Nakai, Ryosuke

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 ( r 2 ≥ 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.

AbstractTo verify the parasitic lifestyle ofCandidatusPatescibacteria in the enrichment cultures derived from a methanogenic bioreactor, we applied multifaceted approaches combining cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses. Cultivation experiments with the addition of exogenous methanogenic archaea with acetate, amino acids, and nucleoside monophosphates and 16S rRNA gene sequencing confirmed the increase in the relative abundance ofCa. Patescibacteria and methanogens. The predominantCa. Patescibacteria wereCa. Yanofskybacteria and 32-520 lineages (to which belongs to classCa. Paceibacteria) and positive linear relationships (r2≥ 0.70) between the relative abundance ofCa. Yanofskybacteria andMethanothrix, suggesting that the tendency of the growth rate is similar to that of the host. By fluorescencein situhybridization (FISH) observations, the FISH signals ofMethanothrixandMethanospirillumcells withCa. Yanofskybacteria and with 32-520 lineages, respectively, were significantly lower than those of the methanogens withoutCa. Patescibacteria, suggesting their parasitic interaction. The TEM and SEM observations also support parasitism in that the cell walls and plugs of these methanogens associated with submicron cells were often deformed. In particular, someMethanothrix-like filamentous cells were dented where the submicron cells were attached. Metatranscriptomic and protein structure prediction analyses identified highly expressed secreted genes from the genomes ofCa. Yanofskybacteria and 32-520, and these genes contain adhesion-related domains to the host cells. Considering the results through the combination of microscopic observations, gene expression, and computational protein modeling, we propose that the interactions betweenCa. Yanofskybacteria and 32-520 belonging to classCa. Paceibacteria and methanogenic archaea are parasitism.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria , it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia . Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria . Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria , and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria ; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria . The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.