Symbiosis between Candidatus Patescibacteria and Archaea Discovered in Wastewater-Treating Bioreactors

One major lineage of the domain Bacteria, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) (1, 2), is a highly diverse group of bacteria that widely inhabits natural (3, 4) and artificial ecosystems (5–7) and is characterized by its small cell and genome sizes (8) and by its poor (genomically predicted) abilities to synthesize cellular building blocks (4), which is suggestive of a symbiotic dependency for cell growth. Most members remain uncultured, leaving major knowledge gaps in the range and nature of their symbioses (i.e., commensal, mutualistic, and parasitic relationships) (3), although a few cultivation-based and microscopy-based studies have demonstrated host-specific symbiotic and parasitic interactions between Ca. Patescibacteria and other bacteria; e.g., Ca. Saccharimonadia with Actinomycetota (formerly known as Actinobacteria) (9) and Ca. Gracilibacteria with Gammaproteobacteria (10, 11). This suggested a specialization toward bacteria-bacteria symbioses, especially given the parallels with DPANN, an archaeal analog which has only been observed to interact with other archaea (4, 12); however, one study has reported symbiosis between Ca. Patescibacteria and eukarya (13), indicating that the Ca. Patescibacteria host range reaches beyond bacteria. Here, based on our previous observations of predominant Ca. Patescibacteria members and methanogenic archaea (“methanogens”) (6, 7), we hypothesize that some Ca. Patescibacteria may symbiotically interact with archaea and use exogenous archaea to culture potential archaea-dependent Ca. Patescibacteria.

To create conditions conducive to the growth of archaea-dependent Ca. Patescibacteria, we took a strategy similar to that of virus or phage cultivation, in which exogenous methanogenic archaea were grown in the presence of Ca. Patescibacteria that would presumably grow using molecules derived from these active hosts (i.e., through symbiosis and parasitism). We chose acetate-utilizing methanogens as the partners, as they ubiquitously inhabit methanogenic ecosystems (5, 6, 14), form symbiotic interactions with bacteria (14), utilize an energy source (acetate) that is generally noninhibitory to organotrophs (unlike other methanogen substrates, such as H₂ or formate [15]), and conveniently have a highly distinguishable cell morphology/structure that is easily differentiable from those of other organisms (i.e., easily traceable under a microscope). To culture Ca. Patescibacteria that may interact with archaea, we used microbial community samples from a bioreactor (“sludge”) that was particularly abundant in Ca. Patescibacteria (7) as starting material and amended them with acetate-utilizing methanogens (Methanothrix soehngenii GP6 and Methanosarcina barkeri MS) as symbiotic partners and acetate as an energy source for the archaea (Text S1). Potential growth factors (yeast extract, various amino acids, and nucleoside monophosphates) were also provided, as Ca. Patescibacteria are known to have poor biosynthetic capacities (4).

In the cultivation experiments, we performed serial dilutions (10⁻¹, 10⁻³, 10⁻⁴, and 10⁻⁶, defined as d1–d4) of the sludge-methanogen mixture to help eliminate low-abundance bacteria that may have interfered with the Ca. Patescibacteria growth. In some cultures with confirmed gas production, we detected the enrichment (increased relative abundances up to 12.1% in A-d2 on day 33) of a population of an uncultured clade of Ca. Patescibacteria, namely, Ca. Yanofskybacteria OTU0011, which belongs to class Ca. Paceibacteria (formerly known as Parcubacteria/OD1 [1]) (Fig. 1A), and we also observed many small cells (<1 μm in diameter) that were consistently attached to cells with a morphology characteristic of Methanothrix (long rods of approximately 0.8 μm in diameter with blunt ends strung together, forming multicellular filaments), with the number of attached cells increasing as the culture aged (Fig. S1A and S1B). To further eliminate other nontarget populations in the culture (i.e., “enrich” the target organisms), we subcultured those abundant in small cells and microscopically confirmed the continued physical attachment of small cells with Methanothrix-like cells (Fig. S1C and S1D). Three independent subcultures (defined as A-d2-d1, B-d1-d1, and C-d2-d1) retaining Ca. Yanofskybacteria (each amended with acetate and/or yeast extract) with high abundance (1.7 to 13.1%) (Fig. 1A) were used for further microscopy observation.

Through fluorescence in situ hybridization (FISH), which allowed for the differentiation of target populations with fluorescence microscopy, we successfully verified that the small cells attached to the surfaces of the Methanothrix filaments were indeed Ca. Yanofskybacteria (with probes MX825 and Pac_683, respectively) (Fig. 1B and C; also see Fig. S2–S4). Across all subcultures, most Methanothrix filaments (59.3 ± 5.3%) were physically associated with Ca. Yanofskybacteria, and, more importantly, nearly all of the Ca. Yanofskybacteria cells (95.7 ± 2.1%) were attached to Methanothrix filaments, with none attached to other hosts and only a small fraction remaining (4.2 ± 2.1%) unattached, showing that the physical attachment and symbiosis are Methanothrix-specific (Fig. 1D).

Compared to the Methanothrix filaments that were free of ectosymbionts in the cultures, those physically associated with more than 5 Ca. Yanofskybacteria cells contained significantly larger areas (P < 0.05 for all cultures) with low ribosomal activity (based on FISH fluorescence) (Fig. S5). Moreover, a major fraction of the Ca. Yanofskybacteria cells (18.8 ± 1.9%) were associated with these low-activity Methanothrix cells. Though highly qualitative, many of the Ca. Yanofskybacteria cells (39.6 ± 6.1%) were attached to Methanothrix cells with weak fluorescence (e.g., Fig. S2H, S3H, and S4H), which may reflect the negative influence of attachment. Using transmission electron microscopy (TEM), we further observed that Methanothrix (sheathed filamentous cells) (16, 17) often had deformed cell walls where submicron coccoid-like cells (presumably Ca. Yanofskybacteria; 0.46 ± 0.13 μm long and 0.36 ± 0.07 μm wide, 0.0377 ± 0.0200 μm³ calculated cell volumes) were attached (Fig. 2A–C). The clearly negative influence of Ca. Yanofskybacteria implies that the symbiosis between Ca. Yanofskybacteria and Methanothrix is parasitic.

Through shotgun metagenomic analysis, we successfully recovered a metagenome-assembled genome of Ca. Yanofskybacteria (PMX_810_sub; 0.8 Mb in total) and nearly full-length 16S rRNA gene sequences (Fig. 2D; Fig. S6; Tables S1 and S2). Based on 43 marker genes for Ca. Patescibacteria (1), the completeness and contamination of PMX_810_sub were estimated to be 90.7% and 0%, respectively. Phylogenetic classification based on SILVA v138.1 and GTDB r207 taxonomy confirmed the classification of the Ca. Yanofskybacteria (99.7% similarity with FPLM01004990) (Fig. S6; Table S2) and the Ca. Patescibacteria family UBA5738 of the order Paceibacterales (Fig. 2D; Table S1). The metagenome-assembled genome PMX_810_sub lacks many biosynthetic pathways (e.g., those for the biosynthesis of amino acids and fatty acids) (Table S3), suggestive of a host-dependent or partner-dependent lifestyle, as was also observed for other Ca. Patescibacteria members (4). Previously, gene arrays that contain small signal peptides have been found in pathogenic bacterial genomes and in Ca. Patescibacteria (18), which may be related to their parasitic potential (19). Indeed, all of the metagenome-assembled genomes that were affiliated with Ca. Yanofskybacteria/UBA5738 contained genes for protein secretion systems (e.g., SecADEFGY) (Table S3) and universally conserved three unique gene arrays that contained multiple genes with signal peptides that could be recognized by the aforementioned systems (and one additional gene array conserved in the two deep-branching genomes) (Fig. 2D and E). Interestingly, all of the genes included in these arrays encode hypothetical proteins, suggesting that, if these genes are involved in interactions with the host, the mechanism is unrelated to known forms of parasitism. Further investigation using transcriptomics and proteomics is necessary to clarify the mechanisms behind the parasitism by this organism and lineage.

In total, through the first successful cultivation and enrichment of the Ca. Patescibacteria class Ca. Paceibacteria (to which Ca. Yanofskybacteria/UBA5738 belongs), we discovered that Ca. Patescibacteria/CPR can symbiotically interact with the domain Archaea. The obtained results suggest that the observed interaction between Ca. Yanofskybacteria/UBA5738 and Methanothrix is a host-specific parasitism. Although the host range and the preference of Ca. Yanofskybacteria/UBA5738 remain unclear, the observed ability of Ca. Patescibacteria to interact with methanogenic archaea, a central group of organisms in anaerobic ecosystems, warrants further investigation into how parasitism may influence ecology and carbon cycling. As the presented archaea cocultivation strategy was effective in culturing Ca. Patescibacteria that were inhabiting methanogenic environments, we anticipate that the further refinement of cocultivation combined with gene and protein expression will allow for the characterization of the details of the symbiosis between Ca. Patescibacteria and Archaea, the determination of the diversity of archaea-dependent Ca. Patescibacteria, and, ultimately, the elucidation of the influence of these organisms’ interactions on anaerobic ecology.

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 by research grants from the Institute for Fermentation, Osaka (G-2019-1-052 and G-2022-1-014). We thank Riho Tokizawa, Yuki Ebara, and Tomoya Ikarashi at AIST for their technical assistance.

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

We declare no conflict of interest.