Biology subjects

Miyata, M.

Publications and source records attributed to Miyata, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Cryo-EM of cytoskeletal 'fibril' protein involved in Spiroplasma swimming

Spiroplasma, parasitic or commensal bacteria, can swim by switching the handedness of its helical cell body. A helical cell body is formed by an internal ribbon of MreB, an actin superfamily, and Spiroplasma-specific fibril proteins. Here we have solved the structure of the fibril filament by single-particle cryo-electron microscopy at 3.6 [A] resolution and built its atomic structure. The structure is composed of repeated rings and cylinders. The N-terminal cylinder of the fibril protein shows a structure similar to that of S-adenosylhomocysteine nucleosidase, while the C-terminal ring does not show similarity to other proteins. The filament is nonpolar and flexible, possessing a helical pitch of 700 nm, consistent with cell helicity. Cryo-electron tomography revealed aligned several MreB filaments in the center of the ribbon, flanked by membrane-binding fibril filaments through electrostatic interactions. This study discusses the evolution and roles of the fibril filament.

microbiology↗

bioRxiv2023

Assembly properties of Spiroplasma MreB involved in swimming motility

Bacterial actin MreB forms filaments in which the unit of the structure is an antiparallel double strand. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), a part of which is composed of an intra-cellular ribbon for driving its swimming motility. The interaction modes of each ribbon component are unclear, although these are clues for understanding Spiroplasma swimming. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), which forms sheets and is a component protein of the ribbon. Electron microscopy (EM) revealed that sheet formation was inhibited under acidic conditions and paracrystal structures were formed under acidic and neutral conditions with low ionic strength. Solution assays found four properties of paracrystals as follows: (I) their formation followed sheet formation, (II) electrostatic interactions were required for their formation, (III) the positively charged and unstructured C-terminal region contributed to the nucleation of their formation, and (IV) their formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic conditions. During these studies, we found two aggregation modes of SpeMreB5, with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

biophysics↗

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↗

bioRxiv2022

Comparison of CRISPR/Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells

The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters, promoter-enhancer loops, and topologically associating domains. To analyze these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Kruppel-associated box zinc finger protein (KRAB-ZFP) gene cluster on chromosome 4 in mouse embryonic stem cells using CRISPR/Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN) with 70-bp homology arms; and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.

genomics↗

bioRxiv2021

Reconstitution of Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium

Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium. One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium.

synthetic biology↗

bioRxiv2021

Core and rod structures of a thermophilic cyanobacterial light-harvesting phycobilisome

Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.

biochemistry↗

bioRxiv2021

Force and step size of gliding motility in human pathogenic bacterium Mycoplasma pneumoniae

Mycoplasma pneumoniae, a human pathogenic bacterium, binds to sialylated oligosaccharides and glides on host cell surfaces via a unique mechanism. Gliding motility is essential for initiating the infectious process. In the present study, we measured the stall force of an M. pneumoniae cell carrying a bead that was manipulated using optical tweezers on two strains. The stall forces of M129 and FH strains were averaged to be 23.7 and 19.7 pN, respectively, much weaker than those of other bacterial surface motilities. The binding activity and gliding speed of the M129 strain on sialylated oligosaccharides were eight and two times higher than those of the FH strain, respectively, showing that binding activity is not linked to gliding force. Gliding speed decreased when cell binding was reduced by addition of free sialylated oligosaccharides, indicating the existence of a drag force during gliding. We detected stepwise movements, likely caused by a single leg under 0.2-0.3 mM free sialylated oligosaccharides. A step size of 14-19 nm showed that 25-35 propulsion steps per second are required to achieve the usual gliding speed. The step size was reduced to less than half with the load applied using optical tweezers, showing that a 2.5 pN force from a cell is exerted on a leg. The work performed in this step was 16%-30% of the free energy of the hydrolysis of ATP molecules, suggesting that this step is linked to the elementary process of M. pneumoniae gliding. IMPORTANCEHuman mycoplasma pneumonia is caused by the bacterium Mycoplasma pneumoniae. This tiny bacterium, shaped like a missile, binds to human epithelial surfaces and spreads using a unique gliding mechanism to establish infection. Here, we analyzed the movements and force of this motility using a special setup: optical tweezers. We then obtained detailed mechanical data to understand this mechanism. Furthermore, we succeeded in detecting small steps of nanometers in its gliding, which is likely linked to the elementary process of the core reaction: chemical to mechanical energy conversion. These data provide critical information to both control this human pathogen and explore new ideas for artificial molecular machines.

microbiology↗

bioRxiv2021

Distinct nociresponsive region in mouse primary somatosensory cortex

Nociception, somatic discriminative aspects of pain, is represented in the primary somatosensory cortex (S1), as is touch, but the separation and the interaction of the two modalities within S1 remain unclear. Here, we show the spatially-distinct tactile and nociceptive processing in the granular barrel field (BF) and the adjacent dysgranular region (Dys) in mouse S1. Simultaneous recording of the multiunit activity across subregions reveals that Dys responses are selective to noxious input whereas those of BF are to tactile input. At the single neuron level, nociceptive information is represented separately from the tactile information in Dys layer 2/3. In contrast, both modalities are converged in a layer 5 neuron in each region. Interestingly, the two modalities interfere with each other in both regions. We further demonstrate that Dys, but not BF, activity is critically involved in neuropathic pain and pain behavior, and thus provide evidence that Dys is a center specialized for nociception in S1.

neuroscience↗

bioRxiv2021

Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. It polymerises into an antiparallel double-stranded filament that generally functions in cell shape determination by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are likely to be involved in swimming motility. Here, we investigated the structure, ATPase activity, and polymerisation dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerised into an antiparallel double-stranded filament, and SpeMreB5 formed sheets, including the antiparallel filament, upon the binding of a nucleotide. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our crystal structures of SpeMreB3 and analyses of the mutant variants showed that the amino acid motif most likely plays a role in eliminating the proton of the nucleophilic water for ATP hydrolysis. Our sedimentation assay suggests that SpeMreB3 has a lower polymerisation activity than SpeMreB5, while their polymerisation dynamics are qualitatively similar to those of other actin superfamily proteins, in which ATP hydrolysis stabilises the filament, and Pi release leads to depolymerisation.

biochemistry↗

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

Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo- SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and disrupt specific membranes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/438527v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@17ef3f4org.highwire.dtl.DTLVardef@1c24d41org.highwire.dtl.DTLVardef@15e071aorg.highwire.dtl.DTLVardef@24740e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones. C_LIO_LICas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes. C_LIO_LIVulnerability of the specific cytoplasmic membranes to the selective Cas toxin that is the most remarkable feature of cytotoxicity of Cas toxin on plant cells. C_LI

biochemistry↗

bioRxiv2021

Elucidation of fibril structure responsible for swimming in Spiroplasma using electron microscopy

Spiroplasma, which are known pathogens and commensals of arthropods and plants, are helical-shaped bacteria that lack a peptidoglycan layer. Spiroplasma swim by alternating between left- and right-handed helicity. Of note, this system is not related to flagellar motility, which is widespread in bacteria. A helical ribbon running along the inner side of the helical cell should be responsible for cell helicity and comprises the bacterial actin homolog, MreB, and a protein specific to Spiroplasma, fibril. Here, we isolated the ribbon and its major component, fibril filament, for electron microscopy (EM) analysis. Single-particle analysis of the fibril filaments using the negative-staining EM revealed a three-dimensional chain structure composed of rings with a size of 11 nm wide and 6 nm long, connected by a backbone cylinder with an 8.7 nm interval with a twist along the filament axis. This structure was verified through EM tomography of quick-freeze deep-etch replica sample, with a focus on its handedness. The handedness and pitch of the helix for the isolated ribbon and fibril filament agreed with those of the cell in the resting state. Structures corresponding to the alternative state were not identified. These results suggest that the helical cell structure is supported by fibril filaments; however, the helical switch is caused by the force generated by the MreB proteins. The isolation and structural outline of the fibril filaments provide crucial information for an in-depth clarification of the unique swimming mechanism of Spiroplasma.

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↗

bioRxiv2020

Updated phylogeny and sequence features of MreB from the Wall-less bacteria Spiropaslma

Spiroplasma are wall-less bacteria which belong to the phylum Tenericutes that evolved from Firmicutes including Bacillus subtilis. Spiroplasma swim by a mechanism unrelated to widespread bacterial motilities, such as flagellar motility, and caused by helicity switching with kinks traveling along the helical cell body. The swimming force is likely generated by five classes of bacterial actin homolog MreBs (SMreBs 1-5) involved in the helical bone structure. We analyzed sequences of SMreBs to clarify their phylogeny and sequence features. The maximum likelihood method based on around 5,000 MreB sequences showed that the phylogenetic tree was divided into several radiations. SMreBs formed a clade adjacent to the radiation of MreBH, an MreB isoform of Firmicutes. Sequence comparisons of SMreBs and Bacillus MreBs were also performed to clarify the features of SMreB. Catalytic glutamic acid and threonine were substituted to aspartic acid and lysine, respectively, in SMreB3. In SMreBs 2 and 4, amino acids involved in inter- and intra-protofilament interactions were significantly different from those in Bacillus MreBs. A membrane-binding region was not identified in most SMreBs 1 and 4 unlike many walled-bacterial MreBs. SMreB5 had a significantly longer C-terminal region than the other MreBs, which possibly forms a protein-protein interaction. These features may support the functions responsible for the unique mechanism of Spiroplasma swimming.

bioinformatics↗

bioRxiv2020

Persistence of a Wolbachia-driven sex ratio bias in an island population of Eurema butterflies

It is generally believed that when maternally inherited sex ratio distorters become predominant, either the host population goes extinct or nuclear suppressors evolve in the host. Here, we show an empirical case where all-female-producing Wolbachia is likely to be stably maintained at a high frequency. On an island population of the butterfly Eurema mandarina, a Wolbachia strain wFem, which makes female hosts produce all-female offspring without sibling lethality (female drive), is highly prevalent. We found that, with some fluctuations, wFem appeared to be stably maintained for at least 12 years at a high frequency, resulting in the existence of an abnormally high number of virgin females. Interestingly, comparison between sex ratios of captive individuals and sex ratios deduced from wFem frequencies suggested a plastic behavioral change of males and females in response to the shift of sex ratios. wFem presence does not affect brood size but has a slightly negative effect on body size. Stable coexistence of wFem-positive and -negative females in the population may be explained via mate choice by males, which keeps wFem in check. Taken together, this butterfly population is an attractive model for future studies on the population dynamics of sex ratios and mating behavior.

evolutionary biology↗

bioRxiv2019

Refined mechanism of Mycoplasma mobile gliding based on structure, ATPase activity, and sialic acid binding of machinery

Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal jellyfish -like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal tentacle -like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the - and {beta}-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and shown that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.\n\nIMPORTANCEThe genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human walking pneumonia, is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three-dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

microbiology↗

bioRxiv2019

Layer-specific sensory processing impairment in the primary somatosensory cortex after motor cortex infarction

Primary motor cortex (M1) infarction occasionally causes sensory impairment. Because sensory signal plays an important role in motor control, sensory impairment compromises recovery and rehabilitation from motor disability. Despite the importance of sensory-motor integration for rehabilitation after M1 infarction, the neural mechanism of the sensory impairment is poorly understood. We show that the sensory processing in the primary somatosensory cortex (S1) was impaired in the acute phase of M1 infarction and recovered in a layer-specific manner in the subacute phase. This layer dependent recovery process and the anatomical connection pattern from M1 to S1 suggested the functional connectivity from M1 to S1 plays a key role in the impairment of sensory processing in S1. The simulation study demonstrated that the loss of inhibition from M1 to S1 in the acute phase of M1 infarction could cause the sensory processing impairment in S1, and the complementation of inhibition could recover the temporal coding. Taken together, we revealed how focal stroke of M1 alters cortical network activity of sensory processing, in which inhibitory input from M1 to S1 may be involved.

physiology↗

bioRxiv2019

Behaviors and Energy Source of Mycoplasma gallisepticum Gliding

Mycoplasma gallisepticum, an avian-pathogenic bacterium, glides on host tissue surfaces by using a common motility system with Mycoplasma pneumoniae. In the present study, we observed and analyzed the gliding behaviors of M. gallisepticum in detail by using optical microscopes. M. gallisepticum glided at a speed of 0.27 {+/-} 0.09 {micro}m/s with directional changes relative to the cell axis of 0.6 {+/-} 44.6 degrees/5 s without the rolling of the cell body. To examine the effects of viscosity on gliding, we analyzed the gliding behaviors under viscous environments. The gliding speed was constant in various concentrations of methylcellulose but was affected by Ficoll. To investigate the relationship between binding and gliding, we analyzed the inhibitory effects of sialyllactose on binding and gliding. The binding and gliding speed sigmoidally decreased with sialyllactose concentration, indicating the cooperative binding of the cell. To determine the direct energy source of gliding, we used a membrane-permeabilized ghost model. We permeabilized M. gallisepticum cells with Triton X-100 or Triton X-100 containing ATP and analyzed the gliding of permeabilized cells. The cells permeabilized with Triton X-100 did not show gliding; in contrast, the cells permeabilized with Triton X-100 containing ATP showed gliding at a speed of 0.014 {+/-} 0.007 m/s. These results indicate that the direct energy source for the gliding motility of M. gallisepticum is ATP.\n\nIMPORTANCEMycoplasmas, the smallest bacteria, are parasitic and occasionally commensal. Mycoplasma gallisepticum is related to human pathogenic Mycoplasmas--Mycoplasma pneumoniae and Mycoplasma genitalium--which causes so-called walking pneumonia and non-gonococcal urethritis, respectively. These Mycoplasmas trap sialylated oligosaccharides, which are common targets among influenza viruses, on host trachea or urinary tract surfaces and glide to enlarge the infected areas. Interestingly, this gliding motility is not related to other bacterial motilities or eukaryotic motilities. Here, we quantitatively analyze cell behaviors in gliding and clarify the direct energy source. The results provide clues for elucidating this unique motility mechanism.

microbiology↗