Biology subjects

Tahara, Y. O.

Publications and source records attributed to Tahara, Y. O..

10 recordsLinked to original sources

bioRxiv2025

Eukaryotes' closest relatives are internally simple syntrophic archaea

Eukaryotes are theorized to have originated from an archaeal phylum Promethearchaeota (formerly Asgard archaea)1,2. The first cultured representatives revealed valuable insight3,4 but are distantly related to the first eukaryotic common ancestor (FECA), leaving many unknowns regarding this archaeons biology. Here, we report isolation of two strains belonging to the order proposed as FECAs closest relative, Hodarchaeales,5 as members of a pure co- and tri-culture with methanogenic partners. Both are obligately anaerobic, syntrophic, peptide-degrading, and mesophilic archaea that have simple internal cell structure and produce protrusions and vesicles, like Promethearchaeales3,4. All strains demonstrate behavior focused on cell construction rather than division, unlike typical prokaryotes6. Strain HC1 possesses genes associated with aerobic lifestyles but lacks complete pathways for aerobic respiration and co-cultures cannot grow under (micro)aerobic conditions, suggesting the genes support oxygen detoxification rather than respiration. Reflecting this, HC1 can survive and grow under microaerobic conditions only when aerobic organisms are present. Phylogenetic analyses indicate FECA may have possessed these genes and thus some aerotolerance. Physiological, genomic, and phylogenetic observations indicate FECA was a simple-celled anaerobic syntrophic peptide-degrading archaeon with a non-growth-centric lifestyle and potential adaptations towards an oxygenated planet--the archaea-eukaryote transition was steep in both cell structure and aerobiosis.

microbiology↗

bioRxiv2025

Visualization of peptidoglycan layer isolated from gliding diderm bacteria, Flavobacterium johnsoniae and Myxococcus xanthus, by quick-freeze deep-etch replica electron microscopy

The bacterial peptidoglycan layer plays an important role in protecting the bacteria from turgor pressure, viruses, and predators. However, it also acts as a barrier in transmitting forces generated on the cell membrane to adhesion proteins on the surface during gliding locomotion. In this study, peptidoglycan layers were isolated from two species of gliding diderm, i.e., Gram-negative bacteria, and their structures were visualized by quick-freeze deep-etch replica electron microscopy. The diameter of pores in the peptidoglycan layer of M. xanthus and the area of surface pores were 51 nm and 14.6%, respectively, which were significantly larger than those of E. coli (32 nm and 5.8%) and F. johnsoniae (29 nm and 7.0%). Based on this, we discussed the mechanism by which diderm bacteria transmit forces across the PG layer to the bacterial surface. SignificancePeptidoglycan is a rigid meshwork that protects bacterial cells from turgor stress, virus, and predators. Bacteria move to get nutrients and escape from predators. The force for motility is generated in the cell membrane and transmitted to the surface across the peptidoglycan layer. In this study, the peptidoglycan structures of two representative gliding Gram-negative bacteria were visualized to understand the transmission mechanism.

microbiology↗

bioRxiv2024

FtsZ of wall-less bacteria forms ring-like structures

The FtsZ protein is involved in bacterial cell division. In cell-walled bacteria, such as Bacillus subtilis, FtsZ forms a ring-like structure, called the Z ring, at the cell division site and acts as a scaffold for cell wall synthesis. The inhibition of cell wall synthesis in B. subtilis has been shown to interfere with the function of the Z ring, causing a loss in cell division control. Spiroplasma, a cell wall-less bacterium, lacks most of the genes involved in cell division; however, the ftsZ gene remains conserved. The function of Spiroplasma eriocheiris FtsZ (SeFtsZ) remains to be determined. In the present study, we analyzed the biochemical characteristics of SeFtsZ. Purified SeFtsZ demonstrated lower polymerization capacity and GTPase activity than FtsZ from E. coli and B. subtilis. We also investigated the relationship between SeFtsZ and SeSepF, which anchors FtsZ to the cell membrane, and found that SeSepF did not contribute to the stability of FtsZ filaments, unlike the B. subtilis SepF. SeFtsZ and SeSepF were produced in E. coli L-forms, where cell wall synthesis was inhibited. SeFtsZ formed ring-like structures in cell wall-less E. coli cells, suggesting that SeFtsZ forms Z rings and is involved in cell division independently of cell wall synthesis.

microbiology↗

bioRxiv2024

Visualization of Bacillus subtilis Spore Structure and Germination using Quick-Freeze Deep-Etch Electron Microscopy

Bacterial spores, known for their complex and resilient structures, have been the focus of visualization using various methodologies. In this study, we applied quick-freeze and replica electron microscopy techniques, allowing observation of Bacillus subtilis spores in high-contrast and three-dimensional detail. This method facilitated visualization of the spore structure with enhanced resolution and provided new insights into the spores and their germination processes. We identified and described five distinct structures: (i) hair-like structures on the spore surface, (ii) spike formation on the surface of lysozyme-treated spores, (iii) the fractured appearance of the outer spore cortex during germination, (iv) potential connections between small vesicles and the core membrane, and (v) the evolving surface structure of nascent vegetative cells during germination.

microbiology↗

bioRxiv2024

Internal structure of Mycoplasma mobile gliding machinery analyzed by negative staining electron tomography

Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9-8.3 nm and length of 5-30 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units. SignificanceMycoplasma mobile, a parasitic bacterium, glides on solid surfaces at speeds of up to 4.0 m per second through a specialized mechanism. The gliding machinery, located at one pole of the cell, is composed of surface legs and internal motor structures. The force generation unit within the internal structure evolved from ATP synthase. This study aimed to clarify the entire architecture of the gliding machinery using electron tomography.

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↗

bioRxiv2022

Structure and Function of Gli123 Involved in Mycoplasma mobile Gliding

Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of a unique mechanism. The gliding machinery of M. mobile is composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a non-binding and non-gliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that the molecules changed their shape between globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of 20.0, 14.5, and 16.0 nm. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm length and 4 nm width. Both molecular structures were suggested to be dimers based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired clumping role of surface proteins. IMPORTANCEMycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities to escape host immunity, such as surface variation and gliding. Here, we focused on a surface-localizing protein that is essential for Mycoplasma mobile gliding. The findings of this study suggested that the protein undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for this unique mycoplasma gliding mechanism has also been suggested in the present study.

microbiology↗

bioRxiv2021

Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called chains that are each comprised of 17 repeating protein units called particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

microbiology↗

bioRxiv2021

Movements of Mycoplasma mobile gliding machinery detected by high-speed atomic force microscopy

Mycoplasma mobile, a parasitic bacterium, glides on solid surfaces, such as animal cells and glass by a special mechanism. This process is driven by the force generated through ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells are unclear. In this study, we detected the movements of the internal structure by scanning cells immobilized on a glass substrate using high-speed atomic force microscopy (HS-AFM). By scanning the surface of a cell, we succeeded in visualizing particles, 2 nm in hight and aligned mostly along the cell axis with a pitch of 31.5 nm, consistent with previously reported features based on electron microscopy. Movements of individual particles were then analyzed by HS-AFM. In the presence of sodium azide, the average speed of particle movements was reduced, suggesting that movement is linked to ATP hydrolysis. Partial inhibition of the reaction by sodium azide enabled us to analyze particle behavior in detail, showing that the particles move 9 nm right, relative to the gliding direction, and 2 nm into the cell interior in 330 ms, then return to their original position, based on ATP hydrolysis. IMPORTANCEThe Mycoplasma genus contains bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide by a special mechanism linked to their infection and survival. The special machinery for gliding can be divided into surface and internal structures that have evolved from rotary motors represented by ATP synthases. This study succeeded in visualizing the real-time movements of the internal structure by scanning from the outside of the cell using an innovative high-speed atomic force microscope, and then analyzing their behaviors.

microbiology↗

bioRxiv2019

Peptidoglycan layer and disruption processes in Bacillus subtilis cells visualized using quick-freeze, deep-etch electron microscopy

Peptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands crosslinked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface in Bacillus subtilis at the nanometer resolution, using quick-freeze, deep-etch electron microscopy. Filamentous structures were observed on the entire surface of the cell, where filaments about 11-nm wide formed concentric circles on cell poles, filaments about 13-nm wide formed a circumferential mesh-like structure on the cylindrical part, and a \"piecrust\" structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during electron microscopy. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using electron microscopy suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

microbiology↗