Wide Diversity of Crenarchaeota Temperature Organisms8•9

SCIENTIFIC CORRESPONDENCE

a high-temperature ancestry for the low Wide diversity of Crenarchaeota temperature organisms8•9. This correlation also suggests that the ability to grow at SrR - The traditional perception of the sediment and marsh samples (5-32 °C) col low temperatures has arisen within the diversity of the bacteria-like Archaea based lected from Lakes Griffy and Lemon near Crenarchaeota in at least three separate on cultivation studies has been that the Bloomington, Indiana, and used it as tem instances. kingdom Euryarchaeota is a physiologically plate in the polymerase chain reaction The occurrence of diverse low-temper variable group including halophiles, (PCR) to amplify small-subunit rRNA ature Crenarchaeota in marine and terres thermophiles and methanogens, whereas genes (rDNA), as previously described4• trial environments is unexpected, and the kingdom Crenarchaeota is more homo We used an Archaea-specific forward shows that members of this kingdom of genous, consisting exclusively of sulphur primer (4Pa, see figure legend) and a uni the phylogenetic domain Archaea are dependent, extreme thermophiles 1• This versal reverse primer with a polylinker tail phenotypically more varied than was pre has led to the notion that the Crenarchaeo (1492RPL) for initial amplification to en viously thought. Because neither these ta are not widespread, but are restricted to rich for archaeal rRNA genes, and then organisms nor their close, high-tempera specialized habitats. Using sequence-based used forward primers specific to marine ture relatives have been cultivated, we techniques that sidestep the cultivation crenarchaeal sequences2•5 (89F), or to all have no information about their meta of organisms, crenarchaeal small-subunit known Crenarchaeota (542F), in conjunc bolic properties. Considering the general ribosomal RNA (rRNA) genes have been tion with 1492RPL to reamplify the initial metabolic capacities of other Archaea, detected in open marine waters2•3• We now PCR products. We cloned and screened however, we predict that the low-tempera report the detection, in terrestrial lake and the rDNA copies from both rounds of PCR ture Crenarchaeota can use molecular marsh sediments, of three novel, deeply by restriction fragment length polymor hydrogen as an energy source. The oxida divergent lineages of low-temperature Cre phism (RFLP) analysis of rDNA inserts, tion of hydrogen, generated abiotically narchaeota, one of which includes the and sequenced a representative of each and biotically, is a significant theme in marine types. This finding indicates that RFLP type either partially or completely microbial ecosystems 10• The ubiquity such organisms are globally distributed and (-500 or 1,500 nucleotides, respectively). of low-temperature Crenarchaeota in have an important role in the biosphere. On phylogenetic analysis, summarized marine and terrestrial environments sug We isolated DNA directly from shallow- in the figure, we found that the collection gests that they are widely distributed in describes three deeply nature. The rRNA sequences provide ARCHAEA Euryarchaeota BACTERIA divergent clades within tools for the study of their distribution Haloferax E.coli mitochondria the Crenarchaeota. We and role in the global ecosystem. termed the three clades Karen L. Hershberger Crenarchaeota Chlorobium Cytophaga of low-temperature Cre Department of Biology and Institute for Epufopiscium narchaeota group 1 (17 Molecular and Cellular Biology, Bacillus Group 2 _,c-:::;.._=--- chloroplast different sequences de Indiana University, Group3 Synechococcus Planctomyces Thermus tected, represented by Bloomington, Indiana 47405, USA Thermotoga pGrfA4 in the figure), Susan M. Barns Aquifex Group 1 pGrlA4 EM 17 which includes the mar- Department of Environmental Molecular 2•5 MarineGp.1 ine sequences and two Biology,

0.1 changes per nucleotide short (200-nucleotide) M888 Life Sciences Division, sequences encountered Los Alamos National Laboratory, in a survey of DNA from Los Alamos, New Mexico 87545, USA soils6; group 2 (14 novel Anna-Louise Reysenbach EUKARYA sequences detected, rep Department of Biochemistry and resented by pGrfC26); Microbiology, and group 3 (one instance Lipman Hall, Cook College, detected, pGrfB286). Rutgers University, Groups 2 and 3 Crenar New Brunswick, New Jersey 08903, USA chaeota have not been Scott C. Dawson detected previously. Low Norman R. Pace*

Encephalitozoon bootstrap values (not Department of Plant and Microbial Biology, shown) for specific place Kosh/and Hall 111, Phylogenetic analysis of terrestrial Crenarchaeota. DNA was puri• ment of the pGrfB286 University of California, Berkeley, fied directly from sediments collected from Lakes Griffy and line (group 3) indicate California 94720-3102, USA Lemon , and used for PCR as outlined in the text and detailed previ• that its branch-point in e-mail: nrpace@nature. berkeley.edu 5 '-GGCTCAGTAACG• ously". Forward primers used were: 89F, the tree is not resolved, 1 . Woese , C. R. , Kandler, 0. & Wheeli s, M. L. Proc. Natl CGTAGTC-3 '; 542F, 5 '-CGCGGTAATACCAGCYC-3' ; and 4Fa , 5'-TCC• however, this lineage Acad. Sci. USA 87, 4576--4579 (1990). 2. Delong, E. F. Proc. Natl Acad. Sci. USA 89. 5685-5689 CGGTTGATCCTGCCRG-3 '. All PCR products were generated using clearly is highly divergent the universal reverse primer 1492RPL, 5'-GGCTCGAGCGGC• (1992). Each ofthe three new 3. Fuhrman, J. A., McCallu m, K. & Davis, A. A. Nature 356, CGCCCGGGTTACCTTGTTACGACTT-3 '. PCR products were cloned as 148- 149 (1992). described, screened for unique types by restriction analysis and clades detected in this 4. Barns, S. M., Fundyga, R. E. , Jeffries, M. W. & Pace , unique types were fully or partially sequenced. Sequences were survey is specifically N. R. Proc. Natl Acad. Sci. USA 91, 1609- 1613 (1994). , 11 5. Del ong, E. F. , Wu , K. Y. , Prezelin , B. B. & Jovine aligned with others from the rRNA sequence database and phylo• associated with sequen R. V. M. Nature 371, 695- 697 (199 4). genetic analyses were done. The phylogenetic tree shown is a com• ces originally cloned 6. Ueda. T., Suga, Y. & Matsuguchi, T. Eur. J. Soil Sci. 46, posite tree based on evolutionary distances12 and full sequences from Obsidian Pool, a 415-421 (1995). of representatives of the three clades discussed in the text. 7. Barns, S. M., Delwiche, C. F., Palmer, J. D. & Pace, N. R. hydrothermal (73- 93 °C) Proc. Natl Acad. Sci. USA 93, 9188--9193 ( 1996). Clones pGrfA4, pGrfC26 and pGrfB286 represent groups 1, 2 and spring in Yellowstone 8. Woese, C. R. Microbial. Rev. 51, 221- 271 (1987). 3 Crenarchaeota, respectively. All sequences determined (31 par• National Park4.7 . The 9. Pace, N. R. Ce// 65, 531-533 (1991). tial and full sequences) have been deposited in the GenBank 10. Stevens, T. 0. & McKinley, J. P. Science 270, 450-454 . Sequence align• nesting of the low (1995). database, accession numbers U59968-U59999 11. Maid ak, B. L. et al. Nucleic Acids Res. 24, 82- 85 ments and bootstrap analyses of phylogenetic trees were as previ• temperature sequences (1996). ously described4 . Additional details of methods are available within groups from hot 12. DeSoete, G. Psychometrika 48, 621-626 (1983). electronically (http://crab2.berkeley.edu/ -pacelab/ cren.htm1) . springs is consistent with * To whom corre spondence should be addressed. 420 NATURE · VOL 384 · 5 DECEMBER 1996