Supporting InformationKoch et al. 10.1073/pnas.1506533112SI Results and DiscussionUrea Transport and Accessory Proteins of Urease in N. moscoviensis.Urea is a small uncharged molecule that diffuses readily throughthe lipid bilayers of bacterial membranes (52). Aside from passivediffusion, the transport of urea into bacterial cells is mediated byurea-specific channels such as UreI of Helicobacter (53) or by ATP-dependent ABC transporters such as UrtABCDE in Cyanobacteria(54). ABC transporters for urea consist of a periplasmic substrate-binding protein (UrtA), a dimer of the transmembrane proteinsUrtB and UrtC, which form a membrane-crossing pore, and theATP-binding and -hydrolyzing proteins UrtD and UrtE (54, 55).These ABC transporters show a high affinity for urea (52, 54) andmay represent an adaptation to environments with urea concen-trations in the micromolar range. Their expression is tightly regu-lated in dependence on N availability due to the energy demand ofthis transport system (54, 56). In the genome of N. lenta, the wholegene set (urtABCDE) of the urea ACB transport system is locatedupstream of the urease genes (Fig. S1A and Dataset S1), suggestingthat N. lenta possesses a high-affinity uptake system for urea and,thus, is adapted to habitats where low urea concentrations prevail.In contrast, in N. moscoviensis, only urtA, which encodes the peri-plasmic urea-binding protein UrtA, is located in close vicinity of theurease genes (Fig. S1A). Whether N. moscoviensis can replace thelacking UrtBCDE proteins with the respective subunits of otherABC transporters encoded in the genome, or whether urea is takenup by passive diffusion only, remains to be determined.The accessory proteins UreD, UreE, UreF, and UreG are

required for the formation of the Ni2+-containing metallocenterin the UreABC apoenzyme during the biosynthesis of urease(27). In addition, the nickel transporter UreH provides Ni2+

(27). The genome of N. moscoviensis contains the ureD,ureF, and ureG genes (Fig. S1A) and ureH (NITMOv2_1657).However, only a 180-nt-long (59 aa) gene fragment of ureE(NITMOv2_1661) was identified, which is unlikely to encode afunctional UreE protein because homologs in other organismsare approximately 200 aa in length. Although UreE is requiredfor a functional urease in Helicobacter (57), various other mi-croorganisms lacking ureE genes express active ureases (58). Inureolytic microbes without UreE, this nickel-binding metal-lochaperone (59) may be substituted by chaperones of othernickel-dependent enzymes (58). Interestingly, in Helicobacterpylori two accessory proteins of nickel-dependent hydrogenase,HypA and HypB, are required for the incorporation of Ni2+ intourease (60). These hydrogenase maturation factors are presentin N. moscoviensis, which possesses an active [NiFe] hydrogenase(23). Hence, hydrogenase chaperones might be involved in theassembly of urease and substitute UreE in this organism as well.

Utilization of Organic Substrates by N. moscoviensis. Aside fromformate (see Results and Discussion in the main text), we testedalso whether N. moscoviensis can use other simple organic com-pounds (acetate, fumarate, succinate, citrate, and pyruvate) incombination with nitrate as terminal electron acceptor. Acetatecould be provided by fermenting organisms in the spatial prox-imity of Nitrospira in hypoxic or anoxic habitats, whereas the othercompounds are key metabolites that could be released by lysedcells within a biofilm. The genetic repertoire of N. moscoviensisincludes the degradation pathways and the respiratory chainneeded to use these organic compounds (Fig. S1B). Transmem-brane transporters for these substrates were not identified in thegenome, but N. moscoviensis encodes permeases of unknownspecificities, and we could not exclude the possibility that such

transporters may facilitate the uptake of one or more of the testedorganics. However, no nitrate reduction was observed duringanoxic incubations with any of these substrates (Fig. S4B).

Core Metabolism of Nitrospira for Chemolithoautotrophic NitriteOxidation. A syntenic gene arrangement is conserved in rela-tively large parts of the N. moscoviensis and N. defluvii genomes(Fig. S7A). Shared genomic features with a highly conservedsynteny are the enzymatic repertoire for nitrite oxidation, theelectron transport chains for aerobic respiration and reverseelectron transport from nitrite to NAD+, and the reductive tri-carboxylic acid (rTCA) cycle for CO2 fixation and the oxidativeTCA cycle (Fig. S7 B–E). The high degree of similarity in thesepathways strongly supports the previous reconstruction of theNitrospira core metabolism for chemolithoautotrophic nitriteoxidation, which was based on only one sequenced genome (25).The few genetic differences in the core pathways include addi-tional (third) copies of respiratory complexes I and III, a secondcytochrome bd oxidase, and five paralogous copies of nitriteoxidoreductase (NXR) subunits NxrA and NxrB in N. mosco-viensis, whereas N. defluvii has only two paralogs of these NXRsubunits (25) (Fig. S7 C and D).NXR, the key enzyme for nitrite oxidation, belongs to

the complex iron–sulfur molybdoenzyme family with amolybdo-bis(pyranopterin guanine dinucleotide) cofactor-containing catalytic subunit (61). The NXR of Nitrospira is lo-cated in the periplasmic space and consists of at least twosubunits (NxrA and NxrB). The third (NxrC) subunit may an-chor the NXR complex in the cytoplasmic membrane andmediate the transfer of electrons from NXR to the membrane-bound electron transport chain (25). The five paralogs of nxrAand nxrB are clustered in three genomic regions of N. moscoviensis,whereas five putative nxrC genes are located elsewhere in thegenome (Fig. S7C). NxrA contains the substrate-binding sitewith the Mo cofactor (25). Like in N. defluvii, all NxrA paralogsof N. moscoviensis contain an N-terminal twin-arginine motiffor export via the twin-arginine protein translocation (Tat)pathway. The presence of this motif is consistent with theperiplasmic localization of the active site of NXR in N. moscoviensis(62) and N. defluvii (25). The periplasmic NXR is energeticallyadvantageous and likely explains the strong competitiveness undernitrite-limited conditions of Nitrospira compared with otherNOB such as Nitrobacter, whose NXR is located on the cytoplasmicside of the cell membrane (25).The amino acid similarities among the NXR subunits of

N. moscoviensis range from 95.7 to 98.5% for NxrA, from 99.5 to100% for NxrB, and from 18.6 to 64.1% for the putative NxrCcandidates. All five NxrA copies are more similar to one of thetwo NxrA paralogs (CDS tag Nide3255) (25) in N. defluvii (87.1–87.9%) than to the other one (Nide3237) (83.6–84.2%). Inter-estingly, the similarity between the two NxrA copies in N. defluviiis only 86.9% and, thus, lower than the similarity between allNxrA copies of N. moscoviensis and one NxrA (Nide3255) ofN. defluvii. It is tempting to speculate that the lower similaritybetween the two NxrA subunits of N. defluvii reflects a functionaldifferentiation, and that all NxrA of N. moscoviensis are func-tionally more similar to one of the NxrA paralogs (Nide3255) ofN. defluvii. Consistently, four of the five nxrA/B gene clusters inN. moscoviensis are preceded by transcriptional regulator genes,which are homologous to a regulator in N. defluvii that occursdirectly upstream of the gene encoding NxrA Nide3255 (Fig.S7C). The amino acid similarities between these regulators are

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relatively high (47–73%). If the genomic localization next tonxrA genes reflects a role of these regulators in the transcrip-tional control of NXR, then the regulation of these four NXRparalogs in N. moscoviensis may resemble the regulation ofNide3255 in N. defluvii. However, one of these nxr gene clustersin N. moscoviensis also contains a second transcriptional regu-lator, which is homologous to a regulator upstream of the secondNxrA copy of N. defluvii (Nide3237) (Fig. S7C). Hence, in bothorganisms, at least two different regulation mechanisms for NXRseem to be present that await confirmation and further analysisin future studies.Each of the five NxrC candidates in N. moscoviensis has a

homolog among the four putative NxrC subunits in N. defluvii(25) (Dataset S1), with two of the N. moscoviensis proteins(NITMOv2_3617 and NITMOv2_4208) being homologous toone candidate NxrC in N. defluvii (Nide3271). All NxrC candi-dates in both Nitrospira genomes have been identified based onsequence similarities to the membrane subunits of other DMSOreductase type II family enzymes (25), but their actual functionalroles and the composition of the NXR protein complex in Ni-trospira remain to be determined.

Assimilatory Nitrite Reduction by a Putative Octaheme Cytochrome cNitrite Reductase. The genome of N. moscoviensis lacks any genefor assimilatory nitrite reductase (NirA). The only gene relatedto nirA most likely encodes a ferredoxin-dependent sulfite re-ductase (NITMOV2_0334) and is involved in sulfur assimilationaccording to its localization close to the genes of sulfate ad-enylyltransferase.However, N. moscoviensis contains a gene for an octaheme

cytochrome c (OCC) protein, which is located in a region thatcontains various other genes for N acquisition including theurease (Fig. S1A). Some OCC proteins from other organisms areoctaheme nitrite reductases (ONRs) that reduce nitrite to am-monia (63–65). These ONRs may represent an evolutionary linkfrom pentaheme nitrite reductases (Nrf) to the OCC with oxi-dizing activities, hydroxylamine oxidoreductase (HAO) of aero-bic ammonia-oxidizing bacteria and hydrazine oxidoreductase(HZO) of anaerobic ammonium-oxidizing (anammox) organisms(63). The OCC of N. moscoviensis does not belong to this ONRgroup, but most closely resembles members of another clade (II.2)of the OCC family, which consists mostly of uncharacterized pro-teins. Interestingly, clade II.2 OCCs seem to be functionally versa-tile because a protein from this group from Beggiatoa had theactivities of ONR and also of HAO and HZO in vitro (65). As aHAO, the enzyme of N. moscoviensismight allow the detoxificationof hydroxylamine. Because Nitrospira usually share their habitatwith aerobic ammonia-oxidizing bacteria, they may easily encounterhydroxylamine in their direct environment.Interestingly, a clade II.1 OCC from Nautilia profundicola

is involved in the reverse hydroxylamine:ubiquinone reductasemodule (HURM) pathway that is used to reduce nitrate to am-monia (45). In this pathway, nitrite reduction to hydroxylamine bythe OCC is linked to quinol oxidation and proton dislocationacross the cell membrane by a cytochrome cM552-like protein(45). N. moscoviensis might use a similar pathway to link nitritereduction to ammonia with proton translocation. Two genes en-coding a Rieske/cytochrome b complex are located upstream ofthe OCC gene in its genome (Fig. S1A). It is tempting to speculatethat this Rieske/cytochrome b complex might generate protonmotive force by channeling electrons from the quinol pool to theOCC, similar to the reverse-HURM pathway. Assuming that it hasONR activity, the OCC would then reduce nitrite to ammonia.

Reactive Oxygen Defense. In contrast to most other aerobic bac-teria, N. defluvii lacks SOD and catalase, and its genome doesnot code for superoxide reductase either (25). The predictedalternative mechanisms for protection against ROS in N. defluvii

include ROS detoxification by manganese or polyamines, H2O2degradation by peroxidases and thioredoxin-dependent peroxir-edoxins, binding of free iron by bacterioferritin to reduce the riskof ROS generation, and free radical scavenging by carotenoids(25). In contrast to N. defluvii, N. moscoviensis possesses a ca-nonical SOD and a catalase (Fig. S1B and Dataset S1). The SODof N. moscoviensis (NITMOv2_2805) binds Fe or Mn based onits overall amino acid sequence similarity to other SODs thatrequire these metal cofactors. The Fe and Mn SODs are difficultto distinguish from each other by sequence analysis, but specificfingerprint residues (66) indicate that the enzyme of N. mosco-viensis may be a tetrameric Fe SOD. N. moscoviensis possesses atypical monofunctional, heme-containing catalase that is encoded bytwo identical gene copies (NITMOv2_0085 and NITMOv2_4696).Either catalase gene belongs to one of two identical copies of a42-kbp-large Tn7 mobile element. In addition to SOD and cat-alase, N. moscoviensis possesses the putative ROS defense mecha-nisms as predicted for N. defluvii (25) except that it lacks apolyamine transporter (Fig. S1B).

SI Materials and MethodsGenome Sequencing and Analysis. DNA was isolated from a pureculture of N. moscoviensis strain NSP M-1 (24) by followingthe hexadecyltrimethylammonium bromide (CTAB) protocol asdescribed (34). Following extraction, RNA was removed byRNase I digestion (Lucigen).Paired-end sequencing libraries were prepared by using the

NexteraDNASample Preparation Kit (Illumina) according to themanufacturer’s instructions. Mate-pair sequencing libraries wereprepared by using the Nextera Mate-Pair Sample PreparationKit (Illumina) using the gel-free protocol according to themanufacturer’s instructions. The sequencing libraries were se-quenced on an Illumina MiSeq DNA sequencer with 2 × 301 bpbyusing the MiSeq Reagent Kit v3 (Illumina). The paired-endreads were imported into the CLC genomics workbench software(version 6.5.1, CLCbio; Qiagen) and quality trimmed by requiring aminimum phred score of 20 and a minimum read length of 50 bp.Nextera adapters were removed if found. The trimmed paired-endreads were de novo assembled by using CLC genomics workbenchv. 6.5.1 using a kmer of 64 and otherwise default parameters. Thede novo assembly was manually inspected by using CLC genomicsworkbench v. 6.5.1 and the Circos (67) tools implemented in themultimetagenome workflow (68). The initial inspection resulted incleaning of misassembled contigs and identification of five copies ofthe NxrAB operon, which proved difficult to assemble because ofhigh similarity between the variants. To assemble the individualNxrAB operons, the nxrA and nxrB genes were separately PCR-amplified with region-specific primer sets, cloned in E. coli, andSanger-sequenced. The partially assembled NxrAB genes were re-placed with their Sanger-sequenced complete counterparts. Themate-pair reads were cleaned by using NextClip v.0.8 (69) withdefault parameters. Reads from Category A were imported intoCLC genomics workbench v. 6.5.1 and mapped to the assembly byusing the “map reads to reference” function with 95% similarityover 70% of the read length as cutoff. The mapping was visualizedby using the Circos (67) tools implemented in the multimetagenomeworkflow (68) and used to manually scaffold the initial assemblyinto a single scaffold. The gaps in the final scaffold were resolvedmanually by using CLC genomics workbench v.6.5.1 by mapping thetrimmed paired-end reads (minimum 2 × 250 bp) to the assemblywith 95% similarity over 30% of the read length as cutoff.The genome annotation platform MicroScope (49) was used

for the automated prediction and annotation of CDS. Homolo-gous proteins present in the N. moscoviensis and N. defluvii ge-nomes were identified by using the phyloprofile exploration toolof MicroScope. Only proteins sharing an amino acid sequenceidentity ≥ 30% over at least 80% of the sequence length wereconsidered as homologs. The annotation of all CDS discussed in

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this study was refined manually based on the same annotationcriteria that were already applied for the annotation of theN. defluvii genome (25). The whole genomes of N. defluvii andN. moscoviensis were aligned by using the PROmer tool ofthe MUMmer 3.23 software package (70). Matches < 333 aa(1,000 nt) were ignored to reduce the number of spurious matchesbetween partial protein sequences. Syntenic regions were iden-tified by the respective tools of MicroScope and visualized byusing Circos (67).

Metagenomic Screening for Nitrospira-Like ureC Sequences andPhylogenetic Analysis of the Urease Alpha Subunit. A large meta-genome from the Aalborg West Wastewater treatment plant inDenmark (MG-RAST ID: 4611649.3) was screened for the pres-ence of Nitrospira-like UreC proteins by first calling genes usingprodigal with the metagenome option (71) and then searching theresulting proteins using blastp.In total, 3,217 publicly available metagenomes in the integrated

microbial genomes (IMG) database (50) were screened by usingHMMER3 hmmsearch (72) with the hmm model (length = 121)for urease_alpha (PFAM PF00449; ref. 73) and default param-eters. Amino acid sequences identified with hmmsearch wererequired to match the hmm over at least 100 aa positions andscreened for similarity (>70%) to the UreC sequence from Ni-trospira moscoviensis. Sequences passing these criteria were thenclustered at 90% similarity by using Usearch 7.0 (74). Usearchcentroids (46 sequences) were used in an exploratory phyloge-netic analysis by using five independent chains of 1,100 iterations(600 burnin) with a randomized initial alignment in Bali-phy(51). The UreC protein sequences from N. moscoviensis andN. lenta formed a well-supported cluster (>99% posterior support)with seven centroids derived from metagenomes. All meta-genomic sequences that matched any of these seven centroidswith >90% amino acid identity were retained for phylogeneticanalysis. Additional genomic UreC sequences were identifiedwith blastp by using known ammonium-oxidizing and nitrite-oxidizing bacteria as query and default parameters. The 100 tophits were purged from highly similar sequences and clustered at90% similarity by using usearch 7.0 and centroids were retainedfor phylogenetic analysis. Phylogenetic reconstruction was car-ried out by using five independent chains of 1,100 iterations (600burn-in, randomized initial alignment) in Bali-phy (51), whichsimultaneously infers alignment and phylogeny from a set ofsequences. The 102 amino acid sequences ranged from 109 aa(C687J26615_103352331 from metagenome 3300002121) to595 aa (Microcoleus sp. WP_015182704). Posterior tree pools fromall five independent runs were combined to determine consensustopology and posterior support for bipartitions. Alignment lengthranged from 736 to 817 (mean = 767) in the posterior pool ofalignments.

Cultivation of N. moscoviensis and N. europaea. To obtain biomassfor experiments, N. moscoviensis was grown at 37 °C in mineralnitrite medium (23) without agitation and in the dark. This NOBmedium had the following composition: 1,000 mL of distilledwater; 0.01 g of CaCO3; 0.5 g of NaCl; 0.05 g of MgSO4·7H2O;0.15 g of KH2PO4; 34.4 μg of MnSO4·H2O; 50 μg of H3BO3; 70 μgof ZnCl2; 72.6 μg Na2MoO4·2H2O; 20 μg of CuCl2·2H2O; 24 μgof NiCl2·6H2O; 80 μg of CoCl2·6H2O; 1 mg of FeSO4·7H2O. Ifnot stated otherwise, 0.01 g of NH4Cl was added to the mediumas additional N source. After autoclaving, filter-sterilized NaNO2was added to a final concentration of 1 mM. The nitrite con-centration in the medium was regularly checked by using nitritetest stripes (Merkoquant; Merck), and nitrite was replenishedwhen completely consumed. N. europaea was grown at 30 °C on arotary shaker (100 rpm) in a modified AOB medium described(75). The modified medium composition and preparation protocolwere as follows: 900 mL of distilled water; 3.3 g of (NH4)2SO4;

0.41 g of KH2PO4; 0.184 g of MgSO4·7H2O; 0.022 g of CaCl2;0.12 mg of CuSO4·7H2O and 0.33 mL of a 30 mM FeSO4·7H2Osolution in 50 mM Na2EDTA·2H2O. After autoclaving, 100 mLof a sterile buffer solution (pH 8.0) containing 6.8 g of KH2PO4and 0.6 g of NaH2PO4 and 4 mL of a sterile 10% (wt/vol) NaCO3solution were added separately to adjust the pH to a value of 7.9.The purity of all nitrifier cultures, including incubated aliquots

before and after experiments, was checked by FISH with theEUB338 probe mix (76, 77) that targets most known Bacteria, theNitrospira lineage II-specific probe Ntspa1151 (78) for N. mosco-viensis cultures, the Nitrospira lineage I-specific probe Ntspa1431(78) for N. defluvii cultures, or the betaproteobacterial AOB-specific probe Nso1225 (79) for N. europaea cultures. For thispurpose, aliquots of the biomass were fixed in formaldehyde andFISH was carried out as described (80). Contaminations by otherbacteria were not detected in any experiment.

Incubation of N. moscoviensis with Urea. N. moscoviensis biomasswas harvested by centrifugation (8,228 × g, 10 min, 25 °C). Thecell pellet was resuspended in 30 mL of ammonium-free NOBmedium and 5-mL aliquots of the biomass were added to 300-mLSchott bottles containing 100 mL of ammonium-free NOB me-dium. To ensure activity of the cells, NaNO2 in the final con-centration of 0.35 mM was added to all incubations and thecultures were incubated at 37 °C in the dark. After 2 d, allNaNO2 was consumed and the respective substrates for thedifferent incubation conditions were added to the incubations inthe following final concentrations: 0.35 mM NaNO2 and 1 mMurea; only 1 mM urea; and as an activity control, only 0.35 mMNaNO2. All incubation conditions were performed in duplicates.In control experiments, medium containing 0.35 mM NaNO2and 1 mM urea and medium containing 1 mM urea was in-cubated without biomass. All incubations were performed for 6 hat 37 °C. Every 2 h, aliquots of the incubations were sampled andcentrifuged (17,949 × g, 10 min, 4 °C) to remove cells. In thesupernatant, ammonium was measured photometrically by thesalicylic acid assay (81).

Incubation of N. defluvii with Urea. N. defluvii biomass was har-vested by centrifugation (6,300 × g, 10 min, 25 °C). The cellpellet was resuspended in ammonium-free NOB medium, andequal aliquots of the biomass were added to 300-mL Schottbottles containing 100 mL of ammonium-free NOB medium. Toall incubations, 0.5 mM NaNO2 (final concentration) was addedand the cultures were incubated at 30 °C in the dark. After 3 d,all nitrite was consumed. Subsequently, 0.5 mM NaNO2 and1 mM urea, only 1 mM urea, or only 0.5 mM NaNO2 was addedto the respective incubations. All incubations were performed induplicates. In addition, medium containing 0.5 mM NaNO2 and1 mM urea and medium containing 1 mM urea was incubatedwithout biomass in control experiments. All incubations wereperformed at 30 °C for 6 h. Every 2 h, aliquots of all incubationswere sampled and centrifuged (17,949 × g for 10 min at 4 °C) toremove cells. In the supernatant, ammonium was measuredphotometrically by the salicylic acid assay (81).

Incubation of N. europaea with Urea. N. europaea biomass washarvested by centrifugation (6,300 × g for 20 min at 25 °C) andwashed by resuspending the cells in ammonium-free AOB me-dium. The biomass was centrifuged again, and the cell pellet wasresuspended in ammonium-free AOB medium. By using syrin-ges, aliquots (1 mL) of this mixture were added to 300-mL Schottflasks containing 50 mL of ammonium-free AOB medium. Thesebottles had already been plugged with butyl rubber stoppers,which were fixed with screw caps. The culture was incubated inthe presence of 1 mM urea for 7 d at 30 °C in the dark in du-plicates. During the incubations, aliquots of the cultures weresampled by using syringes and were centrifuged (14,000 × g for

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10 min at 4 °C) to remove cells. In the supernatant, nitrite wasmeasured photometrically by the Griess assay (82).

Coincubation of N. moscoviensis and N. europaea. N. moscoviensisand N. europaea biomass was harvested by centrifugation (6.300 × g,20 min, 25 °C), resuspended in AOB medium (see above) that didnot contain NH4

+, and centrifuged again. After this washing step,the cells were resuspended in ammonium-free AOB medium andmixed in a cell number ratio of 1:1 (details of the cell quantificationare provided below). By using syringes, aliquots (1 mL) of thismixture were added to 300-mL Schott flasks containing 50 mL ofammonium-free AOB medium. These bottles had already beenplugged with butyl rubber stoppers, which were fixed with screwcaps. The mixture was incubated in the presence of 1 mM or 50 μMurea as sole N and energy source for 7 d at 30 °C in duplicates.During the incubations, aliquots of the cultures were sampled byusing syringes and centrifuged (14,000 × g, 10 min, 4 °C) to removecells. In the supernatant, N compounds were measured photomet-rically by the salicylic acid assay (ammonium) (81) or the Griessassay (nitrite and nitrate) (82). Nitrate was reduced to nitrite beforethe measurements by addition of vanadium(III) (83), and additionalnitrite standards were included to account for inaccuracies in nitratedetermination at high nitrite concentrations.

Cell Counts for the Urea Coincubation Experiments with N. moscoviensisand N. europaea. To achieve a 1:1 ratio of N. moscoviensis andN. europaea in the coincubation experiments, the cell densities inthe axenic cultures of each organism were determined. Subse-quently, aliquots of each culture containing highly similar amountsof cells were washed and mixed (see above).For counting, cells were homogeneously suspended by vor-

texing, and aliquots of each pure culture (5 μL of N. moscoviensisand 25 μL of N. europaea) were diluted separately in 15 mL of1×PBS. The diluted cell suspensions were filtered separatelyonto black 0.2-μm polycarbonate GTBP-type membrane filters(Millipore). After washing the cells once in 1×PBS, the cellswere stained by incubation for 5 min in 400 μL of a 0.1 μg·mL−1

DAPI (4’,6-Diamidino-2-phenylindole) solution. Subsequently,the filters were washed again in 1×PBS and air-dried. The stainedcells were visualized on the dried filters by epifluorescence mi-croscopy (AXIO-Imager M1; Zeiss) at 1,000× magnification andwere counted by using a counting grid (Zeiss) with an area of0.015 mm2. The cell densities were calculated as follows:

C=n · 15124.7

V, [S1]

where C is the cell density, n the average cell number per count-ing grid, V the volume of the filtered culture aliquot, and15,124.7 the microscope factor (226.87 mm2 as the filter area,which contained cells, divided by the grid area of 0.015 mm2).For each culture, two aliquots of cell suspension were filteredand 10 grid areas per filter were analyzed.

Oxic Incubation of N. moscoviensis with Formate. For all short-termincubations of N. moscoviensis under oxic conditions with for-mate, nitrite-grown biomass was harvested by centrifugation(7,232 × g, 20 min, 25 °C), washed in nitrite-free NOB medium,and was centrifuged again. The biomass was again resuspendedin nitrite-free NOB medium, and 1-mL aliquots were added byusing syringes to 300-mL Schott flasks containing 100 mL ofnitrite-free NOB medium. These bottles had already been pluggedwith butyl rubber stoppers, which were fixed with screw caps. Totest whether N. moscoviensis uses formate as the sole substrate,the cells were incubated for 7 d at 37 °C in the presence of sodiumformate (initial concentration 5 mM) in duplicates. In a controlexperiment, nitrite-free NOB medium containing 5 mM sodiumformate was incubated without biomass. During all incubations,

aliquots were sampled by using syringes, centrifuged (17,949 × g,10 min, 4 °C), and the formate concentrations in the supernatantwere quantified by capillary electrophoresis on a P/ACE MDQMolecular Characterization System (Beckman Coulter). Anionswere separated by using the CEofix Anions 5 kit on an anionexchange column (AS11; 250 × 4 mm; Thermo Scientific Dionex)using a linear KOH gradient (0.1–40 mM in 6 min). To testwhether N. moscoviensis would reduce nitrate under oxic condi-tions with formate as electron donor, cells were incubated for 8 dat 37 °C in the presence of sodium formate (initial concentration5 mM) and NaNO3 (initial concentration 1.5 mM), or only with1.5 mM NaNO3 in triplicates. During the incubations, aliquots weresampled by using syringes, centrifuged (17,949 × g, 10 min, 4 °C),and the concentrations of nitrite, nitrate, and formate in the su-pernatant were measured as described above.To quantify the growth of N. moscoviensis under oxic condi-

tions with formate or nitrite, respectively, nitrite-grown biomasswas harvested by centrifugation (6,300 × g, 20 min, 25 °C) andthe cells were washed in nitrite-free NOB medium. Centrifuga-tion and washing of the biomass were repeated until no nitriteand nitrate were detected by NO2

− and NO3− test stripes

(Merkoquant; Merck) in the discarded supernatant. The biomasswas again resuspended in 10 mL of nitrite-free NOB medium,and 1-mL aliquots were distributed to 300-mL Schott flaskscontaining 50 mL of nitrite-free NOB medium. The biomassaliquots were then incubated with 5 mM formate (final con-centration) or 5 mM nitrite (final concentration) in triplicates for10 d at 37 °C. In a control experiment, N. moscoviensis biomasswas incubated in NOB medium without any energy source intriplicates for 10 d at 37 °C. During the incubations, culturealiquots (500 μL) were sampled, centrifuged (17,949 × g, 10 min,4 °C), and the concentrations of nitrite and formate in the super-natant were measured as described above. In addition, total cellprotein was measured by using the BCA protein assay (ThermoFisher Scientific) according to the manufacturer’s instructions.For all long-term incubations of N. moscoviensis under oxic

conditions with formate and nitrate, nitrite-grown biomass washarvested by centrifugation (6,300 × g, 20 min, 25 °C), and thecell pellet was resuspended in nitrite-free NOB medium. Thebiomass was added in equal proportions to 300-mL Schott flaskscontaining 70 mL of nitrite-free NOB medium. To all incuba-tions, 0.5 mM NaNO2 (final concentration) was added and thecultures were incubated at 37 °C in the dark in duplicates. After3 d, all nitrite was oxidized to nitrate and sodium formate (initialconcentration 5 mM) was added to all incubations. For controlexperiments, to test the nitrite-oxidizing activity in absence offormate, 0.5 mM NaNO2 was added instead of formate. Cellswere incubated for 23 d at 37 °C. During all incubations, aliquotswere sampled, centrifuged (17,949 × g, 10 min, 4 °C) and theformate, nitrite, and nitrate concentrations in the supernatantwere quantified as described above.

Respirometry. Substrate-dependent O2 consumption rates weremeasured by using a respiration cell RC-350 (Warner Instrum-ents), equipped with an oxygen sensor (Model 1302) and con-nected to a picoammeter PA2000 (Unisense). Respiration rateswere recorded in SensorTrace Basic (version 3.0.2, Unisense).Measurements were performed by using approximately 20×concentrated N. moscoviensis biomass from actively nitrite-oxi-dizing pure cultures. Biomass was harvested by centrifugation(1,181 × g, 15 min) and washed twice in NOB medium containingno nitrite or nitrate. For measurements, the cell chamber wasfilled with 1.5 mL of biomass suspension and closed with theelectrode inserted into the general electrode holder EH-100(Warner Instruments) without inclosing air bubbles. Measurementswere performed at 39 °C. Substrates (1 mM Na-formate or 5 mMNaNO3) were added with a 100-μL gas-tight Hamilton syringe(model 1710 RN, Hamilton Laboratory Products) from stock

Koch et al. www.pnas.org/cgi/content/short/1506533112 4 of 13

solutions, and samples were taken with the same syringe. Nitriteconcentrations were determined photometrically by the Griessassay (82).

Anoxic Incubation of N. moscoviensis with Organic Substrates. Ace-tate (C2H3O2Na), formate (CHNaO2), fumarate (C4H2Na2O4),nitrate (NaNO3), and succinate (C4H4Na2O4) stock solutionswere prepared in mineral medium in serum bottles and weremade anoxic by sequential application of underpressure (with avacuum pump) and flushing with N2. Subsequently, anoxic stocksolutions were autoclaved and stored at 4 °C in the dark. Heat-labile substrate stocks of citrate (C6H5O73Na·2H2O), nitrite(NaNO2), and pyruvate (C3H3NaO3) were prepared in mineralmedium, sterile filtered, and injected into sterile serum bottles.Solutions were made anoxic as described above under sterileconditions and stored at 4 °C in the dark. Nitrite-free NOBmedium was prepared by strictly anoxic techniques as describedby Widdel and Bak (84). Aliquots of 10 mL of the anoxic nitrite-free NOB medium were injected into sterile, N2-flushed 30-mLserum bottles, which were closed with butyl rubber stoppers andaluminum crimps. The respective organic substrate and NaNO3,or NaNO3 only, were added to the respective serum bottles toa final concentration of 1 mM each. Additionally, 3 mL of aN2:CO2 (80:20) mixture was added to the headspace of eachserum bottle to provide a carbon source. N. moscoviensis biomasswas harvested by centrifugation (8,228 × g, 20 min, 25 °C) and

washed twice in anoxic nitrite-free NOB medium. After re-suspension in the medium, 1 mL of cell suspension was added asinoculum to each serum bottle. The inoculated bottles were in-cubated at 37 °C in the dark for 34 d. During the incubation, sampleswere taken by using syringes and were centrifuged (20,817 × g,20 min, 4 °C). Nitrite, nitrate, and formate concentrations in thesupernatant were determined as described above. The concen-trations of other organic substrates were determined by capillaryelectrophoresis as described above for formate.The anoxic incubation of N. moscoviensis with formate and

nitrate was repeated as described above with the followingmodifications. Aliquots (100 mL) of anoxic nitrite-free NOBmedium were injected into sterile, N2-flushed 300-mL Schottflasks, which were closed with butyl rubber stoppers and screwcaps. To provide a carbon source, 5 mL of a N2:CO2 (80:20) gasmixture was added to the headspace. After addition of washed(see above) N. moscoviensis biomass, all bottles were incubatedfor 1 d before the addition of substrates. Formate and nitratewere added to the incubations to final concentrations of 4.5 mMformate and 1.2 mM NaNO3. All incubations were performed at37 °C for 15 d in the dark. Samples were taken by using syringesand centrifuged (17,949 × g, 10 min, 4 °C). The formate andnitrite concentrations in the supernatant were quantified as de-scribed above.

Koch et al. www.pnas.org/cgi/content/short/1506533112 5 of 13

5 kb

N. moscoviensis

N. lenta

Ca. N. defluvii

putative urea ABC transporter, u

rea binding protein UrtA

urease gamma subunit

urease beta subunit

urease alpha subunit

putative accessory urease protein UreF

accessory urease protein UreG

putative accessory urease protein UreD

glutamine-dependent NAD+ synthase

ammonium transporter

nitrogen regulatory protein PII

putative (protein-PII) uridylyltra

nsferase

glutamine synthetase

putative nitrate ABC tra

nsporter, periplasmic component

nitrate ABC tra

nsporter, integral m

embrane subunit

ABC transporter, A

TP-binding protein

cyanate hydratase

putative quinol cytochrome c reductase, FeS subunit

putative quinol cytochrome c reductase, cytochrome b subunit

putative octaheme cytochrome c

ammonium transporter

ammonium transporter

N-compounds transportNH3 productionNH3 assimilation and its regulation

other functionunknown function

NITMOv2_1249 NITMOv2_1283

NITLEN_v1_110041 NITLEN_v1_110011

8631EDIN4531EDIN ferredoxin-dependent nitrite reductase

cyanate hydratase

nitrite transporter

glutamine synthetase

putative (protein-PII) uridylyltransferase

nitrogen regulatory protein PII

ammonium transporter

glutamine-dependent NAD + synthase

A

S-FDH

HCOOH

CO2

NAD+

NADH +H+

Acetate

Acetyl-CoA

Pyruvate

Phoshoenolpyruvate

Glyceraldehyde-3P

Pentose phosphatepathway

Glycerone-PFructose-1,6PFructose-6PGlucose-6P

Glycogenstorage

SuccinateFumarate

Succinyl-CoA2-Oxoglutarate

IsocitrateCitrate

OxalacetateMalate

TCAcycle

Glycerol-3-P

Phospholipids

III

NAD+ NADH +H+

III

NO2-

NO3-

1/2 O2+ 2H+

H2O

IV V

ADP+ Pi

ATP

NirK

NO2-

NO

urease

urea

NH3+CO2

H2

2 H+

Formate NH4+ NO3

- K+ Sugars

Miscellaneous transporter family

ZnMn

ABC transporter

MoureaBranchedamino acids PO4

3- Fe3+

N. moscoviensis specific

present in both Nitrospira

N. defluvii specific

Flagellum

Secretion system

Tat proteintranslocation Sec protein

translocationType I

secretionType II secretion /

Type IV piliType VI

secretion

FeBacterio-

ferritin

Ferric citrate / siderophorereceptor (TonB)

FecR

CLD

ClO2-

O2

Cl

PolyaminesNO2-

NirANO2- NH4

+

SOD2 O2

- + 2 H+ O2 + H2O2

Cat2 H2O + 2 O2

2 H2O2

CAHCO3- CO2

Peptidesiderophores

RND transporter

Multidrug effluxCd Zn Co

MCPs

poly-P

PO43-

CynSNCO-

+ HCO3-

NH3+ 2 CO2

DefenseCRISPR

Sugars

Cofactor synthesis:Biotin(Ribo)flavinThiamineFolateCobalaminHemeCoenzyme A

Stress resistance:Phytoene, CarotenePeroxidase, PeroxiredoxinThioredoxin systemArsenic resistanceMercuric reductase

-LactamaseAcriflavin/Heavy metals

*

NXR

B

Fig. S1. Key genomic and metabolic features of Nitrospira. (A) Schematic representation of the genomic regions in N. moscoviensis and N. lenta that containthe urease genes, the urea ABC transporter, and various other genes involved in the acquisition and metabolism of N compounds. The respective locus inN. defluvii that lacks urease and the urea transporter is shown for comparison. Solid lines connect homologous genes that encode proteins sharing sequencesimilarities above 50%. Dashed lines connect genes that encode proteins sharing sequence similarities between 30% and 50%. (B) Cell metabolic cartoon

Legend continued on following page

Koch et al. www.pnas.org/cgi/content/short/1506533112 6 of 13

constructed from the annotations of the N. moscoviensis and N. defluvii genomes. Core functions, which are shared by both Nitrospira members (gray), andstrain-specific features (yellow and blue) are shown. CA, carbonic anhydrase; Cat, catalase; CLD, chlorite dismutase; CRISPR, clustered regularly interspacedshort palindromic repeats; CynS, cyanate hydratase (cyanase); MCPs, methyl-accepting chemotaxis proteins; S-FDH, soluble formate dehydrogenase. Enzymecomplexes of the electron transport chain are labeled by Roman numerals. The TCA cycle depicts both directions (oxidative and reductive), with the reductiveTCA cycle being used by Nitrospira for CO2 fixation. *, N. defluvii possesses a canonical CA, whereas N. moscoviensis has only a putative CA-like protein(NITMOv2_0219) that contains the metal binding sites, but lacks some catalytic residues of canonical CA.

Fig. S2. Full nitrification of urea by reciprocal feeding. (A) Absence of ureolytic activity in N. defluvii. Incubation of N. defluvii cells in medium containing1 mM urea or 0.5 mM nitrite or both 1 mM urea and 0.5 mM nitrite. No release of free ammonium was observed in any incubation. Control experiments withcell-free medium containing either 1 mM urea or 1 mM urea and 0.5 mM nitrite confirmed that chemical urea degradation did not affect the results. Twobiological replicates are shown for all incubations with N. defluvii. (B) Absence of ureolytic activity in N. europaea. Incubation of N. europaea cells in mediumcontaining 1 mM urea as the sole source of ammonia. No ammonia oxidation (production of nitrite) was observed in the two biological replicates. Aliquots ofthe same N. europaea biomass were used in the coincubation experiment with N. moscoviensis (see Results and Discussion in the main text). (C) Coincubationof N. moscoviensis and urease-negative N. europaea in presence of 50 μM urea as the source of ammonia. The concentrations of free ammonium, nitrite, andnitrate in the culture supernatant during 7 d of incubation are shown. At the start of the incubation, the medium contained some ammonium, most likely dueto carryover with the N. europaea inoculum. Full nitrification occurred in each of the two biological replicates.

Koch et al. www.pnas.org/cgi/content/short/1506533112 7 of 13

Fig. S3. Phylogenetic affiliation of the urease alpha subunits (UreC) from Nitrospira, Nitrospina, and other nitrifiers. A Bayesian 80% consensus amino acid tree is shown. The degree ofposterior support of a branch is indicated by a single asterisk for >90% posterior probability (PP), a double asterisk for >99% PP or a triple asterisk for >99.9% PP. For metagenomic UreCsequences, the gene ID is followed by the IMG metagenome ID (for UreC received from IMG) and the description of the source habitat. The scale bar shows 7% estimated sequencedivergence.

Koch et al. www.pnas.org/cgi/content/short/1506533112 8 of 13

Fig. S4. Incubation experiments of N. moscoviensis with organic substrates in anoxia. (A) Anaerobic consumption of formate (initial concentration 4.5 mM)with nitrate (initial concentration 1.2 mM) as terminal electron acceptor. Nitrate was nearly stoichiometrically reduced to nitrite. The consumption of formate,which was provided in excess, ceased when all nitrate had been reduced. The results of two biological replicates are shown. The divergence of the formateconcentrations measured on days 0 and 1 was caused by technical problems with formate measurement. The increase in nitrite indicates that formate wasconsumed by N. moscoviensis in both replicates during this period. (B) Incubations with various organic compounds under anoxic conditions. Nitrate (1 mM)was provided as terminal electron acceptor in absence of O2. The initial concentration of each organic substrate was 1 mM. The consumption of nitrate andproduction of nitrite, which would indicate the utilization of the respective organic substrate as electron donor, was not observed in any incubation. Theconcentrations of the organic substrates at the beginning and end of the incubations were identical (not plotted). A control experiment with nitrite (1 mM)and no organic substrate confirmed the absence of nitrite-oxidizing activity under the anoxic conditions applied. The results of two biological replicates areshown for all incubations.

Koch et al. www.pnas.org/cgi/content/short/1506533112 9 of 13

Fig. S5. Aerobic utilization of formate or nitrite by N. moscoviensis. (A) Aerobic use of formate with O2 as terminal electron acceptor by a pure culture ofN. moscoviensis. Data points show the means of biological replicates (n = 3). Error bars represent SD and are not shown if smaller than symbols. This experimentrepresents an independent replication of the experiment shown in Fig. 3B. (B) Aerobic use of nitrite with O2 as terminal electron acceptor by a pure culture ofN. moscoviensis. Data points show the means of biological replicates (n = 3). Error bars represent SD and are not shown if smaller than symbols. This experimentwas performed with the same amount of biomass from the same inoculum as the experiment in A. (C) Aerobic growth of N. moscoviensis on formate or nitrite,respectively. Data points show the means of biological replicates (n = 3). Error bars represent SD and are not shown if smaller than symbols. Total biomassprotein was measured during the incubations shown in A and B to follow the growth of the cultures in these experiments. In the control experiment, the sameamount of N. moscoviensis biomass was incubated in mineral medium without addition of formate or nitrite. Here, total protein decreased likely because ofendogenous respiration in the absence of any external electron donor. (D) Long-term incubation of N. moscoviensis with formate and nitrate under oxicconditions. The initial concentrations of formate and nitrate were 5 mM and 0.5 mM, respectively. This graph shows the formate concentration in the culturesupernatant. The results of two biological replicates are shown. (E) Nitrite concentrations in the culture supernatant during the incubation experiment shownin D. The initial net increase of the nitrite concentration was caused by nitrate reduction. The following net decrease of nitrite demonstrates the concomitantutilization of nitrite and formate (also see D) from day 6 to the end of the experiment. The results of two biological replicates are shown. (F) Nitrate con-centrations in the culture supernatant during the incubation experiment shown in D and E. The initial net decrease and subsequent net increase of the nitrateconcentration are consistent with the nitrate-reducing and nitrite-oxidizing activities (D and E) of N. moscoviensis in this experiment. (G) Nitrite oxidation byN. moscoviensis in absence of formate. The rate of nitrite oxidation was considerably higher than in presence of formate (E). Highly similar amounts ofN. moscoviensis biomass were used in these incubation experiments (D–G).

Koch et al. www.pnas.org/cgi/content/short/1506533112 10 of 13

Fig. S6. Utilization of O2 and nitrate as terminal electron acceptors by N. moscoviensis. (A–D) Microrespirometric measurements of O2 consumption withformate (1 mM initial concentration) as electron donor and in presence or absence of nitrate (5 mM initial concentration) are shown. Curves without symbolsdepict the O2 concentrations in the supernatant of a N. moscoviensis pure culture. Curves with symbols depict the nitrite concentrations in the supernatant.Each graph represents an independent experiment, and all experiments were performed with highly similar amounts of biomass. Black arrows indicate theaddition of formate to the cultures. Purple arrows indicate the addition of nitrate to cultures containing only formate. The reduction in the O2 consumptionrates in presence of both electron acceptors, and the production of nitrite, show that electrons from formate were distributed to both O2 and nitrate. Pleasenote that experiments with formate in the total absence of nitrate (blue curves) were carried out only twice (A and B).

Koch et al. www.pnas.org/cgi/content/short/1506533112 11 of 13

500

1000

1500

2000

2500

3000

3500

4000

500 1000 1500 2000 2500 3000 3500 4000 4500

Nitr

ospi

ra d

eflu

vii

Nitrospira moscoviensis

A

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

D10

D11

D14D12

D13

D15

D16

D17

D18

D19

D20

D21

D22

D23

D1

D2

D3

D4

D5

D6

D7

D8

D9

M10

M12

M11

M13

M14

M15

M16

M17

M20

M21

M22

M23

M24

M25

M26

M27

M19

M18

M28

M1

M2

M4

M5

M6

M3

M7

M8

M9

D10

D11

D14D12

D13

D15

D16

D17

D18

D19

D20

D21

D22

D23

D1

D2

D3

D4

D5

D6

D7

D8

D9

M10

M12

M11

M13

M14

M15

M16

M17

M20

M21

M22

M23

M24

M25

M26

M27

M19

M18

M28

M1

M2

M4

M5

M6

M3

M7

M8

M9

Nitrospiradefluvii

Nitrospiramoscoviensis

Electron transport and respirationNitrite oxidation / nitrate reductionCarbon fixation

B

M15

M16

M17

M18

M19

D14

D13

Sec-independent protein translocase TatA

membrane protein (unknown function)

membra

ne pr

otein

(unkn

own f

uncti

on)

put. Mo cofactor guanylyltransferase

preprotein translocase subunit YajCprotein translocase subunit SecDprotein translocase subunit SecF

tRNA-guanine transglycosylase

ferredoxin-type protein NapG

put. NXR membrane subunit

put. NXR membrane subunit

put. NXR membrane subunit

protein of unknown function

protein of unknown functionprotein of unknown function

protein of unknown function

protein of unknown functionprotein of unknown function

protein of unknown fu

nction

protein of unkn

own functio

n

protei

n of u

nkno

wn fun

ction

protei

n of u

nkno

wn fun

ction

put. glycosyl transferase

transcriptional regulator

transcriptional regulator

put. cyt.

bd-like oxid

ase

put. c

yt. bd

-like o

xidas

e

arginine--tRNA ligase

put. histidine kinase

put. NXR chaperone

response regulator

NXR subunit alpha (Nide3237)N

XR subunit beta

NXR subunit beta

put. cyt. c

put. cyt. c

Sec-independent protein translocase TatA

membrane protein (unknown function)

mem

bran

e pr

otei

n (u

nkno

wn

func

tion)

put. Mo cofactor guanylyltransferase

preprotein translocase subunit YajC

protein translocase subunit SecD

protein translocase subunit SecF

tRNA-guanine transglycosylase

ferredoxin-type protein NapG

put. NXR membrane subunit

put. NXR membrane subunit

put. NXR

mem

brane subunit

put. NXR membrane subunitprotein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

noit

cnuf

nwo

nknu

fo

niet

orp

prot

ein

of u

nkno

wn

func

tionnoitcnuf n

wonknu fo nietorpprot

ein

of u

nkno

wn

func

tion

prot

ein

of u

nkno

wn

func

tion

prot

ein

of u

nkno

wn

func

tion

prote

in of

unkn

own f

uncti

on

protein of unknown fu

nction

protein of unknown function

put. glycosyl transferase

trans

crip

tiona

l reg

ulat

or

trans

cripti

onal

regula

tor

transcr

iptional re

gulator

transcriptional regulator

transcriptional regulator

put. cyt. bd-like oxidaseput.

cyt.

bd-li

ke o

xida

se

arginine--tRN

A ligase

put. histidine kinase

put.

hist

idin

e kin

ase

put. NXR chaperoneresponse regulator

NXR

sub

unit

alph

a

NXR su

bunit

alph

a

NXR subunit alpha

NXR subunit alpha

NXR subunit alpha

NXR

sub

unit

beta

NXR

subu

nit b

eta

NXR subunit b

eta

NXR subunit beta

NXR subunit beta

put. cyt. c

put. cyt. cD12

Putative cytochrome bd-like terminal oxidaseVarious cytochromesOther or unknown functions

Nitrite oxidoreductase (NXR)Putative NXR transcriptional regulators

Nitrospiradefluvii

Nitrospiramoscoviensis

NXR subunit alpha (Nide3255)

C

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

M11M12

M13M14

D11

D10

D9

D8

D7

D6

D5

D4

D3

D2

D1

membrane protein (function unknown)

put. A

TP sy

nthas

e F1 s

ubun

it delt

a

complex III fused cyt. b/c subunit

ATP

synth

ase F

1 sub

unit g

amma

ATP

synt

hase

F1

subu

nit e

psilo

n

complex II flavoprotein subunit

complex II flavoprotein subunit

ATP

synth

ase F

1 sub

unit a

lpha

put. cyt. b

d-like cyt. c oxidase

ATP

synt

hase

F1

subu

nit b

eta

put. complex III cyt. c subunit

put. NXR m

embrane subunit

protein of unknown function

complex I subunits C and D

ATP

synt

hase

F0

subu

nit C

ATP

synt

hase

F0

subu

nit B

ATP

synt

hase

F0

subu

nit A

complex III cyt. b subunit

cyt. b

d oxid

ase s

ubun

it II

complex III Fe-S subunit

complex III Fe-S subunit

cyt. b

d oxid

ase s

ubun

it I

complex II Fe-S subunit

put. cyt. bd-like oxidase

complex II ferredoxin

complex II subunit C

complex I subunit M

complex I subunit M

complex I subunit M

complex I subunit M

complex I subunit N

complex I subunit G

complex I subunit D

complex I subunit C

complex I subunit Ncomplex I subunit H

complex I subunit G

complex I subunit K

complex I subunit B

complex I subunit A

complex I subunit K

complex I subunit B

complex I subunit A

complex I subunit L

complex I subunit Lcomplex I subunit F

complex I subunit E

complex I subunit L

complex I subunit Fcom

plex I subunit J

complex I subunit J

complex I subunit I

complex I subunit I

put. cyt. c

put. cyt. c

put. cyt. c

put. cyt. c

put. cyt.

c

put. cyt.

c

mem

brane protein (function unknown)

complex II cytochrome b558 subunit

put. ATP synthase F1 subunit delta

complex III fused cyt. b/c subunit

ATP synthase F1 subunit gamm

a

ATP synthase F1 subunit epsilon

complex II flavoprotein subunitcomplex II flavoprotein subunit

ATP synthase F1 subunit alpha

put. cyt. bd-like cyt. c oxidase

put. complex III cyt. b subunit

put. complex III cyt. b subunit

ATP synthase F1 subunit beta

put. complex III cyt. c subunit

put. cyt. bd oxidase subunit IIput. cyt. bd oxidase subunit II

put. complex III Fe-S subunit

put. complex III Fe-S subunit

put. complex III Fe-S subunit

put. NXR mem

brane subunit

complex I subunits

C and D

complex I subunits C and D

ATP

synt

hase

F0

subu

nit C B tinubus 0F esahtnys PTAAT

P sy

ntha

se F

0 su

buni

t A

put. complex I subunit M

put. complex I subunit M

cyt. bd oxidase subunit Icyt. bd oxidase subunit I

put. complex I subunit L

put. complex I subunit L

complex II Fe-S subunit

put. cyt. bd-like oxidase

complex II ferredoxin

com

plex

I su

buni

t Mco

mpl

ex I

subu

nit M

comple

x I su

bunit

M

complex I subunit M

com

plex

I su

buni

t N

com

plex

I su

buni

t G

com

plex

I su

buni

t D

com

plex

I su

buni

t Cco

mple

x I su

bunit

N

comple

x I su

bunit

H

complex I

subunit G

complex I subunit G

complex I subunit H

complex I subunit N

com

plex

I su

buni

t K

com

plex I

subu

nit B

com

plex I

subu

nit A

comple

x I su

bunit

K

complex I subunit B

complex I subunit A

complex I subunit A

complex I subunit B

complex I subunit K

com

plex

I su

buni

t L

comple

x I su

bunit

L

complex I subunit F

complex I subunit E

complex I subunit E

complex I subunit F

complex I subunit L

complex I subunit F

com

plex

I su

buni

t J

comple

x I su

bunit

J

complex I subunit J

com

plex

I su

buni

t I

comple

x I su

bunit

I

complex I subunit I

put. cyt. c

put. cyt. cput. cyt. c

put. cyt. c

put. cyt. c

NADH-quinone oxidoreductase (complex I)Succinate dehydrogenase / fumarate reductase (complex II)Quinol-cytochrome c oxidoreductase (complex III)Cytochrome bd quinol oxidase

Putative cytochrome bd-like terminal oxidaseVarious cytochromesNitrite oxidoreductase (NXR)Other or unknown functions

F0F1 ATP synthase (complex V)

Nitrospiradefluvii

Nitrospiramoscoviensis

D

M20

M21

M22

M23

M24

M25

M26

M27

M28D23D22

D21

D20

D19

D18

D17

D16

D15

2-oxoglutarate:ferredoxin oxidoreductase subunit epsilon

2-oxoglutarate:ferredoxin oxidoredutase subunit gamm

a

2-oxoglutarate:ferredoxin oxidoredutase subunit alpha

2-oxoglutarate:ferredoxin oxidoredutase subunit delta

2-oxoglutarate:ferredoxin oxidoredutase subunit beta

pyruvate:ferredoxin oxidoreductase subunit gammapyruvate:ferredoxin oxidoreductase subunit epsilon

2-oxoacid:ferredoxin oxidoreductase subunit beta

pyruvate:ferredoxin oxidoreductase subunit alpha

pyruvate:ferredoxin oxidoreductase subunit delta

pyruvate:ferredoxin oxidoreductase subunit beta

pyruva

te dehydrogenase

E1 subunit a

lpha

pyru

vate

deh

ydro

gena

se E

1 su

buni

t alp

ha

pyruv

ate de

hydro

gena

se E1 s

ubun

it beta

pyru

vate

deh

ydro

gena

se E

1 su

bunit

bet

a

DNA-formamidopyrimidine glycosylase

succinyl-CoA synthetase subunit alpha

succinyl-CoA synthetase subunit beta

heat shock protein (Hsp20 family)

complex II flavoprotein subunit

ATP citrate lyase subunit alpha

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

protein of unknown function

pyruv

ate de

hydro

gena

se E

2

pyru

vate

deh

ydro

gena

se E

2

pyru

vate

deh

ydro

gena

se E

3py

ruva

te d

ehyd

roge

nase

E3

isocitrate dehydrogenase

malate dehydrogenase

aconitate hydratase

fumarate hydratasecitrate synthase

ferredoxin

2-oxoacid:ferredoxin oxidoreductase subunit alpha (fragment)

2-ox

oglu

tara

te:fe

rredo

xin

oxid

ored

ucta

se s

ubun

it ep

silo

n

2-ox

oglu

tara

te:fe

rredo

xin

oxid

ored

utas

e su

buni

t gam

ma

2-ox

oglu

tara

te:fe

rredo

xin

oxid

ored

utas

e su

buni

t alp

ha

2-ox

oglu

tara

te:fe

rredo

xin

oxid

ored

utas

e su

buni

t del

ta

2-ox

oglu

tara

te:fe

rredo

xin

oxid

ored

utas

e su

buni

t bet

a

pyruvate:ferredoxin oxidoreductase subunit gamma

pyruvate:ferredoxin oxidoreductase subunit epsilon

2-oxoacid:ferredoxin oxidoreductase subunit beta

pyruvate:ferredoxin oxidoreductase subunit alpha

pyruvate:ferredoxin oxidoreductase subunit delta

pyruvate:ferredoxin oxidoreductase subunit beta

put. pyruvate dehydrogenase E1 subunit alpha

put. pyruvate dehydrogenase E1 subunit beta

pyruvate dehydrogenase E1 subunit beta

DNA-

form

amido

pyrim

idine

glyc

osyla

se

succinyl-CoA synthetase subunit alpha

succinyl-CoA synthetase subunit beta

heat shock protein (Hsp20 family)

put. pyruvate dehydrogenase E3

put.pyruvate dehydrogenase E2

complex II flavoprotein subunit

ATP ci

trate

lyase

subu

nit al

pha

ATP-

citra

te ly

ase

subu

nit b

eta

prot

ein

of u

nkno

wn

func

tion

prot

ein

of u

nkno

wn fu

nctio

n

prot

ein

of u

nkno

wn fu

nctio

n

prot

ein

of u

nkno

wn fu

nctio

n

prot

ein

of u

nkno

wn fu

nctio

n

prot

ein o

f unk

nown

func

tion

protein of unknown function

pyruvate dehydrogenase E2

pyruvate dehydrogenase E3

pyruvate dehydrogenase E3

isocitr

ate dehydrogenase

malate dehydrogenase

aconita

te hydratase

fumarate hydratasecitrate synthase

ferredoxin

Other or unknown functions

rTCA and oTCA cycle enzymes

Nitrospiradefluvii

Nitrospiramoscoviensis

E

Fig. S7. Whole-genome comparison and core metabolism of N. moscoviensis and Nitrospira defluvii. (A) Whole-genome alignment showing the positions of homologous genes in N. moscoviensis and N. defluvii. Sequence matches with the same orientation are plotted blue, whereas inversions are plotted red. (B) Localizationof regions encoding the Nitrospira core metabolism for nitrite oxidation, electron transport, and inorganic carbon fixation in the genomes of N. defluvii (Left) and N. moscoviensis (Right). Each semicircle depicts one full genome. Ribbons connect regions containing homologous core metabolism genes. Tags (D1 to D23 forN. defluvii, M1 to M28 for N. moscoviensis) identify the genomic regions shown in C–E. (C) Highly conserved, syntenic gene arrangements within regions encoding nitrite oxidoreductase (NXR) in the genomes of N. defluvii (Left) and N. moscoviensis (right). Regions are separated by spaces and ribbons connect homologousgenes. The tags, which identify the regions, are the same as in B. The order of the regions shown here facilitates the synteny comparison and does not reflect the true genomic localization of these regions, which is shown in B. (D) Highly conserved, syntenic gene arrangements within regions encoding the electron transportchains for nitrite oxidation, reverse electron transport, and the utilization of organic substrates in the genomes of N. defluvii (Left) and N. moscoviensis (Right). Regions are separated by spaces and ribbons connect homologous genes. The tags, which identify the regions, are the same as in B. The order of the regions shownhere facilitates the synteny comparison and does not reflect the true genomic localization of these regions, which is shown in B. (E) Highly conserved, syntenic gene arrangements within regions encoding the reductive (rTCA) and oxidative (oTCA) tricarboxylic acid cycles in the genomes of N. defluvii (Left) and N. moscoviensis(Right). The rTCA cycle is the CO2 fixation pathway of Nitrospira. Regions are separated by spaces and ribbons connect homologous genes. The tags, which identify the regions, are the same as in B. The order of the regions shown here facilitates the synteny comparison and does not reflect the true genomic localization ofthese regions, which is shown in B.

Koch et al. www.pnas.org/cgi/content/short/1506533112 12 of 13

Table S1. General genome characteristics of N. moscoviensisand N. defluvii

Genome feature N. moscoviensis N. defluvii

Genome size, bp 4,589,485 4,317,083Average G+C content, % 62 59No. of CDS 4,863 4,274Coding density, % 90.6 89.4CDS with predicted function 2,391 (56%) 2,154 (50%)rRNA operon 1 1tRNA genes 47 46Species-specific CDS* 2,161 (44%) 1,695 (40%)Species-specific CDS with

unknown function1,547 1,216

CDS, coding sequences.*CDS with no homolog in the respective other Nitrospira genome wereconsidered to be species-specific. Homologous proteins were defined as≥30% identical over ≥80% of the amino acid sequence length.

Other Supporting Information Files

Dataset S1 (PDF)

Koch et al. www.pnas.org/cgi/content/short/1506533112 13 of 13