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

Abstract

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r² ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.

IMPORTANCE

Culture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

OBSERVATION

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.

FIG 1 (A) Cell length proportions of clear and weak fluorescence of Methanothrix filamentous cells calculated based on the fluorescence in situ hybridization (FISH) signals using the Methanothrix-targeting MX825-FITC probe and Candidatus Yanofskyibacterium-targeting Pac_683-Cy3 probe. The Methanothrix cells attached with >5 Ca. Yanofskyibacterium cells were chosen for calculation. (B) Proportions of detected Ca. Yanofskyibacterium cells attached to the different fluorescence of Methanothrix cells: attached to Methanothrix filamentous cells with clear fluorescence, attached to Methanothrix with weak fluorescence, and attached to Methanothrix with no fluorescence. (C) The fluorescence of Methanospirillum cells with or without Ca. Minisyncoccaceae cells. (D) FISH brightness of measured Methanothrix filamentous cells based on 8-bit grayscale images. (A–D) Different letters in the figure indicate significant differences among the values of the proportions based on Tukey’s test (P < 0.05). (E–I) Transmission electron micrographs of small submicron cells attached to (E–H) Methanothrix-like cells and (I and J) Methanospirillum-like cells in culture system C-1 on day 33. Orange arrows indicate extracellular substances at the attachment sites.

FIG 2 (A–D) Scanning electron micrographs of small submicron cells (yellow arrows) attached to (A and B) Methanothrix-like cells and (C and D) Methanospirillum-like cells in culture system C-1 on day 33. White arrows indicate extracellular substances at the attachment sites. (E) Gene expression heatmap of the five most highly expressed genes with signal peptides in the genome of Ca. Yanofskyibacterium (PMX.810_sub) and Ca. Minisyncoccaceae (PMX.50 and PMX.108) in culture systems from C-2 to C-4 on day 14 and C-6 and C-7 on day 31. The color scale from white to orange shows the gene expression level based on the normalized reads per kilobase of transcript per million mapped reads (RPKM) value (see Text S1). “Days 31/14” indicates the difference in gene expression between days 31 and 14. (F) Predicted protein structures of the highly expressed genes of Ca. Patescibacteria using the AlphaFold2 software package (14). The overlaying domain was predicted through the InterPro database (http://www.ebi.ac.uk/interpro/). PGBD, Ig-like, and PKD are the peptidoglycan-binding domain, immunoglobulin-like domain, and polycystic kidney disease domain, respectively.

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.

Description of Candidatus Yanofskyibacterium parasiticum sp. nov.

Yanofskyibacterium parasiticum (pa.ra.si.ti.cum. L. neut. adj. parasiticum, parasitic).

Cells are obligate parasitic and coccoid-like and are grown under the anaerobic conditions with the specific host archaeon Methanothrix spp. Growth occurs with acetate, various amino acids, and nucleoside monophosphate in a co-culture with the host archaeon. DNA G+C content is 46.7% based on the genomic sequence. The species was obtained from Candidatus Patescibacteria-enriched culture from an anaerobic bioreactor-treating purified terephthalate- and dimethyl terephthalate-manufacturing wastewater in Sapporo, Hokkaido, Japan. The species belongs to the genus Candidatus Yanofskyibacterium of the family Candidatus Yanofskyibacteriaceae. The nearly full-length of 16S rRNA gene sequence of Candidatus Yanofskyibacterium parasiticum PMX_810_sub has been deposited in the DDBJ/GenBank/EMBL under the accession number LC715099. The delineation of the species has been proposed by phylogenetic information from genomic sequences. We designate the MAG (BTXX01000000) as the type material of this species.

Description of Candidatus Yanofskyibacterium gen. nov.

Yanofskyibacterium (Ya.nof.sky.i.bac.te’ri.um. N.L. neut. n. bacterium, rod or staff and, in biology, a bacterium; N.L. neut. n. Yanofskyibacterium, named after Charles Yanofsky, who received the ASM Lifetime Achievement Award in 1998).

The genus belongs to the family Candidatus Yanofskyibacteriaceae of the order Candidatus Paceibacterales. The delineation of the genus has been proposed by phylogenetic information from genomic sequences.

Description of Candidatus Yanofskyibacteriaceae fam. nov.

Yanofskyibacteriaceae (Ya.nof.sky.i.bac.te.ri.a.ce’ae. N.L. neut. n. Yanofskyibacterium, type genus of the family; -aceae, ending to denote a family; N.L. fem. pl. n. Yanofskyibacteriaceae, the family of the genus Yanofskyibacterium).

The family belongs to the order Candidatus Paceibacterales of the class Candidatus Paceibacteria. The delineation of the family has been proposed by phylogenetic information from genomic sequences.

Description of Candidatus Minisyncoccus archaeophilus sp. nov.

Minisyncoccus archaeophilus (ar.chae.o’phi.lus. Gr. masc. adj. archaîos, ancient; N.L. masc. adj. suff. -philus, friend, loving; N.L. masc. adj. archaeophilus, archaea loving).

Cells are obligate parasitic and are grown under the anaerobic conditions with the specific host archaeon Methanospirillum spp. Growth occurs with acetate, various amino acids, and nucleoside monophosphate in a co-culture with the host archaeon. DNA G+C content is 36.4% based on the genomic sequence. The species was obtained from Candidatus Patescibacteria-enriched culture from an anaerobic bioreactor treating purified terephthalate- and dimethyl terephthalate-manufacturing wastewater in Sapporo, Hokkaido, Japan. The species belongs to the genus Candidatus Minisyncoccus of the family Candidatus Minisyncoccaceae. The nearly full-length of 16S rRNA gene sequence of Candidatus Minisyncoccus archaeophilus PMX.108 has been deposited in the DDBJ/GenBank/EMBL under the accession number LC715100. The delineation of the species has been proposed by phylogenetic information from genomic sequences. We designate the MAG (BTXZ01000000) as the type material of this species.

Description of Candidatus Minisyncoccus gen. nov.

Minisyncoccus (Mi.ni.syn.coc’cus. L. comp. masc. adj. minor, smaller, inferior; Gr. prep. syn, together; N.L. masc. n. coccus, coccus; from Gr. masc. n. kokkos, grain, seed; N.L. masc. n. Minisyncoccus, small coccus which lives together with another species).

The genus belongs to the family Candidatus Minisyncoccaceae of the order Candidatus Paceibacterales. The delineation of the genus has been proposed by phylogenetic information from genomic sequences.

Description of Candidatus Microsyncoccus archaeolyticus sp. nov.

Microsyncoccus archaeolyticus (ar.chae.o.ly’ti.cus. Gr. masc. adj. archaîos, ancient; N.L. masc. adj. lyticus, able to loose, able to dissolve; from Gr. masc. adj. lytikos, able to loosen; N.L. masc. adj., archaeolyticus, archaea-dissolving).

Cells are obligate parasitic and are grown under the anaerobic conditions with the specific host archaeon Methanospirillum spp. Growth occurs with acetate, various amino acids, and nucleoside monophosphate in a co-culture with the host archaeon. DNA G+C content is 31.1% based on the genomic sequence. The species was obtained from Candidatus Patescibacteria-enriched culture from an anaerobic bioreactor treating purified terephthalate- and dimethyl terephthalate-manufacturing wastewater in Sapporo, Hokkaido, Japan. The species belongs to the genus Candidatus Microsyncoccus of the family Candidatus Minisyncoccaceae. The nearly full-length of 16S rRNA gene sequence of Candidatus Microsyncoccus archaeolyticus PMX.50 has been deposited in the DDBJ/GenBank/EMBL under the accession number LC715109. The delineation of the species has been proposed by phylogenetic information from genomic sequences. We designate the MAG (BTXY01000000) as the type material of this species.

Description of Candidatus Microsyncoccus gen. nov.

Microsyncoccus (Mi.cro.syn.coc’cus. Gr. masc. adj. mikros, small, little; Gr. prep. syn, together; N.L. masc. n. coccus, coccus; from Gr. masc. n. kokkos, grain, seed; N.L. masc. n. Microsyncoccus, small coccus which lives together with another species).

The genus belongs to the family Candidatus Minisyncoccaceae of the order Candidatus Paceibacterales. The delineation of the genus has been proposed by phylogenetic information from genomic sequences of the MAG. The delineation of the genus has been proposed by phylogenetic information from genomic sequences.

Description of Candidatus Minisyncoccaceae fam. nov.

Minisyncoccaceae (Mi.ni.syn.coc.ca.ce’ae. N.L. masc. n. Minisyncoccus, type genus of the family; -aceae, ending to denote a family; N.L. fem. pl. n. Minisyncoccaceae, the family of the genera Minisyncoccus).

The family belongs to the order Candidatus Paceibacterales of the class Candidatus Paceibacteria. The delineation of the family has been proposed by phylogenetic information from genomic sequences.

ACKNOWLEDGMENTS

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.

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