Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea C. B. Walkera,b, J. R. de la Torrea, M. G. Klotzc, H. Urakawaa, N. Pinela, D. J. Arpd, C. Brochier-Armanete, P. S. G. Chainf,g,h, P. P. Chani, A. Gollabgirj, J. Hempk, M. Hüglerl,m, E. A. Karrn, M. Könnekeo, M. Shinf,g, T. J. Lawtonp, T. Lowei, W. Martens- Habbenaa, L. A. Sayavedra-Sotod, D. Langf,g, S. M. Sievertq, A. C. Rosenzweigp, G. Manningj, and D. A. Stahla,1 aDepartment of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195; bGeosyntec Consultants, Seattle, WA 98101; cDepartment of Biology, University of Louisville, Louisville, KY 40292; dDepartment of Botany and Plant Pathology, Oregon State University, Corvalis, OR 97331; eUniversité de Provence Aix-Marseille I, Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique Unité Propre de Recherche, Marseille, 13402 France; fBiosciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550; gMicrobial Program, Joint Genome Institute, Walnut Creek, CA 94598; hCenter for Microbial Ecology, Michigan State University, East Lansing, MI 48824; iDepartment of Biomolecular Engineering, University of California, Santa Cruz, CA 95064; jRazavi Newman Center for Bioinformatics, Salk Institute for Biological Studies, La Jolla, CA 92037; kSchool of Chemical Sciences, University of Illinois, Urbana, IL 61801; lLeibniz-Institut für Meereswissenschaften, Kiel, 24105 Germany; mWater Technology Center, Karlsruhe, 76139 Germany; nDepartment of Botany and Microbiology, University of Oklahoma, Norman, OK 73019; oInstitut für Chemie und Biologie des Meeres, Universität Oldenburg, Oldenburg, 26129 Germany; pDepartments of Biochemistry, Molecular Biology and Cell Biology, and Chemistry, Northwestern University, Evanston, IL 60208; and qBiology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Edited by David Karl, University of Hawaii, Honolulu, HI, and approved April 2, 2010 (received for review December 6, 2009) Ammonia-oxidizing archaea are ubiquitous in marine and terres- the highest substrate affinities yet observed (13). Among charac- trial environments and now thought to be significant contributors terized ammonia oxidizers, only N. maritimus is capable growing at to carbon and nitrogen cycling. The isolation of Candidatus “Nitro- the extremely low concentrations of ammonia generally found in sopumilus maritimus” strain SCM1 provided the opportunity for the open ocean (7, 13). This strain therefore provided an excellent linking its chemolithotrophic physiology with a genomic inventory opportunity to investigate the core genetic inventory for ammonia- of the globally distributed archaea. Here we report the 1,645,259- based chemoautotrophy by Group I crenarchaea. bp closed genome of strain SCM1, revealing highly copper-depen- The gene content and gene order of N. maritimus is highly dent systems for ammonia oxidation and electron transport that similar to environmental populations represented in marine bac- are distinctly different from known ammonia-oxidizing bacteria. terioplankton metagenomes, confirming on a genomic level its Consistent with in situ isotopic studies of marine archaea, the close relationship to many oceanic crenarchaea. Thus, an evalu- N. maritimus genome sequence indicates grows autotrophically ation of the genomic inventory of N. maritimus should offer using a variant of the 3-hydroxypropionate/4-hydroxybutryrate a framework to identify features shared among ammonia-oxidizing pathway for carbon assimilation, while maintaining limited capac- Group I crenarchaea, resolve physiological diversity among AOA, ity for assimilation of organic carbon. This unique instance of ar- and refine understanding of their ecology in relationship to the chaeal biosynthesis of the osmoprotectant ectoine and an larger assemblage of marine archaea—not all of which are am- unprecedented enrichment of multicopper oxidases, thioredoxin- monia oxidizers. In support of this expectation, the physiological like proteins, and transcriptional regulators points to an organism fi “ responsive to environmental cues and adapted to handling reac- and genomic pro les together show that many of the non- extreme” archaea identified in metagenomic studies, and currently tive copper and nitrogen species that likely derive from its distinc- Crenarchaeota tive biochemistry. The conservation of N. maritimus gene content assigned to the kingdom, are AOA that contribute and organization within marine metagenomes indicates that the to global carbon and nitrogen cycling, possibly determining rates fi unique physiology of these specialized oligophiles may play a sig- of nitri cation in a variety of environments (6, 8, 9, 13). nificant role in the biogeochemical cycles of carbon and nitrogen. Results and Discussion N. maritimus ammonia oxidation | marine microbiology | archaea | nitroxyl Primary Sequence Characteristics. strain SCM1 con- tains a single chromosome of 1,645,259 bp encoding 1,997 pre- dicted genes and no extrachromosomal elements or complete arine Group I archaea are among the most abundant prophage sequences (Table 1). No unambiguous origin of rep- microorganisms in the global oceans (1–3). Originally dis- M lication could be determined on the basis of local gene content covered through ribosomal RNA gene sequencing (3, 4), recent or GC skew, as commonly observed for other archaeal genomes metagenomic, biogeochemical, and microbiological studies (14). Approximately 61% of the N. maritimus open-reading established the capacity of these organisms to oxidize ammonia, thus linking this abundant microbial clade to one of the key steps of the global nitrogen cycle (5–9). For a century following the dis- Bacteria Author contributions: C.B.W., J.R.d.l.T., P.S.G.C., and D.A.S. designed research; C.B.W., covery of autotrophic ammonia oxidizers, only were J.R.d.l.T., M.G.K., H.U., N.P., C.B-A., P.P.C., A.G., M.H., E.A.K., M.K., M.S., T.L., W.M-H., thought to catalyze this generally rate-limiting transformation in M.S., D.L., S.M.S., A.C.R., G.M., and D.A.S. performed research; C.B.W. and J.R.d.l.T. con- the two-step process of nitrification (10). Despite recent enrich- tributed new reagents/analytic tools; C.B.W., J.R.d.l.T., M.G.K., H.U., N.P., D.J.A., C.B.-A., P.P.C., A.G., J.H., M.H., E.A.K., M.K., T.J.L., T.L., W.M.-H., L.A.S.-S., S.M.S., A.C.R., G.M., and ment of mesophilic as well as thermophilic ammonia-oxidizing D.A.S. analyzed data; and C.B.W., J.R.d.l.T., M.G.K., H.U., N.P., C.B.-A., J.H., M.H., E.A.K., archaea (AOA) (6, 11, 12), only a single Group I-related strain, M.K., T.L., S.M.S., A.C.R., G.M., and D.A.S. wrote the paper. isolated from a gravel inoculum from a tropical marine aquarium, The authors declare no conflict of interest. has thus far been successfully obtained in pure culture (7). This article is a PNAS Direct Submission. Nitrosopumilus maritimus The isolation of strain SCM1 ulti- Data deposition: The sequence reported in this paper has been deposited in the NCBI mately confirmed an archaeal capacity for chemoautotrophic database (accession no. NC_010085). growth on ammonia. More detailed characterization of this strain 1To whom correspondence should be addressed. E-mail: [email protected]. revealed cytological and physiological adaptations critical for life This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in an oligotrophic open ocean environment, most notably one of 1073/pnas.0913533107/-/DCSupplemental. 8818–8823 | PNAS | May 11, 2010 | vol. 107 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.0913533107 Table 1. Genome features of N. maritimus SCM1, C. symbiosum, sequenced AOB, and crenarchaeal genome fragments Nitrosococcus Nitrosomonas Nitrosospira Nitrosopumilus Cenarchaeum oceani europaea Nitrosomonas multiformis Fosmid Cosmid Fosmid maritimus SCM1 symbiosum ATCC 19707 ATCC 19718 eutropha C91 ATCC 25196 4B7 DeepAnt-EC39 74A4 Size (bp) 1,645,259 2,045,086 3,481,691 2,812,094 2,661,057 3,184,243 39,297 33,347 43,902 Percent coding 91.90% 91.20% 86.80% 88.40% 85.60% 85.60% 89.10% 86.10% 84.00% GC content 34.20% 57.70% 50.30% 50.70% 48.50% 53.90% 34.40% 34.10% 32.60% ORFs 1,997 2,066 3,186 2,628 2,578 2,827 41 41 51 ORF density (ORF/kb) 1.19 0.986 0.889 0.876 0.952 0.86 0.992 1.17 1.12 Avg. ORF length (bp) 757 924 964 1009 890 980 898 737 753 Standard tRNAs 44 45 45 41 41 43 0 0 0 rRNAs 1 1 2 1 1 1 1 1 1 Plasmids 0 0 1 0 2 3 NA NA NA NA, not analyzed. frames (ORFs) could be assigned to clusters of orthologous actions with the MCOs (and other predicted redox proteins) are groups of proteins (COGs), a lower percentage than for genomes likely mediated by eight soluble and nine membrane-anchored of ammonia-oxidizing bacteria (AOB) (Table S1) but similar to copper-binding proteins containing plastocyanin-like domains Cenarchaeum symbiosum (15). The genome possesses a relatively (Table S2). The corresponding genes appear to be the result of high coding density (91.9%), with a larger fraction dedicated to a series of duplications within the N. maritimus lineage (Fig. S1). energy production/conservation, coenzyme transport/metabolism, A second family of predicted redox active periplasmic pro- and translation genes than other characterized Crenarchaeota,but teins, composed of 11 thiol-disulfide oxidoreductases from the similar to two common species of photoautotrophic marine thioredoxin family (Nmar_0639, _0655, _0829, _0881, _1140, Bacteria, Prochlorococcus, and Synechococcus. _1143, _1148, _1150, _1181, _1658, and Nmar_1670), show low (but recognizable) identity with the better characterized disulfide Energy Metabolism. The stoichiometry of ammonia oxidation to bond oxidases/isomerases found in Bacillus subtilis (BdbD) and nitrite is similar to that of characterized aerobic, obligate che- Escherichia coli (DsbA, DsbC, and DsbG).