Abstract
Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 18901, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle2. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms3. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.
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Accession codes
Primary accessions
European Nucleotide Archive
Data deposits
Metagenomic data is available in the European Nucleotide Archive (ENA) under accession numbers CZQA01000001–CZQA01000015 and CZPZ01000001–CZPZ01000036.
References
Winogradsky, S. Recherches sur les organismes de la nitrification. Ann. Inst. Pasteur (Paris) 4, 213–231 (1890)
Vlaeminck, S. E., Hay, A. G., Maignien, L. & Verstraete, W. In quest of the nitrogen oxidizing prokaryotes of the early Earth. Environ. Microbiol. 13, 283–295 (2011)
Costa, E., Pérez, J. & Kreft, J. U. Why is metabolic labour divided in nitrification? Trends Microbiol. 14, 213–219 (2006)
Crab, R., Avnimelech, Y., Defoirdt, T., Bossier, P. & Verstraete, W. Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture 270, 1–14 (2007)
Albertsen, M. et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nature Biotechnol. 31, 533–538 (2013)
Richter, M. & Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 19126–19131 (2009)
Lücker, S. et al. A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proc. Natl Acad. Sci. USA 107, 13479–13484 (2010)
Rotthauwe, J. H., Witzel, K. P. & Liesack, W. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 63, 4704–4712 (1997)
Könneke, M. et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543–546 (2005)
El Sheikh, A. F., Poret-Peterson, A. T. & Klotz, M. G. Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707. Appl. Environ. Microbiol. 74, 312–318 (2008)
Berube, P. M. & Stahl, D. A. The divergent AmoC3 subunit of ammonia monooxygenase functions as part of a stress response system in Nitrosomonas europaea . J. Bacteriol. 194, 3448–3456 (2012)
Klotz, M. G. & Stein, L. Y. Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiol. Lett. 278, 146–156 (2008)
Koch, H. et al. Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira . Proc. Natl Acad. Sci. USA 112, 11371–11376 (2015)
Lupo, D. et al. The 1.3-Å resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins. Proc. Natl Acad. Sci. USA 104, 19303–19308 (2007)
Daims, H. et al. Complete nitrification by Nitrospira bacteria. Nature http://dx.doi.org/10.1038/nature16461 (2015)
McTavish, H., Fuchs, J. A. & Hooper, A. B. Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea . J. Bacteriol. 175, 2436–2444 (1993)
Hyman, M. R. & Arp, D. J. 14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase. J. Biol. Chem. 267, 1534–1545 (1992)
Taylor, A. E. et al. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria. Appl. Environ. Microbiol. 79, 6544–6551 (2013)
Ginestet, P., Audic, J.-M., Urbain, V. & Block, J.-C. Estimation of nitrifying bacterial activities by measuring oxygen uptake in the presence of the metabolic inhibitors allylthiourea and azide. Appl. Environ. Microbiol. 64, 2266–2268 (1998)
Zumft, W. G. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616 (1997)
Strous, M., Kuenen, J. G. & Jetten, M. S. Key physiology of anaerobic ammonium oxidation. Appl. Environ. Microbiol. 65, 3248–3250 (1999)
Alonso-Sáez, L. et al. Role for urea in nitrification by polar marine Archaea. Proc. Natl Acad. Sci. USA 109, 17989–17994 (2012)
Burton, S. A. Q. & Prosser, J. I. Autotrophic ammonia oxidation at low pH through urea hydrolysis. Appl. Environ. Microbiol. 67, 2952–2957 (2001)
Solomon, C., Collier, J., Berg, G. & Glibert, P. Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review. Aquat. Microb. Ecol. 59, 67–88 (2010)
Wagner, M., Nielsen, P. H., Loy, A., Nielsen, J. L. & Daims, H. Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17, 83–91 (2006)
Daims, H., Nielsen, J. L., Nielsen, P. H., Schleifer, K. H. & Wagner, M. In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67, 5273–5284 (2001)
Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008)
Stoecker, K. et al. Cohn’s Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase. Proc. Natl Acad. Sci. USA 103, 2363–2367 (2006)
Luesken, F. A. et al. Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge. Appl. Microbiol. Biotechnol. 92, 845–854 (2011)
Glass, E. M., Wilkening, J., Wilke, A., Antonopoulos, D. & Meyer, F. Using the metagenomics RAST server (MG-RAST) for analyzing shotgun metagenomes. Cold Spring Harb. Protoc. http://dx.doi.org/10.1101/pdb.prot5368 (2010)
Zhou, J., Bruns, M. A. & Tiedje, J. M. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322 (1996)
Albertsen, M. mmgenome: tools for extracting individual genomes from metagenomes http://madsalbertsen.github.io/mmgenome/ (2015)
Leggett, R. M., Clavijo, B. J., Clissold, L., Clark, M. D. & Caccamo, M. NextClip: an analysis and read preparation tool for Nextera Long Mate Pair libraries. Bioinformatics 30, 566–568 (2014)
Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010)
Dupont, C. L. et al. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186–1199 (2012)
Eddy, S. R., Wheeler, T. J. & the HMMER development team. HMMER: biosequence analysis using profile hidden Markov models. http://hmmer.janelia.org/ (2015)
Huson, D. H., Mitra, S., Ruscheweyh, H. J., Weber, N. & Schuster, S. C. Integrative analysis of environmental sequences using MEGAN4. Genome Res. 21, 1552–1560 (2011)
Prjibelski, A. D. et al. ExSPAnder: a universal repeat resolver for DNA fragment assembly. Bioinformatics 30, i293–i301 (2014)
Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013)
Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015)
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009)
Vallenet, D. et al. MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. Nucleic Acids Res. 41, D636–D647 (2013)
Vallenet, D. et al. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res. 34, 53–65 (2006)
Spieck, E. & Lipski, A. in Methods in Enzymology Vol. 486 (ed Martin, G. K. ) 109–130 (Academic Press, 2011)
Taylor, S., Ninjoor, V., Dowd, D. M. & Tappel, A. L. Cathepsin B2 measurement by sensitive fluorometric ammonia analysis. Anal. Biochem. 60, 153–162 (1974)
Griess, P. Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt „Ueber einige Azoverbindungen”. Ber. Dtsch. Chem. Ges. 12, 426–428 (1879)
Daims, H., Stoecker, K. & Wagner, M. in Molecular Microbial Ecology (eds Osborn, A. M. & Smith, C. J. ) Ch. 9, 213–239 (Taylor & Francis, 2005)
Daims, H., Lücker, S. & Wagner, M. daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8, 200–213 (2006)
Daims, H. & Wagner, M. Quantification of uncultured microorganisms by fluorescence microscopy and digital image analysis. Appl. Microbiol. Biotechnol. 75, 237–248 (2007)
Lee, N. et al. Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289–1297 (1999)
Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596 (2013)
Ludwig, W. et al. ARB: a software environment for sequence data. Nucleic Acids Res. 32, 1363–1371 (2004)
Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003)
Meyer, F. et al. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9, 386 (2008)
Buchfink, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. Nature Methods 12, 59–60 (2015)
Rasko, D. A., Myers, G. S. & Ravel, J. Visualization of comparative genomic analyses by BLAST score ratio. BMC Bioinformatics 6, 2 (2005)
Schmid, M. et al. Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst. Appl. Microbiol. 23, 93–106 (2000)
Stahl, D. A. & Amann, R. in Nucleic Acid Techniques in Bacterial Systematics (eds Stackebrandt, E. & Goodfellow, M. ) (Wiley, 1991)
Amann, R. I. et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925 (1990)
Daims, H., Brühl, A., Amann, R., Schleifer, K. H. & Wagner, M. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22, 434–444 (1999)
Wagner, M., Rath, G., Amann, R., Koops, H.-P. & Schleifer, K.-H. In situ identification of ammonia-oxidizing bacteria. Syst. Appl. Microbiol. 18, 251–264 (1995)
Juretschko, S. et al. Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 64, 3042–3051 (1998)
Mobarry, B. K., Wagner, M., Urbain, V., Rittmann, B. E. & Stahl, D. A. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl. Environ. Microbiol. 62, 2156–2162 (1996)
Alm, E. W., Oerther, D. B., Larsen, N., Stahl, D. A. & Raskin, L. The oligonucleotide probe database. Appl. Environ. Microbiol. 62, 3557–3559 (1996)
Acknowledgements
We would like to thank K. Stultiens, T. van Alen, J. Frank, P. Klaren, L. Pierson and L. Claessens-Joosten for technical assistance, T. Spanings for biofilter maintenance and C. Herbold for the ANI analysis. We are grateful for the use of the confocal microscope from the Microscopic Imaging Centre (MIC, Radboud UMC, Nijmegen) and would like to thank H. Croes and M. Willemse for technical assistance. The LABGeM team and the National Infrastructure “France Genomique” are acknowledged for support within the MicroScope annotation platform. We are thankful to C. Dupont, A. Santoro and M. Saito for consenting to our use of the Nitrospira marina nxrA sequences, which were produced by the US Department of Energy Joint Genome Institute. M.A.H.J.v.K was supported by the Technology Foundation STW (grant 13146), D.R.S. by the BE-Basic Foundation (grant fs7-002), M.A. and P.H.N. by the Danish Council for Independent Research (DFF 4005-00369), M.S.M.J. by the European Research Council (ERC Advanced Grant projects anammox 232937 and Eco_MoM 339880) and the Dutch Ministry of Education, Culture and Science (Gravitation grant SIAM 024002002), B.K. and S.L. by the Netherlands Organization for Scientific Research (NWO VENI grants 863.11.003 and 863.14.019, respectively). The Radboud Excellence Initiative is acknowledged for support to S.L.
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M.A.H.J.v.K and S.L. executed experiments and analysed data. D.R.S. and M.A. contributed to metagenomic data analyses. M.A. and P.H.N. performed sequencing, assembly and binning. M.A.H.J.v.K., H.J.M.O.d.C., B.K., M.S.M.J. and S.L. planned research. M.A.H.J.v.K., B.K. and S.L. wrote the paper. All authors discussed results and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Ammonium and nitrite conversion by the enrichment culture.
a, b, Inorganic nitrogen load of the enrichment culture per 24 h cycle (filled symbols) and effluent concentrations (open symbols) for ammonium (a, diamonds) and nitrite (b, triangles). Effluent nitrite concentrations were below the detection limit (<5 μM) at all time points. Data points represent the mean of three technical replicates, error bars the standard deviations of these triplicates. Nitrate concentration in the medium varied between 0.5 and 2.0 mM and total organic carbon (TOC) content between 1.30 and 1.44 ppm, which was due to medium preparation with water obtained directly from the recirculation aquaculture system.
Extended Data Figure 2 Metagenome binning.
a, b, Extraction of the Nitrospira sp.1 (a) and sp.2 (b) genome sequences from the metagenome using differential coverage binning. Each circle represents a metagenomic scaffold, with size proportional to scaffold length; the plots contain a total of 47,584 scaffolds. The inlay of each figure shows the secondary binning based on tetranucleotide frequencies, with a total of 331 (a) and 281 (b) scaffolds included. Taxonomic classification is indicated by colour; a total of 3,158 essential marker genes were detected. The extracted bins are enclosed by a dashed line. c, d, Genome contaminations were excluded by generating linkage maps of the final bins of sp.1 (c, 25 scaffolds) and sp.2 (d, 86 scaffolds) using mate-pair sequencing data.
Extended Data Figure 3 Phylogenetic analysis of NXR.
Bayesian interference tree (s.d. = 0.0099) showing the affiliation of the Nitrospira sp.1 and sp.2 nxrA sequences in comparison to other genome-sequenced Nitrospira, Nitrospina and anammox bacteria. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. NCBI protein accession numbers for all publicly available sequences are indicated, numbers with an asterisk are IMG gene IDs. The described Nitrospira sublineages are indicated by coloured boxes and roman numbers. The scale bar represents 10% sequence divergence. Note the different affiliation of the “Candidatus N. nitrosa” (sp.1) nxrA sequences. The tree contains 25 sequences from 12 species, belonging to 3 different phyla. Sequences from closely related bacterial putative nitrate reductases were used as outgroup (n = 4); the outgroup position is indicated by the arrow.
Extended Data Figure 4 16S rRNA-based phylogenetic analysis.
Bayesian interference tree (s.d. = 0.0098) showing the affiliation of the Nitrospira sp.1 and sp.2 16S rRNA sequences within Nitrospira sublineage II. Posterior probabilities ≥70% and ≥90% are indicated by open and filled circles, respectively. The strongly supported sequence group containing the novel Nitrospira spp. catalysing complete nitrification is shaded in grey, the two subgroups containing Nitrospira sp.1 and sp.2 (in bold) are highlighted by green and red boxes, respectively. N. moscoviensis is depicted in bold for comparison. The curly bracket indicates the target group of the newly designed FISH probe Ntspa476 (see Extended Data Table 2). Scale bar indicates 10% sequence divergence. The tree contains a total of 181 sequences; the size of sequence groups is indicated in brackets. Sequences from members of Nitrospira sublineages I and IV were used as outgroup (n = 24); the outgroup position is indicated by the arrow.
Extended Data Figure 5 Control experiments of AMO-labelling.
a, Cells incubated with the fluorescent dye FTCP (green) were stained by FISH using probes specific for Nitrospira (Ntspa662, red) and all bacteria (EUB338mix, blue). A small cell cluster was stained by FTCP and targeted by both probes (resulting in a white overlay signal), while all other bacteria (in blue) were not or only slightly stained by FTCP. The green signal is due to autofluorescence and unspecific FTCP binding to the floc matrix. b, Anammox cells (Amx820, blue) showed minor staining by FTCP (green), but to a much lesser degree than Nitrospira (Ntspa662, red; yellow overlay). c and d, Positive controls: ammonium oxidizing bacteria (c, Nso1225 and Nso190, red) in an aerobic enrichment culture and a Nitrosomonas europaea pure culture (d, NEU, red, and EUB338mix, blue) were stained by FTCP (resulting in yellow and white overlays, respectively). e and f, Negative controls: canonical Nitrospira in an aerobic enrichment culture (e, Ntspa662, blue) and a Nitrospira moscoviensis pure culture (f, Ntspa662, red, and EUB338mix, blue; magenta overlay) did not show any labelling with FTCP (green). The two bright green structures in (c) and the bright pink signal in (e) are due to autofluorescence. Images are representative of two (a and b) or one (c to f) individual experiments, with three technical replicates each. Scale bars in all panels represent 10 μm.
Extended Data Figure 6 Batch incubations with nitrite, urea and without substrate.
a, b, Nitrite (triangles) oxidation by the enrichment culture to nitrate (squares) in the absence (a) and in the presence (b) of ATU. The ammonia (diamonds) in b presumably stems from biomass decay and is not oxidized owing to ATU inhibition. c, Urea conversion to ammonium (diamonds) and subsequent oxidation to nitrate (squares). d, No-substrate control; minor amounts of ammonium (diamonds) presumably stem from mineralisation of degrading biomass, leading subsequently to nitrate (squares) formation. Symbols in all plots represent averages of three independent incubations; ammonium was determined in single measurements, nitrite and nitrate in duplicate (a and b) or triplicate (c and d). Error bars represent standard deviations of three biological replicates.
Extended Data Figure 7 Ammonium and nitrite-dependent CO2 fixation shown by FISH-MAR.
a–d, FISH with probes for all bacteria (EUB338mix, blue), and probes specific for Nitrospira (Ntspa662, red; resulting in magenta) and anammox bacteria (Amx820, green; resulting in cyan). a, Ammonia-dependent carbon fixation. Only Nitrospira cells were active, as indicated by silver grain deposition. Note the inactive anammox cells on the left side of the smaller floc, co-localizing with highly active Nitrospira cells on the right side of the same floc. b, Inhibition of ammonia-dependent carbon fixation by ATU. c, Nitrite-dependent carbon fixation. Only Nitrospira cells incorporated 14CO2. d, No-substrate control. Images are representative of two individual experiments, with two technical replicates each. Scale bars in all panels represent 10 μm.
Supplementary information
Supplementary Table 1
Nitrospira sp.1 and sp.2 genes discussed in this study. (XLSX 16 kb)
Supplementary Table 2
Marker HMMs used by CheckM. (XLSX 20 kb)
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van Kessel, M., Speth, D., Albertsen, M. et al. Complete nitrification by a single microorganism. Nature 528, 555–559 (2015). https://doi.org/10.1038/nature16459
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DOI: https://doi.org/10.1038/nature16459
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