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Methane-dependent complete denitrification by a single Methylomirabilis bacterium

Nature Microbiology volume 9, pages 464–476 (2024)Cite this article

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Abstract

Methane-dependent nitrate and nitrite removal in anoxic environments is thought to rely on syntrophy between ANME-2d archaea and bacteria in the genus ‘Candidatus Methylomirabilis’. Here we enriched and purified a single Methylomirabilis from paddy soil fed with nitrate and methane, which is capable of coupling methane oxidation to nitrate reduction via nitrite to dinitrogen independently. Isotope labelling showed that this bacterium we name ‘Ca. Methylomirabilis sinica’ stoichiometrically performed methane-dependent complete nitrate reduction to dinitrogen gas. Multi-omics analyses collectively demonstrated that ‘M. sinica’ actively expressed a well-established pathway for this process, especially including nitrate reductase Nap. Furthermore, ‘M. sinica’ exhibited a higher nitrate affinity than most denitrifiers, implying its competitive fitness under oligotrophic nitrogen-limited conditions. Our findings revise the paradigm of methane-dependent denitrification performed by two organisms, and the widespread presence of ‘M. sinica’ in public databases suggests that the coupling of methane oxidation and complete denitrification in single cells substantially contributes to global methane and nitrogen budgets.

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Fig. 1: Abundance dynamics of ‘Ca. Methylomirabilis’ bacteria fed with only nitrate and methane.
Fig. 2: Phylogenomic and phylogenetic affiliation of ‘Ca. M. sinica’.
Fig. 3: Isotope labelling batch tests demonstrating methane-dependent complete denitrification by ‘Ca. M. sinica’.
Fig. 4: Expression levels of diagnostic genes involved in methane-dependent denitrification pathways of ‘Ca. M. sinica’ based on transcriptomic and proteomic analyses.
Fig. 5: Kinetic analyses revealing the nitrate/nitrite affinity and the competitive fitness of ‘Ca. M. sinica’ under oligotrophic nitrogen-limited conditions.

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Data availability

The following databases/datasets were used in this study: GTDB (v2.2.1, https://github.com/Ecogenomics/GTDBNCBI), NCBI (https://www.ncbi.nlm.nih.gov/), SRA (https://www.ncbi.nlm.nih.gov/sra) and KEGG (http://www.kegg.jp/kegg/).

Raw data of the 16S rRNA gene sequencing have been submitted to the Sequence Read Archive (SRA) with accession numbers SRR21143259–SRR21143272 and SRR23318916–SRR23318920. The metagenomic and metatranscriptomic sequencing data and MAGs generated in this study have been deposited in the NCBI database under BioProject number PRJNA869304. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE77 partner repository with the dataset identifier PXD047070. Representative images of FISH and microscopy have been deposited in Figshare. Source data are provided with this paper.

References

  1. Kuypers, M. M. M., Marchant, H. K. & Kartal, B. The microbial nitrogen-cycling network. Nat. Rev. Microbiol. 16, 263–276 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Raghoebarsing, A. A. et al. A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440, 918–921 (2006).

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Conrad, R. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). Microbiol. Rev. 60, 609–640 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Segarra, K. E. A. et al. High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions. Nat. Commun. 6, 7477 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Hu, B. L. et al. Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands. Proc. Natl Acad. Sci. USA 111, 4495–4500 (2014).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ettwig, K. F. et al. Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543–548 (2010).

    Article  PubMed  Google Scholar 

  7. Haroon, M. F. et al. Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 500, 567–570 (2013).

    Article  PubMed  Google Scholar 

  8. Graf, J. S. et al. Bloom of a denitrifying methanotroph, ‘Candidatus Methylomirabilis limnetica’, in a deep stratified lake. Environ. Microbiol. 20, 2598–2614 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Versantvoort, W. et al. Complexome analysis of the nitrite-dependent methanotroph Methylomirabilis lanthanidiphila. Biochim. Biophys. Acta Bioenerg. 1860, 734–744 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Versantvoort, W. et al. Comparative genomics of Candidatus Methylomirabilis species and description of Ca. Methylomirabilis lanthanidiphila. Front. Microbiol. 9, 1672 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Li, J., Liu, T., McIlroy, S. J., Tyson, G. W. & Guo, J. Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings. ISME Commun. 3, 39 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mosley, O. E. et al. Nitrogen cycling and microbial cooperation in the terrestrial subsurface. ISME J. 16, 2561–2573 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Su, G. et al. Water column dynamics control nitrite-dependent anaerobic methane oxidation by Candidatus “Methylomirabilis” in stratified lake basins. ISME J. 17, 693–702 (2023).

  14. Wang, Z., Li, J., Xu, X., Li, K. & Chen, Q. Denitrifying anaerobic methane oxidation and mechanisms influencing it in Yellow River Delta coastal wetland soil, China. Chemosphere 298, 134345 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Reimann, J., Jetten, M. S. M. & Keltjens, J. T. Metal enzymes in “impossible” microorganisms catalyzing the anaerobic oxidation of ammonium and methane. Met. Ions Life Sci. 15, 257–313 (2015).

    CAS  PubMed  Google Scholar 

  16. Costa, E., Perez, J. & Kreft, J. U. Why is metabolic labour divided in nitrification? Trends Microbiol. 14, 213–219 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. van Kessel, M. et al. Complete nitrification by a single microorganism. Nature 528, 555–559 (2015).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  18. Vaksmaa, A. et al. Enrichment of anaerobic nitrate-dependent methanotrophic ‘Candidatus Methanoperedens nitroreducens’ archaea from an Italian paddy field soil. Appl. Microbiol. Biotechnol. 101, 7075–7084 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu, M. L. et al. Ultrastructure of the denitrifying methanotroph “Candidatus Methylomirabilis oxyfera,” a novel polygon-shaped bacterium. J. Bacteriol. 194, 284–291 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gambelli, L. et al. The polygonal cell shape and surface protein layer of anaerobic methane-oxidizing Methylomirabilis lanthanidiphila bacteria. Front. Microbiol. 12, 766527 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  21. He, Z. F. et al. A novel denitrifying methanotroph of the NC10 phylum and its microcolony. Sci. Rep. 6, 32241 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Goris, J. et al. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 57, 81–91 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Richter, M. & Rossello-Mora, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 19126–19131 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nie, W. B. et al. Anaerobic oxidation of methane coupled with dissimilatory nitrate reduction to ammonium fuels anaerobic ammonium oxidation. Environ. Sci. Technol. 55, 1197–1208 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Wu, M. et al. Anaerobic oxidation of propane coupled to nitrate reduction by a lineage within the class Symbiobacteriia. Nat. Commun. 13, 6115 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cao, Q., Liu, X., Ran, Y., Li, Z. & Li, D. Methane oxidation coupled to denitrification under microaerobic and hypoxic conditions in leach bed bioreactors. Sci. Total Environ. 649, 1–11 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Liu, T., Hu, S., Yuan, Z. & Guo, J. High-level nitrogen removal by simultaneous partial nitritation, anammox and nitrite/nitrate-dependent anaerobic methane oxidation. Water Res. 166, 115057 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Wang, W. et al. A novel sulfide-driven denitrification methane oxidation (SDMO) system: operational performance and metabolic mechanisms. Water Res. 222, 118909 (2022).

    Article  CAS  PubMed  Google Scholar 

  29. VanCleemput, O. & Samater, A. H. Nitrite in soils: accumulation and role in the formation of gaseous N compounds. Fertilizer Res. 45, 81–89 (1996).

    Article  Google Scholar 

  30. Wang, J. Q. et al. Nitrogen input promotes denitrifying methanotrophs’ abundance and contribution to methane emission reduction in coastal wetland and paddy soil. Environ. Pollut. 302, 19090 (2022).

    Article  Google Scholar 

  31. Guerrero-Cruz, S. et al. Response of the anaerobic methanotroph “Candidatus Methanoperedens nitroreducens” to oxygen stress. Appl. Environ. Microbiol. 84, e01832-18 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Luo, J. H., Chen, H., Yuan, Z. G. & Guo, J. H. Methane-supported nitrate removal from groundwater in a membrane biofilm reactor. Water Res. 132, 71–78 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Wang, V. C. C. et al. Alkane oxidation: methane monooxygenases, related enzymes, and their biomimetics. Chem. Rev. 117, 8574–8621 (2017).

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Kits, K. D. et al. Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle. Nature 549, 269–272 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Oshiki, M., Satoh, H. & Okabe, S. Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environ. Microbiol. 18, 2784–2796 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Wan, Y. et al. Rapid dissimilatory nitrate reduction to ammonium conserves bioavailable nitrogen in organic deficient soils. Soil Biol. Biochem. 177, 108923 (2023).

    Article  CAS  Google Scholar 

  38. Lu, P. L. et al. Growth kinetics of Candidatus ‘Methanoperedens nitroreducens’ enriched in a laboratory reactor. Sci. Total Environ. 659, 442–450 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  39. Abdul-Talib, S., Hvitved-Jacobsen, T., Vollertsen, J. & Ujang, Z. Half saturation constants for nitrate and nitrite by in-sewer anoxic transformations of wastewater organic matter. Water Sci. Technol. 46, 185–192 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Feng, L. et al. Characterisation of Pseudomonas stutzeri T13 for aerobic denitrification: stoichiometry and reaction kinetics. Sci. Total Environ. 717, 135181 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Medhi, K., Singhal, A., Chauhan, D. K. & Thakur, I. S. Investigating the nitrification and denitrification kinetics under aerobic and anaerobic conditions by Paracoccus denitrificans ISTOD1. Bioresour. Technol. 242, 334–343 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Middelburg, J., Soetaert, K., Herman, P. & Heip, C. Denitrification in marine sediments: a model study. Glob. Biogeochem. Cycles 10, 661–673 (1996).

    Article  ADS  CAS  Google Scholar 

  43. Dai, T. et al. Nutrient supply controls the linkage between species abundance and ecological interactions in marine bacterial communities. Nat. Commun. 13, 175 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bertagnolli, A. D. & Stewart, F. J. Microbial niches in marine oxygen minimum zones. Nat. Rev. Microbiol. 16, 723–729 (2018).

    Article  CAS  PubMed  Google Scholar 

  45. Hutchins, D. A. & Capone, D. G. The marine nitrogen cycle: new developments and global change. Nat. Rev. Microbiol. 20, 401–414 (2022).

    Article  CAS  PubMed  Google Scholar 

  46. Mayr, M. J., Zimmermann, M., Guggenheim, C., Brand, A. & Burgmann, H. Niche partitioning of methane-oxidizing bacteria along the oxygen-methane counter gradient of stratified lakes. ISME J. 14, 274–287 (2020).

    Article  CAS  PubMed  Google Scholar 

  47. Padilla, C. C. et al. NC10 bacteria in marine oxygen minimum zones. ISME J. 10, 2067–2071 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Reis, P. C. J., Thottathil, S. D., Ruiz-Gonzalez, C. & Prairie, Y. T. Niche separation within aerobic methanotrophic bacteria across lakes and its link to methane oxidation rates. Environ. Microbiol. 22, 738–751 (2020).

    Article  CAS  PubMed  Google Scholar 

  49. Yao, X., Wang, J. & Hu, B. How methanotrophs respond to pH: a review of ecophysiology. Front. Microbiol. 13, 1034164 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Liu, C. S. et al. Rapid formation of granules coupling n-DAMO and anammox microorganisms to remove nitrogen. Water Res. 194, 16963 (2021).

    Article  ADS  Google Scholar 

  51. Li, H. Z. et al. Active antibiotic resistome in soils unraveled by single-cell isotope probing and targeted metagenomics. Proc. Natl Acad. Sci. USA 119, e2201473119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Shi, L. D. et al. Methane-dependent selenate reduction by a bacterial consortium. ISME J. 15, 3683–3692 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. McIlroy, S. J. et al. Anaerobic methanotroph ‘Candidatus Methanoperedens nitroreducens’ has a pleomorphic life cycle. Nat. Microbiol. 8, 321–331 (2023).

    Article  CAS  PubMed  Google Scholar 

  54. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  55. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  56. Agulleiro, J. I. & Fernandez, J. J. Tomo3D 2.0–exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction. J. Struct. Biol. 189, 147–152 (2015).

    Article  PubMed  Google Scholar 

  57. Imachi, H. et al. Isolation of an archaeon at the prokaryote–eukaryote interface. Nature 577, 519–525 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  58. He, Z. F. et al. Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria. Appl. Microbiol. Biotechnol. 100, 5099–5108 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. He, Z. F. et al. Effect of inoculum sources on the enrichment of nitrite-dependent anaerobic methane-oxidizing bacteria. Appl. Microbiol. Biotechnol. 99, 939–946 (2015).

    Article  CAS  PubMed  Google Scholar 

  60. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Li, D. H. et al. MEGAHIT v1.0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods 102, 3–11 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Dong, X. L. & Strous, M. An integrated pipeline for annotation and visualization of metagenomic contigs. Front. Genet. 10, 999 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Parks, D. H. et al. A complete domain-to-species taxonomy for bacteria and archaea. Nat. Biotechnol. 38, 1079–1086 (2020).

  65. Chaumeil, P. A., Mussig, A. J., Hugenholtz, P. & Parks, D. H. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics 36, 1925–1927 (2020).

    Article  CAS  Google Scholar 

  66. Leu, A. O. et al. Anaerobic methane oxidation coupled to manganese reduction by members of the Methanoperedenaceae. ISME J. 14, 1030–1041 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47, W256–W259 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Kopylova, E., Noe, L. & Touzet, H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012).

    Article  CAS  PubMed  Google Scholar 

  70. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Zhou, Z. et al. Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species. Nature 601, 257–262 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  72. Kruger, N. J. in Protein Protocols Handbook 3rd edn (ed. Walker, J. M.) 17–24 (Springer, 2009).

  73. Orsburn, B. C. Proteome Discoverer—a community enhanced data processing suite for protein informatics. Proteomes 9, 15 (2021).

  74. Harman, J. C., Guidry, J. J. & Gidday, J. M. Comprehensive characterization of the adult ND4 Swiss Webster mouse retina: using discovery-based mass spectrometry to decipher the total proteome and phosphoproteome. Mol. Vis. 24, 875–889 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. McBride, J. D. et al. Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane. Stem Cell Res. Ther. 12, 328 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Lagkouvardos, I. et al. Imngs: a comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies. Sci. Rep. 6, 33721 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  77. Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the Magigene Biotechnology and Qinglian Bio sequencing teams for meta-omics analysis; Y. J. Chang and X. Zhang from the Center of Cryo-Electron Microscopy (School of Medicine, Zhejiang University) for providing the platform for Cryo-ET data acquisition and image processing; and F. M. Fang (School of International Studies, Zhejiang University) for linguistic assistance on this manuscript. This work was funded by Zhejiang Province ‘Leading Talents Program’ R&D Plan (Number 2022C03010) and National Natural Science Foundation of China (Number 41773074 and Number 51478415). M.S.M.J. was supported by SIAM NOW/OCW 024002002 and ERC Synergy MARIX 854088. J.W. was supported by the National Natural Science Foundation of China (Number 42107132).

Author information

Authors and Affiliations

  1. Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education, College of Environmental Resource Sciences, Zhejiang University, Hangzhou, China

    Xiangwu Yao, Zishu Liu, Ping Zheng & Baolan Hu

  2. College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China

    Xiangwu Yao, Jiaqi Wang, Mingyue He, Zishu Liu, Yuxiang Zhao, Yufen Li, Taolve Chi, Lin Zhu, Ping Zheng & Baolan Hu

  3. Department of Microbiology, Radboud Institute for Biological and Environmental Sciences (RIBES), Radboud University Nijmegen, Nijmegen, the Netherlands

    Mike S. M. Jetten

  4. Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou, China

    Baolan Hu

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Contributions

X.Y. and B.H. conceptualized the project, developed the methodology, performed visualization and wrote the original draft; B.H., J.W. and M.S.M.J. reviewed and edited the manuscript; M.H., Z.L., Y.Z., Y.L., T.C. and L.Z. developed the methodology and conducted the investigation; P.Z. procured resources; B.H. supervised the project and acquired funding.

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Correspondence to Baolan Hu.

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Nature Microbiology thanks Gunter Wegener, Lisa Stein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Fluorescence in situ hybridization (FISH) micrographs of the highly purified culture (a-f) and enrichment culture (g-o) of ‘Ca. Methylomirabilis sinica’.

Cells of ‘M. sinica’ were targeted with probe S-*-DBACT-0193−a-A-18 (cy3, red) and bacterial cells were hybridized by EUB I-III (FITC, blue). ‘M. sinica’ appeared in magenta for the double hybridization. a-f, The culture purity was also confirmed based on failure to detect archaeal cells with ARCH915 (cy5, green). g-o, The biomass for FISH was collected from the GLSBR on day 380. m-o, The probe S-*-DARCH-0872−a-A-18 (cy5, green) was used to confirm whether ANME-2d archaea appeared in enrichment cultures. And no obvious signal was detected. The FISH experiments were performed three times with similar results. Scale bars, 5μm.

Extended Data Fig. 2 Transmission electron (a, b) and scanning electron (c, d) micrographs of ‘Ca. Methylomirabilis’ cells in enrichment cultures.

a, b, The polygonal cell shape of Methylomirabilis bacteria was observed. Representative of n = 45 recorded images. c, d, As white arrows showed, longitudinal ridges along cells of this strain were similar to the features of ‘Ca. Methylomirabilis oxyfera’. Representative of n = 23 recorded images. Scale bars, 500 nm.

Extended Data Fig. 3 Other representative photomicrographs of purified ‘Ca. Methylomirabilis sinica’.

a, Phase-contrast image of highly purified culture of ‘M. sinica’. Representative of n = 12 recorded images. b, c, Representative Cryo-ET images of ‘M. sinica’. Arrows from outside to inside of the cell in c show S-layer, outer membrane and cytoplasmic membrane, respectively. d, Enlarged view of the dash framed region in c showing the cell envelope structure. The arrows show S-layer sheets protruding from the cell. b-d, Cryo-EM images are representative of n = 15 recorded images.

Extended Data Fig. 4 Isotope labelling batch tests demonstrating methane-dependent complete denitrification by ‘Ca. Methylomirabilis sinica’.

a, c, Stoichiometrically balanced conversion of 13CH4 to 13CO2 revealed by two biological replicates. b, d, Stoichiometrically balanced conversion of 15NO3− to 30N2 and 15NO2− pulse-fed with nitrate revealed by two biological replicates. Data from replicated tests also demonstrate that ‘M. sinica’ nearly stoichiometrically reduce nitrate to dinitrogen gas coupled to methane oxidation without transitory formation of nitrite.

Source data

Extended Data Fig. 5 Profiles of 13CH4, 13CO2, 15NO3−, 15NO2- and 30N2 in the control batch incubations.

a, b, No methane consumption or carbon dioxide production was observed in two replicated control incubations without nitrate addition. c, d, No nitrate consumption or nitrite and dinitrogen gas production was observed in two replicated control incubations without methane addition.

Source data

Extended Data Fig. 6 Contributions of ‘Ca. Methylomirabilis sinica’ to complete denitrification in enrichment cultures based on analyses of transcriptome (a) and proteome (b).

a, Relative transcript abundances of nitrate reductase (NapAB), nitrite reductase (NirS) and putative nitric oxide dismutase genes affiliated with ‘M. sinica’ among all respective related enzymes. b, Relative protein abundances of nitrate reductase (NapA), nitrite reductase (NirS) and putative nitric oxide dismutase affiliated with ‘M. sinica’ among all respective related enzymes. Biologically independent samples n = 2 and n = 3 were used for transcriptome and proteome analyses, respectively. Data are presented as mean values and individual data points are shown by black circles. Calculation details are shown in the Source Data file.

Source data

Extended Data Fig. 7 Global distribution of ‘Ca. Methylomirabilis sinica’ based on the analysis of 16 S rRNA gene (identity ≥ 98%).

The presence of ‘M. sinica’ is detected by searching the SRA with representative 16 S rRNA gene sequences and locations of natural samples are indicated by red stars. Geographical details are shown in the Source Data file.

Source data

Extended Data Fig. 8 Putative pathways of the nitrite-dependent methane oxidation (a) and methane-dependent complete denitrification (b).

Reactions catalyzed by enzymes are highlighted by white squares. The electrons generated or consumed are highlighted by grey (previous study) and pink (this study) circles. The proposed reactions of methane oxidation to methanol are shown in the grey (previous study) and pink (this study) boxes. Abbreviations: pMMO, particulate methane monooxygenase; Mdh, methanol dehydrogenase; Fdh, formate dehydrogenase; Nap, periplasmic nitrate reductase; Nir, nitrite reductase; Nod, nitric oxide dismutase.

Extended Data Fig. 9

The schematic illustration of the sampling strategy at the enrichment (day 0-380) and purification (day 380-1330) stage.

Supplementary information

Supplementary Information

Supplementary Notes 1–3, Figs. 1–7 and Tables 1 and 2.

Reporting Summary

Supplementary Table 1

Supplementary Tables 3–8.

Supplementary Data 1

Source Data for Supplementary Figs. 2–4 and 6, and mass spectrometry data for isotope labelling tests.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

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Yao, X., Wang, J., He, M. et al. Methane-dependent complete denitrification by a single Methylomirabilis bacterium. Nat Microbiol 9, 464–476 (2024). https://doi.org/10.1038/s41564-023-01578-6

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