A korarchaeal genome reveals insights into the evolution of the Archaea James G. Elkinsa,b, Mircea Podarc, David E. Grahamd, Kira S. Makarovae, Yuri Wolfe, Lennart Randauf, Brian P. Hedlundg, Ce´ line Brochier-Armaneth, Victor Kunini, Iain Andersoni, Alla Lapidusi, Eugene Goltsmani, Kerrie Barryi, Eugene V. Koonine, Phil Hugenholtzi, Nikos Kyrpidesi, Gerhard Wannerj, Paul Richardsoni, Martin Kellerc, and Karl O. Stettera,k,l aLehrstuhl fu¨r Mikrobiologie und Archaeenzentrum, Universita¨t Regensburg, D-93053 Regensburg, Germany; cBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; dDepartment of Chemistry and Biochemistry, University of Texas, Austin, TX 78712; eNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894; fDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520; gSchool of Life Sciences, University of Nevada, Las Vegas, NV 89154; hLaboratoire de Chimie Bacte´rienne, Unite´ Propre de Recherche 9043, Centre National de la Recherche Scientifique, Universite´de Provence Aix-Marseille I, 13331 Marseille Cedex 3, France; iU.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598; jInstitute of Botany, Ludwig Maximilians University of Munich, D-80638 Munich, Germany; and kInstitute of Geophysics and Planetary Physics, University of California, Los Angeles, CA 90095 Communicated by Carl R. Woese, University of Illinois at Urbana–Champaign, Urbana, IL, April 2, 2008 (received for review January 7, 2008) The candidate division Korarchaeota comprises a group of uncul- and sediment samples from Obsidian Pool as an inoculum. The tivated microorganisms that, by their small subunit rRNA phylog- cultivation system supported the stable growth of a mixed commu- eny, may have diverged early from the major archaeal phyla nity of hyperthermophilic bacteria and archaea including an or- Crenarchaeota and Euryarchaeota. Here, we report the initial ganism with a SSU rDNA sequence displaying 99% identity to characterization of a member of the Korarchaeota with the pro- pJP27. The organism was identified as an ultrathin filament be- posed name, ‘‘Candidatus Korarchaeum cryptofilum,’’ which ex- tween 0.16 and 0.18 ␮m in diameter and variable in length. hibits an ultrathin filamentous morphology. To investigate possi- Whole-genome shotgun (WGS) sequencing was used to assemble ble ancestral relationships between deep-branching Korarchaeota an intact composite genome from purified cells originating from and other phyla, we used whole-genome shotgun sequencing to the enrichment culture. The complete genome sequence of construct a complete composite korarchaeal genome from en- ‘‘Candidatus (Ca.) Korarchaeum cryptofilum’’ provides a look into riched cells. The genome was assembled into a single contig 1.59 the biology of these deeply branching archaea and their evolution- Mb in length with a G ؉ C content of 49%. Of the 1,617 predicted ary relationships with Crenarchaeota and Euryarchaeota. protein-coding genes, 1,382 (85%) could be assigned to a revised set of archaeal Clusters of Orthologous Groups (COGs). The pre- dicted gene functions suggest that the organism relies on a simple Results mode of peptide fermentation for carbon and energy and lacks the Cultivation and in Situ Identification. An enrichment culture was ability to synthesize de novo purines, CoA, and several other inoculated with sediment and hot spring samples taken from cofactors. Phylogenetic analyses based on conserved single genes Obsidian Pool, YNP. The enrichment was maintained under strictly and concatenated protein sequences positioned the korarchaeote anaerobic conditions at 85°C, pH 6.5, and continuously fed a dilute as a deep archaeal lineage with an apparent affinity to the organic medium. A stable community of hyperthermophilic ar- Crenarchaeota. However, the predicted gene content revealed that chaea and bacteria with a total cell density of Ϸ1.0 ϫ 108 cells per several conserved cellular systems, such as cell division, DNA ml was supported for nearly 4 years. Sequences from SSU rDNA replication, and tRNA maturation, resemble the counterparts in the clone libraries derived from the enrichment were closely related to Euryarchaeota. In light of the known composition of archaeal other known isolates or environmental sequences from Obsidian genomes, the Korarchaeota might have retained a set of cellular Pool [see supporting information (SI) Text and Fig. S1]. The features that represents the ancestral archaeal form. Korarchaeota were represented by the SSU rDNA clone pOPF࿝08, which is 99% identical to pJP27 from Obsidian Pool, YNP (2), and ͉ ͉ ͉ ͉ microbial cultivation genomics hyperthermophiles Korarchaeota pAB5 from Calcite Springs, YNP (9). FISH analysis allowed the phylogeny positive identification of cells with the pOPF࿝08 SSU rRNA se- quence. Cells hybridizing to Cy3-labeled, Korarchaeota-specific wo established phyla, the Crenarchaeota and Euryarchaeota, probes, KR515R and KR565R, were ultrathin filaments Ͻ0.2 ␮m Tdivide the archaeal domain based on fundamental differences in diameter with an average length of 15 ␮m, although cells were in translation, transcription and replication (1). Yet hydrothermal observed with lengths up to 100 ␮m (Fig. 1A, Fig. S2, and SI Text). environments have yielded small subunit (SSU) rRNA gene se- quences that form deep-branching phylogenetic lineages, which potentially lie outside of these major groups. These uncultivated Author contributions: J.G.E., P.R., M.K., and K.O.S. designed research; J.G.E., M.P., B.P.H., organisms include members of the Korarchaeota (2–4), the Ancient A.L., E.G., K.B., and G.W. performed research; J.G.E., M.P., D.E.G., K.S.M., Y.W., L.R., C.B.-A., Archaeal Group (5), and the Marine Hydrothermal Vent Group (5, V.K., I.A., E.V.K., P.H., N.K., and K.O.S. analyzed data; and J.G.E., D.E.G., E.V.K., and K.O.S. wrote the paper. 6). The Nanoarchaeota have also been suggested to hold a basal phylogenetic position (7), but this placement has been debated (8). The authors declare no conflict of interest. The Korarchaeota comprise the largest group of deep-branching Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession no. CP000968). unclassified archaea and have been detected in several geograph- bPresent address: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN ically isolated terrestrial and marine thermal environments (2, 4, 5, 37831. 9–17). lTo whom correspondence should be addressed at: Lehrstuhl fu¨r Mikrobiologie, Universita¨t To gain insights into the Korarchaeota, we revisited the original Regensburg, Universita¨tstrasse 31, 93343 Regensburg, Germany. E-Mail: karl.stetter@ site where Barns et al. (2) collected korarchaeal environmental SSU biologie.uni-regensburg.de. rDNA sequences (pJP78 and pJP27) from Obsidian Pool, Yellow- This article contains supporting information online at www.pnas.org/cgi/content/full/ stone National Park (YNP), Wyoming. Continuous enrichment 0801980105/DCSupplemental. cultures were established at 85°C by using a dilute organic medium © 2008 by The National Academy of Sciences of the USA 8102–8107 ͉ PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801980105 Downloaded by guest on September 23, 2021 Table 1. General features of the Ca. K. cryptofilum genome Genome feature Value Total number of bases 1,590,757 Coding density, % 90.7 G ϩ C content, % 49.0 Total number of predicted genes 1,665 Protein coding genes 1,617 Average ORF length, bp 870 rRNA genes* 3 tRNA genes 45 Genes assigned to COGs 1,401 Genes assigned to arCOGs 1,382 Genes with function prediction 998 Genes with biochemical prediction only 246 Genes with unknown function or activity 373 *16S, 23S, and 5S rRNA. biological function to 998 (62%) predicted proteins; for another 246 proteins (15%), biochemical activity but not biological function could be predicted, and for the remaining 373 (23%) proteins, no functional prediction was possible, although many of these are conserved in some other archaea and/or bacteria. arCOGs. The predicted proteins were assigned to arCOGs (19) (see Fig. 1. Microscopy of Ca. K. cryptofilum. (A) FISH analysis with Korarchaeota- specific Cy3-labeled oligonucleotide probes KR515R/KR565R. The undulated SI Text, Dataset S1]. Of the 1,617 annotated proteins, 1,382 (85%) cell shape results from drying of the specimen on gelatin coated slides before were found to belong to the arCOGs, a coverage that is slightly MICROBIOLOGY hybridization. (Scale bar, 5 ␮m.) (B) Phase-contrast image of korarchaeal lower than the mean coverage of 88% for other archaea and much filaments after physical enrichment. (Scale bar, 5 ␮m.) (C) Scanning electron greater than the lowest coverage obtained for Nanoarchaeum micrograph of purified cells. (D) Transmission electron micrograph after neg- equitans (72%) and Cenarchaeum symbiosum (58%). When the ative staining with uranyl acetate displaying the paracrystalline S layer. Cells gene complement was compared with the strictly defined core gene are flattened, which increases their apparent thickness. sets for the Euryarchaeota and Crenarchaeota (i.e., genes that are represented in all sequenced genomes from the respective division, with the possible exception for C. symbiosum in the case of the Cell Preparation and Genome Sequencing. It was observed that Crenarchaeota, but that are missing in at least some organisms of the filamentous cells