Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems

Candidate phyla radiation (CPR) or the phylum Candidatus Patescibacteria is a lineage of ultrasmall bacteria widely distributed in various natural and artificial environments (1–5). To date, several Ca. Patescibacteria-bacteria intradomain parasitism and predatory lifestyles have been observed (e.g., class Ca. Saccharimonadia with phylum Actinobacteria (6, 7) and class Ca. Gracilibacteria with class Gammaproteobacteria (8, 9), respectively). Recently, cross-domain interactions with archaea have also been discovered for three lineages of the class Ca. Paceibacteria (formerly, Parcubacteria/OD1) using cultivation and microscopic observations: Ca. Yanofskybacteria/UBA5738 (proposed as family Ca. Yanofskyibacteriaceae in this study) (10), Ca. Nealsonbacteria (11), and 32-520/UBA5633 (proposed as family Ca. Minisyncoccaceae) (12). In all cases, the hosting archaea are methanogens—Methanothrix for the former two and Methanospirillum for the latter. Host cells with attached Ca. Paceibacteria showed markedly low ribosomal activity (based on fluorescence in situ hybridization [FISH]) and deformations at the attachment sites (based on transmission electron microscopy [TEM]) (10, 12), suggesting that the Ca. Paceibacteria are parasitic. In addition, several genetic features that may contribute to parasitism have been identified in the metagenome-assembled genomes (MAGs) of Ca. Paceibacteria (10–12). Here, through successful sustained growth of cultures containing the Ca. Paceibacteria and archaea pairs, we were able to couple microscopy and metatranscriptomics to characterize the behavior/mechanisms facilitating the parasitism. Based on our observations, we propose Ca. Yanofskyibacterium parasiticum gen. nov. sp. nov. belonging to Ca. Yanofskyibacteriaceae, Ca. Minisyncoccus archaeophilus gen. nov. sp. nov., and Ca. Microsyncoccus archaeolyticus gen. nov. sp. nov. (which belong to Ca. Minisyncoccaceae).

To set up the experimental design for these analyses, we prepared seven parallel cultures (termed C-1 to C-7) transferred from the culture C-d2-d1 with high abundances of Ca. Patescibacteria described previously (10), which contains acetate, amino acids, and nucleoside monophosphates as potential growth factors for Ca. Patescibacteria (see Text S1). The cultures showed production of methane on days 14 and 31. The microbial community structures of the cultures on days 7, 14, 21, and 31 were analyzed using 16S ribosomal RNA gene amplicon sequencing. The abundances of Ca. Yanofskyibacterium parasiticum (OTU0011; PMX_810_sub as the corresponding MAG), Ca. Minisyncoccus archaeophilus (OTU0014; PMX.108), and Ca. Microsyncoccus archaeolyticus (OTU0072; PMX.50) (Fig. S1A and B) during cultivation were, respectively, in the ranges of 0.15%–12.5%, 0.6%–2.3%, and 0.1%–0.87% (Fig. S2A through D; Text S1).

The physiological and morphological characteristics of the Ca. Paceibacteria–methanogen interactions were confirmed by microscopic observations based on FISH, TEM, and scanning electron microscopy (SEM). On day 31, the FISH fluorescence of Methanothrix filaments with more than five Ca. Yanofskyibacterium cells was significantly lower than that of Methanothrix cells without Ca. Yanofskyibacterium cells because of the significantly larger areas with no fluorescence (Fig. 1A; P < 0.05). Likewise, comparing Methanothrix filaments with different levels of fluorescence showed clear association of the Ca. Yanofskyibacterium cells with Methanothrix showing no fluorescence (35 ± 25 cells per Methanothrix filament) (Fig. 1B and D; Fig. S3A and S4). Methanospirillum cells with Ca. Minisyncoccaceae cells (from 1.1 ± 0.3 to 1.3 ± 0.5 cells per Methanospirillum cell; Fig. S3B) also had significantly lower FISH signals than those without Ca. Minisyncoccaceae (Fig. 1C; Fig. S5; P < 0.05). Taken together, we conclude that the studied Ca. Paceibacteria parasitize methanogenic archaea, strongly supporting previous predictions with statistical evidence (10, 12). As further indirect evidence for parasitism, the cell walls of Methanothrix (sheathed filamentous cells) (13) were often deformed where the submicron cells were attached (Fig. 1E through H) in TEM images. We also observed extracellular substances between the submicron cells and hosting Methanothrix (Fig. 2A and B) and Methanospirillum cells (Fig. 2C and D), suggesting that such materials are important for parasitism by Ca. Paceibacteria. High-resolution imaging techniques such as cryo-electron microscopy are necessary to further clarify details of the mechanisms/structures underlying attachment to the host.

To confirm their interactions based on gene expression levels, we performed metatranscriptomics for the enrichment cultures on days 14 (triplicate) and 31 (duplicate). A total of 6.0–10.8 Gb sequences were obtained and mapped to the previously reconstructed Paceibacterales MAGs of Ca. Yanofskyibacterium (PMX_810_sub) (10), Ca. Microsyncoccus (PMX.50), and Ca. Minisyncoccus (PMX.108) (12) (Fig. S1B; Text S1). Previous studies have suggested that the competence protein ComEC, secretion systems, pilus, and several transporters are important for the parasitism or predatory lifestyles of ultrasmall microbes, including Ca. Patescibacteria (15–18). Accordingly, these genes were highly expressed in the genome of Ca. Yanofskyibacterium PMX_810_sub on day 14 (Table S4). In addition, F-type H⁺-transporting ATPase proteins were highly expressed in Ca. Yanofskyibacterium and Ca. Microsyncoccus PMX.50 (Tables S2 and S4), which are encoded by type IV pilus assembly proteins (Table S2). In a previous study, ATPase and type IV pili were predicted to function in attachment and motility on larger host surfaces (18). Furthermore, some active peptidase-like proteins with signal peptides (PMX_810_sub_00385, PMX_810_sub_00508, PMX.108_00125, PMX.108_00310, PMX.108_00457, PMX.108_00476, and PMX.50_00413) (Table S2) and substrate-binding proteins of amino acid/metal transport systems were found in the three Ca. Patescibacteria genomes (Table S4). Although the detailed functions remain unclear, the addition of external sources of amino acids and trace elements may be key factors for the successful enrichment of Ca. Patescibacteria. In the gene expression of the methanogens, there were no significant differences between cultivation days 14 and 31 and different co-existing species (Table S3). Further refinement of the cultures is required to elucidate the details of Ca. Patescibacteria parasitism (see the supportlemental material in detail).

We computationally predicted the structures of extracellular enzymes encoded by the five most highly expressed genes with unknown function (Fig. 2F; Table S2). These genes possessed peptidoglycan-binding domains (PMX_810_sub_00350, PMX.50_00411, and PMX.108_00302), immunoglobulin-like folds (PMX_810_sub_00465, PMX108_00787, and PMX.108_01191), galactose-binding domain folds (PMX.50_00003), thioredoxin-like domains (PMX.108_00787), polycystic kidney disease domains (PMX.108_01191), and type IV secretion system pilins (PMX.108_00341, PMX.50_00571, PMX.50_00607, and PMX.50_00608). These domains are known to be host adhesion-related proteins, such as membrane-anchored proteins that bind host the membrane (19–21). Of these, the peptidoglycan-binding domain is found at the N- or C-terminus of several enzymes involved in cell wall degradation (e.g., membrane-bound lytic murein transglycosylase B and zinc-containing D-alanyl-D-alanine-cleaving carboxypeptidase) (22). Interestingly, the enzymes containing peptidoglycan-binding domains showed relatively similar structures among the three Ca. Patescibacteria (Fig. 2F), suggesting that these proteins may be important in parasitizing archaeal hosts.

In summary, we found that the interactions between the Ca. Patescibacteria class Ca. Paceibacteria and methanogenic archaea are parasitisms and uncover overlooked physical and mechanistic details of the interaction through the combination of FISH, TEM, and SEM observations and the first successful gene expression analysis of class Ca. Paceibacteria. In addition, we identified highly expressed extracellular enzymes with peptidoglycan-binding domains that have similar structures among the three archaea-parasitizing Ca. Paceibacteria. Establishment of purified co-cultures of Ca. Paceibacteria and methanogens and further detailed characterization of their cell‒cell interactions are essential to clarify the ecological roles of ultrasmall bacteria on anaerobic ecosystems.

The authors thank Riho Tokizawa, Yuki Ebara, Tomoya Ikarashi, and Maho Takai at AIST for technical assistance.

This study was partly supported by the Japan Society for the Promotion of Science KAKENHI JP16H07403 and JP21H01471, a matching fund between the National Institute of Advanced Industrial Science and Technology (AIST) and Tohoku University, and research grants from the Institute for Fermentation, Osaka (G-2019-1-052 and G-2022-1-014).

K. Kuroda and T.N. designed this study. K. Kuroda and M.N. performed sampling, cultivation, microscopy, and sequence analysis. K. Kuroda, M.N., R.N., Y.H., S.K., K. Kubota, T.Q.P.N., K.Y., H.S., M.K.N., and T.N. interpreted the data. K. Kuroda, M.N., and T.N. wrote the manuscript with input from all co-authors. All authors have read and approved the manuscript submission.