Skip to main content
SpringerLink
Log in

Molecular mechanism of anaerobic ammonium oxidation

  • Letter
  • Published:

From Nature

View current issue Submit your manuscript

Abstract

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N2) to the atmosphere1,2,3,4. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known5. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N2H4)6,7. Here we show that N2H4 is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N2H4. We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N2H4 synthesis and its oxidation to N2. These results present a new biochemical reaction forging an N–N bond and fill a lacuna in our understanding of the biochemical synthesis of the N2 in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Biochemical pathway and enzymatic machinery of K. stuttgartiensis.
Figure 2: Determination of nitric oxide (NO) as an intermediate.
Figure 3: Hydrazine turnover.
Figure 4: 29 N 2 production by hydrazine synthase complex and kustc1061 from 15 NH 4 + and NO.

Similar content being viewed by others

Accession codes

Data deposits

The metatranscriptome and peptidome sequences are deposited in Gene Expression Omnibus under accession numbers GSE15408 and PSE111, respectively.

References

  1. Arrigo, K. R. Marine microorganisms and global nutrient cycles. Nature 437, 349–355 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Brandes, J. A., Devol, A. H. & Deutsch, C. New developments in the marine nitrogen cycle. Chem. Rev. 107, 577–589 (2007)

    Article  CAS  PubMed  Google Scholar 

  3. Payne, W. J. Reduction of nitrogenous oxides by microorganisms. Bacteriol. Rev. 37, 409–452 (1973)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Strous, M. et al. Missing lithotroph identified as new planctomycete. Nature 400, 446–449 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Strous, M. et al. Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440, 790–794 (2006)

    Article  ADS  PubMed  Google Scholar 

  7. Van de Graaf, A. A., deBruijn, P., Robertson, L. A., Jetten, M. S. M. & Kuenen, J. G. Metabolic pathway of anaerobic ammonium oxidation on the basis of N-15 studies in a fluidized bed reactor. Microbiology 143, 2415–2421 (1997)

    Article  CAS  PubMed  Google Scholar 

  8. Kartal, B. et al. Candidatus ‘Brocadia fulgida’: an autofluorescent anaerobic ammonium oxidizing bacterium. FEMS Microbiol. Ecol. 63, 46–55 (2008)

    Article  CAS  PubMed  Google Scholar 

  9. Kartal, B., Geerts, W. & Jetten, M. S. M. Cultivation, detection and ecophysiology of anaerobic ammonium-oxidizing bacteria. Methods Enzymol. 486, 89–108 (2011)

    Article  CAS  PubMed  Google Scholar 

  10. Van der Star, W. R. L. et al. The membrane bioreactor: a novel tool to grow anammox bacteria as free cells. Biotechnol. Bioeng. 101, 286–294 (2008)

    Article  CAS  PubMed  Google Scholar 

  11. Keller, M. & Hettich, R. Environmental proteomics: a paradigm shift in characterizing microbial activities at the molecular level. Microbiol. Mol. Biol. Rev. 73, 62–70 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Akaike, T. & Maeda, H. Quantitation of nitric oxide using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Methods Enzymol. 268, 211–221 (1996)

    Article  CAS  PubMed  Google Scholar 

  13. Guo, F. Q., Okamoto, M. & Crawford, N. M. Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302, 100–103 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Nagano, T. Practical methods for detection of nitric oxide. Luminescence 14, 283–290 (1999)

    Article  CAS  PubMed  Google Scholar 

  15. Gilch, S., Vogel, M., Lorenz, M. W., Meyer, O. & Schmidt, I. Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea . Microbiology 155, 279–284 (2009)

    Article  CAS  PubMed  Google Scholar 

  16. Hyman, M. R. & Wood, P. M. Suicidal inactivation and labeling of ammonia mono-oxygenase by acetylene. Biochem. J. 227, 719–725 (1985)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hooper, A. B. & Nason, A. Characterization of hydroxylamine-cytochrome c reductase from chemoautotrophs Nitrosomonas europaea and Nitrosocystis oceanus . J. Biol. Chem. 240, 4044–4057 (1965)

    CAS  PubMed  Google Scholar 

  19. Hooper, A. B., Vannelli, T., Bergmann, D. J. & Arciero, D. M. Enzymology of the oxidation of ammonia to nitrite by bacteria. Anton. Leeuw. Int. J. G. Microbiology 71, 59–67 (1997)

    Article  CAS  Google Scholar 

  20. Klotz, M. G. et al. Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria. Environ. Microbiol. 10, 3150–3163 (2008)

    Article  CAS  PubMed  Google Scholar 

  21. Shimamura, M. et al. Isolation of a multiheme protein with features of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizing enrichment culture. Appl. Environ. Microbiol. 73, 1065–1072 (2007)

    Article  CAS  PubMed  Google Scholar 

  22. Shimamura, M. et al. Another multiheme protein, hydroxylamine oxidoreductase, abundantly produced in an anammox bacterium besides the hydrazine-oxidizing enzyme. J. Biosci. Bioeng. 105, 243–248 (2008)

    Article  CAS  PubMed  Google Scholar 

  23. Van Niftrik, L. et al. Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’. Mol. Microbiol. 77, 701–715 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ducluzeau, A. L. et al. Was nitric oxide the first deep electron sink? Trends Biochem. Sci. 34, 9–15 (2009)

    Article  CAS  PubMed  Google Scholar 

  25. Watt, G. W. & Chrisp, J. D. A spectrophotometric method for the determination of hydrazine. Anal. Chem. 24, 2006–2008 (1952)

    Article  CAS  Google Scholar 

  26. Strous, M., Heijnen, J. J., Kuenen, J. G. & Jetten, M. S. M. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589–596 (1998)

    Article  CAS  Google Scholar 

  27. Kartal, B. et al. Effect of nitric oxide on anammox bacteria. Appl. Environ. Microbiol. 76, 6304–6306 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003)

    Article  CAS  PubMed  Google Scholar 

  29. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Audrieth, L. F. & Ackerson Ogg, B. The Chemistry of Hydrazine (Wiley, 1951)

    Google Scholar 

  31. 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)

    Article  CAS  PubMed  Google Scholar 

  32. Schmid, M. C. et al. Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria. Appl. Environ. Microbiol. 71, 1677–1684 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Van de Graaf, A. A., de Bruijn, P., Robertson, L. A., Jetten, M. S. M. & Kuenen, J. G. Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142, 2187–2196 (1996)

    Article  CAS  Google Scholar 

  34. Laemmli, U. K. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227, 680–685 (1970)

    Article  ADS  CAS  PubMed  Google Scholar 

  35. Candiano, G. et al. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327–1333 (2004)

    Article  CAS  PubMed  Google Scholar 

  36. Farhoud, M. H. et al. Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry. Mol. Cell. Proteomics 4, 1653–1663 (2005)

    Article  CAS  PubMed  Google Scholar 

  37. Ishihama, Y., Rappsilber, J., Andersen, J. S. & Mann, M. Microcolumns with self-assembled particle frits for proteomics. J. Chromatogr. A 979, 233–239 (2002)

    Article  CAS  PubMed  Google Scholar 

  38. Weatherly, D. B. et al. A heuristic method for assigning a false-discovery rate for protein identifications from mascot database search results. Mol. Cell. Proteomics 4, 762–772 (2005)

    Article  CAS  PubMed  Google Scholar 

  39. Neuhoff, V., Arold, N., Taube, D. & Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric-focusing gels with clear background at nanogram sensitivity using coomassie brilliant blue G-250 and R-250. Electrophoresis 9, 255–262 (1988)

    Article  CAS  PubMed  Google Scholar 

  40. Mortz, E., Krogh, T. N., Vorum, H. & Gorg, A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1, 1359–1363 (2001)

    Article  CAS  PubMed  Google Scholar 

  41. Calvaruso, M. A., Smeitink, J. & Nijtmans, L. Electrophoresis techniques to investigate defects in oxidative phosphorylation. Methods 46, 281–287 (2008)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

B.K. was supported by a grant (05987) from the Dutch Foundation for Applied Research. W.J.M. was supported by a grant (142161201) from the Darwin Center for Biogeosciences. N.M.d.A. and I.C. were supported by a grant (81802015) from the Netherlands Organization for Scientific Research. M.S. was supported by a VIDI grant from the Netherlands Organization for Scientific Research and a European Research Council grant MASEM (242635). The anammox research of M.S.M.J. is supported by an advanced grant (232987) from the ERC. The authors acknowledge R. Klefoth for the initial tests for protein purification procedures.

Author information

Authors and Affiliations

  1. Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Heyendaalseweg 135, 6525AJ Nijmegen, The Netherlands,

    Boran Kartal, Wouter J. Maalcke, Naomi M. de Almeida, Irina Cirpus, Wim Geerts, Huub J. M. Op den Camp, Harry R. Harhangi, Jan T. Keltjens, Mike S. M. Jetten & Marc Strous

  2. Department of Laboratory Medicine, Nijmegen Centre for Mitochondrial Disorders, Nijmegen Proteomics Facility, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6500HB Nijmegen, The Netherlands,

    Jolein Gloerich

  3. Department of Molecular Biology, Radboud University, Nijmegen Centre for Molecular Life Sciences, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands,

    Eva M. Janssen-Megens, Kees-Jan Francoijs & Hendrik G. Stunnenberg

  4. Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands,

    Mike S. M. Jetten

  5. Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany ,

    Marc Strous

Authors
  1. Boran Kartal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wouter J. Maalcke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naomi M. de Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Irina Cirpus
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jolein Gloerich
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wim Geerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Huub J. M. Op den Camp
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Harry R. Harhangi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eva M. Janssen-Megens
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kees-Jan Francoijs
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hendrik G. Stunnenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jan T. Keltjens
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mike S. M. Jetten
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Marc Strous
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Physiological experiments were conceived, designed and performed by B.K., Kuenenia stuttgartiensis was grown by B.K. and W.G., two-dimensional gel electrophoresis was performed by N.M.A. and I.C., one-dimensional gel electrophoresis was performed by W.J.M. and B.K., MALDI–TOF analysis was performed by B.K., W.J.M. and H.J.M.O.d.C., nanoLC-MS/MS by J.G., RNA extraction and reverse transcription by H.R.H., Illumina sequencing by E.M.J.-M., K.-J.F. and H.S., and protein purification and activity tests were designed by W.J.M., B.K. and J.T.K. and performed by W.J.M. Proteomic and transcriptomic data processing was performed by J.G., M.S., K.-J.F., B.K., M.S.M.J. and H.J.M.O.d.C. The manuscript was written by B.K. with input from J.T.K., M.S. and M.S.M.J.

Corresponding author

Correspondence to Boran Kartal.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-3 with legends and Supplementary Table 1. (PDF 1410 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kartal, B., Maalcke, W., de Almeida, N. et al. Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130 (2011). https://doi.org/10.1038/nature10453

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10453

  • Springer Nature Limited

This article is cited by

Search

Navigation