Supporting Information

Koch et al. 10.1073/pnas.1506533112 SI Results and Discussion transporters may facilitate the uptake of one or more of the tested Urea Transport and Accessory Proteins of Urease in N. moscoviensis. organics. However, no nitrate reduction was observed during Urea is a small uncharged molecule that diffuses readily through anoxic incubations with any of these substrates (Fig. S4B). the lipid bilayers of bacterial membranes (52). Aside from passive Nitrospira diffusion, the transport of urea into bacterial cells is mediated by Core Metabolism of for Chemolithoautotrophic Nitrite urea-specific channels such as UreI of Helicobacter (53) or by ATP- Oxidation. A syntenic gene arrangement is conserved in rela- dependent ABC transporters such as UrtABCDE in Cyanobacteria tively large parts of the N. moscoviensis and N. defluvii genomes (54). ABC transporters for urea consist of a periplasmic substrate- (Fig. S7A). Shared genomic features with a highly conserved binding protein (UrtA), a dimer of the transmembrane proteins synteny are the enzymatic repertoire for nitrite oxidation, the electron transport chains for aerobic respiration and reverse UrtB and UrtC, which form a membrane-crossing pore, and the + ATP-binding and -hydrolyzing proteins UrtD and UrtE (54, 55). electron transport from nitrite to NAD , and the reductive tri- These ABC transporters show a high affinity for urea (52, 54) and carboxylic acid (rTCA) cycle for CO2 fixation and the oxidative – may represent an adaptation to environments with urea concen- TCA cycle (Fig. S7 B E). The high degree of similarity in these trations in the micromolar range. Their expression is tightly regu- pathways strongly supports the previous reconstruction of the lated in dependence on N availability due to the energy demand of Nitrospira core metabolism for chemolithoautotrophic nitrite this transport system (54, 56). In the genome of N. lenta, the whole oxidation, which was based on only one sequenced genome (25). gene set (urtABCDE) of the urea ACB transport system is located The few genetic differences in the core pathways include addi- upstream of the urease genes (Fig. S1A and Dataset S1), suggesting tional (third) copies of respiratory complexes I and III, a second that N. lenta possesses a high-affinity uptake system for urea and, cytochrome bd oxidase, and five paralogous copies of nitrite thus, is adapted to habitats where low urea concentrations prevail. oxidoreductase (NXR) subunits NxrA and NxrB in N. mosco- In contrast, in N. moscoviensis, only urtA, which encodes the peri- viensis, whereas N. defluvii has only two paralogs of these NXR plasmic urea-binding protein UrtA, is located in close vicinity of the subunits (25) (Fig. S7 C and D). urease genes (Fig. S1A). Whether N. moscoviensis can replace the NXR, the key enzyme for nitrite oxidation, belongs to – lacking UrtBCDE proteins with the respective subunits of other the complex iron sulfur molybdoenzyme family with a ABC transporters encoded in the genome, or whether urea is taken molybdo-bis(pyranopterin guanine dinucleotide) cofactor- up by passive diffusion only, remains to be determined. containing catalytic subunit (61). The NXR of Nitrospira is lo- The accessory proteins UreD, UreE, UreF, and UreG are cated in the periplasmic space and consists of at least two + required for the formation of the Ni2 -containing metallocenter subunits (NxrA and NxrB). The third (NxrC) subunit may an- in the UreABC apoenzyme during the biosynthesis of urease chor the NXR complex in the cytoplasmic membrane and + (27). In addition, the nickel transporter UreH provides Ni2 mediate the transfer of electrons from NXR to the membrane- (27). The genome of N. moscoviensis contains the ureD, bound electron transport chain (25). The five paralogs of nxrA ureF, and ureG genes (Fig. S1A) and ureH (NITMOv2_1657). and nxrB are clustered in three genomic regions of N. moscoviensis, However, only a 180-nt-long (59 aa) gene fragment of ureE whereas five putative nxrC genes are located elsewhere in the (NITMOv2_1661) was identified, which is unlikely to encode a genome (Fig. S7C). NxrA contains the substrate-binding site functional UreE protein because homologs in other organisms with the Mo cofactor (25). Like in N. defluvii, all NxrA paralogs are approximately 200 aa in length. Although UreE is required of N. moscoviensis contain an N-terminal twin-arginine motif for a functional urease in Helicobacter (57), various other mi- for export via the twin-arginine protein translocation (Tat) croorganisms lacking ureE genes express active ureases (58). In pathway. The presence of this motif is consistent with the ureolytic microbes without UreE, this nickel-binding metal- periplasmic localization of the active site of NXR in N. moscoviensis lochaperone (59) may be substituted by chaperones of other (62) and N. defluvii (25). The periplasmic NXR is energetically nickel-dependent enzymes (58). Interestingly, in Helicobacter advantageous and likely explains the strong competitiveness under pylori two accessory proteins of nickel-dependent hydrogenase, nitrite-limited conditions of Nitrospira compared with other + HypA and HypB, are required for the incorporation of Ni2 into NOB such as Nitrobacter, whose NXR is located on the cytoplasmic urease (60). These hydrogenase maturation factors are present side of the cell membrane (25). in N. moscoviensis, which possesses an active [NiFe] hydrogenase The amino acid similarities among the NXR subunits of (23). Hence, hydrogenase chaperones might be involved in the N. moscoviensis range from 95.7 to 98.5% for NxrA, from 99.5 to assembly of urease and substitute UreE in this organism as well. 100% for NxrB, and from 18.6 to 64.1% for the putative NxrC candidates. All five NxrA copies are more similar to one of the Utilization of Organic Substrates by N. moscoviensis. Aside from two NxrA paralogs (CDS tag Nide3255) (25) in N. defluvii (87.1– formate (see Results and Discussion in the main text), we tested 87.9%) than to the other one (Nide3237) (83.6–84.2%). Inter- also whether N. moscoviensis can use other simple organic com- estingly, the similarity between the two NxrA copies in N. defluvii pounds (acetate, fumarate, succinate, citrate, and pyruvate) in is only 86.9% and, thus, lower than the similarity between all combination with nitrate as terminal electron acceptor. Acetate NxrA copies of N. moscoviensis and one NxrA (Nide3255) of could be provided by fermenting organisms in the spatial prox- N. defluvii. It is tempting to speculate that the lower similarity imity of Nitrospira in hypoxic or anoxic habitats, whereas the other between the two NxrA subunits of N. defluvii reflects a functional compounds are key metabolites that could be released by lysed differentiation, and that all NxrA of N. moscoviensis are func- cells within a biofilm. The genetic repertoire of N. moscoviensis tionally more similar to one of the NxrA paralogs (Nide3255) of includes the degradation pathways and the respiratory chain N. defluvii. Consistently, four of the five nxrA/B gene clusters in needed to use these organic compounds (Fig. S1B). Transmem- N. moscoviensis are preceded by transcriptional regulator genes, brane transporters for these substrates were not identified in the which are homologous to a regulator in N. defluvii that occurs genome, but N. moscoviensis encodes permeases of unknown directly upstream of the gene encoding NxrA Nide3255 (Fig. specificities, and we could not exclude the possibility that such S7C). The amino acid similarities between these regulators are

Koch et al. www.pnas.org/cgi/content/short/1506533112 1of13 relatively high (47–73%). If the genomic localization next to include ROS detoxification by manganese or polyamines, H2O2 nxrA genes reflects a role of these regulators in the transcrip- degradation by peroxidases and thioredoxin-dependent peroxir- tional control of NXR, then the regulation of these four NXR edoxins, binding of free iron by bacterioferritin to reduce the risk paralogs in N. moscoviensis may resemble the regulation of of ROS generation, and free radical scavenging by carotenoids Nide3255 in N. defluvii. However, one of these nxr gene clusters (25). In contrast to N. defluvii, N. moscoviensis possesses a ca- in N. moscoviensis also contains a second transcriptional regu- nonical SOD and a catalase (Fig. S1B and Dataset S1). The SOD lator, which is homologous to a regulator upstream of the second of N. moscoviensis (NITMOv2_2805) binds Fe or Mn based on NxrA copy of N. defluvii (Nide3237) (Fig. S7C). Hence, in both its overall amino acid sequence similarity to other SODs that organisms, at least two different regulation mechanisms for NXR require these metal cofactors. The Fe and Mn SODs are difficult seem to be present that await confirmation and further analysis to distinguish from each other by sequence analysis, but specific in future studies. fingerprint residues (66) indicate that the enzyme of N. mosco- Each of the five NxrC candidates in N. moscoviensis has a viensis may be a tetrameric Fe SOD. N. moscoviensis possesses a homolog among the four putative NxrC subunits in N. defluvii typical monofunctional, heme-containing catalase that is encoded by (25) (Dataset S1), with two of the N. moscoviensis proteins two identical gene copies (NITMOv2_0085 and NITMOv2_4696). (NITMOv2_3617 and NITMOv2_4208) being homologous to Either catalase gene belongs to one of two identical copies of a one candidate NxrC in N. defluvii (Nide3271). All NxrC candi- 42-kbp-large Tn7 mobile element. In addition to SOD and cat- dates in both Nitrospira genomes have been identified based on alase, N. moscoviensis possesses the putative ROS defense mecha- sequence similarities to the membrane subunits of other DMSO nismsaspredictedforN. defluvii (25) except that it lacks a reductase type II family enzymes (25), but their actual functional polyamine transporter (Fig. S1B). roles and the composition of the NXR protein complex in Ni- trospira remain to be determined. SI Materials and Methods Genome Sequencing and Analysis. DNA was isolated from a pure Assimilatory Nitrite Reduction by a Putative Octaheme Cytochrome c culture of N. moscoviensis strain NSP M-1 (24) by following Nitrite Reductase. The genome of N. moscoviensis lacks any gene the hexadecyltrimethylammonium bromide (CTAB) protocol as for assimilatory nitrite reductase (NirA). The only gene related described (34). Following extraction, RNA was removed by to nirA most likely encodes a ferredoxin-dependent sulfite re- RNase I digestion (Lucigen). ductase (NITMOV2_0334) and is involved in sulfur assimilation Paired-end sequencing libraries were prepared by using the according to its localization close to the genes of sulfate ad- Nextera DNA Sample Preparation Kit (Illumina) according to the enylyltransferase. manufacturer’s instructions. Mate-pair sequencing libraries were However, N. moscoviensis contains a gene for an octaheme prepared by using the Nextera Mate-Pair Sample Preparation cytochrome c (OCC) protein, which is located in a region that Kit (Illumina) using the gel-free protocol according to the contains various other genes for N acquisition including the manufacturer’s instructions. The sequencing libraries were se- urease (Fig. S1A). Some OCC proteins from other organisms are quenced on an Illumina MiSeq DNA sequencer with 2 × 301 bp octaheme nitrite reductases (ONRs) that reduce nitrite to am- byusing the MiSeq Reagent Kit v3 (Illumina). The paired-end monia (63–65). These ONRs may represent an evolutionary link reads were imported into the CLC genomics workbench software from pentaheme nitrite reductases (Nrf) to the OCC with oxi- (version 6.5.1, CLCbio; Qiagen) and quality trimmed by requiring a dizing activities, hydroxylamine oxidoreductase (HAO) of aero- minimum phred score of 20 and a minimum read length of 50 bp. bic ammonia-oxidizing bacteria and hydrazine oxidoreductase Nextera adapters were removed if found. The trimmed paired-end (HZO) of anaerobic ammonium-oxidizing (anammox) organisms reads were de novo assembled by using CLC genomics workbench (63). The OCC of N. moscoviensis does not belong to this ONR v. 6.5.1 using a kmer of 64 and otherwise default parameters. The group, but most closely resembles members of another clade (II.2) de novo assembly was manually inspected by using CLC genomics of the OCC family, which consists mostly of uncharacterized pro- workbench v. 6.5.1 and the Circos (67) tools implemented in the teins. Interestingly, clade II.2 OCCs seem to be functionally versa- multimetagenome workflow (68). The initial inspection resulted in tile because a protein from this group from Beggiatoa had the cleaning of misassembled contigs and identification of five copies of activities of ONR and also of HAO and HZO in vitro (65). As a the NxrAB operon, which proved difficult to assemble because of HAO,theenzymeofN. moscoviensis might allow the detoxification high similarity between the variants. To assemble the individual of hydroxylamine. Because Nitrospira usually share their habitat NxrAB operons, the nxrA and nxrB genes were separately PCR- with aerobic ammonia-oxidizing bacteria, they may easily encounter amplified with region-specific primer sets, cloned in E. coli,and hydroxylamine in their direct environment. Sanger-sequenced. The partially assembled NxrAB genes were re- Interestingly, a clade II.1 OCC from Nautilia profundicola placed with their Sanger-sequenced complete counterparts. The is involved in the reverse hydroxylamine:ubiquinone reductase mate-pair reads were cleaned by using NextClip v.0.8 (69) with module (HURM) pathway that is used to reduce nitrate to am- default parameters. Reads from Category A were imported into monia (45). In this pathway, nitrite reduction to hydroxylamine by CLC genomics workbench v. 6.5.1 and mapped to the assembly by the OCC is linked to quinol oxidation and proton dislocation using the “map reads to reference” function with 95% similarity across the cell membrane by a cytochrome cM552-like protein over 70% of the read length as cutoff. The mapping was visualized (45). N. moscoviensis might use a similar pathway to link nitrite by using the Circos (67) tools implemented in the multimetagenome reduction to ammonia with proton translocation. Two genes en- workflow (68) and used to manually scaffold the initial assembly coding a Rieske/cytochrome b complex are located upstream of into a single scaffold. The gaps in the final scaffold were resolved the OCC gene in its genome (Fig. S1A). It is tempting to speculate manually by using CLC genomics workbench v.6.5.1 by mapping the that this Rieske/cytochrome b complex might generate proton trimmed paired-end reads (minimum 2 × 250 bp) to the assembly motive force by channeling electrons from the quinol pool to the with 95% similarity over 30% of the read length as cutoff. OCC, similar to the reverse-HURM pathway. Assuming that it has The genome annotation platform MicroScope (49) was used ONR activity, the OCC would then reduce nitrite to ammonia. for the automated prediction and annotation of CDS. Homolo- gous proteins present in the N. moscoviensis and N. defluvii ge- Reactive Oxygen Defense. In contrast to most other aerobic bac- nomes were identified by using the phyloprofile exploration tool teria, N. defluvii lacks SOD and catalase, and its genome does of MicroScope. Only proteins sharing an amino acid sequence not code for superoxide reductase either (25). The predicted identity ≥ 30% over at least 80% of the sequence length were alternative mechanisms for protection against ROS in N. defluvii considered as homologs. The annotation of all CDS discussed in

Koch et al. www.pnas.org/cgi/content/short/1506533112 2of13 this study was refined manually based on the same annotation 0.41 g of KH2PO4; 0.184 g of MgSO4·7H2O; 0.022 g of CaCl2; criteria that were already applied for the annotation of the 0.12 mg of CuSO4·7H2O and 0.33 mL of a 30 mM FeSO4·7H2O N. defluvii genome (25). The whole genomes of N. defluvii and solution in 50 mM Na2EDTA·2H2O. After autoclaving, 100 mL N. moscoviensis were aligned by using the PROmer tool of of a sterile buffer solution (pH 8.0) containing 6.8 g of KH2PO4 < the MUMmer 3.23 software package (70). Matches 333 aa and 0.6 g of NaH2PO4 and 4 mL of a sterile 10% (wt/vol) NaCO3 (1,000 nt) were ignored to reduce the number of spurious matches solution were added separately to adjust the pH to a value of 7.9. between partial protein sequences. Syntenic regions were iden- The purity of all nitrifier cultures, including incubated aliquots tified by the respective tools of MicroScope and visualized by before and after experiments, was checked by FISH with the using Circos (67). EUB338 probe mix (76, 77) that targets most known Bacteria, the Nitrospira lineage II-specific probe Ntspa1151 (78) for N. mosco- Metagenomic Screening for Nitrospira-Like ureC Sequences and viensis cultures, the Nitrospira lineage I-specific probe Ntspa1431 Phylogenetic Analysis of the Urease Alpha Subunit. A large meta- (78) for N. defluvii cultures, or the betaproteobacterial AOB- genome from the Aalborg West Wastewater treatment plant in specific probe Nso1225 (79) for N. europaea cultures. For this Denmark (MG-RAST ID: 4611649.3) was screened for the pres- purpose, aliquots of the biomass were fixed in formaldehyde and ence of Nitrospira-like UreC proteins by first calling genes using FISH was carried out as described (80). Contaminations by other prodigal with the metagenome option (71) and then searching the bacteria were not detected in any experiment. resulting proteins using blastp. In total, 3,217 publicly available metagenomes in the integrated Incubation of N. moscoviensis with Urea. N. moscoviensis biomass microbial genomes (IMG) database (50) were screened by using was harvested by centrifugation (8,228 × g, 10 min, 25 °C). The HMMER3 hmmsearch (72) with the hmm model (length = 121) cell pellet was resuspended in 30 mL of ammonium-free NOB for urease_alpha (PFAM PF00449; ref. 73) and default param- medium and 5-mL aliquots of the biomass were added to 300-mL eters. Amino acid sequences identified with hmmsearch were Schott bottles containing 100 mL of ammonium-free NOB me- required to match the hmm over at least 100 aa positions and dium. To ensure activity of the cells, NaNO2 in the final con- screened for similarity (>70%) to the UreC sequence from Ni- centration of 0.35 mM was added to all incubations and the trospira moscoviensis. Sequences passing these criteria were then cultures were incubated at 37 °C in the dark. After 2 d, all clustered at 90% similarity by using Usearch 7.0 (74). Usearch NaNO2 was consumed and the respective substrates for the centroids (46 sequences) were used in an exploratory phyloge- different incubation conditions were added to the incubations in netic analysis by using five independent chains of 1,100 iterations the following final concentrations: 0.35 mM NaNO2 and 1 mM (600 burnin) with a randomized initial alignment in Bali-phy urea; only 1 mM urea; and as an activity control, only 0.35 mM (51). The UreC protein sequences from N. moscoviensis and NaNO2. All incubation conditions were performed in duplicates. > N. lenta formed a well-supported cluster ( 99% posterior support) In control experiments, medium containing 0.35 mM NaNO2 with seven centroids derived from metagenomes. All meta- and 1 mM urea and medium containing 1 mM urea was in- genomic sequences that matched any of these seven centroids cubated without biomass. All incubations were performed for 6 h with >90% amino acid identity were retained for phylogenetic at 37 °C. Every 2 h, aliquots of the incubations were sampled and analysis. Additional genomic UreC sequences were identified centrifuged (17,949 × g, 10 min, 4 °C) to remove cells. In the with blastp by using known ammonium-oxidizing and nitrite- supernatant, ammonium was measured photometrically by the oxidizing bacteria as query and default parameters. The 100 top salicylic acid assay (81). hits were purged from highly similar sequences and clustered at 90% similarity by using usearch 7.0 and centroids were retained Incubation of N. defluvii with Urea. N. defluvii biomass was har- for phylogenetic analysis. Phylogenetic reconstruction was car- vested by centrifugation (6,300 × g, 10 min, 25 °C). The cell ried out by using five independent chains of 1,100 iterations (600 pellet was resuspended in ammonium-free NOB medium, and burn-in, randomized initial alignment) in Bali-phy (51), which equal aliquots of the biomass were added to 300-mL Schott simultaneously infers alignment and phylogeny from a set of bottles containing 100 mL of ammonium-free NOB medium. To sequences. The 102 amino acid sequences ranged from 109 aa all incubations, 0.5 mM NaNO2 (final concentration) was added (C687J26615_103352331 from metagenome 3300002121) to and the cultures were incubated at 30 °C in the dark. After 3 d, 595 aa (Microcoleus sp. WP_015182704). Posterior tree pools from all nitrite was consumed. Subsequently, 0.5 mM NaNO2 and all five independent runs were combined to determine consensus 1 mM urea, only 1 mM urea, or only 0.5 mM NaNO2 was added topology and posterior support for bipartitions. Alignment length to the respective incubations. All incubations were performed in ranged from 736 to 817 (mean = 767) in the posterior pool of duplicates. In addition, medium containing 0.5 mM NaNO2 and alignments. 1 mM urea and medium containing 1 mM urea was incubated without biomass in control experiments. All incubations were Cultivation of N. moscoviensis and N. europaea. To obtain biomass performed at 30 °C for 6 h. Every 2 h, aliquots of all incubations for experiments, N. moscoviensis was grown at 37 °C in mineral were sampled and centrifuged (17,949 × g for 10 min at 4 °C) to nitrite medium (23) without agitation and in the dark. This NOB remove cells. In the supernatant, ammonium was measured medium had the following composition: 1,000 mL of distilled photometrically by the salicylic acid assay (81). water; 0.01 g of CaCO3; 0.5 g of NaCl; 0.05 g of MgSO4·7H2O; N. europaea 0.15 g of KH2PO4;34.4μg of MnSO4·H2O; 50 μgofH3BO3;70μg Incubation of with Urea. N. europaea biomass was × of ZnCl2;72.6μgNa2MoO4·2H2O; 20 μgofCuCl2·2H2O; 24 μg harvested by centrifugation (6,300 g for 20 min at 25 °C) and of NiCl2·6H2O; 80 μg of CoCl2·6H2O; 1 mg of FeSO4·7H2O. If washed by resuspending the cells in ammonium-free AOB me- not stated otherwise, 0.01 g of NH4Cl was added to the medium dium. The biomass was centrifuged again, and the cell pellet was as additional N source. After autoclaving, filter-sterilized NaNO2 resuspended in ammonium-free AOB medium. By using syrin- was added to a final concentration of 1 mM. The nitrite con- ges, aliquots (1 mL) of this mixture were added to 300-mL Schott centration in the medium was regularly checked by using nitrite flasks containing 50 mL of ammonium-free AOB medium. These test stripes (Merkoquant; Merck), and nitrite was replenished bottles had already been plugged with butyl rubber stoppers, when completely consumed. N. europaea was grown at 30 °C on a which were fixed with screw caps. The culture was incubated in rotary shaker (100 rpm) in a modified AOB medium described the presence of 1 mM urea for 7 d at 30 °C in the dark in du- (75). The modified medium composition and preparation protocol plicates. During the incubations, aliquots of the cultures were were as follows: 900 mL of distilled water; 3.3 g of (NH4)2SO4; sampled by using syringes and were centrifuged (14,000 × g for

Koch et al. www.pnas.org/cgi/content/short/1506533112 3of13 10 min at 4 °C) to remove cells. In the supernatant, nitrite was aliquots were sampled by using syringes, centrifuged (17,949 × g, measured photometrically by the Griess assay (82). 10 min, 4 °C), and the formate concentrations in the supernatant were quantified by capillary electrophoresis on a P/ACE MDQ Coincubation of N. moscoviensis and N. europaea. N. moscoviensis Molecular Characterization System (Beckman Coulter). Anions and N. europaea biomass was harvested by centrifugation (6.300 × g, were separated by using the CEofix Anions 5 kit on an anion 20 min, 25 °C), resuspended in AOB medium (see above) that did exchange column (AS11; 250 × 4 mm; Thermo Scientific Dionex) + not contain NH4 , and centrifuged again. After this washing step, using a linear KOH gradient (0.1–40 mM in 6 min). To test the cells were resuspended in ammonium-free AOB medium and whether N. moscoviensis would reduce nitrate under oxic condi- mixed in a cell number ratio of 1:1 (details of the cell quantification tions with formate as electron donor, cells were incubated for 8 d are provided below). By using syringes, aliquots (1 mL) of this at 37 °C in the presence of sodium formate (initial concentration mixture were added to 300-mL Schott flasks containing 50 mL of 5 mM) and NaNO3 (initial concentration 1.5 mM), or only with ammonium-free AOB medium. These bottles had already been 1.5 mM NaNO3 in triplicates. During the incubations, aliquots were plugged with butyl rubber stoppers, which were fixed with screw sampled by using syringes, centrifuged (17,949 × g,10min,4°C), caps. The mixture was incubated in the presence of 1 mM or 50 μM and the concentrations of nitrite, nitrate, and formate in the su- urea as sole N and energy source for 7 d at 30 °C in duplicates. pernatant were measured as described above. During the incubations, aliquots of the cultures were sampled by To quantify the growth of N. moscoviensis under oxic condi- using syringes and centrifuged (14,000 × g, 10 min, 4 °C) to remove tions with formate or nitrite, respectively, nitrite-grown biomass cells. In the supernatant, N compounds were measured photomet- was harvested by centrifugation (6,300 × g, 20 min, 25 °C) and rically by the salicylic acid assay (ammonium) (81) or the Griess the cells were washed in nitrite-free NOB medium. Centrifuga- assay (nitrite and nitrate) (82). Nitrate was reduced to nitrite before tion and washing of the biomass were repeated until no nitrite − − the measurements by addition of vanadium(III) (83), and additional and nitrate were detected by NO2 and NO3 test stripes nitrite standards were included to account for inaccuracies in nitrate (Merkoquant; Merck) in the discarded supernatant. The biomass determination at high nitrite concentrations. was again resuspended in 10 mL of nitrite-free NOB medium, and 1-mL aliquots were distributed to 300-mL Schott flasks Cell Counts for the Urea Coincubation Experiments with N. moscoviensis containing 50 mL of nitrite-free NOB medium. The biomass and N. europaea. To achieve a 1:1 ratio of N. moscoviensis and aliquots were then incubated with 5 mM formate (final con- N. europaea in the coincubation experiments, the cell densities in centration) or 5 mM nitrite (final concentration) in triplicates for the axenic cultures of each organism were determined. Subse- 10 d at 37 °C. In a control experiment, N. moscoviensis biomass quently, aliquots of each culture containing highly similar amounts was incubated in NOB medium without any energy source in of cells were washed and mixed (see above). triplicates for 10 d at 37 °C. During the incubations, culture For counting, cells were homogeneously suspended by vor- aliquots (500 μL) were sampled, centrifuged (17,949 × g, 10 min, texing, and aliquots of each pure culture (5 μLofN. moscoviensis 4 °C), and the concentrations of nitrite and formate in the super- and 25 μLofN. europaea) were diluted separately in 15 mL of natant were measured as described above. In addition, total cell 1×PBS. The diluted cell suspensions were filtered separately protein was measured by using the BCA protein assay (Thermo onto black 0.2-μm polycarbonate GTBP-type membrane filters Fisher Scientific) according to the manufacturer’s instructions. (Millipore). After washing the cells once in 1×PBS, the cells For all long-term incubations of N. moscoviensis under oxic − were stained by incubation for 5 min in 400 μL of a 0.1 μg·mL 1 conditions with formate and nitrate, nitrite-grown biomass was DAPI (4’,6-Diamidino-2-phenylindole) solution. Subsequently, harvested by centrifugation (6,300 × g, 20 min, 25 °C), and the the filters were washed again in 1×PBS and air-dried. The stained cell pellet was resuspended in nitrite-free NOB medium. The cells were visualized on the dried filters by epifluorescence mi- biomass was added in equal proportions to 300-mL Schott flasks croscopy (AXIO-Imager M1; Zeiss) at 1,000× magnification and containing 70 mL of nitrite-free NOB medium. To all incuba- were counted by using a counting grid (Zeiss) with an area of tions, 0.5 mM NaNO2 (final concentration) was added and the 0.015 mm2. The cell densities were calculated as follows: cultures were incubated at 37 °C in the dark in duplicates. After 3 d, all nitrite was oxidized to nitrate and sodium formate (initial n · 15124.7 concentration 5 mM) was added to all incubations. For control C = , [S1] V experiments, to test the nitrite-oxidizing activity in absence of formate, 0.5 mM NaNO2 was added instead of formate. Cells where C is the cell density, n the average cell number per count- were incubated for 23 d at 37 °C. During all incubations, aliquots ing grid, V the volume of the filtered culture aliquot, and were sampled, centrifuged (17,949 × g, 10 min, 4 °C) and the 15,124.7 the microscope factor (226.87 mm2 as the filter area, formate, nitrite, and nitrate concentrations in the supernatant which contained cells, divided by the grid area of 0.015 mm2). were quantified as described above. For each culture, two aliquots of cell suspension were filtered and 10 grid areas per filter were analyzed. Respirometry. Substrate-dependent O2 consumption rates were measured by using a respiration cell RC-350 (Warner Instrum- Oxic Incubation of N. moscoviensis with Formate. For all short-term ents), equipped with an oxygen sensor (Model 1302) and con- incubations of N. moscoviensis under oxic conditions with for- nected to a picoammeter PA2000 (Unisense). Respiration rates mate, nitrite-grown biomass was harvested by centrifugation were recorded in SensorTrace Basic (version 3.0.2, Unisense). (7,232 × g, 20 min, 25 °C), washed in nitrite-free NOB medium, Measurements were performed by using approximately 20× and was centrifuged again. The biomass was again resuspended concentrated N. moscoviensis biomass from actively nitrite-oxi- in nitrite-free NOB medium, and 1-mL aliquots were added by dizing pure cultures. Biomass was harvested by centrifugation using syringes to 300-mL Schott flasks containing 100 mL of (1,181 × g, 15 min) and washed twice in NOB medium containing nitrite-free NOB medium. These bottles had already been plugged no nitrite or nitrate. For measurements, the cell chamber was with butyl rubber stoppers, which were fixed with screw caps. To filled with 1.5 mL of biomass suspension and closed with the test whether N. moscoviensis uses formate as the sole substrate, electrode inserted into the general electrode holder EH-100 the cells were incubated for 7 d at 37 °C in the presence of sodium (Warner Instruments) without inclosing air bubbles. Measurements formate (initial concentration 5 mM) in duplicates. In a control were performed at 39 °C. Substrates (1 mM Na-formate or 5 mM experiment, nitrite-free NOB medium containing 5 mM sodium NaNO3)wereaddedwitha100-μL gas-tight Hamilton syringe formate was incubated without biomass. During all incubations, (model 1710 RN, Hamilton Laboratory Products) from stock

Koch et al. www.pnas.org/cgi/content/short/1506533112 4of13 solutions, and samples were taken with the same syringe. Nitrite washed twice in anoxic nitrite-free NOB medium. After re- concentrations were determined photometrically by the Griess suspension in the medium, 1 mL of cell suspension was added as assay (82). inoculum to each serum bottle. The inoculated bottles were incubated at 37 °C in the dark for 34 d. During the incubation, samples N. moscoviensis Anoxic Incubation of with Organic Substrates. Ace- were taken by using syringes and were centrifuged (20,817 × g, tate (C H O Na), formate (CHNaO ), fumarate (C H Na O ), 2 3 2 2 4 2 2 4 20 min, 4 °C). Nitrite, nitrate, and formate concentrations in the nitrate (NaNO3), and succinate (C4H4Na2O4) stock solutions were prepared in mineral medium in serum bottles and were supernatant were determined as described above. The concen- made anoxic by sequential application of underpressure (with a trations of other organic substrates were determined by capillary vacuum pump) and flushing with N2. Subsequently, anoxic stock electrophoresis as described above for formate. solutions were autoclaved and stored at 4 °C in the dark. Heat- The anoxic incubation of N. moscoviensis with formate and labile substrate stocks of citrate (C6H5O73Na·2H2O), nitrite nitrate was repeated as described above with the following (NaNO2), and pyruvate (C3H3NaO3) were prepared in mineral modifications. Aliquots (100 mL) of anoxic nitrite-free NOB medium, sterile filtered, and injected into sterile serum bottles. medium were injected into sterile, N2-flushed 300-mL Schott Solutions were made anoxic as described above under sterile flasks, which were closed with butyl rubber stoppers and screw conditions and stored at 4 °C in the dark. Nitrite-free NOB caps. To provide a carbon source, 5 mL of a N2:CO2 (80:20) gas medium was prepared by strictly anoxic techniques as described mixture was added to the headspace. After addition of washed by Widdel and Bak (84). Aliquots of 10 mL of the anoxic nitrite- (see above) N. moscoviensis biomass, all bottles were incubated free NOB medium were injected into sterile, N -flushed 30-mL 2 for 1 d before the addition of substrates. Formate and nitrate serum bottles, which were closed with butyl rubber stoppers and were added to the incubations to final concentrations of 4.5 mM aluminum crimps. The respective organic substrate and NaNO3, formate and 1.2 mM NaNO3. All incubations were performed at or NaNO3 only, were added to the respective serum bottles to a final concentration of 1 mM each. Additionally, 3 mL of a 37 °C for 15 d in the dark. Samples were taken by using syringes × N2:CO2 (80:20) mixture was added to the headspace of each and centrifuged (17,949 g, 10 min, 4 °C). The formate and serum bottle to provide a carbon source. N. moscoviensis biomass nitrite concentrations in the supernatant were quantified as de- was harvested by centrifugation (8,228 × g, 20 min, 25 °C) and scribed above.

Koch et al. www.pnas.org/cgi/content/short/1506533112 5of13 A ubunit cytochrome b subunit

+ synthase

cytochromecytochrome c reductase, c reductase, FeS s ter, ATP-binding protein

putativeurease ureaurease ABCgamma beta transporter, subunit subunitureaseputative alphaureaaccessory putativebinding subunitaccessory urease glutamine-dependentproteinaccessory urease protein UrtA ammoniumproteinurease UreGnitrogen proteinUreF putativeNAD transporter regulatory UreDglutamine (protein-PII) proteinputative synthetasenitrate uridylyltransferase PII nitrateABC ABCcyanate transpor ABC transporter,ammonium transporter,hydrataseammoniumputative integraltransporterputative periplasmic transporterputative quinol membrane quinol octaheme component subunit cytochrome c N. moscoviensis NITMOv2_1249 NITMOv2_1283

N. lenta NITLEN_v1_110041 NITLEN_v1_110011

Ca. N. defluvii NH3 assimilation and its regulation NIDE1354 glutamine-dependentammoniumnitrogenputative NADtransporter regulatory glutamine(protein-PII)nitrite proteincyanate transportersynthetase uridylyltransferaseferredoxin-dependentPII hydrataseNIDE1368 nitrite reductase NH3 production N-compounds transport other function 5 kb + synthase unknown function

Ferric citrate / siderophore B receptor (TonB)

+ - + - Branched - Formate NH NO Sugars NO Zn 3+ NO NO 4 3 K 2 Sugars 3- Fe 2 urea Mn Mo amino acids PO4 Polyamines NO - NirK I II III 3 IV V NXR - + + NO 1/2 O H O 3- 2 NAD NADH +H 2 2 PO + 4 FecR + 2H ADP ATP + P Defense poly-P Peptide H i + siderophores 2 NAD HCOOH CRISPR Bacterio- ferritin 2 H+ NO - NH + Fe 2 NirA 4 S-FDH - NCO NH urea + HCO - CynS 3 3 + 2 CO + 2 NADH +H CO Glycogen Cofactor synthesis: * 2 Biotin HCO - storage urease 3 CA CO2 (Ribo)flavin Thiamine Flagellum Glucose-6P Pentose phosphate Folate NH +CO Phospholipids 3 2 Fructose-6P pathway Cobalamin

- + Glycerol-3-P Fructose-1,6P Heme MCPs 2 O + 2 H O + H O 2 2 2 2 Fumarate Succinate Coenzyme A SOD Glycerone-P Glyceraldehyde-3P TCA Malate Succinyl-CoA Stress resistance: 2 H O Cat Phoshoenolpyruvate cycle Phytoene, Carotene 2 2 Oxalacetate 2-Oxoglutarate 2 H2O + 2 O2 Peroxidase, Peroxiredoxin Pyruvate Multidrug efflux Citrate Isocitrate Thioredoxin system Arsenic resistance Cd Zn Co Acetyl-CoA Mercuric reductase Acetate -Lactamase Cl Acriflavin/Heavy metals O2

Tat protein CLD translocation Sec protein Type I Type II secretion / Type VI ClO - translocation secretion Type IV pili secretion 2

Miscellaneous transporter family Secretion system ABC transporter RND transporter N. moscoviensis specific

present in both Nitrospira

N. defluvii specific

Fig. S1. Key genomic and metabolic features of Nitrospira.(A) Schematic representation of the genomic regions in N. moscoviensis and N. lenta that contain the urease genes, the urea ABC transporter, and various other genes involved in the acquisition and metabolism of N compounds. The respective locus in N. defluvii that lacks urease and the urea transporter is shown for comparison. Solid lines connect homologous genes that encode proteins sharing sequence similarities 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 6of13 constructed from the annotations of the N. moscoviensis and N. defluvii genomes. Core functions, which are shared by both Nitrospira members (gray), and strain-specific features (yellow and blue) are shown. CA, carbonic anhydrase; Cat, catalase; CLD, chlorite dismutase; CRISPR, clustered regularly interspaced short palindromic repeats; CynS, cyanate hydratase (cyanase); MCPs, methyl-accepting chemotaxis proteins; S-FDH, soluble formate dehydrogenase. Enzyme complexes of the electron transport chain are labeled by Roman numerals. The TCA cycle depicts both directions (oxidative and reductive), with the reductive

TCA 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 containing 1 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 with cell-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. Two biological replicates are shown for all incubations with N. defluvii.(B) Absence of ureolytic activity in N. europaea. Incubation of N. europaea cells in medium containing 1 mM urea as the sole source of ammonia. No ammonia oxidation (production of nitrite) was observed in the two biological replicates. Aliquots of the same N. europaea biomass were used in the coincubation experiment with N. moscoviensis (see Results and Discussion in the main text). (C) Coincubation of N. moscoviensis and urease-negative N. europaea in presence of 50 μM urea as the source of ammonia. The concentrations of free ammonium, nitrite, and nitrate in the culture supernatant during 7 d of incubation are shown. At the start of the incubation, the medium contained some ammonium, most likely due to 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 7of13 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 of posterior 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 UreC sequences, 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 sequence divergence.

Koch et al. www.pnas.org/cgi/content/short/1506533112 8of13 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 formate concentrations measured on days 0 and 1 was caused by technical problems with formate measurement. The increase in nitrite indicates that formate was consumed 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 and production of nitrite, which would indicate the utilization of the respective organic substrate as electron donor, was not observed in any incubation. The concentrations 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 are shown for all incubations.

Koch et al. www.pnas.org/cgi/content/short/1506533112 9of13 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 of N. moscoviensis. Data points show the means of biological replicates (n = 3). Error bars represent SD and are not shown if smaller than symbols. This experiment represents 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 of N. moscoviensis. Data points show the means of biological replicates (n = 3). Error bars represent SD and are not shown if smaller than symbols. This experiment was 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 biomass protein 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 same amount of N. moscoviensis biomass was incubated in mineral medium without addition of formate or nitrite. Here, total protein decreased likely because of endogenous respiration in the absence of any external electron donor. (D) Long-term incubation of N. moscoviensis with formate and nitrate under oxic conditions. The initial concentrations of formate and nitrate were 5 mM and 0.5 mM, respectively. This graph shows the formate concentration in the culture supernatant. The results of two biological replicates are shown. (E) Nitrite concentrations in the culture supernatant during the incubation experiment shown in D. The initial net increase of the nitrite concentration was caused by nitrate reduction. The following net decrease of nitrite demonstrates the concomitant utilization 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 concentrations in the culture supernatant during the incubation experiment shown in D and E. The initial net decrease and subsequent net increase of the nitrate concentration are consistent with the nitrate-reducing and nitrite-oxidizing activities (D and E)ofN. moscoviensis in this experiment. (G) Nitrite oxidation by N. moscoviensis in absence of formate. The rate of nitrite oxidation was considerably higher than in presence of formate (E). Highly similar amounts of N. 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 with formate (1 mM initial concentration) as electron donor and in presence or absence of nitrate (5 mM initial concentration) are shown. Curves without symbols depict 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 the addition of formate to the cultures. Purple arrows indicate the addition of nitrate to cultures containing only formate. The reduction in the O2 consumption rates in presence of both electron acceptors, and the production of nitrite, show that electrons from formate were distributed to both O2 and nitrate. Please note 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 A

4000

3500

3000

2500

2000 Nitrospira defluvii 1500

1000

500

500 1000 1500 2000 2500 3000 3500 4000 4500 Nitrospira moscoviensis

Nitrite oxidoreductase (NXR) Putative NXR transcriptional regulators Electron transport and respiration Putative cytochrome bd-like terminal oxidase Nitrite oxidation / nitrate reduction B C Carbon fixation M25 Various cytochromes

M17 M13 Other or unknown functions

D1 M2 0.2 D12 M15 D10 Nitrospira Nitrospira 0.4 M20 Nitrospira M8 Nitrospira M10 NXR subunit alpha (Nide3237) 0.2 M21 defluvii moscoviensis defluvii 0.6 moscoviensis

D2 0.4 0.8 NXR subunit beta

D15 0.6 transcriptional regulator 1.0 D6 D13 D8 transcriptional regulator 0.8 D16 1.2 NXR subunit beta NXR subunit alpha (Nide3255) NXR subunit alpha

protein of unknown function transcriptional regulator

put. histidine kinase 1.0 1.4 NXR subunit beta

NXR subunit alpha

1.2 1.6 NXR subunit beta protein of unknown function M28 Sec-independent put.protein NXR translocase membrane subunit transcriptional regulator

D17 protein of unknown function M22 transcriptional regulator 1.4 1.8 membrane proteinprotein (unknownof unknown function) function protein of unknown function M16 NXR subunit beta M23

1.6 2.0 NXR subunit alpha protein of unknown functionTatA protein of unknown function protein of unknown function put. NXR membrane subunit transcriptional regulator 1.8 2.2 NXR subunit beta put. NXR chaperone ferredoxin-type protein NapG 2.4 NXR subunit alpha 2.0 M12 protein of unknown function protein of unknown function transcriptional regulator 2.6 2.2 response regulator M3 D14 NXR subunit beta

2.8 put. histidine kinase 2.4 NXR subunit alpha D18 M11 M17 M5 D5 3.0 protein translocase subunit SecF D9 2.6 ajC put. NXR membrane subunit protein translocase subunit SecD put. NXR membrane subunit D23 3.2 2.8 preprotein translocase subunit Y protein of unknown function M18 tRNA-guanine transglycosylase Sec-independent protein translocase arginine--tRNA ligase protein of unknown function 3.4 membrane protein (unknown function) 3.0 put. glycosyl transferase protein of unknown function M19 protein of unknown function put. NXR membrane subunit 3.6 put. cyt. c 3.2 put. NXR chaperone TatA put. Mo cofactor guanylyltransferaseput. cyt. c ferredoxin-type protein NapG put. NXR membrane subunit protein of unknown function D12 3.8 protein of unknown function D14 3.4 M9 protein of unknown function D13 M15 response regulator 4.0 M27 M6 3.6 put. histidine kinase M14 M24 put. cyt. bd-like oxidase M18 4.2 protein translocase subunit SecF D19 3.8 protein translocase subunit SecD protein of unknown function preprotein translocase subunit D4 1 4.4 tRNA-guanine transglycosylase protein of unknown function arginine--tRNA D1 4.0 protein of unknown function M16 put. glycosyl transferase protein of unknown function put. Mo cofactor guanylyltransferase D7 put. NXR membrane subunit 4.2 M7 put. cyt. c put. cyt. c

put. cyt. bd-like oxidase

D20 D21 n M26 protein ofput. unknown cyt. bd-like function oxidase r o

M4 o

t t

n n D3 membrane protein (unknown function)

D22 u

o f

f ligase

n n

f M19

f o YajC

n n

i put. cyt. bd-like oxidase

t t

n protein of unknown function

protein of unknown function

p protein of unknown function

NADH-quinone oxidoreductase (complex I) Succinate dehydrogenase / fumarate reductase (complex II) Quinol-cytochrome c oxidoreductase (complex III) membrane protein (unknown function) Cytochrome bd quinol oxidase F F ATP synthase (complex V) D 0 1 Putative cytochrome bd-like terminal oxidase Various cytochromes Nitrite oxidoreductase (NXR) rTCA and oTCA cycle enzymes

Other or unknown functions 2-oxoglutarate:ferredoxin oxidoredutase subunit gamma Other or unknown functions 2-oxoglutarate:ferredoxin 2-oxoglutarate:ferredoxinoxidoreductase subunit oxidoredutase epsilon subunit beta

M1 2-oxoglutarate:ferredoxin oxidoredutase subunit delta E 2-oxoglutarate:ferredoxin oxidoredutase subunit alpha D1

complex I subunit N Nitrospira complex I subunit M Nitrospira complex I subunit M complex I subunit L defluvii complex I subunit K moscoviensis Nitrospira Nitrospira complex I subunit J complexcomplex I subunit I subunit G I defluvii moscoviensis complex I subunit D complex I subunit C A complex I subunit B complex I subunit complex I subunit N complex I subunit M complex I subunit M protein of unknown function complex I subunit N complex I subunit L complex I subunit K protein of unknown function M20 complex I subunit M complex I subunit J DNA-formamidopyrimidine glycosylase complex I subunit I protein of unknown function complex I subunit G complex I subunit L complex I subunit D M2 D15 protein of unknown function D2 complex I subunit C protein of unknown function complex I subunit K complex I subunit B complex I subunit complex I subunit J complex I subunit N

A complex I subunit M 2-oxoglutarate:ferredoxin oxidoredutase subunit beta 2-oxoglutarate:ferredoxin oxidoredutase subunit gamma complex I subunit I 2-oxoglutarate:ferredoxin oxidoreductase subunit epsilon 2-oxoglutarate:ferredoxin oxidoredutase subunit delta complex I subunit H complex I subunit L protein of unknown function 2-oxoglutarate:ferredoxin oxidoredutase subunit alpha complex I subunit K protein of unknown function complex I subunit J protein of unknown function complex I subunit G A protein of unknown function complex I subunit I T protein of unknown function complex I subunit H P citrate lyase subunit alpha protein of unknown function complex I subunit F DNA-formamidopyrimidine glycosylase complex I subunits C and D complex I subunit G A proteinTP-citrate of unknown lyase function subunit beta complex I subunit E complex I subunit F A A P citrate lyase subunit alpha complex I subunit E T complex I subunits C and D A complex I subunit B complex I subunit B complex I subunit complex I subunit aconitate hydratase aconitate hydratase complex I subunit D3 complex I subunit B complex I subunit L complex I subunits C and D A isocitrate dehydrogenase complex I subunit E isocitrate dehydrogenase complex I subunit M complex I subunit F M3 ferredoxin D4 complex I subunit G complex II flavoprotein subunit complex I subunit F complex I subunit H A synthetase subunit beta complex I subunit I complex II flavoproteinferredoxin subunit D5 complex I subunit J succinyl-Co synthetase subunit alpha complex II Fe-S subunit complex I subunit K succinyl-CoA complex I subunit L succinyl-Co complex II ferredoxin complex I subunit M A synthetase subunit beta D6 complex I subunit N pyruvate:ferredoxin oxidoreductase subunit epsilon complex II flavoprotein subunit succinyl-Co pyruvate:ferredoxin oxidoreductase subunit gamma A synthetase subunit alpha put. complex I subunit L pyruvate:ferredoxin oxidoreductase subunit beta M21 put. complex I subunit M M4 pyruvate:ferredoxin oxidoreductase subunit alpha pyruvate:ferredoxin oxidoreductase subunit epsilon complex II flavoprotein subunit pyruvate:ferredoxin oxidoreductase subunit delta put. complex I subunit M pyruvate:ferredoxin oxidoreductase subunit gamma put. complex I subunit L 2-oxoacid:ferredoxin oxidoreductase subunit alpha (fragment) complex II subunit C pyruvate:ferredoxin oxidoreductase subunit beta M5 2-oxoacid:ferredoxin oxidoreductase subunit beta put. complex III cyt. c subunit complex I subunit F D16 put. cyt. c protein of unknown function pyruvate:ferredoxin oxidoreductase subunit alpha M22 complex II Fe-S subunit fumarate hydratase complex II flavoprotein subunit D7 M6 pyruvate:ferredoxin oxidoreductase subunit delta heat shock protein (Hsp20 family) put. cyt. c complex II cytochrome b558 subunit complex IIIcomplex fused cyt.III Fe-S b/c subunit subunit citrate synthase put. cyt. c complex II ferredoxin complex II flavoprotein subunit malate dehydrogenase put. cyt. bd-like oxidase D17 2-oxoacid:ferredoxin oxidoreductase citratesubunit synthase beta put. complex III cyt. c subunit M7 M23 complex III fused cyt. b/c subunit complex III Fe-S subunit put. complex III Fe-S subunit put.pyruvate dehydrogenase E2 put. cyt. c protein of unknown function complex III cyt. b subunit put. pyruvate dehydrogenase E1 subunit beta put. cyt. c put. complex III Fe-S subunit put. complex III cyt. b subunit heat shock proteinfumarate (Hsp20 hydratasefamily) put. pyruvate dehydrogenase E1 subunit alpha D8 put. cyt. c put. cyt. c put. cyt. bd-like cyt. c oxidase put. cyt. bd-like oxidase M8 D18 membrane protein (function unknown) put. cyt. c put. pyruvate dehydrogenase E3 put. complex III Fe-S subunit put. complex III cyt. b subunit membrane protein (function unknown) malate dehydrogenase M24 put. cyt. bd-like cyt. c oxidase put. NXR membrane subunit put. cyt. c pyruvate dehydrogenase E1 subunit beta put. cyt. c A put. NXR membrane subunit put. cyt. c M9 pyruvate dehydrogenase E2 put. cyt. bd oxidase subunit II cyt. bd oxidase subunit I protein of unknown function cyt. bd oxidase subunit II pyruvate dehydrogenase E3 cyt. bd oxidase subunit I cyt. bd oxidase subunit I put. cyt. bd oxidase subunit II put. A pyruvate dehydrogenase E3 A ATP synthase F1 subunit beta ATP synthase F1 subunit epsilon

T T A

P P synthase F1 subunit gamma D19 t AT

i synthase F1 subunit alpha

synthase F1 subunit delta n P M25 u pyruvate dehydrogenase E2 T synthase F1 subunit alpha P synthase F1 subunit delta pyruvate dehydrogenase E1 subunit alpha D9 A P b T u

synthase F1 subunit beta s

A put. synthase F1 subunit synthase gamma F0 subunit B pyruvate dehydrogenase E1 subunit beta P P synthase F0 subunit C 0 T pyruvate dehydrogenase E2 T ATP P synthase F0 subunit F

synthase F1 subunit epsilon A A T P

P e A T D20

T s A A a

h M10 t M26

D10 n

y pyruvate dehydrogenase E3

synthase F0 subunit C

P synthase F0 subunit

P pyruvate dehydrogenase E1 subunit beta pyruvate dehydrogenase E3 T

T TP

A A pyruvate dehydrogenase E1 subunit alpha D11 D21 M11 M27 M12 D22 M14 M13 D23 M28

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) Localization of 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 for N. 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)andN. 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 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 transport chains 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 shown here 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)andN. 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 of these 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. moscoviensis and N. defluvii Genome feature N. moscoviensis N. defluvii

Genome size, bp 4,589,485 4,317,083 Average G+C content, % 62 59 No. of CDS 4,863 4,274 Coding density, % 90.6 89.4 CDS with predicted function 2,391 (56%) 2,154 (50%) rRNA operon 1 1 tRNA genes 47 46 Species-specific CDS* 2,161 (44%) 1,695 (40%) Species-specific CDS with 1,547 1,216 unknown function

CDS, coding sequences. *CDS with no homolog in the respective other Nitrospira genome were considered 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