Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Systems-Level Insights into the Physiology of Methane-Fueled Syntrophy Between Anaerobic Methanotrophic Archaea and Sulfate-Reducing Bacteria


Magdalena Mayr1*(, Philip Woods1* (, Ranjani Murali1, Felipe Wang Liu2, Andrew P. Freiburger2, José P. Faria2, Nidhi Gupta2, Janaka Edirisinghe2, Robert Hettich3, Christopher S. Henry2, Victoria Orphan1



1California Institute of Technology; 2Argonne National Laboratory; 3Oak Ridge National Laboratory


(1) Develop a mechanistic understanding of anaerobic oxidation of methane (AOM) syntrophic interactions; (2) define and functionally characterize the microbial community, including viruses, associated with methanotrophic consortia under changing environmental conditions; and (3) create an integrative modeling framework to explore the ecophysiology of AOM consortia and their community interactions in an environmental context.


AOM is a geologically important process that impacts methane utilization in marine sediments. AOM is mediated by anaerobic methanotrophic archaea (ANME) and is made energetically feasible by coupling methane oxidation with the reduction of electron acceptors such as nitrate, metals, or sulfate. In sulfate-coupled AOM, ANME are found in an obligate syntrophic partnership with sulfate reducing bacteria (SRB). This syntrophic association is driven by direct interspecies electron transfer between partners co-localized in a multicellular consortium.

Using comparative phylogenomics, the research team and others have shown that ANME archaea are polyphyletic, evolving multiple times from methanogenic ancestors (Evans et al. 2019; Chadwick et al. 2022). Analysis of gene phylogenies, locus organization, and sequence alignments suggests that during this process, each ANME clade has convergently evolved to encode distinctive genes often involved in central metabolic pathways. In particular, studies of the most recently evolved ANME-3 clade have produced a step-by-step view of this process. Results suggests that evolution of convergent modifications to proteins involved in carbon and energy metabolism precedes later optimization through horizontal acquisition of multi-heme cytochromes and genes involved in nutrient acquisition and cell-cell interaction. Sulfate-reducing syntrophic bacterial partners have also convergently evolved from free-living sulfate reducers to syntrophic SRB by adapting their energy metabolism and acquiring genes by lateral gene transfer that promote interspecies interaction and biofilm formation (Murali et al. 2023).

In this work, environmental metaproteomics and metabolic modeling were used to test phylogenomics-derived inferences. Model results highlight differences in electron transport pathways that are critical to the differences between ANME and methanogens. Metaproteomics analyses of environmental ANME–SRB consortia demonstrated that previously identified pathways associated with ANME and SRB energy metabolism (e.g., Mcr, Dsr, Apr, Rnf) and interspecies interactions (e.g., eCIS, adhesins) are actively expressed.

Additionally, the research team identified several highly expressed proteins that currently have no characterized function, highlighting additional unexplored aspects of AOM physiology. Targets were identified from highly expressed proteins for further heterologous expression (e.g., putative fibronectin binding matrix proteins, Rnf).

Further physiological insights of these slow-growing microbes will be gained by stable isotope probing metaproteomics (e.g., 13C-CH4, 13C-NaHCO3, 15N-NH4Cl). Preliminary analysis shows uptake of labeled substrates by ANME-SRB and upcoming analysis will provide insight into protein turnover, growth rates, and carbon uptake.


Chadwick, G. L., et al. 2022. “Comparative Genomics Reveals Electron Transfer and Syntrophic Mechanisms Differentiating Methanotrophic and Methanogenic Archaea,” PLOS Biology, 20(1), e3001508. DOI:10.1371/journal.pbio.3001508.

Evans, P. N., et al. 2019. “An Evolving View of Methane Metabolism in the Archaea,” Nature Reviews Microbiology, 17(4), 219–32. DOI:10.1038/s41579-018-0136-7.

Murali, R., et al. 2023. “Physiological Potential and Evolutionary Trajectories of Syntrophic Sulfate-Reducing Bacterial Partners of Anaerobic Methanotrophic Archaea,” PLoS Biology, 21(9). DOI:10.1371/journal.pbio.3002292.

Funding Information

This work was supported by the DOE Office of Science, BER Program, GSP under award no. DE-SC0022991.