Asgard archaea modulate potential methanogenesis substrates in wetland soil

Abstract

The roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems remain unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and a complete genome of Freyarchaeia, and predicted their metabolism in situ. Metatranscriptomics reveals expression of genes for [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway. Also expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to include non-methanogenic acetogens, highlighting their potential role in carbon cycling in terrestrial environments.

Fig. 1. Archaea dominate deep regions of wetland soil and host novel Asgard archaea.

a Photograph of the vernal pool that was sampled for metagenomics analyses in this study, in Lake County, California, USA. b Archaeal genomic abundance excluding bacterial genomes. c Phylogenetic distribution of Asgard Archaea complete genomes. The maximum-likelihood phylogeny was generated with IQ-TREE v1.6.1, utilizing 47 concatenated archaeal Clusters of Orthologous Groups of proteins (arCOGs). The best-fit model was determined as LG + F + R10 based on the Bayesian Information Criterion. Non-parametric bootstrapping was conducted with 1000 replicates for robustness. The filled-in square, circle, and triangle indicate closed complete genomes from short reads, published complete genomes from long reads, and genomes from co-isolated cultured representatives, respectively. The Pentagon highlights the long-read draft genomes from this site (PacBio or Nanopore). d Indicators of bidirectional replication in Atabeyarchaeia complete genomes. The GC skew is shown as a gray plot overlaying the cumulative GC skew, presented as a green line. The blue lines mark the predicted replication terminus. Source data are provided as a Source Data file.

Fig. 2. Metabolic capacities of terrestrial Atabeyarchaeia and Freyarchaeia for overall implications for biogeochemical cycling in wetlands.

Inference of the pathways from the complete genomes is based on the comparison of predicted proteins with a variety of functional databases (“see methods”). The extraction depth location within the cores is shown on the left. All reactions are numbers and correspond to Supplementary Data 7. EC/TCDB numbers shaded fully or partially in blue or green are unique to the lineages and complete genomes, whereas the dashed boxes distinguish oxygen-sensitive enzymes. The multi-functional aldehyde ferredoxin oxidoreductase is shown with a star. Proteins marked with a triangle have generated phylogenies to determine their evolutionary histories and substrate specificity. Reactions with mapped transcripts are denoted with red text and arrows. Created using BioRender.com.

Fig. 3. Metatranscriptomic profiling of soil-associated Asgard archaeal genomes.

a Heatmap visualization of normalized Reads Per Kilobase per Million mapped reads (RPKM) values for ORFs with high sequence similarity (≥ 95%) to the genomes of Atabeyarchaeia-1, Atabeyarchaeia-2, and Freyarchaeia, across various soil depths. It is important to note that these samples are homogenized and do not represent biological replicates, thus they do not reflect variation across depths “see methods”. A total of 2191 open reading frames (ORFs) were categorized using the Clusters of Orthologous Groups (COG) database, with Atabeya-1, Atabeya-2, and Freya expressing 465, 804, and 922 unique ORFs, respectively. The ORFs were annotated and assigned to 15 COG categories, indicating the functional potential of each archaeal genome in situ. Columns represent metatranscriptomes from different soil depths, highlighting the spatial variability in the expression of key metabolic and cellular processes. b Expanded heatmap of Atabeyarchaeia-1 and Freyarchaeia expressed genes under the category C: Energy production and conversion. Key genes of the Wood-Ljungdahl Pathway (CODH/ACS, carbon monoxide dehydrogenase/acetyl-CoA synthase; fwdB, formate dehydrogenase; mtd, 5,10-methylene-H4-methanopterin dehydrogenase), hydrogenases and associated genes (hdrA, heterodisulfide reductase and group 3c NiFe-hydrogenase; mvh, methyl viologen reducing hydrogenase); hyaD (maturation factor); hycE and nuo-like subunits denoted with an asterisks, (group 4 NiFe-hydrogenase), ATP synthase (atpE, V/A-type H⁺/Na⁺-transporting ATPase subunit K; ntpD, V/A-type H⁺/Na⁺ transporting ATPase subunit D) and aldehyde metabolism (gor, aldehyde:ferredoxin oxidoreductases), pyruvate oxidation (porABCD, 2-pyruvate:ferredoxin oxidoreductase; pflD, pyruvate-formate lyase). Source data are provided as a Source Data file.

Fig. 4. Phylogeny, genetic organization and AlphaFold predicted structure of the novel group 4 energy-conservation complex I-like NiFe-hydrogenase from Asgard archaea.

a Genetic organization of the group 4 [NiFe]-hydrogenase module, the proton-translocating membrane module, and ATP synthase from the Freyarchaeia genome. b Maximum likelihood phylogeny of group 4 [NiFe]-hydrogenase large subunit from Asgard archaea and reference sequences. The bolded taxonomic groups highlight the clades with genomes from this study used for modeling. Note that group 4 g [NiFe]-hydrogenase is currently polyphyletic based on HydDB. c AlphaFold Multimer models of [NiFe]-hydrogenase module and the proton-translocating membrane module where each candidate subunit is represented by a different color based on the best subunit matched. d AlphaFold Multimer model of Freyarchaeia hydrogenase complex colored by chains, aligned with cryoEM structure of a respiratory membrane-bound hydrogenase (MBH) from Pyrococcus furiosus (PDB ID: 5L8X). e AlphaFold Multimer model of Freyarchaeia hydrogenase complex colored by chains, aligned with the crystal structure of respiratory complex I from Thermus thermophilus (PDB: 4HEA).

Fig. 5. Non-methanogenic MtrA, MtrH and MtrAH fusion methyltransferases.

a Maximum likelihood phylogeny of MtrA and the MtrAH fusion, with reference to Tetrahydromethanopterin S-methyltransferase subunit A (MtrA) with the closest corresponding domains being MtrA from the characterized Tetrahydromethanopterin S-methyltransferase subunit A (MtrA) protein (PDB ID: 5L8X). The coral-colored clade is the novel fusion present in Atabeyarchaeia, Freyarcheia, and other Asgardarchaeota members. b AlphaFold models of Atabeyarchaeia-1 MtrAH (fusion) in coral aligned with the gray corresponding domains of the characterized protein Tetrahydromethanopterin S-methyltransferase subunit A (MtrA) (PDB ID: 5L8X) and Methyltransferase (MtgA) from Desulfitobacterium hafniense in complex with methyl-tetrahydrofolate (PDB ID: 6SK4) at the N terminus. We also modeled the putative MtrA present in Atabeyarchaeia-1 with the closest corresponding domains being MtrA from the characterized Tetrahydromethanopterin S-methyltransferase subunit A (MtrA) protein (PDB ID: 5L8X).

Fig. 6. Overview of the wetland soil dynamics and biogeochemical cycling in Atabeyarchaeia and Freyarchaeia.

Complete genomes for Atabeyarchaeia and Freyarchaeia are shown with green and orange circles, respectively. The Atabeyarchaeia (Atabeya-1 and Atabeya-2) and Freyarchaeia (Freya) genomes were isolated and carefully curated and closed from wetland soil between 60-100 cm. These anaerobic lineages were shown in this study to encode the Wood-Ljungdahl Pathway for CO₂ fixation (e.g., methylated compounds such as quaternary amines) and Embden-Meyerhof-Parnas (EMP) Pathway, components of chemolithotrophy and heterotrophy, producing acetate shown by the arrows (green and orange), corresponding to the taxonomic lineage colors. The metabolic versatility of these soil Asgardarchaeota like their marine counterparts may provide a competitive advantage in wetlands. A detailed description of the specific pathways is found in the main text, Fig. 2, and supplementary materials. Created using BioRender.com.