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Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria

Abstract

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor1,2. In marine sediments, AOM is performed by dual-species consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) inhabiting the methane–sulfate transition zone3,4,5. The biochemical pathways and biological adaptations enabling this globally relevant process are not fully understood. Here we study the syntrophic interaction in thermophilic AOM (TAOM) between ANME-1 archaea and their consortium partner SRB HotSeep-1 (ref. 6) at 60 °C to test the hypothesis of a direct interspecies exchange of electrons7,8. The activity of TAOM consortia was compared to the first ANME-free culture of an AOM partner bacterium that grows using hydrogen as the sole electron donor. The thermophilic ANME-1 do not produce sufficient hydrogen to sustain the observed growth of the HotSeep-1 partner. Enhancing the growth of the HotSeep-1 partner by hydrogen addition represses methane oxidation and the metabolic activity of ANME-1. Further supporting the hypothesis of direct electron transfer between the partners, we observe that under TAOM conditions, both ANME and the HotSeep-1 bacteria overexpress genes for extracellular cytochrome production and form cell-to-cell connections that resemble the nanowire structures responsible for interspecies electron transfer between syntrophic consortia of Geobacter9,10. HotSeep-1 highly expresses genes for pili production only during consortial growth using methane, and the nanowire-like structures are absent in HotSeep-1 cells isolated with hydrogen. These observations suggest that direct electron transfer is a principal mechanism in TAOM, which may also explain the enigmatic functioning and specificity of other methanotrophic ANME–SRB consortia.

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Figure 1: Activity of the TAOM consortia in culture.
Figure 2: Hydrogen in TAOM cultures.
Figure 3: Expression of genes and visualization of structures attributed to interspecies electron transfer in TAOM.

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Primary accessions

BioProject

GenBank/EMBL/DDBJ

Data deposits

Representative full-length 16S rRNA gene sequences of TAOM and HotSeep-1 enrichment have been submitted to NCBI GenBank under accession numbers KT152859KT152885 and KT759143KT759147, functional genes under the accession numbers KT152886, KT152887 and KT795302KT795321, and genomic 16S rRNA genes under the accession numbers KT795322 and KT795323. Sequencing projects are registered at NCBI under the BioProject accession numbers PRJNA286178 (TAOM enrichment) and PRJNA276404 (HotSeep-1 enrichment).

References

  1. Reeburgh, W. S. Oceanic methane biogeochemistry. Chem. Rev. 107, 486–513 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. Boetius, A. & Wenzhöfer, F. Seafloor oxygen consumption fuelled by methane from cold seeps. Nat. Geosci. 6, 725–734 (2013)

    Article  ADS  CAS  Google Scholar 

  3. Hinrichs, K.-U., Hayes, J. M., Sylva, S. P., Brewer, P. G. & DeLong, E. F. Methane-consuming archaebacteria in marine sediments. Nature 398, 802–805 (1999)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Boetius, A. et al. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Orphan, V. J. et al. Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments. Appl. Environ. Microbiol. 67, 1922–1934 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Holler, T. et al. Thermophilic anaerobic oxidation of methane by marine microbial consortia. ISME J. 5, 1946–1956 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Thauer, R. K. & Shima, S. Methane as fuel for anaerobic microorganisms. Ann. NY Acad. Sci. 1125, 158–170 (2008)

    Article  ADS  CAS  PubMed  Google Scholar 

  8. McGlynn, S. E., Chadwick, G. L., Kempes, C. P. & Orphan, V. J. Single cell activity reveals direct electron transfer in methanotrophic consortia. Nature http://dx.doi.org/10.1038/nature15512 (this issue)

  9. Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Summers, Z. M. et al. Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria. Science 330, 1413–1415 (2010)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Knittel, K. & Boetius, A. Anaerobic oxidation of methane: progress with an unknown process. Annu. Rev. Microbiol. 63, 311–334 (2009)

    Article  CAS  PubMed  Google Scholar 

  12. Niemann, H. et al. Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink. Nature 443, 854–858 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Schreiber, L., Holler, T., Knittel, K., Meyerdierks, A. & Amann, R. Identification of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade. Environ. Microbiol. 12, 2327–2340 (2010)

    CAS  PubMed  Google Scholar 

  14. Hoehler, T. M., Alperin, M. J., Albert, D. B. & Martens, C. S. Field and laboratory studies of methane oxidation in an anoxic marine sediment: evidence for a methanogen-sulfate reducer consortium. Glob. Biogeochem. Cycles 8, 451–463 (1994)

    Article  ADS  CAS  Google Scholar 

  15. Krüger, M. et al. A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature 426, 878–881 (2003)

    Article  ADS  PubMed  CAS  Google Scholar 

  16. Hallam, S. J. et al. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305, 1457–1462 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  17. Meyerdierks, A. et al. Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME-1 group. Environ. Microbiol. 12, 422–439 (2010)

    Article  CAS  PubMed  Google Scholar 

  18. Kojima, H., Moll, J., Kahnt, J., Fukui, M. & Shima, S. A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate. Environ. Microbiol. 16, 3431–3442 (2014)

    Article  CAS  PubMed  Google Scholar 

  19. Thauer, R. K. Anaerobic oxidation of methane with sulfate: on the reversibility of the reactions that are catalyzed by enzymes also involved in methanogenesis from CO2 . Curr. Opin. Microbiol. 14, 292–299 (2011)

    Article  CAS  PubMed  Google Scholar 

  20. Milucka, J., Widdel, F. & Shima, S. Immunological detection of enzymes for sulfate reduction in anaerobic methane-oxidizing consortia. Environ. Microbiol. 15, 1561–1571 (2013)

    Article  CAS  PubMed  Google Scholar 

  21. Milucka, J. et al. Zero-valent sulphur is a key intermediate in marine methane oxidation. Nature 491, 541–546 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Schink, B. Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61, 262–280 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Stams, A. J. & Plugge, C. M. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nature Rev. Microbiol. 7, 568–577 (2009)

    Article  CAS  Google Scholar 

  24. Moran, J. J. et al. Methyl sulfides as intermediates in the anaerobic oxidation of methane. Environ. Microbiol. 10, 162–173 (2008)

    CAS  PubMed  Google Scholar 

  25. Stokke, R., Roalkvam, I., Lanzen, A., Haflidason, H. & Steen, I. H. Integrated metagenomic and metaproteomic analyses of an ANME‐1‐dominated community in marine cold seep sediments. Environ. Microbiol. 14, 1333–1346 (2012)

    Article  CAS  PubMed  Google Scholar 

  26. Nagarajan, H. et al. Characterization and modelling of interspecies electron transfer mechanisms and microbial community dynamics of a syntrophic association. Nature Commun. 4, 2809 (2013)

    Article  ADS  CAS  Google Scholar 

  27. Rotaru, A.-E. et al. A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energ. Environ. Sci. 7, 408–415 (2014)

    Article  CAS  Google Scholar 

  28. Malvankar, N. S. et al. Structural basis for metallic-like conductivity in microbial nanowires. MBio 6, e00084–e00015 (2015)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Methé, B. A. et al. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science 302, 1967–1969 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Reitner, J. et al. Concretionary methane-seep carbonates and associated microbial communities in Black Sea sediments. Palaeogeogr. Palaeoclimatol. Palaeoecol. 227, 18–30 (2005)

    Article  Google Scholar 

  31. Widdel, F. & Bak, F. in The Prokaryotes Vol. 4 (eds Trüper, H. G., Balows, A., Dworkin, M., Harder, W. & Schleifer, K. H. ) 3352–3378 (Springer, 1992)

    Book  Google Scholar 

  32. Zhou, J., Bruns, M. A. & Tiedje, J. M. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Muyzer, G., Teske, A., Wirsen, C. O. & Jannasch, H. W. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164, 165–172 (1995)

    Article  CAS  PubMed  Google Scholar 

  34. Massana, R., Murray, A., Preston, C. & DeLong, E. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Appl. Environ. Microbiol. 63, 50–56 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Teske, A. et al. Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities. Appl. Environ. Microbiol. 68, 1994–2007 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pernthaler, A., Pernthaler, J. & Amann, R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 3094–3101 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Steudel, R., Göbel, T. & Holdt, G. The molecular composition of hydrophilic sulfur sols prepared by decomposition of thiosulfate. Z. Naturforsch. B Chem. Sci. 43, 203–218 (1988)

    Article  CAS  Google Scholar 

  40. Cord-Ruwisch, R. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. Microbiol. Meth. 4, 33–36 (1985)

    Article  CAS  Google Scholar 

  41. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012)

    Article  MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  42. Strous, M., Kraft, B., Bisdorf, R. & Tegetmeyer, H. E. The binning of metagenomic contigs for microbial physiology of mixed cultures. Front. Microbiol. 3, 410 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  43. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014)

    Article  CAS  PubMed  Google Scholar 

  44. Meyer, F. et al. GenDB—an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 31, 2187–2195 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Richter, M. et al. JCoast — a biologist-centric software tool for data mining and comparison of prokaryotic (meta) genomes. BMC Bioinformatics 9, 177 (2008)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Richter, M. & Rosselló-Móra, 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 

  47. Eddy, S. HMMER User’s Guide. Biological sequence analysis using profile hidden Markov models (Howard Hughes Medical Institute, 2003)

    Google Scholar 

  48. Finn, R. D., Miller, B. L., Clements, J. & Bateman, A. iPfam: a database of protein family and domain interactions found in the Protein Data Bank. Nucleic Acids Res. 42, D364–D373 (2014)

    Article  CAS  PubMed  Google Scholar 

  49. Haft, D. H., Selengut, J. D. & White, O. The TIGRFAMs database of protein families. Nucleic Acids Res. 31, 371–373 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yu, N. Y. et al. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26, 1608–1615 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Li, B., Ruotti, V., Stewart, R. M., Thomson, J. A. & Dewey, C. N. RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics 26, 493–500 (2010)

    Article  CAS  PubMed  Google Scholar 

  52. Fernandes, A. D. et al. Unifying the analysis of high-throughput sequencing datasets: characterizing RNA-seq, 16S rRNA gene sequencing and selective growth experiments by compositional data analysis. Microbiome 2, 1–13 (2014)

    Article  CAS  Google Scholar 

  53. Liao, Y., Smyth, G. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014)

    Article  CAS  PubMed  Google Scholar 

  54. Studer, D., Michel, M. & Müller, M. High pressure freezing comes of age. Scanning Microsc., Suppl. 3, 253–268 (1989)

  55. Conrad, R. & Wetter, B. Influence of temperature on energetics of hydrogen metabolism in homoacetogenic, methanogenic, and other anaerobic bacteria. Arch. Microbiol. 155, 94–98 (1990)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Harding, I. Kattelmann, A. Ellrott and M. Meiners for technical support, and M. Richter, H. Gruber-Vodicka and P. Luigi Buttigieg for bioinformatic support, and K. Knittel, N. Dubilier, M. M. M. Kuypers and F. Widdel for discussions. Furthermore we thank A. Teske and the RV ATLANTIS and ALVIN team of cruise AT15-56 in 2009 for providing the initial sediment material. The project was funded by the DFG Leibniz program to A.B., and by the DFG excellence cluster MARUM, Center of Marine Environmental Sciences, Bremen. Further support was provided by the Max Planck Society to D.R. and A.B.

Author information

Authors and Affiliations

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Contributions

G.W., V.K. and A.B designed the experiments; G.W., V.K., H.E.T. and D.R. performed experiments and data analyses; G.W., V.K. and A.B. wrote the manuscript with contributions from D.R. and H.E.T.

Corresponding authors

Correspondence to Gunter Wegener or Viola Krukenberg.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Models of possible species interaction mechanisms in TAOM tested in this study.

a, Transfer of molecular intermediates such as hydrogen. b, Incomplete reduction of sulfate in ANME and zero-valent sulfur transfer to the partner bacteria. c, Direct interspecies electron transfer via conductive nanowires.

Extended Data Figure 2 Visualization of and growth experiments with HotSeep-1.

a, Representative fluorescence micrograph of HotSeep-1 culture (probe HotSeep-1-590; 22 similar images obtained). Cells are solitary or form small aggregates. Scale bar, 10 µm. b, c, Semi-logarithmic illustration of the development of sulfide (b) or numbers of cells and resulting doubling times (c) (doubling time = ln(2)/exponential factor of the regression curve) during incubation of the HotSeep-1 culture with hydrogen as the sole energy source and sulfate. Biological replicates n = 3; data is presented as mean ± s.d., lines of best fit defined by least squares method.

Source data

Extended Data Figure 3 Effect of zero-valent elemental sulfur and molybdate additions on TAOM.

a, Sulfide production in response to zero-valent (colloidal) sulfur addition versus TAOM conditions; zero-valent sulfur did not cause sulfide formation. b, c, Monitoring of hydrogen partial pressures at TAOM conditions (open circles) versus extra addition of 10 mM molybdate (filled circles) for either the full times series (b) or the first 10 h (c). Molybdate addition caused tenfold higher hydrogen concentrations than the TAOM condition. d, Inhibition of methane-dependent sulfide production at different molybdate concentrations. Biological replicates n = 3; symbols represent mean values; error bars are s.d.; b and c show a single time series with the same culture.

Source data

Extended Data Figure 4 Effect of hydrogen on microbial methane oxidation.

a, Methane (0.15 MPa) supplied as the sole electron source was steadily consumed over time by TAOM. b, When both methane (0.15 MPa) and hydrogen (0.05 MPa) were added, hydrogen was rapidly consumed (grey bars), whereas methane consumption was reversely inhibited (green line) until hydrogen was fully consumed. Afterwards methane consumption occurred at the same rate as in the control with only methane (a). Methane, technical replicates n = 3; symbols represent mean values; error bars are s.d.; hydrogen, single measurements. Experiment was replicated once in the laboratory.

Source data

Extended Data Figure 5 Relative expression of marker genes of HotSeep-1 in consortial growth on methane (TAOM) versus enrichment on hydrogen.

Genes encoding proteins apparently involved in direct interspecies electron transfer (CytC and PilA) are strongly overexpressed during TAOM (red) compared to hydrogenotrophic growth (green). Gene expression given in terms of TPM (transcripts per million). Biological replicates n = 3; error bars are s.d.

Extended Data Figure 6 Thin-sections of TAOM and dual species Geobacter spp. aggregates.

a, TAOM culture. High-pressure frozen ANME-1 cells (A) have a cylindrical shape and a size of 1.5 × 0.8 µm, appearing circular in cross-section, and rectangular when cut along the axis. Their cell content shows a high contrast. A* indicates cell envelopes. HotSeep-1 cells (H) are smaller (approximately 1 × 0.5 µm), of rod-like shape, and have lower contrast. The matrix between the cells is largely filled with filaments. Representative of 24 images. Scale bar, 3 µm. b, Thin section of Geobacter consortium with intercellular nanowires using the same embedding techniques as for TAOM consortia, representative of 20 images. Scale bar, 300 nm.

Extended Data Table 1 Phylogenetic affiliation of cloned 16S rRNA gene sequences obtained from TAOM enrichments in 2010 (compiled from ref. 6) and after 1.5 years of cultivation in 2012 (this study)
Extended Data Table 2 Pairwise comparison of nucleotide sequences from the HotSeep-1 draft genomes derived from the TAOM culture versus the HotSeep-1 culture with hydrogen
Extended Data Table 3 Effect of potential intermediates in AOM on sulfide production of TAOM culture (n = 3 replicates, 20 days incubation)
Extended Data Table 4 Genes encoding cytochrome c proteins identified in thermophilic ANME-1 and HotSeep-1 draft genomes, and for type IV pili biogenesis identified in the HotSeep-1 draft genome with expression >20 transcripts per million.

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Wegener, G., Krukenberg, V., Riedel, D. et al. Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria. Nature 526, 587–590 (2015). https://doi.org/10.1038/nature15733

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