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Nitrite-driven anaerobic methane oxidation by oxygenic bacteria

Nature volume 464, pages 543–548 (2010)Cite this article

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Abstract

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.

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Figure 1: Significant pathways of Methylomirabilis oxyfera.
Figure 2: Phylogeny of ‘ Methylomirabilis oxyfera ’ pmoA protein sequences.
Figure 3: Coupling of methane oxidation and nitrite reduction in enrichment cultures of ‘ Methylomirabilis oxyfera ’.
Figure 4: Oxygen production from nitrite in ‘ Methylomirabilis oxyfera ’.

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Data deposits

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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Acknowledgements

We thank F. Stams and N. Tan for sharing their ideas on NO decomposition; D. Speth and L. Russ for pilot experiments; N. Kip for providing M. acidophilus cultures; A. Pierik for electron paramagnetic resonance analysis; G. Klockgether and G. Lavik for technical assistance; and B. Kartal, J. Keltjens, A. Pol, J. van de Vossenberg and F. Widdel for helpful discussions. M.M.M.K., F.S., J.Z. and D.d.B. were supported by the Max Planck Society, M.S.M.J. by European Research Council grant 232937, M.S., K.F.E. and M.K.B. by a Vidi grant to M.S. from the Netherlands Organisation for Scientific Research (NWO), and M.L.W. and B.D. by a Horizon grant (050-71-058) from NWO.

Author Contributions Genome sequencing and assembly from enrichment culture ‘Twente’ was performed by D.L.P., E.P., S.M. and J.W. M.S., E.M.J.-M., K.-J.F. and H.S. performed the sequencing and initial assembly of sequence from enrichment culture ‘Ooij’. Mapping of sequences from enrichment culture ‘Ooij’ to ‘Twente’ was performed by B.E.D. and M.S. B.E.D. and E.P. performed SNP and coverage analyses. Genome annotation and phylogenetic analysis were conducted by M.K.B. H.J.M.O.d.C. provided support with alignments. Sample preparation for proteome analysis was performed by M.K.B. and M.W., with LC–MS/MS and protein identification performed by J.G. and H.J.C.T.W. Material for transcriptome analysis was prepared by T.v.A. and F.L., with sequencing performed by E.M.J.-M., K.-J.F. and H.S. Continuous cultures were set up and maintained by K.F.E. and K.T.v.d.P.-S. Experiments for nitrogenous intermediates were designed and performed by K.F.E., M.M.M.K., F.S., D.d.B. and J.Z., and those for methane activation were designed and performed by K.F.E. Pilot experiments were conducted by K.F.E., F.L., M.K.B., K.T.v.d.P.-S, T.A. and M.S. K.F.E., M.K.B., M.S.M.J. and M.S. conceived the research. K.F.E., M.K.B. and M.S. wrote the paper with input from all other authors.

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Author notes

  1. Margaret K. Butler

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

  2. Katharina F. Ettwig and Margaret K. Butler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Radboud University Nijmegen, IWWR, 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. Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Nijmegen Proteomics Facility, Laboratory of Genetic, Endocrine and Metabolic Diseases, Geert Grooteplein-Zuid 10,

    Jolein Gloerich & Hans J. C. T. Wessels

  8. Department of Molecular Biology, Radboud University Nijmegen, 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

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  1. Katharina F. Ettwig
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Correspondence to Katharina F. Ettwig or Marc Strous.

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Ettwig, K., Butler, M., Le Paslier, D. et al. Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543–548 (2010). https://doi.org/10.1038/nature08883

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