Extremophiles and the Search for Extraterrestrial Life

Home , Acidianus, Alvinella pompejana, Extremophile, Halococcus, Methanococcoides, Methanococcus

ASTROBIOLOGY Volume 2, Number 3, 2002 © Mary Ann Liebert, Inc.

RICARDO CAVICCHIOLI

ABSTRACT

Extremophiles thrive in ice, boiling water, acid, the water core of nuclear reactors, salt crystals, and toxic waste and in a range of other extreme habitats that were previously thought to be inhospitable for life. Extremophiles include representatives of all three domains (Bacteria, Archaea, and Eucarya); however, the majority are microorganisms, and a high proportion of these are Archaea. Knowledge of extremophile habitats is expanding the number and types of extraterrestrial locations that may be targeted for exploration. In addition, contemporary biological studies are being fueled by the increasing availability of genome sequences and associated functional studies of extremophiles. This is leading to the identification of new biomarkers, an accurate assessment of cellular evolution, insight into the ability of microorganisms to survive in meteorites and during periods of global extinction, and knowledge of how to process and examine environmental samples to detect viable life forms. This paper evaluates extremophiles and extreme environments in the context of astrobiology and the search for extraterrestrial life. Key Words: Extremophile— Microorganism— Genomics— Methanogen—Lake Vostok—Psychrophile— Hyperthermophile— Extraterrestrial. Astrobiol- ogy 2, 281–292.

DEFINITION AND DESCRIPTION OF about extraterrestrial life. It also incorporates a THE FIELD discussion on the use of genomics to study the biology of extremophiles and illustrates how this XTREMOPHILESAREORGANISMS that not only sur- understanding is likely to provide important in- Evive, but thrive under extreme conditions. sight into the search for extraterrestrial life. This contrasts with an organism that may toler- Numerous comprehensive reviews of specific ex- ate and survive extreme conditions, but grows treme environments can be found in the litera- optimally under less extreme conditions. The ture. A number of reviews and books with a term “extreme” is anthropocentrically derived, broad coverage of extremophiles are also avail- thereby providing a significant scope for what able (Madigan et al., 1997; Atlas and Bartha, 1998; may be considered extreme. On Earth, there ex- Gross, 1998; Horikoshi and Grant, 1998; Cavic- ists a truly remarkable diversity of extreme envi- chioli and Thomas, 2000; Rothschild and ronments that are capable of supporting life. The Mancinelli, 2001). In addition, a compilation of aim of this paper is not just to overview extreme articles describing extremophiles will be pub- environments and their inhabitants, but to dis- lished in the UNESCO Encyclopedia of Life Sup- cuss specific examples that may provide clues port Systems (http://www.eolss.com).

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia.

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durans, which is able to grow in the water core of EXTREME ENVIRONMENTS nuclear reactors. Such is the extent of growth in AND EXTREMOPHILES nuclear reactors that biocides are added to inhibit microbial growth. Deinococcus does not prevent Examples of extreme environments and resi- radiation-induced damage to its DNA, but in- dent extremophiles are provided in Table 1. stead appears to have a novel DNA repair sys- High-temperature environments are most often tem that enables it to perform interchromosomal associated with being extreme. Terrestrial surface DNA recombination to reform a functional environments include hot springs that are close genome (Battista, 1997). The natural selection to neutral pH, or acidic and sulfurous, or rich in pressure for the evolution of this DNA-repair sys- iron. Hot subterranean areas are diverse and tem is thought to be desiccation. Dehydration range from volcanically heated environments to leads to DNA hydrolysis in a similar way to ra- those, such as the Great Artesian Basin in Aus- diation damage. Deinococcus is a natural inhabi- tralia, that are heated by virtue of their depth. tant of desert environments on Earth and sur- Submarine environments include volcanic and vives by repairing DNA damage during hydrothermal vents. The latter are often de- rehydration. Artificial environments, such as ra- scribed as black smokers owing to the precipita- diation and toxic chemical waste dumps, also tion of minerals when hot, mineral-rich volcanic provide intense selection pressure for extremo- fluids come into contact with cold ocean waters philes. A Rhodococcus species isolated from a con- (,5°C). The present record for high-temperature growth is held by the archaeon Pyrolobus fumarii, taminated site at a chemical plant was shown not which can grow in the laboratory at 113°C and is only to survive in water saturated with benzene, restricted to temperatures of $85°C (Blochl et al., but to be capable of using benzene as a sole source 1997). It is likely, however, that microorganisms of carbon for growth (Paje et al., 1997). The abili- capable of growth at higher temperatures will be ties to tolerate and degrade toxic compounds and isolated as growth has been reported on a glass resist high levels of radiation have been combined slide placed in superheated water at 125–140° C in a genetically engineered strain of D. radiodu- inside a fumarole (Madigan et al., 1997). Numer- rans (Lange et al., 1998). The capacity of ex- ous examples of acidophilic thermophiles exist, tremophiles to survive high levels of radiation including species of Acidianus [optimum pH [particularly UV (Rothschild and Cockell, 1999)] (pH ) 2; optimum temperature ( ) 90°C] and and transform toxic compounds has prompted opt Topt (pH 2.5; 80°C) (Stetter, 1996). discussion about their potential role in ter- Sulfolobus opt Topt The most extreme examples of thermophilic, raforming other planets (Slotnick, 2000). acid-loving extremophiles are two species of Pi- The methanogens are a class of microorganism crophilus that were isolated from volcanically often associated with extreme environments. heated, dry soils in Japan. Not only do these Methanogenic Archaea are the only microorgan- Archaea tolerate these conditions, but they have isms presently known to be able to use a variety optimal growth at pH 0.7 (1.2 H SO ) and 60°C of inorganic or organic carbon compounds for M 2 4 (Schleper et al., 1995). In contrast to highly acidic growth, and produce methane as an end product. environments, alkaliphiles have optimal growth They grow strictly anaerobically, and various at pH values .10. They are often isolated from members can tolerate a broad range of salt con- natural environments that also tend to have high centrations. They also span the largest thermal ex- concentrations of salt, and as a consequence they tremes of any class of microorganism, from up may be halophilic. A typical soda lake may be pH to 110°C (Methanopyrus kandleri ) to below 0°C 11 and have concentrations of Na, Cl, SO 22, and (Methanogenium frigidum ). Despite their require- 4 HCO 2 of 140, 150, 20, and 70 gL 21, respectively ment for anaerobic conditions they are ubiquitous 3 (Cavicchioli and Thomas, 2000). Members of on Earth and are evidently extremely active. 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s i h h h h h h i k d s r A h p o x o l c d x w w g g g g g g ( ( t ( ( 1 a o i i i i i i o l a o o o o C H H C H H H R T H L L R 284 CAVICCHIOLI range of thermal extremes and their lack of a re- years (Weiss et al., 2000). Even if the travel time quirement for oxygen or light provide them with was extended, there is evidence to indicate the the capacity to live in a wide range of organically ability of microorganisms to survive for millions deprived environments. of years without growing. The supporting data include the resuscitation of Bacillus spores after preservation in amber for 25–40 million years EXTREME SURVIVAL AND (Cano and Borucki, 1995) and in brine inclusions SPACE TRAVEL within salt crystals for 250 million years (Stan- Lotter et al., 1999; Vreeland et al., 2000). In addi- The ability of microorganisms to live within tion to these factors, there is evidence that rocks and generate a subterranean biosphere microorganisms could withstand the forces gen- (Pedersen, 1993, 1997; Cunningham et al., 1995) erated from an impact sufficiently large to pro- provides further evidence that life can exist with- duce meteorite-sized rocks and eject them into out light or the input of external nutrients. It also space (Melosh, 1993; Mastrapa et al., 2001). These provides a rationale for why microorganisms findings imply that the panspermia hypothesis is may be resident in rock samples, including me- plausible and provides impetus for the continued teorites. Moreover, it provides a mechanism for search for evidence of biological markers in me- life to have survived during cataclysmic events teorites. brought about by large-scale planetary impacts capable of sterilizing the Earth’s surface. A number of these extremophiles have been isolated, al- COLD ENVIRONMENTS AND though they are not well studied. They include EXTRATERRESTRIAL LIFE nitrate-reducing, sulfide-oxidizing (Gevertz et al., 2000), sulfate- and iron-reducing (Nilsen et al., While there are arguments that the last com- 1996; Haveman et al., 1999), iron-oxidizing (Fer- mon ancestor to life on Earth was thermophilic ris et al., 1999), heterotrophic (Adkins et al., 1993; and that extant hyperthermophiles retain prop- Ferris et al., 1999), and methanogenic (Kotel- erties of the last common ancestor (Stetter, 1996), nikova and Pedersen, 1997; Kotelnikova et al., it is also argued that life may have originated in 1998) microorganisms that grow at cold (Voroby- cold environments (Levy and Miller, 1998; Levy ova et al., 1997; Rashid et al., 1999) or high (Lhari- et al., 2000). In addition to a potential role in the don et al., 1995; Huber et al., 2000) temperatures. origin of life, cold-adapted microorganisms may In addition, evidence for possible fossil remains provide insight into the search for extraterrestrial of such microorganisms has been described (Hof- life on Mars and moons such as Europa (Blamont, mann and Farmer, 2000). 2000). The surface of Mars is cold, and life forms The growth characteristics of the hyperther- surviving, or multiplying in or near the surface, mophilic chemolithoautotrophic Archaea, such as would need to be cold-adapted. Recently, the La- Methanococcus jannaschii, have led to speculation belled Release experiments performed aboard the that life may have originated on Earth by the “in- Viking spacecraft in 1976 have been reassessed to oculation” and subsequent colonization of the include the possibility that they may have ocean and subsurfaces by extraterrestrial mi- demonstrated biological activity in the soil sam- croorganisms (Morell, 1996; Davies, 1998). This is ples (Paine, 2001). The potential of the soil to sup- referred to as panspermia and may have derived port life was further demonstrated in a recent pre- from concepts developed by Arrhenius in the liminary report (http://www.spaceflightnow. early 1900s (Arrhenius, 1908). Support for this com/news/n0105/27marsorg), where methano- concept comes from putative fossil evidence of gens were grown in a liquid medium formed by microorganisms and associated biological mark- dissolving Mars soil simulant in water. An even ers in meteorites from Mars such as ALH84001 more provoking possibility for discovering extant (McKay et al., 1996), where the interior tempera- extraterrestrial life is the possibility of subsurface ture of ALH84001 has not exceeded 40°C (Weiss water existing on Europa (Carr et al., 1998; His- et al., 2000). Meteorites would also provide pro- cox, 1999; Chyba and Phillips, 2001). Subsurface tection from the damaging effects of UV light. lakes, even if they receive no light energy, may Furthermore, a significant number of meteorites be able to support lithoautotrophic biological may be able to travel between planets in ,10 processes (McCollom, 1999). It is clear from stud- EXTREMOPHILES AND EXTRATERRESTRIALS 285 ies of polar, alpine, and deep ocean ecosystems on the sampling depth, drill cores have revealed that microbial life proliferates in cold environ- 3,000-year-old viable yeasts and actinomycetes, ments (Cavicchioli and Thomas, 2000; Cavicchi- 38,600-year-old mycelial fungi, 110,000-year-old oli et al., 2000a), and natural microbial metabo- unicellular algae, 180,000-year-old diatom, and lism has been measured at temperatures of at 200,000-year-old spore-forming Bacteria (Ellis- least 217°C (Carpenter et al., 2000). In the Vest- Evans and Wynn-Williams, 1996). From the deep- fold Hills region of Antarctica, a unique ecosys- est core samples ( ,120 m above the lake surface) tem is preserved that contains numerous lakes four genera of Bacteria were identified by 16S ranging in salinity from freshwater to up to eight rRNA sequencing of community DNA (Priscu et times that of seawater, in temperature from up to al., 1999), and microbial respiration was indicated 20°C to below 210°C, and in oxygen content from by the conversion of radiolabeled substrates in ac- aerobic to strictly anaerobic (McMeekin et al., cretion ice samples where cells numbers were on 1 1993). The lakes also vary in nutrient and solute the order of 10 2 cells mL2 (Karl et al., 1999). Re- level from highly ionic to extremely oligotrophic. cently, members of the bacterial genera Sphin- A variety of microorganisms have been isolated gomonas, Methylobacterium, Brachybacteria, and and characterized (McMeekin et al., 1993; Franz- Paenibacillus were cultivated from accretion ice mann, 1996; Franzmann et al., 1997b), and 16S 3,593 m below the glacial ice surface (Christner et rRNA community analyses have been performed al., 2001), thereby demonstrating the ability of mi- (Bowman et al., 2000a,b). The lakes are also the croorganisms to survive and recover after peri- source of the only free-living, cold-adapted Ar- ods in ice of $400,000 years. The importance of chaea characterized (Franzmann et al., 1988, 1992, Lake Vostok (and other Antarctic subsurface 1997a). Of particular note are the cold-adapted lakes) as analogues for Europa and Mars is well methanogens, Methanococcoides burtonii (Franz- recognized (Wynn-Williams and Edwards, 2000; mann et al., 1992) and Methanococcoides frigidum Duxbury et al., 2001), and there is presently a high (Franzmann et al., 1997a). While M. burtonii uti- level of consideration being directed at how to lizes organic forms of carbon (methylamines and penetrate into the pristine environments, and methanol), M. frigidum derives its energy, carbon, what measurements to employ in the search for and nitrogen from inorganic sources. In addition, life (Newton et al., 2000; Price, 2000; Wynn- M. frigidum is truly adapted to growth in the cold, Williams and Edwards, 2000; Blake et al., 2001; displaying a theoretical growth temperature min- Chyba and Phillips, 2001). imum of 210°C and a maximum growth temperature of 18°C. The Antarctic subsurface lake system is an en- METAZOAN EXTREMOPHILES vironment that is even more closely linked to what may be present on Europa. More than 70 While it is clear that most extremophiles are lakes have been identified (Dowdeswell and microorganisms, it is noteworthy that a number Siegert, 1999; Siegert, 2000). The largest and most of metazoans (multicellular eukaryotes) can sur- comprehensively studied is Lake Vostok. It is 230 vive in extreme environments, and a few may be km long and up to 50 km wide (Dowdeswell and considered extremophiles. This implies that the Siegert, 1999; Jouzel et al., 1999) and is 1,000 m search for extraterrestrial life should not be re- deep in places (Gibbs, 2001), giving it a volume stricted to microorganisms. The Pompeii worm in excess of that of Lake Ontario in North Amer- (Alvinella pompejana ) is a polychaetous annelid ica. There are predictions that life in Lake Vostok that lives at deep-sea vents of the East Pacific Rise may have originated 30 million years ago where it may be exposed to a highly variable mix- (Duxbury 2001) and that microorganisms in ture of vent (350°C, anoxic, acidic, and rich in CO et al., 2 400,000-year-old ice may be maintained by grow- and metal sulfides) and deep-sea (2°C, mildly hy- ing in liquid veins that surround ice crystals poxic) waters (Desbruyeres et al., 1998). It has (Price, 2000). However, the most compelling evi- been reported that its posterior may be exposed dence that the upper ice mass (and presumably to temperatures of 81°C (Cary et al., 1998) with lake water) maintains viable and possibly grow- brief periods of exposure .100°C (Chevaldonne ing microorganisms is derived from studies of ice et al., 1992). However, the delicate and intricate cores drilled from the Antarctic surface through nature of the worm tubes and the technical diffi- to the lake’s accretion ice 3,623 m down. Based culty associated with monitoring temperatures 286 CAVICCHIOLI have led to a more cautious appraisal of maxi- of hydrothermal vents were discovered on the mum worm body temperatures of 55°C (Cheval- seafloor in north Iceland (Marteinsson et al., donne et al., 2000). The recent interest in the deep- 2001). Unlike most marine hydrothermal systems, sea worms has led to the discovery of a unique which are acidic and salty as a result of the dis- composition of zinc–iron sulfide crystals within charged fluids originating from seawater seepage the exoskeleton of the Pompeii worm (Zbinden et interacting with heated magma, these giant geo- al., 2001). It has been proposed that these biolog- thermal cones emit freshwater with a pH of 10 ically produced minerals may be useful as bio- and temperature up to 72°C. While it is unclear markers in fossilized paleohydrothermal vent from where the freshwater and indigenous mi- systems. croorganisms originate, they are likely to have Perhaps the best example of metazoans that traveled at least 1.8 km from land. These kinds of can survive extreme conditions are the hy- discoveries will continue to expand the possible drophilous micrometazoans, also known as locations that may be investigated in the search tardigrades. They represent a separate phylum for extraterrestrial life. that is related to arthropods (Nelson and Mar- When contemplating where extraterrestrial life ley, 2000). More than 800 species have been de- may be found, it is clearly useful to consider scribed from marine, freshwater, and terrestrial which environments support life on Earth. How- habitats. One class of tardigrade is able to form ever, the extremophiles themselves provide ad- a stress-resistant “tun” state. They have been ditional levels of information that cannot be ob- recorded to survive 120 years of desiccation, tained simply from physical studies of the 6,000 atm pressure, temperatures as low as environments. Studying the growth and survival 2272°C and as high as 151°C, and x-ray bom- of extremophiles will generate critical insight into bardment 1,000 times greater than a level suffi- developing methods for resuscitating and cultur- cient to kill a human, and have been shown ing extraterrestrial life forms when (if) they are to revive after being photographed using an located. Knowledge of mechanisms of survival in electron microscope [Copley, 1999 (see also extremophiles will also assist the understanding http://www.tardigrades.com); Rothschild and of how extraterrestrial life may survive space Mancinelli, 2001]. Their resistance relates in part travel (e.g., in meteorites). Biological studies will to the ability to stop metabolism and replace in- also provide important information about poten- tracellular water with trehalose, thereby pre- tial biomarkers. The importance of this latter serving cellular integrity and the ability to re- point is highlighted by the controversy sur- sume growth when suitable conditions are rounding nanobes and other structures that re- encountered. These abilities have led to specu- semble extant microorganisms and that have lation that tardigrades may survive transport been found in subterranean rocks 3,400–5,100 m through outer space (Copley, 1999). below the sea bed (Uwins et al., 1998), in the Mar- tian meteorite ALH84001 (McKay et al., 1996), from human kidney stones (Kajander and Cift- SEARCHING FOR EXTRATERRESTRIALS cioglu, 1998), and in a range of other environments (Folk, 1999). The claims have prompted in- What can be learned about extraterrestrial life tense debate, which has led to a search for from studying extant life on Earth? The ability of alternative explanations to the findings (Sears earthly microorganisms to colonize extreme en- and Kral, 1998; Abbott, 1999; Kirland et al., 1999; vironments expands the range of extraterrestrial Cisar et al., 2000; Zolotov and Shock, 2000) and a bodies that may be candidates for extant life, or discussion of the minimum size of a structure that that may have harbored life in the past and there- may support life (Maniloff et al., 1997; Knoll and fore may retain fossil records. The discovery of Osborne, 1999; Cavicchioli and Ostrowski, 2002). Lake Vostok provided a fresh perspective on In particular, the demonstration that mixtures of cold, oligotrophic environments with important inorganic compounds may form structures that implications for equivalent environments on Jo- resemble the size and shape of microorganisms vian moons such as Europa. The discoveries of means that the presence of microorganisms in a hyperthermophiles in volcanically heated hot sample must be substantiated by more than mor- springs has provided a focus for candidate loca- phological evidence (García-Ruiz et al., 2002). tions on Mars (Gulick, 2001). Recently, new types While this has important implications for the in- EXTREMOPHILES AND EXTRATERRESTRIALS 287 terpretation of fossils records such as those of chioli, 2000). Importantly, the capacity of this or- the 3.5-billion-year-old cyanobacteria-like fossils ganism to display a distinct starvation response (Schopf, 1993), it is also relevant to the search for could be rationalized with the ecology of its na- viable extraterrestrial life. tive environment. These examples serve to high- Biomarkers may be biological remnants such light the importance of understanding the phys- as the presence and type of lipid remnants (Sum- iology and genetics of extremophiles in order to mons and Walter, 1990), the chirality of amino confidently predict their potential responses to an acids and sugars, and carbon isotope ratios (Sum- environmental condition, and for identifying ap- ner, 2001). However, it is also important to con- propriate biomarkers. sider what responses might be expected from a microorganism when a sample is tested for the presence of life. It is evident from studies of mi- EXTREMOPHILES, GENOMICS, crobial adaptation that physiological responses AND EXTRATERRESTRIALS and the molecular mechanisms that underpin those responses are often different to what may Identifying adaptation strategies used by ex- have been expected. For example, a high mol per- tremophiles is likely to benefit from the rapid ad- cent G1C content of DNA would be more ther- vances occurring in genomics. The number of mally stable than a high mol percent A 1T. While complete genome sequences has increased from hyperthermophiles may be expected to have a the first published for Haemophilus influenzae in G1C bias, this is not generally the case. For ex- July 1995 (Fleischmann et al., 1995) to the com- ample, Acidianus infernus grows optimally at 90°C plete genome sequences for 63 different microbial but has a 31% G 1C content (Stetter, 1996). If this species in October 2001. The third genome se- DNA were naked it would rapidly melt at this quence completed was of the hyperthermophilic temperature. Halophilic Archaea thrive under archaeon, M. jannaschii (Bult et al., 1996). A large conditions of saturated salt (5.5 M). Unlike Bac- proportion of published genomes are for ex- teria, which adapt to high salt by excluding salt tremophiles, including 11 Archaea and five Bac- from their cytoplasm, Archaea such as Halobac- teria that are hyperthermophilic, thermophilic, terium salinarium accumulate potassium (5.3 M) halophilic, oligotrophic, radiation-resistant, aci- and chloride (3.3 M) (Grant et al., 1998). Rather dophilic, or acid-tolerant. Genome sequences than retain “normal” proteins, these halophilic provide an enormous amount of information Archaea have evolved proteins with an excess of about genetic potential, and appropriate analysis negative surface charges, which facilitate the for- of that information may reveal the basis for the mation of stabilizing salt bridges or attract water specific characteristics of a cell (Pallen, 1999). This and salt to form a strong hydration shell around is well illustrated by analyses of genomes to elu- the protein. This represents a fundamentally dif- cidate the molecular determinants of protein ferent mechanism of adaptation in Archaea that thermostability (Thompson and Eisenberg, 1999; could not have been predicted from studying Cambillau and Claverie, 2000) and the analysis of only bacterial species. The oligotrophic marine the genome of a halophilic archaeon to determine bacterium Sphingomonas alaskensis grows slowly mechanisms of adaptation to high salt (Kennedy irrespective of media richness, retains a constant et al., 2001). However, depending on the cellular cell size regardless of whether it is growing or target of interest, it may still remain a challenge starved, and does not become more stress resis- to identify the genomic basis of a particular char- tant when it is starved (Eguchi et al., 1996). This acteristic. This is illustrated by the efforts to de- growth strategy contrasts with copiotrophic ma- fine the extreme radiation resistance of D. radio- rine Bacteria, which undergo distinct changes in durans (Battista, 2000), which have revealed that growth rate, stress resistance, and cell size. As a the radiation resistance is likely to be due to sev- result of these physiotypes, it may be expected eral different biological mechanisms (Makarova that oligotrophic Bacteria would have relatively et al., 2001). Clearly the genome provides the few changes in gene expression in comparison starting point for comprehensive in silico studies; with their copiotrophic counterparts. This, how- however, the greatest advances will arise from ever, is not the case as it has been shown that star- global analyses of gene function. The utility of vation of S. alaskensis produces a high level of functional studies for examining extremophiles is change in gene expression (Fegatella and Cavic- illustrated by microarray analysis of gene ex- 288 CAVICCHIOLI pression in the acid-tolerant bacterium Helicobac- ACKNOWLEDGMENTS ter pylori (Ang et al., 2001) and cell cycle control in Caulobacter crescentus (Laub et al., 2000). Other I acknowledge the efforts of Jeremy Bailey and functional studies include proteomic analysis of Malcolm Walter for organizing an extremely growth rate control and starvation control of gene stimulating astrobiology workshop. Thanks to expression in the oligotrophic bacterium S. Juergen Wiegel for communication of important alaskensis (Fegatella and Cavicchioli, 2000; Os- data, Tassia Kolesnikow and Torsten Thomas for trowski et al., 2001) and control of flagellin genes critically reviewing the manuscript, and Carl and protein modification in M. jannaschii (Muk- Ruppin for assistance with literature surveys. I hopadhyay et al., 2000; Giometti et al., 2001). A also express my appreciation to the reviewer who further example is the structural genomic analy- provided detailed and extremely valuable feed- sis of proteins from Methanobacterium thermoau- back. totrophicum (Christendat et al., 2000). 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