Methanotrophic Flexibility of 'Ca. Methanoperedens' and Its Interactions With Sulphate-Reducing Bacteria in the Sediment of Meromictic Lake Cadagno

Abstract

The greenhouse gas methane is an important contributor to global warming, with freshwater sediments representing important potential methane sources. Anaerobic methane-oxidising archaea mitigate methane release into the atmosphere by coupling the oxidation of methane to the reduction of extracellular electron acceptors or through interspecies electron transfer with microbial partners. Understanding their metabolic flexibility and microbial interactions is crucial to assess their role in global methane cycling. Here, we investigated anoxic sediments of the meromictic freshwater Lake Cadagno (Switzerland), where 'Ca. Methanoperedens' co-occur with a specific sulphate-reducing bacterium, with metagenomics and long-term incubations. Incubations were performed with different electron acceptors, revealing that manganese oxides supported highest CH₄ oxidation potential but enriched for 'Ca. Methanoperedens' phylotypes that were hardly present in the inoculum. Combining data from the inoculum and incubations, we obtained five 'Ca. Methanoperedens' genomes, each harbouring different extracellular electron transfer pathways. In a reconstructed Desulfobacterota QYQD01 genome, we observed large multi-heme cytochromes, type IV pili, and a putative loss of hydrogenases, suggesting facultative syntrophic interactions with 'Ca. Methanoperedens'. This research deepens our understanding of the metabolic flexibility and potential interspecific interactions of 'Ca. Methanoperedens' in freshwater lakes.

FIGURE 1

(A) Cartoon summarising previous anaerobic oxidation of methane (AOM) data from Lake Cadagno sediment (Su et al. 2020). Top: Depth‐specific in situ anaerobic oxidation of methane oxidation rates (AOM‐R) determined using ¹³C‐CH₄ tracer whole‐core incubations, and profiles of porewater concentrations of dissolved methane, sulphate and sulphide. Bottom: Depth profiles of 16S rRNA gene amplicons (relative abundances) of ‘Ca. Methanoperedens spp.’ and SRBs in the sediment. (B) Overview of long‐term slurry incubation assays presented in the current study, including the conditions employed and the parameters analysed. The sediment samples and incubations selected for metagenomics are coloured in pink.

FIGURE 2

Percentage of mapped reads of top (above 0.5% for all metagenome sources) Metagenome Assembled Genomes (MAGs) analysed across the two sediment depths (23 and 25 cm) and the three incubations: Unamended control, sulphate, and manganese oxides. Subcategories include anaerobic methane oxidizers and canonical sulphate‐reducing bacteria (SRB) (containing dsrABCD) and non‐canonical SRB (containing dsrABC), as described in Diao et al. . The ‘Others’ category is subdivided into all genomes that either exceeded 0.5% (dark grey) across all metagenome sources or fell below this threshold (light grey). The remaining reads constitute the unbinned fraction (not filling up to 100%).

FIGURE 3

Phylogenomic tree of Methanoperedeneacae MAGs with Lake Cadagno representatives highlighted in bold. The tree has been generated using GTDB‐Tk classification tools with multiple sequence alignments of 53 archaeal phylogenetic markers. MAGs that were not in the GTDB database at the time of analysis were additionally included: GCA_018900975.1 and GCA_018899795.1 (Mehrshad et al. 2021), GCA_022013335.1 (Bell et al. 2022) and GCA_020062615.1 and GCA_020062365.1 (Casar et al. 2021). Extracellular electron transfer (EET) and presence/absence of hydrogenases are only indicated for MAGs with shown or suspected EET. Branch lengths represent the average number of amino acid substitutions per site. Bootstrap values are shown for > 70% branching support.

FIGURE 4

Multi‐heme c‐type cytochrome (MHC) protein counts in the Lake Cadagno ‘Ca. Methanoperedens sp.’ MAGs. Annotations are based on similarity (> 70% to domain MHC) to reference ‘Ca. Methanoperedens’ enrichment species that showed MHC expression under different electron accepting conditions—multiple (including electrode), manganese or iron oxides—and expression levels. ‘High’ expression refers to the top 10% of the MHC transcripts, ‘medium’ to the following 40%, and ‘low’ to the remaining MHC transcript expression. OmcZ‐like nanowire subunits are included as a separate category. The “Others” category includes MHC genes from this study that lack domain‐homology‐based expression links to reference ‘Ca. Methanoperedens’ MAGs. Comparing the similarity of MHC proteins among the Lake Cadagno ‘Ca. Methanoperedens’ spp. (Figure S4) revealed that they shared between 9 and 16 homologous MHC proteins. For details, see Figure S4.

FIGURE 5

(A) Phylogenomic tree of the Desulfobacterota phylum based on Metagenome Assembled Genomes (MAG) with collapsed clades except for class QYQD01 and classDTXE01. The tree was generated via GTDB‐Tk classification tools using multiple sequence alignments via concatenation of 120 bacterial phylogenetic markers. One MAG was included in addition to the genomes retrieved from the GTDB database: GCA_020053325.1 (Casar et al. 2021). Bootstrap values are shown for > 70% branching support. Branch length represents the average number of amino acid substitutions per site. (B) From top left to right, sequence read archive (SRA) hits for Methanoperedeneaceae family and Desulfobacterota class QYQD01. Colours indicate percentages of Sequencing Read Archive (SRA) entries where both groups were found in the same data set (pink) or not (brown). Bottom pie‐chart displays SRAsource from where both groups were found present.