Nitrite-driven anaerobic methane oxidation by oxygenic bacteria

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Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named ‘Candidatus Methylomirabilis oxyfera’, was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that ‘M. oxyfera’ bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

At a glance


  1. Significant pathways of Methylomirabilis oxyfera.
    Figure 1: Significant pathways of Methylomirabilis oxyfera.

    Canonical pathways of denitrification (a), aerobic methane oxidation (b) and proposed pathway of methane oxidation with nitrite (c). narGHJI, nitrate reductase; napABCDE, periplasmic nitrate reductase; nirSJFD/GH/L, nitrite reductase; norZ, nitric oxide reductase; nosDFYLZ, nitrous oxide reductase; pmoCAB, particulate methane monooxygenase; mxaFJGIRSACKL/DE, methanol dehydrogenase; fae, formaldehyde-activating enzyme; mtdB, methylene-tetrahydromethanopterin (H4MPT) dehydrogenase; mch, methenyl-H4MPT cyclohydrolase; fhcABCD, formyltransferase/hydrolase; fdhABC, formate dehydrogenase. Genes in red are absent from the genome, those in blue are present in the genome and those genes in green are present in both the proteome and the genome. Asterisk, H4MPT-dependent reactions involve the intermediates methylene-H4MPT, methenyl-H4MPT and formyl-H4MPT.

  2. Phylogeny of ‘Methylomirabilis oxyfera’ pmoA protein sequences.
    Figure 2: Phylogeny of ‘Methylomirabilis oxyfera’ pmoA protein sequences.

    Neighbour-joining tree showing the position of enrichment cultures ‘Twente’ and ‘Ooij’ (in bold) relative to other pmoA and amoA sequences. The distance tree was computed with the Dayhoff matrix-based method, and bootstrapping of 100 replicates was performed within the neighbour-joining, minimum-evolution, maximum-parsimony and maximum-likelihood evolutionary methods. Bootstrapping results are summarized on the tree, with filled circles representing branch points at which all four methods give greater than 70% support. The scale bar represents 50 amino-acid changes per 100 amino acids. See also Supplementary Fig. 3 for more detailed tree and bootstrap values.

  3. Coupling of methane oxidation and nitrite reduction in enrichment cultures of ‘Methylomirabilis oxyfera’.
    Figure 3: Coupling of methane oxidation and nitrite reduction in enrichment cultures of ‘Methylomirabilis oxyfera’.

    Methane is oxidized only after addition of 15N-labelled nitrite (50μM, arrow), which is converted to 15N-labelled dinitrogen gas in the presence of about 2,000μM 14N-nitrate (a) or 2,000μM 14N-nitrate and 135μM 14N-N2O (b). Experiments were performed with 380ml of anoxic, stirred enrichment culture ‘Ooij’ (protein content 147±11mg). Red circles, CH4; dark blue triangles, 15,15N2; light blue triangles, 15,14N2; green squares, total N2O; dark green squares, 14,15N2O and 15,15N2O.

  4. Oxygen production from nitrite in ‘Methylomirabilis oxyfera’.
    Figure 4: Oxygen production from nitrite in ‘Methylomirabilis oxyfera’.

    Whole cells of enrichment culture ‘Ooij’ were incubated in buffer containing nitrite and 25% 18O-labelled water, leading to 90% O exchange within 30min. Total oxygen production from this indirectly labelled N18O2- was inferred from the measured concentration of 16,18O2 and 18,18O2 in the helium headspace with the following additions: propylene (dark blue diamonds), propylene and acetylene (blue triangles), methane (purple squares) and oxygen (light blue circles). Anaerobic control incubations of Methylosinus acidophilus (red asterisks) with 18O-labelled nitrite did not produce measurable amounts of oxygen. Cells were concentrated to obtain similar maximum rates of propylene oxidation activity; 1.15nmolmin-1 (with NO2-, 1.22mg of protein) for ‘M. oxyfera’, and 1.68nmolmin-1 (with O2, 0.046mg of protein) for M. acidophilus.

Author information

  1. These authors contributed equally to this work.

    • Katharina F. Ettwig &
    • Margaret K. Butler


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

    • Katharina F. Ettwig,
    • Margaret K. Butler,
    • Theo van Alen,
    • Francisca Luesken,
    • Ming L. Wu,
    • Katinka T. van de Pas-Schoonen,
    • Huub J. M. Op den Camp,
    • Mike S. M. Jetten &
    • Marc Strous
  2. CEA Genoscope,

    • Denis Le Paslier,
    • Eric Pelletier,
    • Sophie Mangenot &
    • Jean Weissenbach
  3. CNRS-UMR 8030, 2 rue Gaston Crémieux,

    • Denis Le Paslier,
    • Eric Pelletier &
    • Jean Weissenbach
  4. Université d’Evry Val d’Essonne, Boulevard François Mitterrand CP 5706, 91057 Evry, France

    • Denis Le Paslier,
    • Eric Pelletier &
    • Jean Weissenbach
  5. Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany

    • Marcel M. M. Kuypers,
    • Frank Schreiber,
    • Johannes Zedelius,
    • Dirk de Beer &
    • Marc Strous
  6. Radboud University Nijmegen Medical Centre, Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, Geert Grooteplein 28,

    • Bas E. Dutilh
  7. Radboud University Nijmegen Medical Centre, Nijmegen Proteomics Facility, Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and Metabolic Diseases, Geert Grooteplein-Zuid 10,

    • Jolein Gloerich &
    • Hans J. C. T. Wessels
  8. Radboud University Nijmegen, Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Geert Grooteplein-Zuid 26, 6525 GA, Nijmegen, The Netherlands

    • Eva M. Janssen-Megens,
    • Kees-Jan Francoijs &
    • Henk Stunnenberg
  9. Centre for Biotechnology, University of Bielefeld, Postfach 10 01 31, D-33501 Bielefeld, Germany

    • Marc Strous
  10. Present address: Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, 4072, Australia.

    • Margaret K. Butler

Corresponding authors

Correspondence to:

Sequencing and proteomic data are deposited at the National Centre for Biotechnology Information under accession numbers FP565575, SRR023516.1, SRR022749.2, GSE18535, SRR022748.2, PSE127 and PSE128.

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Supplementary information

PDF files

  1. Supplementary Information (530K)

    This file contains Supplementary Tables 1-5 and Supplementary Figures 1-7 with legends.


  1. Report this comment #10611

    Antti Rissanen said:

    Could this pathway also be working in anaerobic oxidation of ammonium?

  2. Report this comment #10617

    Viktor Nardin said:

    The metabolic pathway of anaerobic ammonium oxidation is not elucidated yet. Thus, it could well be. Hopefully, new physiological experiments will enhance our understanding of anaerobic ammonium oxidation.

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