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Home , Archaea, Axinella, Cenarchaeum, Crenarchaeota, Edward DeLong, Euryarchaeota

Oceans of Abundant oceanic appear to derive from thermophilic ancestors that invaded low-temperature marine environments

Edward F. DeLong

arth’s is remarkably per- karyotes), Archaea, and . Although al- vasive, thriving at extremely high ternative taxonomic schemes have been recently temperature, low and high pH, high proposed, whole- and other analyses E , and low water availability. tend to support Woese’s three- concept. One of microbial in par- Well-known and cultivated archaea generally ticular, the Archaea, is especially adept at ex- fall into several major phenotypic groupings: ploiting environmental extremes. Despite their these include extreme , , success in these challenging , the Ar- and extreme and thermoacido- chaea may now also be viewed as a philes. Early on, extremely halo- cosmopolitan lot. These microbes philic archaea () were exist in a wide variety of terres- first noticed as bright-red colonies trial, freshwater, and marine habi- Archaea exist in growing on salted fish or hides. tats, sometimes in very high abun- a wide variety For many years, halophilic isolates dance. The oceanic Marine Group of terrestrial, from salterns, deposits, and I Crenarchaeota, for example, ri- freshwater, and landlocked seas provided excellent val total bacterial in wa- , model systems for studying adap- ters below 100 m. These wide- tations to high salinity. It was only spread Archaea appear to derive sometimes in much later, however, that it was from thermophilic ancestors that very high realized that these salt-loving invaded diverse low-temperature “bacteria” are actually members environments. Novel cultivation of the domain Archaea. strategies, in situ biochemical and Another major archaeal group, geochemical methods, and environmental genom- the strictly anaerobic methanogenic archaea, ics promise to further our understanding of these produce most of the in our . and other ubiquitous and abundant Archaea. Methanogens are one of the most cosmopolitan of cultivated archaeal groups, occupying diverse habitats that include anoxic and rice Brief Overview of Archaea paddies, the of cattle, the guts of , In the late , at the University hydrothermal vents, and deep subterranean of Illinois, Urbana-Champaign, and his collabo- habitats. A third phenotypic group of cultured rators, through their ribosomal RNA analyses, Archaea are the heat- and -loving thermo- first recognized Archaea as one of three major philes and . These unusual monophyletic lineages on . Their analyses Archaea dwell at hydrothermal vents, hot Edward F. DeLong of several microbial groups, including extreme springs, deep subterranean environments, oil is a senior scientist halophiles and methanogens, revealed a unique reservoirs, and even on burning coal refuse piles. at the Monterey prokaryotic lineage that diverges from most fa- Cultivated members within one of Bay Aquarium Re- miliar bacterial groups at the deepest phyloge- the domain Archaea, the Crenarchaeota (Fig. 1), search Institute, netic levels. In recognizing the Archaea as a consist entirely of thermophilic . Re- Landing, separate evolutionary unit, Woese reclassified markably, a recent report by Derek Lovely at the Calif., delong@mbari into three domains: Eucarya (all eu- University of , Amherst, suggests .org.

Volume 69, Number 10, 2003 / ASM News Y 503 FIGURE 1 surveys of mixed microbial popula- tions from open and coastal ma- rine waters. From such studies, Jed Fuhrman of University of Southern California in Los Angeles presented evidence in 1992 for one new type of archaeal ri- bosomal RNA sequence, recovered from planktonic microbes at depths of 500 m in the Pacific Ocean. At about the same , I reported the discovery of two archaeal groups in coastal sea- water, one identical to that found by Fuhrman (the Group I planktonic Cre- narchaeota, Fig. 1), and an additional group (Group II planktonic Eur- yarchaeota, Fig. 1) distantly related to halophiles and methanogens (Fig. 1). We found that these Archaea contrib- Schematic tree showing the relationships of the four major groups of planktonic marine ute significantly to the total extractable Archaea relative to cultivated groups. rRNA in marine surface waters. The surprise at that time was that any Archaea could be found in cold, that some Crenarchaeota can grow at tempera- aerobic habitats of coastal and open ocean wa- tures exceeding 120°C (see p. 484). In addition ters. No cultivated, characterized Archaea were to high-temperature growth, some archaeal then known to grow at the , tempera- thermophiles like oshimae also tures, and concentrations found in tem- thrive at extremely low pH, growing best at perate oceanic waters, shallow or deep. Soon, concentrations approaching 1 M. researchers were reporting finding rRNAs be- longing to divergent archaeal groups in all sorts of habitats, including , freshwater and ma- Archaeal Ubiquity? rine sediments, and anaerobic digestors. Many Despite expanding information on cultivated of the new, nonmarine archaeal types in soils Archaea, only a small fraction of naturally oc- and freshwater sediments were related to the curring archaeal diversity is currently repre- marine Group I Crenarchaeota. Recently, a sur- sented in culture collections. Cultivation-inde- vey of the Great Lakes by Randall Hicks and pendent survey approaches pioneered by colleagues at the University of Minnesota, Du- Norman Pace of the University of Colorado, luth, has also revealed the prevalence of similar Boulder, have greatly expanded our current ap- types of Crenarchaeota. It has been surprising to preciation of natural microbial diversity. Such realize that Archaea, initially thought to be rel- surveys indicate that a significant amount of egated to extreme environments, actually thrive microbial diversity found in natural habitats has all over our planet, including the vast world so far eluded cultivation in the laboratory. Char- , the Great Lakes, and the very be- acterizing these elusive and understudied but neath our feet. ubiquitous remains a critical Among the four major groups of planktonic goal for gaining a deeper and truer appreciation marine Archaea, the most abundant appear to of microbial , , and . be the first groups recognized, Group I Crenar- Although Archaea were thought to preferen- chaeota and Group II (Fig. 1). tially occupy environments that are inhospitable Two other planktonic archaeal groups have also to Eucarya and Bacteria, their ecological and been detected, but appear to be lower in abun- physiological diversity turns out to be far greater dance, according to recent surveys. The Group than previously supposed. Some early hints III planktonic Archaea, found in waters below came from PCR-based, ribosomal RNA- the , are peripherally related to the

504 Y ASM News / Volume 69, Number 10, 2003 Breaking the Ice, Finding New Habitats for Archaea Marine Edward DeLong around the boat, and nothing lone, scuba received an unforgettable lesson budged. Fortunately, the situation dive, and pad- about eight years ago during a wasn’t dire, but it was unlikely dle a kayak. field trip to Antarctica, when the that another icebreaker would be “I fell in love ship he was on became lodged in available to dislodge them. So with the ice. What ordinarily should have they waited, wondering whether ocean as a been a four-day journey from they would have to remain there kid. I really South America to Antarctica until the summer thaw. do love the stretched into two weeks with no When a shift in the led sea,” he says. rescue in sight. “When you’re the ice to break up, they were free “I always tell people that it’s down in Antarctica, calls but also low on fuel. They had to amazing I get paid to do what I the shots and you learn to be turn back, refuel, and start again. do.” adaptable,” says DeLong, a senior “It wasn’t scary, just an interest- After finishing high school, De- scientist with the Monterey Bay ing experience in sociology, to see Long moved to Alaska for a year Aquarium Research Institute in how you and your colleagues “to find myself,” then returned to Moss Landing, Calif. adapt to being stuck on a boat for California to start college. He DeLong and his colleagues two weeks with nothing to do,” studied at Santa Rosa were en route to the Antarctica Delong says. “Actually, it was Junior College, Calif., in 1980, Peninsula in August 1995, the fun.” before obtaining a B.S. degree in austral winter, to study Archaea, DeLong has been having fun bacteriology from the University then newly discovered to inhabit for his entire career. It began of California, Davis (UCD) in cold-ocean waters. They were during his childhood with a love 1982, and a Ph.D. in marine biol- traveling by icebreaker, but that of the sea. Delong, now 45, ogy from the Scripps Institute of didn’t prevent the ship from get- grew up in Sonoma, Calif., where , San Diego, Calif., ting trapped in the ice. Ice formed he learned to skin dive for aba- in 1986. In 1989, he received an Continued

order . Members of the pe- archaeal rRNA in marine yielded some lagic Group IV Archaea, discovered by Fran- unexpected surprises. Perhaps most startling was cisco Rodriguez-Valera and collaborators of the the high relative abundance of Archaea in winter Universidad Miguel Hernandez in Spain, appear surface waters off Antarctica, where planktonic to be related to haloarchaea, and apparently Crenarchaeota sometimes comprise as much as inhabit deep ocean waters. Currently, hundreds 20% of total microbial rRNA. Those coastal of ribosomal RNA sequences derived from ma- waters are Ϫ1.8oC, a far cry from the 80oCor rine planktonic Archaea exist in the public da- higher growth temperature optima of cultivated tabases. These data indicate that substantial ge- Crenarchaeota. Similar surveys in temperate netic variability (e.g., microheterogeneity) exists waters off the coast of California show that the among very closely related sequence types, re- planktonic Crenarchaeota tend to be most flecting unexplored evolutionary, genetic, and abundant in depths below about 100 m or so. ecological complexities. Modified fluorescence in situ hybridization assays have led to better estimates of archaeal Planktonic Archaeal Abundance, concentrations throughout the oceanic wa- Distribution, and Variability ter column. Off the California coast, we showed Archaeal Groups I and II appear to be the most that planktonic Crenarchaeota represent more abundant in the ocean and so far have been the than 20% of the total picoplanton cells in water easiest to study in situ. Early reports using ra- from 80 m to 3,000 m. Our later studies with diolabeled oligonucleotide probes to quantify David M. Karl and colleagues of the University

Volume 69, Number 10, 2003 / ASM News Y 505 independent study award from proaches allow scientists to sur- ocean water, but it turns out they Woods Hole Oceanographic In- vey entire microbial communities are major ocean inhabitants,” he stitute, Woods Hole, Mass., without the requirement of culti- says. “If you would have asked where he met his wife, Kathleen vation in the lab. any 10 years ago Ledyard, a biogeochemist. They “There was a real big impact whether Archaea were abundant have a six-year-old son. when these techniques became in the , they would have Earlier, DeLong thought he available to study naturally occur- justifiably said ‘no—it’s not an would end up becoming a medical ring microbial communities en extreme environment.’ In actual technologist, specializing in mi- masse, rather than studying mi- fact, oceanic Archaea represent crobiology. But at UCD, he met crobial cultures one by one,” one of the major groups of Ar- Paul Baumann, a professor and DeLong says. “Science jumps for- chaea on Earth. What we are up microbiologist who is an expert in ward a lot of in lockstep to now is trying to learn more marine bacterial and with new technologies, and this about their biology and ecology,” . “Paul got me into opens up new world views for us.” he adds. this whole research world of ma- In the late 1970s, microbiolo- The goal of that nearly ill-fated, rine , and I gradually gist Carl Woese had begun to ice-breaking field trip in 1995 was figured out you could make a ca- compare microorganisms by us- to gather samples of cold-sea wa- reer doing this sort of thing,” ing purely genetic information— ter, hoping to find Archaea. When DeLong says. and learned that Archaea are not they arrived on the continent, After finishing his thesis work ordinary bacteria, as originally they crossed the sea ice on skis, with Art Yayanos at Scripps, De- thought. Later, scientists learned drilling holes to extract seawater. Long did his postdoctoral work that these and related microor- “We found, as predicted, that the with Norman Pace, then at Indi- ganisms do not live only in ex- frigid Antarctic waters were ana University, Bloomington, treme environments, such as boil- chock-full of Archaea,” he says. who introduced him to the tech- ing water, acid-laden streams, or Marlene Cimons niques of using DNA sequences to super-salty brine. “We didn’t even Marlene Cimons is a freelance writer identify microbes. These ap- know that they lived in cold- in Bethesda, Md.

of Hawaii, Manoa, show a similar trend in the seem to be more abundant at the surface. This North Pacific Ocean Gyre, with pelagic Crenar- group also shows evidence of seasonal fluctua- chaeota comprising more than 30% of the total tions, reaching highest abundance in summer in microbial cells from 200 m down to 5,000 m the North Sea. Marine Group II Archaea spring (Fig. 2). “blooms” have been observed off the coast of Estimates from these cell counts indicate that California that are restricted to the photic zone. planktonic Crenarchaeota account for a large fraction of the total microbial biomass through- Another Surprise—an Archaeal Symbiont out the world’s oceans—consisting of about 1 ϫ of , symbiosum 28 10 cells, or about 20% or so of all the bacterial While a graduate student at the University of and archaeal cells found in the sea. Recent esti- California, Santa Barbara, Christina Preston mates based on the abundance of archaeal made an unexpected discovery. She found that a in seawater are consistent with these cell counts. single species of cold-loving crenarchaeote In some locations, archaeal abundance appears within the tissues of the coastal marine to vary with the seasons. For instance, off the mexicana (Fig. 3). Dubbed Cenar- Antarctic Peninsula, the pelagic Crenarchaeota chaeum symbiosum, this symbiotic crenarchae- (Group I) appear more abundant in surface win- ote can thrive at low temperatures—in this case, ter waters and subsurface summer waters. In about 10oC. This sponge-archaeal association is temperate seas, Marine Group II Euryarcha- specific, with C. symbiosum detectable only eota, unlike Marine Group I Crenarchaeota, within Axinella mexicana, and not in surround-

506 Y ASM News / Volume 69, Number 10, 2003 ing seawater or other sponges. This sym- biotic archaeon grows well at temper- FIGURE 2 atures of 10°C, more than 60°C below the growth temperature optimum of its closest cultivated relatives in the Cren- archaeota (Fig. 1). Since C. symbiosum can be successfully maintained in lab- oratory aquaria within the tissues of its , it provides a useful source of cold-dwelling crenarchaeotal biomass. Because C. symbiosum is the only ar- chaeon present, archaeal cell prepara- tions from the sponge are useful for biochemical studies—for example, to define the composition of cold- dwelling Crenarchaeota. This ar- chaeon should also be useful to help further define the physiological, bio- chemical, and genomic characteristics of cold-dwelling Crenarchaeota. Simi- lar species from other habitats, includ- ing Mediterranean sponges of the ge- nus Axinella, now also have been reported to specifically harbor archaea closely related to C. symbiosum.

Physiological Ecology of Oceanic Archaea Relatively little is known about the physiology of oceanic Archaea species, due in part to the unavailability of pure cultures. Creative applications of bio- chemical, geochemical, and genomic techniques, however, are now provid- ing valuable insights into planktonic Crenarchaeota, including information about specific , membrane lipid composition, and potential car- bon sources. Cold marine sediments contain high levels of tetraether lipids (Fig. 4), de- rived from the membrane lipids of planktonic Archaea, according to Jaap S. Sinninghe-Damste´ of the Royal Abundance of planktonic Archaea at the Hawaii Ocean Time series station as a function Netherlands Institute for Sea Research of depth, determined by fluorescence in situ hybridization counts. Percentage of Marine (NIOZ), Texel, the Netherlands. Freshly Group I Archaea relative to total microbial counts in 2A is indicated by color; red, 40% of cell total; dark blue, 0% of cell total. Figure 2B shows the distribution of cell types collected marine plankton samples determined by FISH, averaged over one year. Crenarchaeota refers specifically to Marine containing abundant planktonic Cren- Group I Archaea, and Euryarchaeota refers specifically to Marine Group II Archaea. From archaeota also contain high levels of Karner, M., E. F. DeLong, and D. M. Karl. 2001. Archaeal in the of the Pacific Ocean. Nature 409:507–510, with permission. similar tetraether lipid sediments. Fur- ther, , a mem- ber of the Marine Group I archaeal

Volume 69, Number 10, 2003 / ASM News Y 507 FIGURE 3

The marine sponge Axinella mexicana and its archaeal symbiont, Cenarchaeum symbiosum. 3A., Axinella mexicana a bright red found off the California coast. 3B., A. mexicana maintained in laboratory aquaria. 3C., FISH experiment showing C. symbiosum population present in the sponge tissues (in green). Many of the C. symbiosum cells are visibly dividing.

508 Y ASM News / Volume 69, Number 10, 2003 , contains the exact same tet- FIGURE 4 raether lipids as are found in plankton. Taken together, these data establish that Marine Group I Archaea are a main source of the tetraether lipids, al- though additional archaeal sources (e.g., Group II) for these lipids in plank- ton cannot be ruled out. Stefan Schouten and colleagues of NIOZ found structural differences be- tween the lipids of planktonic archaea and those of archaeal thermophiles such as solfataricus. Glyc- erol dibiphytanyl tetraethers (GDGTs) are commonly found ar- chaeal lipid structures in marine sedi- ments and plankton, and they include a novel lipid, called , Archaeal lipid structures found in marine plankton and sediments. GDGT, glycerol containing a six-membered cyclohexyl dibiphytanyl glycerol tetraether. The most common forms found in marine plankton, ring (Fig. 4, structure e). Studies with C. incorporating sidechains a, or d and e, are GDGTs I and VIII. Modified after S. Schouten, E. C. Hopmans, R. D. Pancost, and J. S. S. DamsteЈ. 2001. Widespread occurrence of symbiosum have confirmed that these structurally diverse tetraether membrane lipids: Evidence for the ubiquitous presence of novel lipids are definitely found in low-temperature relatives of . Proc. Natl. Acad. Sci. USA 97:14421– members of the Marine Group I Cren- 14426, Copyright 2001, National Academy of Sciences, U.S.A. archaeota. This uniquely characteristic cyclohexyl ring structure likely has naturally occurring 14C in oceanic organic mat- adaptive significance for the low-temperature ter and CO as a tracer, their results indicated Archaea. The Netherlands group proposes that 2 that deep-water archaea are not using organic this change from the standard, five-membered derived from recent photoautotrophic ring (found in thermophilic archaeal lipids) adds . Instead, these deep-sea a kink in the tetraether chains, rendering the Crenarchaeota apparently are incorporating membrane structures they form more fluid at dissolved CO as their main source. lower temperatures. 2 Meanwhile, the Netherlands group has recently If planktonic archaea are globally abundant, acquired additional data supporting this hy- what niche do they occupy? The Netherlands pothesis, via 13CO isotope tracer-labeling ex- group analyzed stable isotopes in marine sedi- 2 periments. This finding, if true, is quite a sur- ment-derived archaeal lipids in attempting to prise, implying that oceanic Archaea fixCO determine the carbon sources that support ar- 2 and may represent an unexpected source of pri- chaeal growth. Their initial findings suggested mary in the sea. If correct, what are that planktonic archaea use either algal carbo- these Archaea using as an source to fuel hydrates, , or dissolved bicarbonate and and CO fixation? Answering this thus do not indicate whether these archaea are 2 question may provide quite a bit of new infor- or heteroptrophs. Other studies us- mation about carbon and energy flux in the deep ing radiolabeled amino , microautoradiog- waters of the world’s oceans. raphy, and flourescent in situ hybridization (FISH) have provided some evidence indicating that planktonic archaea might incorporate ex- and Evolutionary ogenous amino acids. Origins of Oceanic Archaea In her thesis work Ann Pearson, working with Tim Eglinton and John Hayes at the Woods Modern genomic approaches are now proving Hole Oceanographic Institution, Woods Hole, extremely useful for studying microbes that Mass., purified large amounts of marine ar- have resisted cultivation. In the early 1990s, chaeal lipids from the deep sea, presumably working with Jeff Stein, then at the Agouron derived from planktonic Crenarchaeota. Using Institute, La Jolla, Calif., we isolated large

Volume 69, Number 10, 2003 / ASM News Y 509 genomic fragments from the planktonic Marine high-temperature habitats. It also seems likely, Group I Crenarchaeota. Thus began our explo- based on phylogenetic analyses, that different rations in environmental genomics. Later in lineages within the Crenarchaeota adapted to my lab, Christa Schleper, now at the Technical low-temperature habitats on multiple occasions. University of Darmstadt in Germany, devel- The journey from hot to cold environments oped high-quality genomic libraries from the seems to have been a common trek in the course symbiotic archaeon Cenarchaeum symbiosum. of evolution within different archaeal groups. Schleper next identified C. symbiosum DNA There are a number of outstanding puzzles polymerase in those genomic libraries, and ex- concerning the marine Archaea that remain to pressed, purified, and characterized it biochem- be solved. For instance, the marine Group I ically. Although the DNA polymerase amino Crenarchaeota are eurybathyl, that is, they are acid sequence most closely resembles thermo- distributed throughout the ocean depths from stable DNA polymerases from other hyperther- near-surface waters to the bottom of the abyss. mophilic Crenarchaeota, the itself is not Studies of fine-scale genetic and physiological thermostable. It is inactivated at about 40oC, differences in the group are likely to tell us quite consistent with the lower temperature origins a bit more about deep-sea adaptations. Deter- and of C. symbiosum. We later mining the specific carbon and energy sources of collaborated with a group at Diversa Corpora- Group I Crenarchaeota remains another out- tion, San Diego, Calif., to sequence 100 kbp of standing problem concerning this ubiquitous the C. symbiosum genome and learn more about microbial group. Resolving this mystery will the genome of this archaeon. help us better interpret the ecological roles and More recently, support from the National significance of Archaea in oceanic . Science Foundation has allowed us to embark So, where to next with the marine Archaea? on full-genome sequencing of C. symbiosum. Steve Giovannoni at Oregon State University, With a third of the genome now in hand, new Corvallis, has recently developed novel cultiva- clues about the physiological and genetic archi- tion strategies that have successfully recovered tecture of marine Crenarchaeota are already elusive and difficult-to-cultivate planktonic bac- beginning to emerge. These new genomic data teria (see ASM News, August 2003, p. 370). promise to teach us much about the natural Similar approaches may eventually lead to the history, , evolution, and physiology of of pure cultures of planktonic Archaea. the ubiquitous marine Crenarchaeota. This would aid tremendously in our understand- What extraordinary events led to the wide ing of the physiology and ecology of these distribution of Crenarchaeota in habitats rang- ocean-going microbes. In situ studies using ar- ing from boiling hot springs of 100oC to frigid chaeal-specific biomarkers and geochemical polar waters of –1.8oC ? There are several clues tracers also offer much promise for studying to the natural history of this fascinating group indigenous marine Archaea on their own turf. that come from a variety of disciplines, includ- Finally, genomic studies will provide us with not ing , biochemistry, and geo- only the parts list of marine Archaea, but also chemistry. All of these perspectives tend to sug- clues as to the physiological activities of this gest that the ancestors of modern-day, cold- widespread, abundant, and fascinating compo- adapted marine Archaea once lived in anoxic, nent of microbial life on Earth.

ACKNOWLEDGMENTS I am indebted to the intellectual and creative contributions (and camaraderie) of my laboratory cohorts and collaborators, especially: Oded Be´ja`, Lynne Christianson, Matthew Church, John Hayes, David Karl, Jose´ de la Torre, Peter Girguis, Steven Hallam, Ramon Massana, Alison Murray, Ann Pearson, Christina Preston, Christa Schleper, Jeff Stein, Trent Taylor, and Ke Ying Wu. Carl Woese and Norman Pace have generously provided me with ideas, advice, and inspiration over the past decade. Undergraduate research with Paul Baumann at the University of California, Davis, launched me on my career in science. My thesis advisor Art Yayanos gave me just enough rope to hang myself, for which I will always be grateful. My work on planktonic Archaea has been supported by the National Science Foundation and the David and Lucile Packard Foundation.

SUGGESTED READING Church, M. J., E. F. DeLong, H. W. Ducklow, M. B. Karner, C. M. Preston, and D. M. Karl. 2003. Abundance and distribution of planktonic Archaea and Bacteria in the Western Antarctic Peninsula. Limnol. Oceanogr. 48:1893–1902.

510 Y ASM News / Volume 69, Number 10, 2003 Damste´, J. S., S. Schouten, E. C. Hopmans, A. C. van Duin, and J. A. Geenevasen. 2002. Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic Crenarchaeota. J. Lipid Res. 43:1641–1651. DeLong, E. F., L. L. King, R. Massana, H. Cittone, A. Murray, C. Schleper, and S. G. Wakeham. 1998. Dibiphytanyl ether lipids in nonthermophilic crenarchaeotes. Appl. Environ. Microbiol. 64:1133–1138. DeLong, E. F. 1998. Everything in moderation: Archaea as “nonextremophiles.” Curr. Opin. Genet. Develop. 8:649–654. Fuhrman, J. A., K. McCallum, and A. A. Davis. 1992. Novel major archaebacterial group from marine plankton. Nature 356:148–9. Karner, M., E. F. DeLong, and D. M. Karl. 2001. Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409:507–510. Pearson, A., A. McNichol, B. Benitez-Nelson, J. Hayes, and T. Eglinton. 2001. Origins of lipid biomarkers in Santa Monica Basin surface : a case study using compound-specific delta 14C analysis. Geochimica et Cosmochimica Acta 65:3123–3137. Preston, C. M., K. Y. Wu, T. F. Molinski, and E. F. DeLong. 1996. A psychrophilic crenarchaeon inhabits a marine sponge; Cenarchaeum symbiosum, gen. nov. sp. nov. Proc. Natl. Acad. Sci. USA 93:6241–6246. Schleper, C., R. V. Swanson, E. J. Mathur, and E. F. DeLong. 1997. Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum. J. Bacteriol. 179:7803–7811. Stein, J., T. L. , K. Y. Wu, H. Shizuya, and E. F. DeLong. 1996. Characterization of uncultivated marine : isolation and analysis of a 40-kilobase genome fragment from a planktonic marine archaeon. J. Bacteriol. 178:591–599.

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