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Sulfate differentially stimulates but is not respired by diverse anaerobic methanotrophic archaea

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Sulfate-coupled anaerobic oxidation of methane (AOM) is a major methane sink in marine sediments. Multiple lineages of anaerobic methanotrophic archaea (ANME) often coexist in sediments and catalyze this process syntrophically with sulfate-reducing bacteria (SRB), but the potential differences in ANME ecophysiology and mechanisms of syntrophy remain unresolved. A humic acid analog, anthraquinone 2,6-disulfonate (AQDS), could decouple archaeal methanotrophy from bacterial sulfate reduction and serve as the terminal electron acceptor for AOM (AQDS-coupled AOM). Here in sediment microcosm experiments, we examined variations in physiological response between two co-occurring ANME-2 families (ANME-2a and ANME-2c) and tested the hypothesis of sulfate respiration by ANME-2. Sulfate concentrations as low as 100 µM increased AQDS-coupled AOM nearly 2-fold matching the rates of sulfate-coupled AOM. However, the SRB partners remained inactive in microcosms with sulfate and AQDS and neither ANME-2 families respired sulfate, as shown by their cellular sulfur contents and anabolic activities measured using nanoscale secondary ion mass spectrometry. ANME-2a anabolic activity was significantly higher than ANME-2c, suggesting that ANME-2a was primarily responsible for the observed sulfate stimulation of AQDS-coupled AOM. Comparative transcriptomics showed significant upregulation of ANME-2a transcripts linked to multiple ABC transporters and downregulation of central carbon metabolism during AQDS-coupled AOM compared to sulfate-coupled AOM. Surprisingly, genes involved in sulfur anabolism were not differentially expressed during AQDS-coupled AOM with and without sulfate amendment. Collectively, this data indicates that ANME-2 archaea are incapable of respiring sulfate, but sulfate availability differentially stimulates the growth and AOM activity of different ANME lineages.

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Fig. 1: Metabolic activities of methane-oxidizing consortia in sediment microcosms incubated with methane and different electron acceptors.
Fig. 2: Sulfate stimulates AQDS-coupled anaerobic oxidation of methane (AOM).
Fig. 3: Schematic showing two hypothetical scenarios of anaerobic oxidation of methane coupled to AQDS as the terminal electron acceptor by ANME-2 and SRB consortia.
Fig. 4: Paired FISH-nanoSIMS photomicrographs showing anabolic activity for two ANME-2 lineages and their syntrophic bacterial partners with different electron acceptors.
Fig. 5: Gene expression variation of two ANME-2 lineages and two syntrophic sulfate-reducing bacterial lineages with different electron acceptors after 9 days of incubation.
Fig. 6: Differential expression of key pathways by two ANME-2 lineages from comparative metatranscriptomics.

Data availability

The high-coverage metagenomic assembly and metagenome-assembled genomes (MAGs) from this study can be found in Joint Genome Institute Genome Online Database under Study ID Gs0135232. Also, the metagenomic reads, transcriptomic reads and MAGs can be found under National Center for Biotechnology Information BioProject IDs PRJNA431796 and PRJNA576751. Database IDs for each MAG can be found in Supplementary Table 3.

Change history

  • 18 August 2021

    All instances of “AQDS + Sulfate” are changed to “AQDS+Sulfate” (without the two extra spaces.) In Figure 6 legend (x-axis) is italiczed.


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We thank our colleagues, facilities managers, and staff at Caltech: Stephanie Connon for assistance with iTag sample preparation; Ranjani Murali for assistance with sulfate-reducing bacteria phylogeny; Haley Sapers for comments on the manuscript; Nathan Dalleska for assistance with ion chromatography at the Environmental Analysis Center; Yunbin Guan for assistance with nanoSIMS analyses at the Microanalysis Center in the Division of Geological and Planetary Sciences; Fan Gao for valuable discussions on transcriptome analysis at Bioinformatics Resource Center in the Beckman Institute; David Vander Velde for nuclear magnetic resonance analysis at the Liquid NMR Facility in the Division of Chemistry and Chemical Engineering. We also thank Margaret Butler at University of Queensland for assistance with RNA sample preparation and sequencing. We further acknowledge the support from the Monterey Bay Aquarium Research Institute (MBARI) and the pilots and crew of the R/W Western Flyer and ROV Doc Ricketts who supported the field expedition and sample collection. Special thanks to three anonymous reviewers for their constructive comments on the manuscript. This work was supported by the United States Department of Energy’s Office of Science Biological and Environmental Research Program (DE-SC0016469 and DE-SC0020373), Caltech’s Center for Environmental Microbial Interactions (CEMI), and the Simons Foundation Principles of Microbial Ecosystems (PriME).

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HY, GWT and VJO designed the study. HY performed geochemical analyses. MA developed the ANME-2a FISH probe. HY and GLC performed the FISH-nanoSIMS experiments and analyses. HY, CTS and AOL performed the metagenomic and metatranscriptomic experiments and analyses. HY and VJO wrote the manuscript with contributions from other authors. All authors reviewed, revised, and approved the final manuscript.

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Correspondence to Victoria J. Orphan.

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Yu, H., Skennerton, C.T., Chadwick, G.L. et al. Sulfate differentially stimulates but is not respired by diverse anaerobic methanotrophic archaea. ISME J (2021).

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