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

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.IMPORTANCECulture-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.

Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem.

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.

Diversity of Candidatus Patescibacteria in Activated Sludge Revealed by a Size-|Fractionation Approach.

Uncultivated members of Candidatus Patescibacteria are commonly found in activated sludge treating sewage and are widely distributed in wastewater treatment plants in different regions and countries. However, the phylogenetic diversity of Ca. Patescibacteria is difficult to examine because of their low relative abundance in the environment. Since Ca. Patescibacteria members have small cell sizes, we herein collected small microorganisms from activated sludge using a filtration-based size-fractionation approach (i.e., 0.45-0.22| |µm and 0.22-0.1| |µm fractions). Fractionated samples were characterized using 16S rRNA gene amplicon and shotgun metagenomic sequence ana-lyses. The amplicon ana-lysis revealed that the relative abundance of Ca. Patescibacteria increased to 73.5% and 52.5% in the 0.45-0.22| |µm and 0.22-0.1| |µm fraction samples, respectively, from 5.8% in the unfractionated sample. The members recovered from the two size-fractionated samples included Ca. Saccharimonadia, Ca. Gracilibacteria, Ca. Paceibacteria, Ca. Microgenomatia, class-level uncultured lineage ABY1, Ca. Berkelbacteria, WS6 (Ca. Dojkabacteria), and WWE3, with Ca. Saccharimonadia being predominant in both fraction samples. The number of operational taxonomic units belonging to Ca. Patescibacteria was approximately 6-fold higher in the size-fractionated samples than in the unfractionated sample. The shotgun metagenomic ana-lysis of the 0.45-0.22| |µm fractioned sample enabled the reconstruction of 24 high-quality patescibacterial bins. The bins obtained were classified into diverse clades at the family and genus levels, some of which were rarely detected in previous activated sludge studies. Collectively, the present results suggest that the overall diversity of Ca. Patescibacteria inhabiting activated sludge is higher than previously expected.