Microscopic and metatranscriptomic analyses revealed unique cross-domain symbiosis between Candidatus Patescibacteria/candidate phyla radiation (CPR) and methanogenic archaea in anaerobic ecosystems

To verify the parasitic lifestyle of Candidatus Patescibacteria in the enrichment cultures derived from a methanogenic bioreactor, we applied multifaceted approaches combining cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses. Cultivation experiments with the addition of exogenous methanogenic archaea with acetate, amino acids, and nucleoside monophosphates and 16S rRNA gene sequencing confirmed the increase in the relative abundance of Ca . Patescibacteria and methanogens. The predominant Ca . Patescibacteria were Ca . Yanofskybacteria and 32-520 lineages (to which belongs to class Ca . Paceibacteria) and positive linear relationships ( r 2 ≥ 0.70) between the relative abundance of Ca . Yanofskybacteria and Methanothrix , suggesting that the tendency of the growth rate is similar to that of the host. By fluorescence in situ hybridization (FISH) observations, the FISH signals of Methanothrix and Methanospirillum cells with Ca . Yanofskybacteria and with 32-520 lineages, respectively, were significantly lower than those of the methanogens without Ca . Patescibacteria, suggesting their parasitic interaction. The TEM and SEM observations also support parasitism in that the cell walls and plugs of these methanogens associated with submicron cells were often deformed. In particular, some Methanothrix -like filamentous cells were dented where the submicron cells were attached. Metatranscriptomic and protein structure prediction analyses identified highly expressed secreted genes from the genomes of Ca . Yanofskybacteria and 32-520, and these genes contain adhesion-related domains to the host cells. Considering the results through the combination of microscopic observations, gene expression, and computational protein modeling, we propose that the interactions between Ca . Yanofskybacteria and 32-520 belonging to class Ca . Paceibacteria and methanogenic archaea are parasitism. ### Competing Interest Statement The authors have declared no competing interest.