Complete genomes of Asgard archaea reveal diverse integrated and mobile genetic elements

Abstract

Asgard archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, a wetland Asgard archaea lineage represented by two complete genomes. We used soil depth-resolved population metagenomic data sets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kbp circular plasmid and two family-level groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp integrative conjugative element (ICE) integrates genomically into the Atabeyarchaeum deiterrae-1 chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp ICE encodes an expressed Type IIG restriction-modification system with a sequence specificity matching an active methylation motif identified by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea, Freyarchaeia, which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman, not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility.

Figure 1.

Read mapping to the reference genome provides evidence for integration and excision, illustrated for the case of one mobile genetic element (MGE). The central region of the integrated sequence of Yucahu-i (between black bars) has been deleted to focus on details of reads mapped to the start and end of the region. Read sequences that match the genome sequences are shown as gray bars; small vertical colored bars adjacent to read portions in agreement with the reference indicate bases that disagree with the reference. The yellow, purple, blue, green, and orange arrows indicate distinct nucleotide sequences (same color, same sequence). In the panel demonstrating that some cells that lack the integrated MGE, one read has been split (the black line links the two parts of a single read) to illustrate agreement with the flanking sequence at both ends of the integrated region. Note that the sequence designated by the purple arrow occurs twice when the MGE is integrated. The vertical colored bars indicate single-nucleotide polymorphisms (SNPs) relative to the reference genome sequence and thus do not represent the sequence itself.

Figure 2.

Chromosomally integrated MGEs and defense systems in soil Asgard Archaea genomes. Each panel (A–C) depicts the coverage across each complete genome, as determined by mapping to metagenome reads derived from three different soil depth profiles. Regions exhibiting low coverage suggest strain variations associated with specific soil depths and may indicate the presence of integrated MGEs in only a subset of cells. Notably, the Freyarchaeia genome exhibits even coverage using reads from all sampling depths, with no discernible integrated MGEs identified. Some of the low-coverage regions are not labeled as potential MGEs; these regions are strain variants with sequences so divergent that read mapping is precluded. Oval symbols indicate predicted defense systems.

Figure 3.

Integration and excision of Yucahu. (A) For the 60 cm sample, elevated coverage and paired reads indicate that Yucahu-i is integrated into the genome, excised from some genomes (blue lines), and coexists in circularized form (yellow lines). The red box indicates elevated coverage from a related gene from another genome. (B) For the 165 cm sample, low coverage over the MGE and read sequence discrepancies indicate that most cells in this sample lack the MGE. (C) For the 70 cm sample, coverage and paired read information indicate that Yucahu-i is integrated into essentially all cells. The circularized Yucahu-i is present but rare. Paired reads pointing out internal to the MGE indicate that a 2644 bp element has integrated into the plasmid and coexists in circularized form.

Figure 4.

Evolutionary relationships and structural conservation of Opia virus hallmark proteins. (A–C) Maximum-likelihood phylogenetic trees of capsid (A), terminase large subunit (B), and proliferating cell nuclear antigen (PCNA; C). Trees are midpoint-rooted; circles indicate bootstrap support >70%. Insets show structural models: Opia virus (infecting Atabeyarchaeia, this study) and Nidhogg virus (infecting Helarchaeales) colored by AlphaFold3 confidence (blue to red, high to low), with best FoldSeek match in coral. Reference structures: bacteriophage capsid (PDB: 3BJQ) for A, large terminase from thermophilic bacteriophage D6E (PDB: 5OEB) for B, and P. abyssi PCNA (PDB: 6T8H) for C. Scale bars represent one substitution per site. Opia virus variants cluster with other Asgardarchaeota MGEs, highlighting their evolutionary relationships within this archaeal phylum.

Figure 5.

Comparative genomics of Asgardarchaeota MGEs. (A) Whole-proteome similarity network of MGEs from Atabeyarchaeia and other Asgardarchaeota (Medvedeva et al. 2022; Rambo et al. 2022; Wu et al. 2022). Nodes represent individual MGEs, with edge thickness indicating similarity strength. Yucahu, Opia virus, Zemi, and Guacar form a distinct cluster, separate from other Asgard MGEs. Analysis based on NCBI database entries. (B) Genome size distribution of reported Asgard viruses/MGEs, including those from this study.

Figure 6.

Sequence variation in the cysteine-rich proteins of Opia viruses and comparison to Pacl, a rare-cutting HNH restriction endonuclease. (A) The aligned ∼5.6 kbp variable region of two Opia viral genotypes. Light gray bars indicate perfect nucleotide identity, and thin vertical black lines indicate SNPs. The three genes labeled in brown are present in both Opia-3708 (top) and Opia-19564 (bottom) but are absent in some other genotypes. Light blue genes are cysteine-rich (For 3708L, 10, five, nine, five, 10; for Opia-19564, three, 14, five, nine, four, 10 cysteines per protein). Dark blue genes encode ParB-like proteins that are very divergent between some genotypes (Opia-3708 vs. Opia-66823, 67881). Before the three-gene indel and after the ParB-like gene, the ∼35 kb regions of all genomes are essentially identical. (B) Phylogenetic tree including the 17 larger cysteine-rich proteins from the variable regions of seven Opia genomes (numbers represent genome names) (for context, see Supplemental Material). Proteins with identical sequences occur in five different combinations across the genotypes. (C) Comparison of the active site region of PacI (with nine cysteines) and the structure of Opia-19564 protein 16 (with 10 cysteines; silver). Active site residues of PDB 3m7k (gold) are displayed based on figure 1 of Shen et al. (2010). The critical ββα-metal motifs are well aligned. Tyrosine and histidine residues exist in proximity to the active site, although their locations differ somewhat, possibly owing to fold inaccuracy.

Figure 7.

Phylogenetic placement of Asgard archaeal genomes and distribution of associated MGEs. The phylogeny shows the evolutionary relationships among different Asgard archaeal lineages, with symbols indicating the presence and types of published MGEs in various candidate groups. Descriptions of the phylogenomic analyses have been previously described in detail (Valentin-Alvarado et al. 2024). Our work on Atabeyarchaeia and Freyarchaeia is highlighted, demonstrating the novel MGEs identified in this study and the presence of two novel defense systems in Freyarchaeia. This figure provides a comprehensive overview of the current state of knowledge regarding MGEs across the Asgardarchaeota phylum.