Biology subjects

Ohkuma, M.

Publications and source records attributed to Ohkuma, M..

12 recordsLinked to original sources

bioRxiv2026

Reconstitution in a minimal synthetic bacterium reveals distinct Mollicute MreB-based motility systems driven by orthogonal force directions

MreB, a bacterial actin homolog, is widely conserved in bacteria where it functions as a scaffold for a peptidoglycan synthesis. Intriguingly, members of the cell wall-lacking Mollicutes retain multiple mreB genes despite the absence of peptidoglycan. Previous work demonstrated that two MreB isoforms from Spiroplasma can reconstitute swimming motility in the minimal synthetic bacterium syn3B, indicating that MreB was repurposed for motility. Here, we expressed seven MreBs from Haloplasma contractile, an early-diverging Mollicute, in syn3B. Approximately 50% of cells elongated, and 15% exhibited curving and coiling movements that resembled Haloplasma but differed from Spiroplasma motility. Systematic analysis of isoform combinations revealed that movement requires specific MreB pairs, and that additional isoforms enhance motility efficiency. Cryo-electron tomography showed membrane-associated ribbons composed of MreB filaments. Mathematical modeling demonstrated that Haloplasma- and Spiroplasma-type movements can be reproduced by altering only the direction of force generation. Thus, diversification of force orientation within an actin homolog enabled independent evolution of distinct motility systems in Mollicutes.

Synthetic Biology↗

bioRxiv2026

A Tripartite Co-culture System Reveals Defensive Mutualism Between a Nanobdellati Symbiont and Its Host

Archaeal symbioses remain among the least understood cellular interactions. Ultra-small Nanobdellati (initially called DPANN) archaea rely on larger hosts for survival, yet their effects on host eco-physiology and their interactions with infecting viruses have not yet been examined experimentally. Here, we established the first stable tripartite co-culture comprising a Nanobdellales symbiont (YN4), its archaeal host (YN4HA), and a virus (MTIV4). We analyzed this system using physiological, genomic, transcriptomic, glycoproteomic, and cryo-electron tomography (cryoET) approaches. CryoET analysis revealed that the Nanobdellales archaeon formed cone-like structures to contact the host, as has been observed in other Nanobdellati-host systems. In this system, the host is infected by the virus. The Nanobdellales archaeon mitigated virus-induced growth inhibition of the host without any detectable fitness cost, indicating a defensive mutualism rather than a strictly parasitic relationship. This protective effect may involve Nanobdellati-driven remodeling of host cell surface glycans, suggesting a previously unrecognized glycan-mediated defense strategy. Overall, our tripartite system provides new insight into complex archaeal interactions in extreme environments that cannot be captured through conventional binary co-culture systems.

microbiology↗

bioRxiv2025

Discovery and genomics of H2-oxidizing/O2-reducing Deferribacterota ectosymbiotic with protists in the guts of termites and a Cryptocercus cockroach

Members of the phylum Deferribacterota inhabit diverse environments, but their symbiosis with protists has never been reported. We discovered an ectosymbiotic clade of Deferribacterota specifically associated with spirotrichonymphid protists in the guts of the termites Reticulitermes speratus and Hodotermopsis sjostedti and trichonymphid protists in the gut of the wood-feeding cockroach Cryptocercus punctulatus. The ectosymbiotic Deferribacterota were spiral shaped and attached to 16-91% of the host protist cells. These formed a monophyletic cluster within an uncultured insect gut-associated family-level clade, which is sister to the vertebrate gut-associated family Mucispirillaceae. The complete genome of an ectosymbiotic Deferribacterota was obtained from a Trichonympha acuta cell in a C. punctulatus gut and analyzed together with a single-cell amplified genome of another ectosymbiotic Deferribacterota associated with Holomastigotes sp. in the gut of R. speratus. Genome analyses suggest that these Deferribacterota ferment monosaccharides and conduct fumarate and oxidative respiration with H2 as an electron donor. They thus possibly contribute to the removal of hydrogen and oxygen to protect the fermentative activity of the protist hosts. The ectosymbionts possess reduced signal transduction gene repertoires, implying that the association has provided a relatively stable environment for these bacteria. The ectosymbionts likely possess flagella with an unusually expanded number of flagellin variants up to 40, which may reflect an adaptation to their ectosymbiotic lifestyle. We propose a novel genus, Termitispirillum, for these ectosymbionts and a novel family, Termitispirillaceae, for the insect-gut clade, under SeqCode. Our findings provide new insights into the ecology and evolution of Deferribacterota.

microbiology↗

bioRxiv2025

Whole-genome sequencing of diverse 351 cultured prokaryotes including yet-unsequenced fastidious type strains

Genome sequences provide fundamental information for both basic and applied life sciences. Whole-genome sequencing is now requested for describing novel prokaryotic species and designating their type strains, which serve as representative and well-characterized strains of the species. Indeed, the number of sequenced prokaryotic genomes has been rapidly increasing. However, a considerable number of isolated strains, particularly "fastidious" type strains such as strict anaerobes and slow growers, remain without genome sequence information. Here we report the whole-genome sequencing of 290 bacterial and 61 archaeal strains, including fastidious type strains, obtained from Japan Collection of Microorganisms (JCM) using a combination of short- and long-read sequencing technologies. The dataset includes 284 type strain genomes and 235 complete genomes. Notably, in the dataset, genomes of over 200 strains, including over 150 type strains, had not been made publicly available. Comparative genomic analysis suggests that some strains need to be assigned to novel taxa or reclassified. Functional gene survey indicates that some strains possess previously unrecognized potential for carbon fixation or bioactive secondary metabolite production. Our dataset will contribute to more accurate taxonomic classification, fill gaps in the phylogeny of prokaryotes, and provide insights into their physiology and ecology.

microbiology↗

bioRxiv2025

Exploring the diversity and physiological characteristics of RubisCO-mediated carbon fixation in culturable prokaryotes

The utilization of microbial resources requires their relevant reproducible characteristics, and genome analysis plays a crucial role in discovering valuable strains for future applications. In this study, we analyzed potential carbon-fixing microorganisms via Calvin-Benson-Bassham (CBB) cycle using 6,262 bacterial and 487 archaeal genomes from available cultures in Japan Collection of Microorganisms (JCM), one of the well-established culture collections today. A total of 306 strains (147 genera, eight phyla) carried CBB cycle genes and a literal survey showed that 74 genera had reported evidence of their autotrophic growth, although 73 lacked supporting information. Phylogenetic analysis of RubisCO large subunit (RbcL) identified diverse forms (IA, IB, IC, IE, I+, II, and III) with distinct metabolic associations: form IA associated with sulfur oxidation and form IC with hydrogen oxidation. Genome-based metabolic predictions suggested potential carbon fixation in numerous strains lacking experimental evidence. Our analyses showed members of Actinomycetota harboring form IE RubisCO tend to associate with hydrogen oxidation possibly using oxygen or nitrate as an electron acceptor. Additionally, 12 strains in Pseudomonadota contained pufL and pufM genes, suggesting possible phototrophic capabilities, although some failed to predict their electron donors and they possibly use CBB cycle to regulate intracellular redox balance under photoheterotrophic growth. Our findings highlight unrecognized autotrophic potentials in JCM strains and expand our knowledge of carbon fixation diversity. Future experimental validation will deepen our understanding of these microbes roles in the global carbon cycle, with potential applications in carbon sequestration and environmental sustainability.

microbiology↗

bioRxiv2024

A large attachment organelle mediates interaction between a novel Nanobdellota archaeon YN1 and its host

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here we successfully isolated and cultivated a co-culture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterised the co-culture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (CryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together our results unravelled the molecular and structural basis of ectosymbiotic relationship between YN1 and YNHA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.

microbiology↗

bioRxiv2024

Facultative endosymbiosis between cellulolytic protists and methanogenic archaea in the gut of the Formosan termite Coptotermes formosanus

Anaerobic protists frequently harbour methanogenic archaea, which apparently contribute to the hosts fermentative metabolism by consuming excess H2. However, the ecological properties of endosymbiotic methanogens remain elusive in many cases. Here we investigated the ecology and genome of the endosymbiotic methanogen of the Cononympha protists in the hindgut of the termite Coptotermes formosanus. Microscopic and 16S rRNA amplicon sequencing analyses revealed that a single species, designated here Candidatus Methanobrevibacter cononymphae, is associated with both Cononympha leidyi and Cononympha koidzumii and that its infection rate in Cononympha cells varied from 0.0 to 99.8% among termite colonies. Fine-scale network analysis indicated that multiple 16S rRNA sequence variants coexisted within a single host cell and that identical variants were present in both Cononympha species and also on the gut wall. Thus, Ca. Methanobrevibacter cononymphae is a facultative endosymbiont, transmitted vertically with frequent exchanges with the gut environment. Indeed, transmission electron microscopy showed escape or uptake of methanogens from/by a Cononympha cell. The genome of Ca. Methanobrevibacter cononymphae showed features consistent with its facultative lifestyle: i.e., the genome size (2.7 Mbp) comparable to those of free-living relatives; the pseudogenization of the formate dehydrogenase gene fdhA, unnecessary within the non-formate-producing host cell; the dependence on abundant acetate in the host cell as an essential carbon source; and the presence of a catalase gene, required for colonization on the microoxic gut wall. Our study revealed a versatile endosymbiosis between the methanogen and protists, which may be a strategy responding to changing conditions in the termite gut.

microbiology↗

bioRxiv2023

Cell surface architecture of the cultivated DPANN archaeon Nanobdella aerobiophila

The DPANN archaeal clade includes obligately ectosymbiotic species. Their cell surfaces potentially play an important role in the symbiotic interaction between the ectosymbionts and their hosts. However, little is known about the mechanism of the ectosymbiosis. Here, we show cell surface structures of the cultivated DPANN archaeon Nanobdella aerobiophila strain MJ1T and its host Metallosphaera sedula strain MJ1HA, using a variety of electron microscopy techniques, i.e., negative-staining transmission electron microscopy (TEM), quick-freeze deep-etch (QFDE) TEM, and 3D electron tomography. The thickness, unit size, and lattice symmetry of the S-layer of strain MJ1T were different from those of the host archaeon strain MJ1HA. Genomic and transcriptomic analyses highlighted the most highly expressed MJ1T gene for a putative S-layer protein with multiple glycosylation sites and immunoglobulin-like folds, which has no sequence homology to known S-layer proteins. In addition, genes for putative pectin lyase- or lectin-like extracellular proteins, which are potentially involved in symbiotic interaction, were found in the MJ1T genome based on in silico 3D protein structure prediction. Live cell imaging at the optimum growth temperature of 65{degrees}C indicated that cell complexes of strains MJ1T and MJ1HA were motile, but sole MJ1T cells were not. Taken together, we propose a model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila. ImportanceDPANN archaea are widely distributed in a variety of natural and artificial environments, and may play a considerable role in the microbial ecosystem. All of the cultivated DPANN archaea so far need host organisms for their growth, i.e., obligately ectosymbiotic. However, the mechanism of the ectosymbiosis by DPANN archaea is largely unknown. To this end, we performed a comprehensive analysis of the cultivated DPANN archaeon, Nanobdella aerobiophila, using electron microscopy, live cell imaging, transcriptomics, and genomics including 3D protein structure prediction. Based on the results, we propose a reasonable model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila, which will enhance our understanding of the enigmatic physiology and ecological significance of DPANN archaea.

microbiology↗

bioRxiv2023

Interareal synaptic inputs underlying whisking-related activity in the primary somatosensory barrel cortex

Body movements, especially orofacial movements, are known to influence brain-wide neuronal activity. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials (Vm) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices are still unclear. Using a Gi-coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory cortex (wS1) of awake behaving mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a fraction of wS1 neurons which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of Vm. Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and Vm dynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold Vm dynamics of wS1 neurons. Our findings thus suggest that sensorimotor integration in wS1 during spontaneous whisking is mediated by direct synaptic inputs from wTLM and wS2 rather than from wM1. Significance statementThe traditional viewpoint underscores the importance of motor-sensory projections in shaping movement-induced neuronal activity within sensory cortices. However, this study challenges such established views. We reveal that the synaptic inputs from the whisker primary motor cortex do not alter the dynamics of neuronal activity in the whisker primary somatosensory cortex (wS1) during spontaneous whisker movements. Furthermore, we make a novel observation that inhibiting inputs from the whisker secondary somatosensory cortex (wS2) substantially curtails movement-related activities in wS1. These findings provoke a reconsideration of the role of motor-sensory projections in sensorimotor integration and bring to light a new function for wS2-to-wS1 projections.

neuroscience↗

bioRxiv2023

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

microbiology↗

bioRxiv2019

Soil prokaryotes associated with decreasing pathogen density during anaerobic soil disinfestation

Anaerobic soil disinfestation (ASD) is a chemical-independent method that can reduce pathogens. Although soil microbes play essential roles in ASD, the relationship between the microbial community structure and disinfestation efficiency remains unclear. To this end, we investigated changes in the microbial community and pathogen density during a period of ASD under field conditions for 14 days in a greenhouse using three different substrates. Soil samples were collected at 0, 3, 7, and 14 days after ASD treatment. The pathogen densities were analyzed by real-time polymerase chain reactions, prokaryotic community analysis was conducted using unidirectional pyrosequencing, and the factors related to pathogen density were statistically analyzed. The pathogen density rapidly decreased by >90% at 3 days after treatment and then slowly decreased until day 14, but the rate of decrease differed among the substrates. The microbial communities became altered after 3 days and recovered to their original state on day 14. The dipyridyl reaction, microbial diversity, richness, and community structure were not correlated with pathogen density. The most negatively correlated operational taxonomic units with pathogen density were Clostridia and Bacilli, both belonging to Firmicutes. These results suggested that the growth of specific microbes, but not the changes in microbial community structure, might be important for ASD disinfestation efficiency.

microbiology↗

bioRxiv2019

Comparison of prokaryotic communities between the fields showing different disinfestation effects by anaerobic soil disinfestation

Anaerobic soil disinfestation (ASD) is a chemical-independent fumigation method used for reducing the abundance of pathogens at soil depths of <40 cm. However, its disinfestation efficiency is unstable under field conditions. The microbial community reflects the soil environment and is a good indicator of soil health. Therefore, soil with a good disinfestation efficiency may have a unique microbial community. The aim of the present study was to compare the prokaryotic communities among soils obtained from 17 geographically different greenhouses that experienced tomato bacterial wilt but exhibited different disinfestation efficiencies after ASD treatment with the same substrate. In the present study, soil prokaryotic communities in the field, which indicate difference in disinfestation effects after ASD treatment among several fields, were compared using next-generation sequencing. The prokaryotic communities in the fields showing different disinfestation effects were roughly separated into sampling fields. The relative abundances of Betaproteobacteria and Clostridia were significantly increased in well-disinfested fields. Overall, 25 operational taxonomic units (OTUs) were specifically increased in various well-disinfested soils and 18 OTUs belonged to phylogenetically diversified Clostridia. Other OTUs belonged to aerobic bacteria and were not previously detected in sample collected from ASD-treated fields. The results showed that the changes to the prokaryotic communities did not affect ASD efficiency, whereas changes in the abundance of specific microbes in the community were related to disinfestation.

microbiology↗