研究者詳細 - 森本　雄祐

論文 - 森本　雄祐

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Novel Insights into Conformational Rearrangements of the Bacterial Flagellar Switch Complex 査読有り国際誌

Sakai T., Miyata T., Terahara N., Mori K., Inoue Y., Morimoto Y., Kato T., Namba K., Minamino T.

mBio 10 ( 2 ) 2019年04月

記述言語：英語掲載種別：研究論文（学術雑誌）

Copyright © 2019 Sakai et al. The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.

DOI： 10.1128/mBio.00079-19

Kyutacar

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Effect of the MotA(M206I) mutation on torque generation and stator assembly in the Salmonella H-driven flagellar motor 査読有り国際誌

Suzuki Y., Morimoto Y., Oono K., Hayashi F., Oosawa K., Kudo S., Nakamura S.

Journal of Bacteriology 201 ( 6 ) 2019年02月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Copyright © 2019 American Society for Microbiology. All Rights Reserved. The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella. The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-m beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-m beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly. IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella. We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

DOI： 10.1128/JB.00727-18

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Collective cell migration of Dictyostelium without cAMP oscillations at multicellular stages 査読有り国際誌

Hashimura H., Morimoto Y., Yasui M., Ueda M.

Communications Biology 2 ( 34 ) 2019年01月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

© 2019, The Author(s). In Dictyostelium discoideum, a model organism for the study of collective cell migration, extracellular cyclic adenosine 3’,5’-monophosphate (cAMP) acts as a diffusible chemical guidance cue for cell aggregation, which has been thought to be important in multicellular morphogenesis. Here we revealed that the dynamics of cAMP-mediated signaling showed a transition from propagating waves to steady state during cell development. Live-cell imaging of cytosolic cAMP levels revealed that their oscillation and propagation in cell populations were obvious for cell aggregation and mound formation stages, but they gradually disappeared when multicellular slugs started to migrate. A similar transition of signaling dynamics occurred with phosphatidylinositol 3,4,5-trisphosphate signaling, which is upstream of the cAMP signal pathway. This transition was programmed with concomitant developmental progression. We propose a new model in which cAMP oscillation and propagation between cells, which are important at the unicellular stage, are unessential for collective cell migration at the multicellular stage.

DOI： 10.1038/s42003-018-0273-6

Kyutacar

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A study of wound repair in Dictyostelium cells by using novel laserporation 査読有り国際誌

Pervin M., Itoh G., Talukder M., Fujimoto K., Morimoto Y., Tanaka M., Ueda M., Yumura S.

Scientific Reports 8 ( 1 ) 2018年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

© 2018 The Author(s). We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca 2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 μm. In the absence of Ca 2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca 2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca 2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca 2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.

DOI： 10.1038/s41598-018-26337-0

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Salmonella Flagellum 査読有り国際誌

Tohru Minamino, Yusuke V. Morimoto, Akihiro Kawamoto, Hiroyuki Terashima, Katsumi Imada

Salmonella - A Re-emerging Pathogen ( IntechOpen ) 2018年07月

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export 査読有り国際誌

Terahara N., Inoue Y., Kodera N., Morimoto Y., Uchihashi T., Imada K., Ando T., Namba K., Minamino T.

Science Advances 4 ( 4 ) 2018年04月

記述言語：英語掲載種別：研究論文（学術雑誌）

Copyright © 2018 The Authors. The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhA C ) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhA C (FlhA L ) is required not only for FlhA C ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhA L was required for cooperative ring formation of FlhA C . Alanine substitutions of residues involved in FlhA C ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhA C .

DOI： 10.1126/sciadv.aao7054

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Novel insights into the mechanism of well-ordered assembly of bacterial flagellar proteins in Salmonella 査読有り国際誌

Inoue Y., Morimoto Y., Namba K., Minamino T.

Scientific Reports 8 ( 1 ) 2018年01月

記述言語：英語掲載種別：研究論文（学術雑誌）

© The Author(s) 2018. The FliI ATPase of the flagellar type III protein export apparatus forms the FliH2FliI complex along with its regulator FliH. The FliH2FliI complex is postulated to bring export substrates from the cytoplasm to the docking platform made of FlhA and FlhB although not essential for flagellar protein export. Here, to clarify the role of the FliH2FliI complex in flagellar assembly, we analysed the effect of FliH and FliI deletion on flagellar protein export and assembly. The hook length was not controlled properly in the ΔfliH-fliI flhB(P28T) mutant compared to wild-Type cells, whose hook length is controlled to about 55 nm within 10% error. The FlhA(F459A) mutation increased the export level of the hook protein FlgE and the ruler protein FliK by about 10-fold and 3-fold, respectively, and improved the hook length control in the absence of FliH and FliI. However, the ΔfliH-fliI flhB(P28T) flhA(F459A) mutant did not produce flagellar filaments efficiently, and a large amount of flagellin monomers were leaked out into the culture media. Neither the hook length control nor flagellin leakage was affected by the FlhB(P28T) and FlhA(F459A) mutations. We will discuss a hierarchical protein export mechanism of the bacterial flagellum.

DOI： 10.1038/s41598-018-20209-3

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Structural differences in the bacterial flagellar motor among bacterial species 査読有り国際誌

Terashima Hiroyuki, Kawamoto Akihiro, Morimoto Yusuke V., Imada Katsumi, Minamino Tohru

Biophysics and Physicobiology ( 一般社団法人日本生物物理学会 ) 14 ( 0 ) 191 - 198 2017年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

<p>The bacterial flagellum is a supramolecular motility machine consisting of the basal body as a rotary motor, the hook as a universal joint, and the filament as a helical propeller. Intact structures of the bacterial flagella have been observed for different bacterial species by electron cryotomography and subtomogram averaging. The core structures of the basal body consisting of the C ring, the MS ring, the rod and the protein export apparatus, and their organization are well conserved, but novel and divergent structures have also been visualized to surround the conserved structure of the basal body. This suggests that the flagellar motors have adapted to function in various environments where bacteria live and survive. In this review, we will summarize our current findings on the divergent structures of the bacterial flagellar motor.</p>

DOI： 10.2142/biophysico.14.0_191

CiNii Article

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バクテリア1細胞内における局所pH計測査読有り

森本雄祐, 上池伸徳, 難波啓一, 南野徹

生物物理 ( 一般社団法人日本生物物理学会 ) 57 ( 6 ) 296 - 298 2017年12月

Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R) 招待有り査読有り国際誌

Yusuke V. Morimoto, Nobunori Kami-ike, Keiichi Namba, Tohru Minamino

Bio-protocol ( Bio-protocol LLC ) 7 ( 17 ) e2529 2017年09月

Tunnel Formation Inferred from the I-Form Structures of the Proton-Driven Protein Secretion Motor SecDF 査読有り国際誌

Furukawa A., Yoshikaie K., Mori T., Mori H., Morimoto Y., Sugano Y., Iwaki S., Minamino T., Sugita Y., Tanaka Y., Tsukazaki T.

Cell Reports 19 ( 5 ) 895 - 901 2017年05月

記述言語：英語掲載種別：研究論文（学術雑誌）

© 2017 The AuthorsProtein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.

DOI： 10.1016/j.celrep.2017.04.030

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Straight and rigid flagellar hook made by insertion of the FlgG specific sequence into FlgE 査読有り国際誌

Hiraoka K., Morimoto Y., Inoue Y., Fujii T., Miyata T., Makino F., Minamino T., Namba K.

Scientific Reports 7 2017年04月

記述言語：英語掲載種別：研究論文（学術雑誌）

© The Author(s) 2017. The bacterial flagellar hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The hook protein FlgE shows considerable sequence and structural similarities to the distal rod protein FlgG; however, the hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the hook to suppress axial compression and extension of the protofilaments required for bending flexibility.

DOI： 10.1038/srep46723

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Stoichiometry and turnover of the stator and rotor 招待有り査読有り国際誌

Morimoto Y., Minamino T.

Methods in Molecular Biology 1593 203 - 213 2017年04月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

© Springer Science+Business Media LLC 2017. Fluorescence imaging techniques using green fluorescent protein (GFP) and related fluorescent proteins are utilized to monitor and analyze a wide range of biological processes in living cells. Stepwise photobleaching experiments can determine the stoichiometry of protein complexes. Fluorescence recovery after photobleaching (FRAP) experiments can reveal in vivo dynamics of biomolecules. In this chapter, we describe methods to detect the subcellular localization, stoichiometry, and turnovers of stator and rotor components of the Salmonella flagellar motor.

DOI： 10.1007/978-1-4939-6927-2_16

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Measurements of free-swimming speed of motile Salmonella cells in liquid media 招待有り査読有り国際誌

Yusuke V. Morimoto, Keiichi Namba and Tohru Minamino

Bio-protocol ( Bio-protocol LLC ) 7 ( 1 ) e2093 2017年01月

Bacterial intracellular sodium ion measurement using CoroNa Green 招待有り査読有り国際誌

Yusuke V. Morimoto, Keiichi Namba and Tohru Minamino

Bio-protocol ( Bio-protocol LLC ) 7 ( 1 ) e2092 2017年01月

High-resolution pH imaging of living bacterial cells to detect local pH differences 査読有り国際誌

Morimoto Y., Kami-Ike N., Miyata T., Kawamoto A., Kato T., Namba K., Minamino T.

mBio 7 ( 6 ) 2016年12月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

© 2016 Morimoto et al.Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+ flow to drive protein export. IMPORTANCE The flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed an in vivo pH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.

DOI： 10.1128/mBio.01911-16

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FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella 査読有り

Minamino T., Kinoshita M., Inoue Y., Morimoto Y., Morimoto Y., Ihara K., Koya S., Hara N., Nishioka N., Kojima S., Homma M., Namba K., Namba K.

MicrobiologyOpen 5 ( 3 ) 424 - 435 2016年06月

記述言語：英語掲載種別：研究論文（学術雑誌）

© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN–FlgK chaperone–substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN–FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly.

DOI： 10.1002/mbo3.340

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The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export 査読有り

Tohru Minamino, Yusuke V. Morimoto, Noritaka Hara, Phillip D. Aldridge and Keiichi Namba

PLOS Pathogens 12 e1005495 2016年03月

Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor 査読有り国際誌

Baker M., Baker M., Hynson R., Ganuelas L., Mohammadi N., Liew C., Rey A., Duff A., Whitten A., Jeffries C., Jeffries C., Delalez N., Morimoto Y., Stock D., Armitage J., Turberfield A., Namba K., Berry R., Lee L., Lee L.

Nature Structural and Molecular Biology 23 ( 3 ) 197 - 203 2016年03月

記述言語：英語掲載種別：研究論文（学術雑誌）

© 2016 Nature America, Inc.Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.

DOI： 10.1038/nsmb.3172

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H+ and Na+ are involved in flagellar rotation of the spirochete Leptospira 査読有り

Md. Shafiqul Islam, Yusuke V. Morimoto, Kudo Seishi and Shuichi Nakamura

Biochemical and Biophysical Research Communications 466 196 - 200 2015年10月

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