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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) -oxidizing 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 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 . 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 that likely derive from its distinc- 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. 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 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 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 -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 is similar to that of characterized aerobic, obligate che- Escherichia coli (DsbA, DsbC, and DsbG). The mean percentage molithoautotrophic AOB (13), yet the contributing biochemistry is of sequence identity between the N. maritimus proteins and distinctly unique. All AOB share a common pathway where hy- BdbD is 21 ± 3%. The significantly lower mean percentage of droxylamine, produced by an ammonia monooxygenase (AMO), is sequence identity to DsbA, DsbC, and DsbG (9.2, 10.7, and oxidized to nitrite by a -rich hydroxylamine oxidoreductase 10.4%, respectively) is comparable to that shared between the (HAO) complex; the oxidation of hydroxylamine supplies elec- E. coli proteins (10–11%). Although functional equivalency trons to both the AMO and a typical electron transport chain cannot be established, all but Nmar_0881 preserve the conserved c N. maritimus composed of cytochrome proteins. lacks genes thioredoxin-like active site FX4CXXC sequence (18–20). In encoding a recognizable AOB-like HAO complex and pertinent E. coli, both DsbA and DsbC rectify nonspecific disulfide bonds cytochrome c proteins, indicating an alternative archaeal pathway. catalyzed by copper (21), whereas up-regulation of dsbA by the The numerous copper-containing proteins, including multicopper Cpx regulon occurs during copper stress (22, 23). Eukaryotic oxidases and small blue copper-containing proteins (similar to protein disulfide isomerase (PDI) homologs sequester and/or plasto-, halo-, and sulfocyanins), suggest an alternative electron reduce oxidized Cu(II), perhaps serving as copper acceptors/ transfer mechanism (Table S2). These predicted periplasmic donors for copper-containing proteins (24). Another described proteins likely serve functionally similar roles to soluble cyto- function of PDIs is the capture and transport of (25, chrome c proteins in other Archaea, including Natronomonas 26), a possible intermediate or by-product of ammonia oxidation. pharaonis and such as Sulfolobus (16). This The related protein family in N. maritimus may function in part apparent reliance on copper for redox reactions is a major di- to alleviate copper and nitric oxide toxicity. vergence from the iron-based electron transfer system of AOB. The N. maritimus genome contains genes coding for six soluble Pathways for Ammonia Oxidation and Electron Transfer. The three periplasmic multicopper oxidase (MCO) proteins: two nearly genes (Nmar_1500, _1503, and _1502) annotated as amoA, identical NO-forming nitrite reductase proteins (NirK; amoB, and amoC and coding for a putative ammonia mono- Nmar_1259 and -1667), each with three cupredoxin domains; oxygenase complex are the only recognizable genetic hallmarks two NcgA-like (nirK cluster gene A) MCOs (Nmar_1131 and of ammonia oxidation in the genome sequence. However, the -1663) with two cupredoxin domains; one MCO (Nmar_1136) N. maritimus sequences are no more similar (in either content or with three cupredoxin domains; and one MCO (Nmar_1354) organizational structure) to bacterial amo genes than they are to with two domains fused to a blue copper-containing protein the genes encoding bacterial particulate mono- (Table S2). Two (Nmar_1131 and Nmar_1663) of the three oxygenases (pMMO), suggestive of functional differences be- genes that are classified as belonging to the emerging class of tween the archaeal and bacterial versions of AMO (7, 27). two-domain MCOs (2dMCOs) resemble the general architecture Notably, mapping the sequence encoded by amoB onto the of the 2dMCO NcgA present in Nitrosomonas europaea. Al- pmoB crystal structure of Methylococcus capsulatus (Bath) (28) though the overall sequence identity is low, clustering of NcgA reveals the conservation of the ligands to the pMMO metal with a nitrite reductase suggests it may play a supporting role in centers and the complete absence of both a transmembrane helix nitrite reduction (17). Genes Nmar_1131 and Nmar_1663 are also and a C-terminal cupredoxin domain predicted to be present in colocated with a member of the DtxR family of metal regulators and bacterial AMO (Fig. S2). a member of the ZIP metal transport family, suggesting a role in The structural differences in the archaeal AMO, the lack of metal homeostasis (see below). The third 2dMCO (Nmar_1354), genes encoding the hydroxylamine–ubiquinone redox module

possessing a fused blue copper-containing domain, has not been (29), and a periplasm enriched in redox active proteins together MICROBIOLOGY found in AOB and appears to be unique to AOA. Redox inter- suggest significant divergence from the bacterial pathway of

Walker et al. PNAS | May 11, 2010 | vol. 107 | no. 19 | 8819 ammonia oxidation. There are two hypothetical mechanistic bisphosphate carboxylase/oxygenase (RubisCO) as the key , alternatives (Fig. 1, Table S2): either a unique biochemistry the absence of genes in N. maritimus coding for RubisCO and other exists for the oxidation of hydroxylamine or the divergent AMO of this cycle points to an alternative pathway for carbon does not actually produce hydroxylamine. If the former is true, fixation. The most likely mechanism supported by the genome se- hydroxylamine oxidation may occur via one of the periplasmic quence is the 3-hydroxypropionate/4-hydroxybutyrate pathway elu- MCOs (CuHAO). Given the lack of cytochrome c proteins, the cidated in the thermophilic crenarchaote Metallosphaera sedula and four electrons would then be transferred to a quinone reductase suggested as a potential pathway of carbon fixation in C. symbiosum (QRED) via small blue copper-containing plastocyanin-like (33). The pathway has two parts: a sequence including two carbox- electron carriers. The protein encoded by Nmar_1226, which ylation reactions transforming acetyl-CoA to succinyl-CoA and contains four transmembrane-spanning regions and two plasto- a multistep sequence converting succinyl-CoA into two of cyanin-like domains, may serve as an analog of the membrane- acetyl-CoA. Genes identified in the N. maritimus genome coding for c bound cytochrome M552 quinone reductase present in AOB key enzymes of the pathway (Fig. S3) include a biotin-dependent (29) and is a good candidate for the QRED. acetyl-CoA/propionyl-CoA carboxylase (Nmar_0272–0274), meth- In an alternative scenario, the archaeal AMO produces not ylmalonyl-CoA epimerase and mutase (Nmar_0953, _0954, and hydroxylamine, but the reactive intermediate nitroxyl (nitroxyl _0958), and 4-hydroxybutyrate dehydratase (Nmar_0207). With the hydride, HNO). Nitroxyl is a highly toxic and reactive compound exception of one gene (Nmar_1608), all of the genes implicated in the fi recently recognized as having biological signi cance in a number 3-hydroxyproprionate/4-hydrobutyrate pathway for carbon assimila- of systems (30, 31). During archaeal ammonia oxidation, nitroxyl tion in N. maritimus are present and show highest similarity to the might be formed by a unique monooxygenase function of ar- genes of C. symbiosum (34, 35). Although N. maritimus and M. sedula chaeal AMO. Alternatively, the archaeal AMO may act as most likely use the same CO2-fixation reaction sequences, not all a dioxygenase and insert two atoms into ammonia, individual reactions appear to be catalyzed by identical enzymes. In producing nitroxyl from the spontaneous decay of HNOHOH. one instance, the stepwise reductive transformation of malonyl-CoA Both reaction sequences eliminate the requirement for reductant to propionyl-CoA involves five enzymes in M. sedula (33, 36, 37). recycling during the initial oxygenase reaction, a simplification N. maritimus fi Although the genome lacks any close homologs of offering signi cant ecological advantage (when compared with the M. sedula genes, it contains alternative alcohol dehydrogenases, AOB) in nutrient poor environments. In this pathway, one of the aldehyde dehydrogenases, acyl-CoA synthetases, and enoyl-CoA MCO-like proteins may act as a nitroxyl oxidoreductase (NXOR) hydratases possibly fulfilling the same functions. Similarly, M. sedula and facilitate the oxidation of nitroxyl to nitrite with the extrac- catalyzes the activation of 3-hydroxypropionate to 3-hydroxypropionyl- tion of two protons and two electrons in the presence of . CoA, using an AMP-forming 3-hydroxypropionyl-CoA synthetase The proposed NXOR would relay the two extracted electrons (37). The N. maritimus genome lacks an obvious homolog, although into the quinone pool via the QRED pathway described above. does code for an ADP-forming acyl-CoA synthetase (Nmar_1309) that In this proposed model, the electrons extracted by either fi a CuHAO or a NXOR (and transferred into the quinone pool) suggests a more energy ef cient alternative. In addition to the genes coding for the 3-hydroxypropionate/4- would generate a proton motive force (PMF) through complexes N. maritimus III (plastocyanin-like subunit, Nmar_1542; Rieske-type subunit, hydroxybutyrate pathway, the genome of contains Nmar_1544; transmembrane subunit, Nmar_1543) and IV a number of genes encoding enzymes of the tricarboxylic acid (TCA) cycle. No homologs for genes coding for a citrate-cleaving (Nmar_0182-5), driving the generation of ATP by an F0F1-type – enzyme (ATP citrate lyase or citryl-CoA lyase) (38) were identi- ATP synthase (Nmar_1688 1693). The production of reductant fi (i.e., NADH) would require the reverse operation of complex I ed, permitting exclusion of the reductive TCA cycle as a pathway fi N. mar- (NuoABCDHIJKMLN, Nmar_0276–286) as a quinol oxidase for carbon xation. The lack of these genes suggests that itimus driven by a PMF. The proposed biochemistry involving nitroxyl utilizes either an incomplete (or horseshoe-type) TCA cycle produces the same net gain as bacterial ammonia oxidation, for strictly biosynthetic purposes or possibly a complete oxidative providing two electrons for reduction of the quinone pool and TCA cycle. N. maritimus subsequent linear electron flow and the generation of a PMF. grows on a completely inorganic medium, in- SI The presence of a copper-containing (versus heme) complex III dicating the genes coding for essential biosynthetic capacity ( Materials and Methods and the unique evolutionary placement of terminal oxidase ), yet its genomic inventory also suggests (complex IV) between two of the heme–copper oxygen reductase some flexibility in the utilization of organic sources of phos- families further distinguish this proposed ammonia oxidation phorus and carbon. Two systems for phosphorous acquisition are pathway from that in AOB. suggested: the high-affinity, high-activity phosphate transport system (pstSCAB, Nmar_0479, Nmar_0481–0483) and a phos- Carbon Fixation and Mixotrophy. N. maritimus,likeallknown phonate transporter (Nmar_0873–0875). However, because the AOB, grows chemolithoautotrophically by using inorganic carbon genome lacks genes encoding known C-P lyases and hydrolases as the sole carbon source (7, 32). However, whereas AOB use the (39), and phosphate limitation is not alleviated by supplemen- Calvin–Bassham–Benson cycle with the CO2-fixing enzyme ribulose tation with phosphonates common in the marine environment

Nitrite Reduction

+ H CuNIR CuHAO NO + H O 2 H O + NH OH NXOR 22 4H+ + HNO 2 pcy H2 O + HNO 2H+ + HNO2 4 e- C I NH + O 2 e- H+ 2 H+ 4 H+ 3 2 H+ Fig. 1. Proposed AOA respiratory pathway. Text indi- Out pcy pcy cates the described possible hydroxylamine (blue text Q Q Q and arrows) and nitroxyl (green) pathways. Red arrows NDH I ATPase QH QH QH 2 2 aa indicate electron flow not involved in ammonia oxida- AMO 2 e- 2 QRED 3 tion. Blue shading denotes blue copper-containing pro- In + + 0.5 O NAD + H NADH + 2 H O teins. Pink box indicates possible alternative respiratory H 2 ADP + Pi ATP Ammonia Metabolism + + 2 H H+ 4 H 4 H+ electron sink. Hexagons containing Q and QH2 represent Complex I Complex III Complex IV Complex V the oxidized and reduced quinone pools, respectively.

8820 | www.pnas.org/cgi/doi/10.1073/pnas.0913533107 Walker et al. (e.g., aminoethylphosphonate), there is as yet no support for (TBP) (Table S3) genes required for starting site-specific tran- a functional phosphonate utilization pathway. Numerous organic scription initiation, making N. maritimus among the densest and transport functions are also evident, broadly encompassing trans- richest archaeal genomes for these transcription factors. TFB porters for different amino acids, dipeptides/oligopeptides, sul- and TBP are thought to serve functions similar to the bacterial fonates/taurine, and glycerol. Additional physiological charac- sigma factors (e.g., modulating cellular function in response to terization will likely demonstrate some capacity for mixotrophic fluctuating environmental conditions) in Archaea with genomes growth, as suggested by isotopic studies of natural populations coding for multiple copies, with optimal TFB/TPB partners (47). (40–42). No genomic evidence exists for growth on urea, as Although many other archaeal genomes contain multiple copies N. maritimus lacks the homologs of the putative urease and urea of these transcription factors, only the have more transporter genes identified in C. symbiosum (35). than five TFB genes (47). The functional significance of this exceptionally high density of regulatory factors in an apparently Noncoding RNA Genes. The N. maritimus genome contains a full metabolically specialized organism will likely be informed by complement of essential noncoding RNA (ncRNA) genes, in- future transcriptional analyses of different growth states. Genes cluding one copy each of 5S/16S/23S ribosomal RNAs, RNase P, for two widely distributed types of archaeal chromatin proteins SRP RNA, and 44 transfer RNAs (Table S3). In addition to six are present, an archaeal histone (Nmar_0683) and two Alba normally placed canonical tRNA introns, noncanonical introns genes (Nmar_0255 and Nmar_0933). These are thought to were found at different positions in six of the tRNAs (ValCAC, Met, maintain chromosomal material in a state permitting polymerase Trp, ArgCCT, LeuTAA, and GluTTC), a phenomenon previously accessibility, with differential expression possibly providing for observed only in and C. symbiosum (34, 43). All altered global transcription (48). other sequenced crenarchaea (including C. symbiosum) contain at least 46 tRNAs. N. maritimus lacks tRNA sequences coding for Unique Cell Division Machinery and Previously Uncharacterized Instance ProCGG or ArgCCG, perhaps resulting from (or related to) the low of Archaeal Biosynthesis of Hydroxyectoine. The N. maritimus genome G + C content of the genome and preference for protein codons contains elements homologous with two systems of cell division: ftsZ ending in A/T. Other archaeal genomes with relatively low G + C (Nmar_1262) and cdvABC (Nmar_0700, _0816, and _1088). The content, such as the euryarchaeon Haloquadratum walsbyi, also cdvABC operon, induced at the onset of genome segregation and cell lack these tRNAs, while possessing the exact complement of 44 division, codes for machinery related to the eukaryotic endosomal tRNAs found in N. maritimus (43). This occurrence likely reflects sorting complex (49, 50). With the exception of N. maritimus, C. a difference in posttranscriptional modification of the wobble symbiosum, and the Thermoproteales (where the cell division ma- of tRNAs ProTGG and ArgTCG, allowing more efficient decoding chinery remains uncharacterized), all available archaeal genomes of the rare codons CCG and CGG, respectively (44). have either the FtsZ or the Cdv cell division machinery, but not both. Six candidates for C/D box small RNAs (sRNAs) were identified The two cell division systems may comprise a hybrid mechanism or (Nmar_sR1–sR6). Most C/D box sRNAs guide the precise posi- serve two distinct processes in marine Crenarchaeota. tioning of posttranscriptional 2′-O-methyl group addition to rRNAs ThegenomeofN. maritimus also encodes for synthesis of or tRNAs, a process also occurring in , but not Bacteria.In thecompatiblesolutehydroxyectoine:ectoinesynthase(ectC,Nmar_ N. maritimus, predicted targets of 2′-O-methylation may include the 1344), a L-2,4-diaminobutyrate transaminase (ectB, Nmar_1345), wobble base of the LeuCAA tRNA and two different positions sep- a L-2,4-diaminobutyrate acetyltransferase (ectA, Nmar_1346), and an arated by 26 nucleotides in the 23S rRNA. Before the N. maritimus ectoine hydroxylase (ectD, Nmar_1343). Although widely distributed and C. symbiosum genomes, multiple C/D box sRNAs were found among Bacteria (in particular the genome sequences of marine almost exclusively in hyperthermophilic archaea (45). Detection of organisms), the presence of these genes in N. maritimus represents these conserved, syntenic guide sRNAs in the two mesophilic cren- a unique indication of archaeal biosynthesis. archaeal genomes supports an RNA stabilization/chaperone func- tion not seen in other archaeal mesophiles and possibly more similar Relationship to C. symbiosum. The genome of N. maritimus differs to their predicted function(s) in eukaryotes (46). significantly in G + C content and size from that of the closely re- lated sponge symbiont (∼97% 16S rRNA gene sequence identity). Regulation of Transcription. The genome contains at least eight Despite the differences in overall genomic G + C content (34.2% transcription factor B (TFB) and two TATA-box binding protein for N. maritimus versus 57.7% for C. symbiosum), the G + C content

AB

C

Fig. 2. Synteny plots comparing the N. maritimus genome with (A)theCenarchaeum symbiosum A type genome, (B) crenarchaeal genome fragments, and MICROBIOLOGY (C) Sargasso Sea fosmids. Vertical gray bar indicates location of ribosomal RNA operon.

Walker et al. PNAS | May 11, 2010 | vol. 107 | no. 19 | 8821 conservation of gene content and gene order with numerous ar- chaeal sequences previously recovered in fosmid libraries and re- cent oceanic surveys. The Antarctic genome fragments DeepAnt- EC39 (taken from 500 m depth) (52) and cosmid 74A4 (from sur- face ) (53) both share very high synteny with portions of the N. maritimus genome (Fig. 2B and Table S6) despite significant dif- ferences in rRNA sequence identity (93 and 98%, respectively). Sixteen Sargasso Sea contigs (93% 16S rRNA gene sequence identity with N. maritimus)alsohavehighsynteny(Fig.2C and Table S6). Retrieval of DNA fragments from the Global Ocean Sampling (GOS) database using differential protein sequence similarity showed that N. maritimus-like sequences constituted an average of 1.15% of all sequences across a wide range of temper- atures (9–29 °C), salinities (freshwater to seawater, 0.1–63 practical salinity units), two open oceans, and several coastal environments (Fig. 3A). The Block Island, NY, coastal site and the Lake Gatun, Panama Canal, site (neither of which share any notable physical/ chemical characteristics other than sample depth) both exhibited notably large increases in density (>2.5%). The available GOS sequences provided almost complete and uniform coverage of the N. maritimus genome (Fig. 3B), although at least three significant gaps in coverage exist (possibly corresponding with unique N. maritimus genomic islands). Whereas some of the coverage may result in matches to bacterial sequences, particularly for very highly conserved proteins, the majority of recruited proteins had >50% Fig. 3. Comparison of N. maritimus genome to metagenomic sequence reads sequence identity to N. maritimus proteins. Together, these shared from GOS. (A) Percentage of reads from each GOS site that align to the N. genomic features suggest N. maritimus is representative of many of maritimus genome by protein sequence similarity. (B) Number of GOS reads the globally abundant marine Group I Crenarchaeaota and that it homologous to each N. maritimus protein-coding gene. Counts of 40 are mostly due to highly conserved proteins that include contaminants from other clades. should provide a useful model for developing an understanding of the basic physiology of these abundant and cosmopolitan organisms. A comparison of the sequence content and genome organi- for the rRNAs is largely similar between both organisms (50–52%). zation hints at functionally more divergent marine population The higher ORF density (1.19 ORF/kb) relative to C. symbiosum types. N. maritimus has limited syntenic similarity to a deep- (0.986 ORF/kb) results principally from the 0.4 Mbp smaller ge- water population represented by the North Pacific fosmid 4B7 nome of N. maritimus, not from a large disparity in the number of (93% 16S rRNA sequence identity), but shares proteins highly predicted ORFs. The two genomes share 1,267 genes in common similar to most of those coded on this fosmid. Previous com- (when compared via reciprocal BLAST with expectation cutoff parison of marine crenarchaeal genomic fragments reported − values of 10 4), yet there is little conservation of synteny (Fig. 2C, changes in genomic organization with sampling depths, sugges- Table S4). Most of the increased size of the C. symbiosum genome tive of depth-related habitat types (54). Coupled with recent and much of the divergence in gene content are associated with evidence indicating varied physiological lifestyles along depth discrete regions (“islands”), although no obvious functionality could and latitudinal gradients, distinct crenarchaeal ecotypes likely be assigned to individual islands. Homologs for a majority of genes exist, analogous to that observed in marine cyanobacteria (2, 55). implicated in the archaeal ammonia oxidation pathway (51 of 69 However, no clear correlations currently exist between environ- listed in Table S2) appear present in C. symbiosum. mental parameters and the similarity of recruited fragments. The genome sequence presented here also offers further insight Phylogeny and Evolution. The widely distributed Group I archaeal into the ecological success of AOA. For example, using the likely more lineage with which N. maritimus is affiliated was earlier assigned to energy-efficient 3-hydroxypropionate/4-hydroxybutyrate pathway for the hyperthermophilic Crenarchaeota (3). Questions regarding this CO2 fixation rather than the Calvin–Bassham–Benson cycle used by association arose through phylogenetic analysis of C. symbiosum AOB could provide a growth advantage. Further ecological advantage ribosomal proteins, indicating possible divergence before the may be conferred by their potential capacity for mixotrophic growth or Crenarchaeota–Euryarchaeota split and therefore deserving pro- the use of copper as a major redox-active metal for respiration in visional assignment to a new archaeal kingdom, the Thaumarch- generally iron-limited oceans. However, a deeper understanding of aeota (51). The basal position of the Group I archaea previously the remarkable success of this archaeal lineage will come only from inferred from protein sequences encoded by the C. symbiosum ge- more detailed physiological, biochemical, and genetic characteriza- nome was reexamined by reanalysis of the combined dataset, using tion of N. maritimus and additional environmental isolates. patterns of gene distribution (Table S5) and phylogeny inference. Maximum-likelihood analyses confirmed the basal branching with Materials and Methods significant statistical support (bootstrap value = 90%, Fig. S4). Genome sequencing was performed on high-molecular-weight DNA Bayesian analysis of a selection of species from the same dataset extracted from two cultures of N. maritimus. Whole-genome shotgun se- produced results linking C. symbiosum and N. maritimus as sister quencing of 3-, 8-, and 40-kb DNA libraries by the Joint Genome Institute taxa of Crenarchaeota, albeit with nonsignificant support (posterior produced at least 8× coverage. Annotation of the closed genome was per- probability = 0.88). Although a definitive placement within the formed using Department of Energy (DOE) computational support at Oak fi Ridge National Laboratory and The Institute for Genomic Research (TIGR) Archaea still must be con rmed by inclusion of genomes from more Autoannotation Service in conjunction with Manatee visualization software. distantly related lineages, both analyses strongly support a lineage fi Crenarchaeota Complete details describing high-molecular-weight DNA puri cation and distinct from all other cultivated . sequence analysis are found in SI Materials and Methods.

High Similarity to the Metagenome of the Globally Distributed Marine ACKNOWLEDGMENTS. The authors thank David Bruce and Paul Richardson Group I Archaea. The genome of N. maritimus shares remarkable from the Joint Genome Institute for facilitating genome sequencing. This

8822 | www.pnas.org/cgi/doi/10.1073/pnas.0913533107 Walker et al. work was supported by the Department of Energy Microbial Genome and OCE-0623908 (to S.M.S.), by National Science Foundation Grant EF- Program, by National Science Foundation Microbial Interactions and Processes 0412129 (to M.G.K.), by incentive funds from the University of Louisville VP Grant MCB-0604448 (to D.A.S. and J.R.d.l.T.), by National Science Foundation Research office (to M.G.K.), by the Deutsche Forschungsgemeinschaft (M.K.), Molecular and Cellular Biosciences Grant MCB-0920741 (to D.A.S.), by National by US Department of Agriculture Grant 2010-65115-20380 (to A.C.R.), and by Science Foundation Biological Oceanography Grants OCE-0623174 (to D.A.S.) a Salk Institute Innovation Grant (to G.M.).

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Walker et al. PNAS | May 11, 2010 | vol. 107 | no. 19 | 8823 Supporting Information

Walker et al. 10.1073/pnas.0913533107 SI Materials and Methods with a comparison library generated through WebACT (www. High-Molecular-Weight Genomic DNA Preparation. Two cultures of webact.org/WebACT/home). Orthologous genes shared between Nitrosopumilus maritimus strain SCM1 were grown as previously these two organisms were identified through reciprocal BLAST −4 described in 500 mL of media in 1-L flasks (1). Cells from both searches, with an expectation cutoff value of 10 and a minimum of cultures were harvested in late-exponential phase using Sterivex 75% alignable N. maritimus sequence. Comparisons with the filters for one culture and a 0.1-μm filter for the other. High- Global Ocean Sampling (GOS) and Sargasso Sea metagenomic molecular-weight DNA was isolated as previously described using datasets were performed using several single-copy universal ar- either agarose plugs (2) or a modified guanadinium thiocynate chaeal genes to determine a count of ∼15 archaeal genomes in the protocol (3). Cells from the Sterivex filter were resuspended in GOS dataset. An initial set of candidate N. maritimus-like proteins 1mLof2× STE buffer [1 M NaCl, 0.1 M EDTA (pH 8.0), 10 mM was found by BLASTP searches with each N. maritimus protein- Tris (pH 8.0)], extracted from the filter, and mixed with 1 vol of 1% coding gene, using a cutoff of 100 hits and a maximum expected molten SeaPlaque LMP agarose (FMC). The mixture was cooled value of e = 10. This set consisted of 125,326 peptides drawn from to 40 °C, immediately drawn into a 1-mL syringe, and placed on ice 107,223 scaffolds. Neighboring ORFs on any scaffold with two or for 10 min. The agarose plug was mixed with 10 mL of lysis buffer, more hits were added to make a total of 319,585 peptides, incubated at 37 °C for 1 h, and then transferred to 40 mL of ESP amounting to 5.2% of all ORFs in GOS. These sequences were buffer (1% Sarkosyl–1 mg of Proteinase K per ml in 0.5 M EDTA). filtered by BLAST alignment to four sequence datasets, containing After incubation at 55 °C for 16 h, the solution was replaced with 24 complete proteomes from diverse euryarchaeota, crenarchaeota, fresh ESP buffer and incubated at 55 °C for another hour. DNA and bacteria. Sequences scoring more highly to N. maritimus and/or was purified using phenol:chloroform:isoamyl alcohol (24:24:1) C. symbiosum proteins than any other entry in these datasets were and recovered by precipitation with isopropanol. retained, giving a filtered dataset of 21,278 ORFs. Coverage of the Cells collected on the 0.1-μm filter were resuspended in 100 μLof N. maritimus proteome was measured by bidirectional BLASTP of Tris-EDTA (pH 8.0) and 100 mg/mL lysozyme before incubating at N. maritimus proteins against the filtered dataset to assign putative 37 °C for 30 min. Then, 3.0 mL of a solution containing 5 M guani- orthologs. The average coverage was ∼11×, although some highly dinium thiocyanate, 100 mM EDTA (pH 8.0), and 0.5% (vol/vol) conserved genes had a much greater number of hits, probably due to sarkosyl was added. The solution was mixed gently for 15 min before recruitment of nonarchaeal homologs: 48 ORFs had >30 hits, of being cooled on ice for 10 min. After cooling, an equal volume of cold which most were highly conserved. Searches for CRISPR regions 7.5 M acetate was added, and the solution was mixed were performed using the Java-based CRISPR recognition tool gently and cooled on ice. Purification and precipitation of DNA were (CRT) with least stringent settings (4). performed with chloroform:isoamyl alcohol (24:1) and isopropanol. Maximum-Likelihood and Bayesian Trees of the Archaeal Domain. Genome Sequencing. A completely sequenced and closed genome The trees are based on the concatenation of ribosomal proteins of N. maritimus was obtained through collaboration with the used by Brochier-Armanet et al. (5) but including sequences from N. Joint Genome Institute. Whole-genome shotgun sequencing of maritimus and from “Candidatus Korarchaeum cryptofilum” OPF8. 3-, 8-, and 40-kb DNA libraries produced at least 8× coverage of Sequences were aligned using MUSCLE (6). Resulting alignments the entire genome. Specifics of clone library generation, se- were visually inspected and improved with the MUST software (7). quencing, and assembly strategies may be found at the DOE JGI Regions where homology between sites was doubtful were removed website (www.jgi.doe.gov/sequencing/index.html). from further phylogenetic analyses. A total of 6,142 positions were kept for the phylogenetic analyses. The maximum-likelihood tree Genome Sequence Analysis. Autoannotation of the closed genome was computed with PHYML, using the WAG model corrected by sequence was performed by both the Computational Biology a gamma law to take into account evolutionary rate among site group at Oak Ridge National Laboratory (http://genome.ornl. variations (8). The parameteralpha of the gamma distribution asthe gov/microbial/nmar/02jul07/) and the TIGR Autoannotation proportion of invariable sites was estimated from the dataset. The Service (now hosted by JCVI; details available from http://www. robustness of each branch was estimated by the bootstrap procedure jcvi.org/cms/research/projects/annotation-service/overview/). The implemented in PHYML. A Bayesian tree analysis on a subset of 29 genome visualization software Manatee (release 2.4.1; latest taxa was performed using MrBayes 3.2 (9) with a mixed model of version available from http://manatee.sourceforge.net/) was used amino acid substitution and a gamma distribution (eight discrete for manual curation. Analysis of potential transporter genes was categories and an estimated proportion of invariant sites) to take performed using the Transporter Automatic Annotation Pipe- into account among-site rate variation. MrBayes was run with four line (TransAAP) through the TransportDB genomic comparison chains for 1 million generations and trees were sampled every 100 tool (membranetransport.org). generations. To construct the consensus tree, the first 1,500 trees Direct comparisons with the C. symbiosum genome and genome were discarded as ‘‘burn-in.’’ The reduction of the taxonomic sam- fragments were performed using the Artemis Comparison Tool (3) pling was necessary to reduce the computation time.

1. Könneke M, et al. (2005) Isolation of an autotrophic ammonia-oxidizing marine 6. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high archaeon. Nature 437:543–546. throughput. Nucleic Acids Res 32:1792–1797. 2. Stein LY, et al. (2007) Whole-genome analysis of the ammonia-oxidizing bacterium, 7. Philippe H (1993) MUST, a computer package of management utilities for sequences Nitrosomonas eutropha C91: Implications for niche adaptation. Environ Microbiol 9: and trees. Nucleic Acids Res 21:5264–5272. 2993–3007. 8. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large 3. Carver TJ, et al. (2005) ACT: The Artemis comparison tool. Bioinformatics 21:3422–3423. phylogenies by maximum likelihood. Syst Biol 52:696–704. 4. Bland C, et al. (2007) CRISPR recognition tool (CRT): A tool for automatic detection of 9. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8:209. mixed models. Bioinformatics 19:1572–1574. 5. Brochier-Armanet C, Boussau B, Gribaldo S, Forterre P (2008) Mesophilic Crenarchaeota: Proposal for a third archaeal phylum, the Thaumarchaeota. Nat Rev Microbiol 6:245–252.

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Thermoplasma volcanium GSS1 [rusticyanin; gi13542283] Acidithiobacillus ferrooxidans [rusticyanin; gi3282064] Thermobaculum terrenum ATCC BAA-798 [plastocyanin; gi227373287] Sphaerobacter thermophilus DSM 20745 [plastocyanin; gi229876615] Streptomyces sp. AA4 [hypothetical protein; gi256667890] Burkholderia cenocepacia PC184 [plastocyanin; gi254250279] 95 Catenulispora acidiphila DSM 44928 [blue (type 1) copper domain protein; gi256391720] Mycobacterium intracellulare ATCC 13950 [hypothetical protein; gi254819204] 83 Mycobacterium avium subsp. avium ATCC 25291 [copper-binding protein; gi254776845] 99 Mycobacterium avium 104 [copper-binding protein; gi118462975] Nmar0747 A. 96 Nmar0361 Candidatus Koribacter versatillis [plastocyanin-like protein; gi94968815] Nmar0902 Chromobacterium violaceum ATCC 12472 [hypothetical protein; gi34498952] Methylocella silvestris BL2 [blue (type 1) copper domain protein; gi217976580] Methylobacterium nodulans ORS [blue (type 1) copper domain protein; gi220920817] Methylocella silvestris BL2 [blue (type 1) copper domain protein; gi217976580] 61 Nitrobacter hamburgensis X14 [blue (type 1) copper domain protein; gi92118183] Bradyrhizobium japonicum USDA 110 [putative Amicyanin precursor; gi27378126] Paracoccus denitrificans PD1222 [amicyanin; gi119387457] Nmar0190 Methanosarcina mazei Go1 [hypothetical protein; gi21226177] 64 Methanosarcina acetivorans C2A [hypothetical protein; gi20090537] C. symbiosum A [copper-binding protein; gi118575216] 99 Nmar0167 99 Nmar0621 Methanosarcina mazei Go1 [copper-binding protein; gi21228445] Methanosarcina acetivorans C2A [plastocyanin/azurin family copper-binding protein; gi20090218] 62 Methanosarcina mazei Go1 [copper-binding protein; gi21228446] 100 Methanosarcina acetivorans C2A [plastocyanin/azurin family copper-binding protein; gi20090219] sp. CCS1 [blue (type 1) copper domain protein; gi89056161] Jannaschia Nmar0718 Thermobaculum terrenum ATCC BAA-798 [plastocyanin; gi227373254] Deinococcus deserti VCD115 [copper-binding protein; gi226356528] Synechococcus sp. WH 8102 [plastocyanin; gi33866031] 91 Prochlorococcus marinus MIT 9312 [plastocyanin; gi78778966] 88 Anabaena variabilisATCC 29413 [plastocyanin; gi75908957] 79 Gloeobacter violaceus PCC 7421 [plastocyanin; gi35212909] Methanococcus maripaludis C7 [copper-binding protein; gi150402166] 82 Methanococcus maripaludis S2 [copper-binding protein; gi45358560] Nmar1913 82 Nmar1789 Nmar0300 Nmar0762 C. symbiosum A [copper-binding protein; gi118576966] 100 C. symbiosum A [copper-binding protein; gi118576965] Nmar0516 Nmar1087 C. symbiosum A [hypothetical protein; gi118575848] 98 C. symbiosum A [hypothetical protein; gi118575831] C. symbiosum A [copper-binding protein; gi118575244] 79 Nmar0121 uncultured marine Crenarchaeote [putative copper-binding protein; gi167045254] 100 uncultured marine Crenarchaeote [putative copper-binding protein; gi167044521] 100 uncultured marine Crenarchaeote [putative copper-binding protein; gi167042919] C. symbiosum A [copper-binding protein; gi118575724] 100 Nmar0273 Nmar0732 Nmar0121 Nmar0324 79 B. Nmar0167 80 C. symbiosum A [copper-binding protein; gi118576967] Nmar0190

Nmar0273

Nmar0300

Nmar0324

Nmar0361

Nmar0516

Nmar0621

Nmar0718

Nmar0732

Nmar0747

Nmar0762

Nmar1087

Nmar1789

Nmar1913

100 aa

0.1 Bacillus subtilis subsp. subtilis str. 168 [BdbD thiol-disulfide oxidoreductase; gi 16080401] Bacillus amyloliquefaciens FZB42 [BdbD; gi 154687467] Paenibacillus larvae subsp. larvae BRL-230010 [disulfide dehydrogenase D; gi 167461952] Deinococcus geothermalis DSM 11300 [DsbA oxidoreductase; gi 94984799] Thermobaculum terrenum ATCC BAA-798 [protein-disulfide isomerase; gi 227374421] 100 Gemmatimonas aurantiaca T-27 [putative oxidoreductase; gi 226228008] Sphaerobacter thermophilus DSM 20745 [protein-disulfide isomerase; gi 229876989] Thermomicrobium roseum DSM 5159 [DsbA oxidoreductase; gi 221635547] 100 Y51MC23 [DsbA oxidoreductase; gi 218295130] 87 Thermobifida fusca YX [protein-disulfide isomerase; gi 72161925] C. Mycobacterium kansasii ATCC 12478 [DsbA oxidoreductase; gi 240169146] Rubrobacter xylanophilus DSM 9941 [DsbA oxidoreductase; gi 108804655] 82 A3(2) [hypothetical protein SCO5993; gi 21224330] 75 Streptomyces coelicolor 100 Streptomyces ghanaensis ATCC 14672 [hypothetical protein SghaA1_08748; gi 239928300] Symbiobacterium thermophilum IAM 14863 [hypothetical protein STH2058; gi 51893196] Roseiflexus castenholzii DSM 13941 [DsbA oxidoreductase; gi 156741642] 100 Chloroflexus aggregans DSM 9485 [DsbA oxidoreductase; gi 219849651] Chloroflexus aggregans DSM 9485 [DsbA oxidoreductase; gi 219847445] 99 Marinobacter algicola DG893 [DsbA oxidoreductase; gi 149377658] Vibrio harveyi HY01 [DsbA oxidoreductase; gi 153832115] RIMD 2210633 [hypothetical protein VPA0994; gi 28900849] 100 Vibrio parahaemolyticus Moritella sp. PE36 [putative membrane protein; gi 149908466] 99 Shewanella benthica KT99 [hypothetical protein KT99_10483; gi 163751466] Roseiflexus castenholzii DSM 13941 [protein-disulfide isomerase-like protein; gi 156741356] DSM 13941 [DsbA oxidoreductase; gi 156743646] 75 Roseiflexus castenholzii J-10-fl [DsbA oxidoreductase; gi 163848707] Herpetosiphon aurantiacus ATCC 23779 [DsbA oxidoreductase; gi 159896786] J-10-fl [DsbA oxidoreductase; gi 163845898] 72 Chloroflexus aurantiacus 100 Chloroflexus aggregans DSM 9485 [DsbA oxidoreductase; gi 219850456] Stigmatella aurantiaca DW4/3-1 [conserved hypothetical protein; gi 115374845] Solibacter usitatus Ellin6076 [DsbA oxidoreductase; gi 116625220] Solibacter usitatus Ellin6076 [DsbA oxidoreductase; gi 116624599] 82 Syntrophobacter fumaroxidans MPOB [DsbA oxidoreductase; gi 116751066] Anaeromyxobacter dehalogenans 2CP-1 [DsbA oxidoreductase; gi 220919173] DK 1622 [putative lipoprotein; gi 108762810] 100 Myxococcus xanthus Stigmatella aurantiaca DW4/3-1 [disulfide interchange protein; gi 115379912] Archaeoglobus fulgidus DSM 4304 [hypothetical protein AF1354; gi 11498950] Cenarchaeum symbiosum A [protein-disulfide isomerase; gi 118575694] Nmar0752 93 91 Nmar0179 Nmar0218 Nmar0740 73 79 Nmar0175 Natrialba magadii ATCC 43099 ]DsbA oxidoreductase; gi 224823141] Nmar1863 90 Cenarchaeum symbiosum A [protein-disulfide isomerase; gi 118576454] Nmar1805 67 100 Cenarchaeum symbiosum A [protein-disulfide isomerase; gi 118576169] Nmar1606 63 Nmar0164 72 Nmar0169 Nmar1590 88 Cenarchaeum symbiosum A [protein-disulfide isomerase; gi 118575427]

Fig. S1. Phylogeny of plastocyanin-like protein sequences. Sequences with significant matches to COG3794 (PetE: Plastocyanin [Energy production and conversion]) were used to query the non-redundant protein sequence database from NCBI. Sequences from the top 20–30 non-N. maritimus hits were re- trieved and their match to the above conserved domain model verified. Sequences from experimentally characterized proteins were obtained from the available literature and included, aligned with ClustalW and then curated manually. Distance-based phylogenies were inferred in Phylip using the Neighbor- Joining algorithm and 100 bootstrap replicates. Bootstrap support values >60% are displayed. Nodes with <50% bootstrap support were collapsed.

Walker et al. www.pnas.org/cgi/content/short/0913533107 2of5 Fig. S2. Archaeal ammonia monooxygenase AmoB sequence mapped onto the crystal structure of the particulate methane monooxygenase (PDB accession code 1YEW). The pmoA subunit is shown in dark gray, the pmoC subunit in light gray, and the pmoB subunit in red and pink. The red part represents the region of pmoB conserved in the predicted N. maritimus AmoB. The transmembrane helix and C-terminal cupredoxin domain shown in pink are missing in the predicted N. maritimus AmoB. Cyan spheres represent copper ions. The grey sphere represents a zinc ion.

Walker et al. www.pnas.org/cgi/content/short/0913533107 3of5 Acetoacetyl-CoA -ketothiolase (Nmar_0841 or Nmar_1631)

3-Hydroxybutyryl-CoA dehydrogenase (Nmar_1028)

Crotonyl-CoA hydratase (Nmar_1308) Acetyl-CoA carboxylase (Nmar_0272, 0273, 0274) 4-Hydroxybutyryl-CoA dehydratase (Nmar_0207) Malonyl-CoA reductase Malonate semialdehyde reductase (unknow n) 4-Hydroxybutyryl-CoA synthetase (Nmar_0206)

3-Hydroxypropionyl-CoA synthetase 3-Hydroxypropionyl-CoA dehydratase Acryloyl-CoA reductase (unknow n) Succinate semialdehyde reductase (Nmar_1110 or Nmar_0161)

Propionyl-CoA carboxylase Succinyl-CoA reductase (Nmar_0272, 0273, 0274) (Nmar_1608)

Methylmalonyl-CoA epimerase Methylmalonyl-CoA mutase (Nmar_0953, 0954, 0958)

Fig. S3. Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation by N. maritimus.

Walker et al. www.pnas.org/cgi/content/short/0913533107 4of5 A. Giardia lamblia Entamoeba histolytica Leishmania major 100 Trypanosoma cruzi 100 100 Trypanosoma brucei Cryptosporidium parvum 64 100 Theileria parva 100 Plasmodium yoelii Eucarya 100 Plasmodium falciparum 100 Arabidopsis thaliana 100 Oryza sativa 97 Dictyostelium discoideum Homo sapiens 51 100 Anopheles gambiae 86 Saccharomyces cerevisiae 100 Schizosaccharomyces pombe Cenarchaeum symbiosum 100 Candidatus Nitrosopumilus maritimus Thaumarchaeota Candidatus Korarchaeum cryptofilum Korarchaeota Thermofilum pendens 52 100 Caldivirga maquilingensis 100 Pyrobaculum calidifontis Thermoproteales C 100 Pyrobaculum islandicum r 100 94 Pyrobaculum aerophilum e 100 100 Pyrobaculum arsenaticum n 100 Ignicoccus hospitalis a

Staphylothermus marinus rc 61 Aeropyrum pernix Desulfurococcales ha 100 70 Hyperthermus butylicus 90 Metallosphaera sedula 100 Sulfolobus solfataricus e 90 Sulfolobus acidocaldarius Sulfolobales ot 100

Sulfolobus tokodaii a Nanoarchaeum equitans Nanoarchaeota Thermococcus gammatolerans 100 Thermococcus kodakarensis 96 100 Thermococcales 100 Pyrococcus abyssi 100 Pyrococcus horikoshii 97 kandleri Methanopyrales Methanosphaera stadtmanae Methanobacteriales 55 100 Methanothermobacter thermautotrophicus Methanocaldococcus jannaschii 78 100 Methanococcus aeolicus 100 Methanococcus maripaludis Methanococcales 100 Methanococcus vannielii E 38 Picrophilus torridus u

100 Ferroplasma acidarmanus r 100 Thermoplasma acidophilum Thermoplasmatales y 100 Thermoplasma volcanium a Archaeoglobus fulgidus Archaeoglobales rc 95 69 sp h

Natronomonas pharaonis ae 71 Haloarcula marismortui 88 100 Halorubrum lacusprofundi Halobacteriales o 100 Haloquadratum walsbyi 100 Haloferax volcanii ta 100 Methanocorpusculum labreanum 100 Methanoculleus marisnigri 75 Candidatus Methanoregula Methanomicrobiales 61 82 Methanospirillum hungatei Methanosaeta thermophila 100 Methanococcoides burtonii 100 Methanosarcina barkeri Methanosarcinales 0.1 100 Methanosarcina acetivorans 100 Methanosarcina mazei B. Saccharomyces cerevisiae Dictyostelium discoideum Eucarya 1.00 Oryza sativa Candidatus Korarchaeum cryptofilum Korarchaeota Candidatus Nitrosopumilus maritimus Thaumarchaeota 1.00 Cenarchaeum symbiosum 0.88 Pyrobaculum aerophilum C Thermoproteales r 1.00 1.00 Thermofilum pendens e n

1.00 1.00 Aeropyrum pernix Desulfurococcales archa Hyperthermus butylicus 1.00 0.88 Sulfolobus acidocaldarius 1.00 Sulfolobales Metallosphaera sedula e

Nanoarchaeum equitans Nanoarchaeota o t a Thermococcus kodakarensis Thermococcales 1.00 1.00 Pyrococcus abyssi Methanopyrus kandleri Methanopyrales 1.00 1.00 Methanothermobacter thermautotrophicus Methanobacteriales 1.00 Methanosphaera stadtmanae Methanocaldococcus jannaschii E 1.00 Methanococcales u 1.00 Methanococcus maripaludis r y

Thermoplasma volcanium a

1.00 rcha 1.00 Ferroplasma acidarmanus Thermoplasmatales 1.00 Archaeoglobus fulgidus Archaeoglobales Haloarcula marismortui eo 1.00 1.00 Halobacteriales Natronomonas pharaonis t a 1.00 1.00 Methanosarcina mazei Methanosarcinales Methanosaeta thermophila 0.70 Methanospirillum hungatei 0.1 1.00 Methanocorpusculum labreanum Methanomicrobiales

Fig. S4. (A) Maximum-likelihood phylogeny of Group 1 Archaea. The phylogeny was inferred using an alignment of concatenated R-proteins (66 taxa, 6,142 positions). WAG+Inv+Gamma (4 classes); 100 replicates. (B) Bayesian tree of mesophilic Group 1 Archaea inferred using an alignment of concatenated R- proteins (29 taxa, 6,142 positions). Mixed model + Gamma (4 classes); 100 replicates.

Other Supporting Information Files

Table S1 (DOC) Table S2 (DOC) Table S3 (DOC) Table S4 (DOC) Table S5 (DOC) Table S6 (DOC)

Walker et al. www.pnas.org/cgi/content/short/0913533107 5of5