Isolation of an Autotrophic Ammonia-Oxidizing Marine Archaeon

Vol 437|22 September 2005|doi:10.1038/nature03911 LETTERS Isolation of an autotrophic ammonia-oxidizing marine archaeon Martin Ko¨nneke1*†, Anne E. Bernhard1*†, Jose´ R. de la Torre1*, Christopher B. Walker1, John B. Waterbury2 & David A. Stahl1 For years, microbiologists characterized the Archaea as obligate affiliated with the marine group 1 Crenarchaeota in nitrifying extremophiles that thrive in environments too harsh for other dilution cultures developed from Plum Island Sound (Massachusetts) organisms. The limited physiological diversity among cultivated estuary sediment, in nitrifying filtration systems at the Shedd Archaea suggested that these organisms were metabolically Aquarium (Chicago, Illinois), and in gravel from a marine tropical constrained to a few environmental niches. For instance, all fish tank at the Seattle Aquarium (Seattle, Washington). Crenarchaeota that are currently cultivated are sulphur- Further evidence for archaeal nitrifiers resulted from ammonia- metabolizing thermophiles1. However, landmark studies using oxidizing cultures highly enriched in marine group 1 Crenarchaeota. cultivation-independent methods uncovered vast numbers of Filtered aquarium water (0.2-mm polyethersulphone membrane; Crenarchaeota in cold oxic ocean waters2,3. Subsequent molecular Nalgene) supplemented with 1 mM ammonium chloride was inocu- surveys demonstrated the ubiquity of these low-temperature lated with gravel from a tropical marine tank at the Seattle Aquarium. Crenarchaeota in aquatic and terrestrial environments4.The Cultures enriched for Crenarchaeota were incubated at 21–23 8Cin numerical dominance of marine Crenarchaeota—estimated at the dark. Repeated serial transfers of 10% of the culture volume into 1028 cells in the world’s oceans5—suggests that they have a major fresh aquarium-water medium resulted in an enrichment comprised role in global biogeochemical cycles. Indeed, isotopic analyses of of approximately 90% Crenarchaeota and 10% organisms affiliated marine crenarchaeal lipids suggest that these planktonic Archaea with the bacterial domain after six months (data not shown). fix inorganic carbon6. Here we report the isolation of a Characterization of this highly enriched culture revealed that oxi- marine crenarchaeote that grows chemolithoautotrophically by dation rates of ammonia to nitrite corresponded with increasing aerobically oxidizing ammonia to nitrite—the first observation of abundance of Crenarchaeota (measured by quantitative polymerase nitrification in the Archaea. The autotrophic metabolism of this chain reaction (PCR); Supplementary Information) indicating nitri- isolate, and its close phylogenetic relationship to environmental fication (data not shown). marine crenarchaeal sequences, suggests that nitrifying marine After initial enrichment, the Crenarchaeota were isolated in a Crenarchaeota may be important to global carbon and nitrogen defined medium (see Methods) containing bicarbonate and ammo- cycles. nia as the sole carbon and energy sources, suggesting autotrophy. A Since their discovery by Fuhrman et al. and DeLong over a decade pure culture of Crenarchaeota (designated SCM1) was recovered ago2,3, marine Crenarchaeota are now recognized to be a dominant after three serial end-point dilutions in this medium, facilitated by fraction of bacterioplankton in the ocean. These microorganisms can the addition of streptomycin and filtration of the inoculum through a account for up to 40% of the bacterioplankton in deep ocean waters5. 0.45-mm HT Tuffryn membrane syringe filter (Pall). The purity of Although there are no known low-temperature Crenarchaeota in SCM1 was confirmed by quantitative PCR and fluorescent in situ culture, compound-specific D14C analysis of lipid biomarkers6 and hybridization (FISH), and supported by a failure to recover bacterial studies of 13C-bicarbonate tracer uptake by natural populations7 16S rRNA genes by PCR amplification or to promote the growth of have suggested autotrophy. However, another study of natural heterotrophic bacteria by the addition of yeast extract and peptone to populations demonstrated the uptake of tracer levels of tritiated the defined culture medium (data not shown). PCR amplification of amino acids, suggesting some use of fixed carbon8. We expect that the nearly full-length 16S rRNA genes from SCM1 identified only availability of a representative organism in pure culture will facilitate crenarchaeal sequences. The clonal structure of SCM1 was confirmed studies of their physiology and evolutionary origin, and help in by comparing the sequences of PCR-amplified fragments of 1,650 understanding their contribution to oceanic biogeochemical cycles. base pairs (bp) in length and containing most of the 16S rRNA, the Known nitrifying bacteria fall into two distinct physiological complete 16S–23S internal transcribed spacer, and a small portion of groups: those that oxidize ammonia to nitrite, and others that oxidize the 23S rRNA gene (Supplementary Information). nitritetonitrate9. None has been shown to oxidize ammonia Comparative sequence analysis of 16S rRNA genes revealed a high completely to nitrate. Existing genera of ammonia-oxidizing bacteria level of sequence identity (.98%) between SCM1 and marine group (AOB) fall within the Betaproteobacteria and the Gammaproteo- 1 Crenarchaeota sequences recovered from the North Atlantic, the bacteria (ref. 9). Molecular studies of AOB in nitrifying systems, Red Sea, the Antarctic and hydrothermal vents (Fig. 1). Phylogenetic including aquaria10, have been limited to these two phylogenetic analysis indicates that all marine group 1 Crenarchaeota—including groups9. We first suspected an involvement of Archaea in nitrification SCM1, crenarchaeal sequences from the Sargasso Sea11 and Cenarch- after we completed several cultivation-independent ribosomal RNA aeum symbiosum (an uncultured marine sponge symbiont)12—form gene surveys of nitrifying environments. We detected sequences a monophyletic clade sharing .94% rRNA sequence identity (Fig. 1). 1Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195, USA. 2Woods Hole Oceanographic Institute, Woods Hole, Massachusetts 02543, USA. †Present addresses: Institut fu¨r Chemie und Biologie des Meeres, Universita¨t Oldenburg, Oldenburg 26111, Germany (M.K.); Department of Biology, Connecticut College, New London, Connecticut 06320, USA (A.E.B.). *These authors contributed equally to this work. 543 © 2005 Nature Publishing Group LETTERS NATURE|Vol 437|22 September 2005 Figure 1 | Phylogenetic relationships between 16S rRNA sequences from indicated. The sequences from the Shedd Aquarium and Plum Island SCM1 and representatives of the marine group 1 Crenarchaeota. The tree sediment clones are short (674 and 720 bp, respectively) and were added to was constructed using the neighbour-joining algorithm with the Kimura the tree using the parsimony tool in ARB (ref. 25). The scale bar represents two-parameter correction (1,218 positions). Nodes supported by bootstrap 0.1 nucleotide changes per position. values .50% by neighbour-joining and parsimony (in parentheses) are In contrast, members of the marine group 1 Crenarchaeota limit growth in the marine environment, it is possible that the marine share only 84% 16S rRNA sequence identity with low-temperature Crenarchaeota are responsible for keeping ammonia concentrations Crenarchaeota found in soil, and less than 80% sequence identity low. The maximum growth rate of SCM1 in culture (0.78 d21) was with cultivated thermophilic Crenarchaeota. These differences in 16S somewhat higher than the range of rates estimated for natural rRNA sequences are consistent with variations found in genome bacterioplankton communities, which vary between 0.05 and fragments from marine and soil Crenarchaeota13,14. Nevertheless, 0.3 d21 (ref. 17). Notably, the addition of organic compounds, phylogenetic analyses clearly indicate that all low-temperature even in very low concentrations, appeared to inhibit the growth of Crenarchaeota are more closely related to each other than to their SCM1 (data not shown). Thus, organic material excreted by other thermophilic relatives (Fig. 1). organisms (for example, phototrophic primary producers) and a low Cells of SCM1 visualized by electron microscopy appear as straight concentration of ammonium may limit the abundance of marine rods with a diameter of 0.17–0.22 mm and a length of 0.5–0.9 mm Crenarchaeota in the environment. (Fig. 2). Cells occurred individually or in loose aggregates. Neither Growth of SCM1 is correlated with near-stoichiometric conver- flagella nor intracellular compartments were apparent by electron sion of ammonia to nitrite (Fig. 3). Among characterized AOB, microscopy. The size and morphology of SCM1 cells are nearly ammonia monooxygenase (AMO) oxidizes ammonia to hydroxyl- identical to marine Crenarchaeota in natural samples visualized by amine, which is further oxidized to nitrite by hydroxylamine oxido- FISH12,15. Individual SCM1 cells stained with 16S rRNA polyribo- reductase9. The overall reaction is represented by the following nucleotide probes showed strong fluorescence at both poles and weak stoichiometry: staining in the central region corresponding to the location of the þ NH þ 1:5O ! NO2 þ H O þ H ðDG0; ¼ 2235kJmol21Þ nucleoid, identified by staining with the fluorescent DNA-binding 3 2 2 2 dye 4 0 ,6 0 -diamidino-2-phenylindole (DAPI) (Fig. 2a and b; arrows), Recently, AMO-related genes have been reported in environmental giving labelled cells

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