Satoh, Hisashi

In the domain Bacteria, one of the largest, most diverse and environmentally ubiquitous phylogenetic groups, Candidatus Patescibacteria (also known as candidate phyla radiation/CPR), remains poorly characterized, leaving a major knowledge gap in microbial ecology. We recently discovered a novel cross-domain symbiosis between Ca. Patescibacteria and Archaea in highly purified enrichment cultures and proposed Candidatus taxa for the characterized species, including Ca. Minisyncoccus archaeophilus and the corresponding family Ca. Minisyncoccaceae. In this study, we report the isolation of this bacterium, designated strain PMX.108T, in a two-strain co-culture with a host archaeon, Methanospirillum hungatei strain DSM 864T (JF-1T), and hereby describe it as the first representative species of Ca. Patescibacteria. Strain PMX.108T was isolated from mesophilic methanogenic sludge in an anaerobic laboratory-scale bioreactor treating synthetic purified terephthalate- and dimethyl terephthalate-manufacturing wastewater. The strain could not grow axenically and is obligately anaerobic and parasitic, strictly depending on M. hungatei as a host. The genome was comparatively large (1.54 Mbp) compared to other members of the clade, lacked some genes involved in the biosynthesis pathway and encoded type IV pili-related genes associated with the parasitic lifestyle of ultrasmall microbes. The G+C content of the genomic DNA was 36.6 mol%. Here, we report the phenotypic and genomic properties of strain PMX.108T; we propose Minisyncoccus archaeiphilus gen. nov., sp. nov. to accommodate this strain. The type strain of the species is PMX.108T (=JCM 39522T). We also propose the associated family, order, class and phylum as Minisyncoccaceae fam. nov. Minisyncoccales nov., Minisyncoccia class. nov. and Minisyncoccota phyl. nov. within the bacterial kingdom Bacillati.

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.

Summary Anaerobic ammonium‐oxidizing (anammox) bacteria affiliated with the genus ‘ Candidatus Scalindua’ are responsible for significant nitrogen loss in oceans, and thus their ecophysiology is of great interest. Here, we enriched a marine anammox bacterium, ‘ Ca . S. japonica’ from a Hiroshima bay sediment in Japan, and comparative genomic and proteomic analyses of ‘ Ca . S. japonica’ were conducted. Sequence of the 4.81‐Mb genome containing 4019 coding regions of genes (CDSs) composed of 47 contigs was determined. In the proteome, 1762 out of 4019 CDSs in the ‘ Ca . S. japonica’ genome were detected. Based on the genomic and proteomic data, the core anammox process and carbon fixation of ‘ Ca . S. japonica’ were further investigated. Additionally, the present study provides the first detailed insights into the genetic background responsible for iron acquisition and menaquinone biosynthesis in anammox bacterial cells. Comparative analysis of the ‘ Ca . Scalindua’ genomes revealed that the 1502 genes found in the ‘ Ca . S. japonica’ genome were not present in the ‘ Ca . S. profunda’ and ‘ Ca . S. rubra’ genomes, showing a high genomic diversity. This result may reflect a high phylogenetic diversity of the genus ‘ Ca . Scalindua’.

Summary Although metabolic pathways and associated enzymes of anaerobic ammonium oxidation (anammox) of ‘ Ca . Kuenenia stuttgartiensis’ have been studied, those of other anammox bacteria are still poorly understood. reduction to NO is considered to be the first step in the anammox metabolism of ‘ Ca . K. stuttgartiensis’, however, ‘ Ca . Brocadia’ lacks the genes that encode canonical NO‐forming nitrite reductases (NirS or NirK) in its genome, which is different from ‘ Ca . K. stuttgartiensis’. Here, we studied the anammox metabolism of ‘ Ca . Brocadia sinica’. 15 N‐tracer experiments demonstrated that ‘ Ca . B. sinica’ cells could reduce to NH 2 OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N 2 H 4 synthesis, downstream reaction of reduction, was investigated using a purified ‘ Ca . B. sinica' hydrazine synthase (Hzs) and intact cells. Both the ‘ Ca . B. sinica’ Hzs and cells utilized NH 2 OH and , but not NO and , for N 2 H 4 synthesis and further oxidized N 2 H 4 to N 2 gas. Taken together, the metabolic pathway of ‘ Ca . B. sinica’ is NH 2 OH‐dependent and different from the one of ‘ Ca . K. stuttgartiensis’, indicating metabolic diversity of anammox bacteria.

Summary To date, six candidate genera of anaerobic ammonium‐oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus ‘ C andidatus   J ettenia’. Planctomycete KSU ‐1 was found to be a mesophilic (20–42.5°C) and neutrophilic ( pH 6.5–8.5) bacterium with a maximum growth rate of 0.0020 h −1 . Planctomycete KSU ‐1 cells showed typical physiological and structural features of anammox bacteria; i.e. 29 N 2 gas production by coupling of 15 NH 4 + and 14 NO 2 − , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone‐7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl‐ CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU ‐1 cells. On the basis of these experimental results, we proposed the name ‘ C a . J ettenia caeni’ sp. nov. for the bacterial clade of the planctomycete KSU ‐1.

The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m−3day−1. The anammox bacteria were identified as representing ‘CandidatusBrocadia sinica’ on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (μmax) was 0.0041 h−1, corresponding to a doubling time of 7 days. The half-saturation constants (Ks) for ammonium and nitrite of ‘Ca.B. sinica’ were 28±4 and 86±4 µM, respectively, higher than those of ‘CandidatusBrocadia anammoxidans’ and ‘CandidatusKuenenia stuttgartiensis’. The temperature and pH ranges of anammox activity were 25–45 °C and pH 6.5–8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (molNH4+)−1, respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.