Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem

Abstract

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.

Keywords: 32-520/UBA5633; Candidate Phyla Radiation (CPR) superphylum; Candidatus Patescibacteria; Methanospirillum; cross-domain symbiosis.

Fig. 1.

(A) and (B) Phase-contrast micrographs of small cells attached to rod-shaped cells in culture systems A-d2 on days 12 (A) and 23 (B). Yellow arrows indicate small cells attached to rod-shaped cells. (C) Phylogenetic tree of the 32-520 lineage based on 16S rRNA gene sequences. The 16S rRNA gene-based tree was constructed using the neighbor-joining method implemented in the ARB program. The OTUs obtained in the present study are shown in bold type in the tree. Sequences that match the 32-520-1066 probe are shown in blue font. Star symbols indicate the symbiotic partners of these lineages (green: Methanospirillum (this study) and gray: Methanothrix (Kuroda et al., 2022b) (Chen, X., et al., 2022. Candidatus Nealsonbacteria (OD1) in a methanogenic enrichment culture is likely an ectosymbiotic biomass recycler. bioRxiv https://doi.org/10.1101/2022.04.20.488981). Micrographs of (D) phase-contrast micrographs, (E) 4′,6-diamidino-2-phenylindole dihydrochloride staining, and (F) fluorescence in situ hybridization (FISH) by uncultured order-level clade 32-520 targeting the 32-520-1066-Cy3 probe obtained from the culture system B-d1-d1 on day 23. Yellow arrows indicate FISH-detectable 32-520 cells belonging to the class Ca. Paceibacteria (belonging to the phylum Ca. Patescibacteria).

Fig. 2.

(A–F) Transmission electron micrographs of small coccoid sub-micron cells attached to Methanospirillum-like cells in culture system A-d2 on day 40. S, Sh, M, P, ad, div, and PPB indicate the S-layer, sheath structure, cell membrane, plug structure, adhesion substance, dividing cells, and polyphosphate-like body, respectively. Orange arrows indicate extracellular polymeric substances (EPS) or pilin-like substances.

Fig. 3.

(A) Phylogenetic tree of the order Ca. Paceibacterales based on concatenated phylogenetic marker genes of GTDBtk 2.0.0 (ver. r207). The phylogenetic position of the metagenomic bins of PMX.50 and PMX.108 are shown in blue. (B) Gene clusters containing multiple genes with signal peptides in 32-520/UBA5633. P indicates the pilin signal peptide. Blue circles indicate a blastp-based homology (threshold ≤1e–10) with the metagenomic bin PMX.50. No annotated genes are hypothetical proteins (based on the annotation using BlastKOALA in Table S4). Abbreviated locus tags are shown in (B) (e.g., “PMX.50_00679” as “00679” in the row of PMX.50).