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.

The physiological and morphological characteristics of the Ca.Paceibacteria-metha nogen interactions were confirmed by microscopic observations based on FISH, TEM, and scanning electron microscopy (SEM).On day 31, the FISH fluorescence of Methano thrix 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.Minisyncoc caceae (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 mecha nisms/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 (dupli cate).A total of 6.0-10.8Gb 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)(16)(17)(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.Patescibacte ria 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 coexisting species (Table S3).Further refinement of the cultures is required to elucidate the details of Ca.Patescibacteria parasitism (see the supportlemental material in detail).
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 archaeaparasitizing 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.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 Candi datus 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.
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.
The family belongs to the order Candidatus Paceibacterales of the class Candida tus Paceibacteria.The delineation of the family has been proposed by phylogenetic information from genomic sequences.
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 Minisyncoc cus 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.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 Microsyncoc cus 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.
The genus belongs to the family Candidatus Minisyncoccaceae of the order Candida tus 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.

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FIG 1 ( 4 FIG 2
FIG 1 (Continued) 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 (
FIG2 (Continued)    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.
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 Candida tus Paceibacteria.The delineation of the family has been proposed by phylogenetic information from genomic sequences.editing | Kyosuke Yamamoto, Funding acquisition, Validation, Writing -review and editing | Hisashi Satoh, Supervision, Validation, Writing -review and editing | Masaru K. Nobu, Methodology, Project administration, Supervision, Validation, Writing -review and editing | Takashi Narihiro, Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing -review and editing