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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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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).

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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.

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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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