Letter | Published:

Complete nitrification by a single microorganism

Nature volume 528, pages 555559 (24 December 2015) | Download Citation

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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Data deposits

Metagenomic data is available in the European Nucleotide Archive (ENA) under accession numbers CZQA01000001CZQA01000015 and CZPZ01000001CZPZ01000036.

References

  1. 1.

    Recherches sur les organismes de la nitrification. Ann. Inst. Pasteur (Paris) 4, 213–231 (1890)

  2. 2.

    , , & In quest of the nitrogen oxidizing prokaryotes of the early Earth. Environ. Microbiol. 13, 283–295 (2011)

  3. 3.

    , & Why is metabolic labour divided in nitrification? Trends Microbiol. 14, 213–219 (2006)

  4. 4.

    , , , & Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture 270, 1–14 (2007)

  5. 5.

    et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nature Biotechnol. 31, 533–538 (2013)

  6. 6.

    & Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 19126–19131 (2009)

  7. 7.

    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)

  8. 8.

    , & 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)

  9. 9.

    et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543–546 (2005)

  10. 10.

    , & 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)

  11. 11.

    & The divergent AmoC3 subunit of ammonia monooxygenase functions as part of a stress response system in Nitrosomonas europaea. J. Bacteriol. 194, 3448–3456 (2012)

  12. 12.

    & Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiol. Lett. 278, 146–156 (2008)

  13. 13.

    et al. Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira. Proc. Natl Acad. Sci. USA 112, 11371–11376 (2015)

  14. 14.

    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)

  15. 15.

    et al. Complete nitrification by Nitrospira bacteria. Nature (2015)

  16. 16.

    , & Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea. J. Bacteriol. 175, 2436–2444 (1993)

  17. 17.

    & 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)

  18. 18.

    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)

  19. 19.

    , , & 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)

  20. 20.

    Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616 (1997)

  21. 21.

    , & Key physiology of anaerobic ammonium oxidation. Appl. Environ. Microbiol. 65, 3248–3250 (1999)

  22. 22.

    et al. Role for urea in nitrification by polar marine Archaea. Proc. Natl Acad. Sci. USA 109, 17989–17994 (2012)

  23. 23.

    & Autotrophic ammonia oxidation at low pH through urea hydrolysis. Appl. Environ. Microbiol. 67, 2952–2957 (2001)

  24. 24.

    , , & Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review. Aquat. Microb. Ecol. 59, 67–88 (2010)

  25. 25.

    , , , & Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17, 83–91 (2006)

  26. 26.

    , , , & In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67, 5273–5284 (2001)

  27. 27.

    et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008)

  28. 28.

    et al. Cohn’s Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase. Proc. Natl Acad. Sci. USA 103, 2363–2367 (2006)

  29. 29.

    et al. Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge. Appl. Microbiol. Biotechnol. 92, 845–854 (2011)

  30. 30.

    , , , & Using the metagenomics RAST server (MG-RAST) for analyzing shotgun metagenomes. Cold Spring Harb. Protoc. (2010)

  31. 31.

    , & DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322 (1996)

  32. 32.

    mmgenome: tools for extracting individual genomes from metagenomes (2015)

  33. 33.

    , , , & NextClip: an analysis and read preparation tool for Nextera Long Mate Pair libraries. Bioinformatics 30, 566–568 (2014)

  34. 34.

    et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010)

  35. 35.

    et al. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186–1199 (2012)

  36. 36.

    , & the HMMER development team. HMMER: biosequence analysis using profile hidden Markov models. (2015)

  37. 37.

    , , , & Integrative analysis of environmental sequences using MEGAN4. Genome Res. 21, 1552–1560 (2011)

  38. 38.

    et al. ExSPAnder: a universal repeat resolver for DNA fragment assembly. Bioinformatics 30, i293–i301 (2014)

  39. 39.

    , , & QUAST: quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013)

  40. 40.

    , , , & CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015)

  41. 41.

    et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009)

  42. 42.

    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)

  43. 43.

    et al. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res. 34, 53–65 (2006)

  44. 44.

    & in Methods in Enzymology Vol. 486 (ed ) 109–130 (Academic Press, 2011)

  45. 45.

    , , & Cathepsin B2 measurement by sensitive fluorometric ammonia analysis. Anal. Biochem. 60, 153–162 (1974)

  46. 46.

    Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt „Ueber einige Azoverbindungen”. Ber. Dtsch. Chem. Ges. 12, 426–428 (1879)

  47. 47.

    , & in Molecular Microbial Ecology (eds & ) Ch. 9, 213–239 (Taylor & Francis, 2005)

  48. 48.

    , & daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8, 200–213 (2006)

  49. 49.

    & Quantification of uncultured microorganisms by fluorescence microscopy and digital image analysis. Appl. Microbiol. Biotechnol. 75, 237–248 (2007)

  50. 50.

    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)

  51. 51.

    et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596 (2013)

  52. 52.

    et al. ARB: a software environment for sequence data. Nucleic Acids Res. 32, 1363–1371 (2004)

  53. 53.

    & MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003)

  54. 54.

    et al. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9, 386 (2008)

  55. 55.

    , & Fast and sensitive protein alignment using DIAMOND. Nature Methods 12, 59–60 (2015)

  56. 56.

    , & Visualization of comparative genomic analyses by BLAST score ratio. BMC Bioinformatics 6, 2 (2005)

  57. 57.

    et al. Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst. Appl. Microbiol. 23, 93–106 (2000)

  58. 58.

    & in Nucleic Acid Techniques in Bacterial Systematics (eds & ) (Wiley, 1991)

  59. 59.

    et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925 (1990)

  60. 60.

    , , , & 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)

  61. 61.

    , , , & In situ identification of ammonia-oxidizing bacteria. Syst. Appl. Microbiol. 18, 251–264 (1995)

  62. 62.

    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)

  63. 63.

    , , , & Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl. Environ. Microbiol. 62, 2156–2162 (1996)

  64. 64.

    , , , & The oligonucleotide probe database. Appl. Environ. Microbiol. 62, 3557–3559 (1996)

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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.

Author information

Affiliations

  1. Department of Microbiology, IWWR, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands

    • Maartje A. H. J. van Kessel
    • , Daan R. Speth
    • , Huub J. M. Op den Camp
    • , Boran Kartal
    • , Mike S. M. Jetten
    •  & Sebastian Lücker
  2. Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark

    • Mads Albertsen
    •  & Per H. Nielsen
  3. Laboratory for Microbiology, University of Gent, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

    • Boran Kartal
  4. Department of Biotechnology, TU Delft, Julianalaan 67, 2628 BC Delft, the Netherlands

    • Mike S. M. Jetten

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sebastian Lücker.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Nitrospira sp.1 and sp.2 genes discussed in this study.

  2. 2.

    Supplementary Table 2

    Marker HMMs used by CheckM.

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DOI

https://doi.org/10.1038/nature16459

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