Updated on 2024/03/26

写真a

 
KOJIMA, Seiji
 
Organization
Graduate School of Science Professor
Graduate School
Graduate School of Science
Undergraduate School
School of Science Department of Biological Science
Title
Professor
Contact information
メールアドレス

Degree 1

  1. 理学博士 ( 1999.2   名古屋大学 ) 

Research Interests 3

  1. 膜タンパク質

  2. 細菌べん毛

  3. 分子モーター

Research Areas 6

  1. Life Science / Biophysics

  2. Life Science / Bacteriology

  3. Life Science / Functional biochemistry

  4. Life Science / Functional biochemistry

  5. Life Science / Bacteriology

  6. Life Science / Biophysics

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Current Research Project and SDGs 2

  1. 細菌べん毛モーターの回転機構

  2. 細菌べん毛の本数および形成位置制御機構

Research History 9

  1. Nagoya University   Graduate School of Science Division of Biological Science Cell Regulation   Professor

    2020.10

  2. Nagoya University   Graduate School of Science Division of Biological Science Supramolecular Biology   Associate professor

    2013.11 - 2020.9

  3. Nagoya University   Graduate School of Science Division of Biological Science Supramolecular Biology   Lecturer

    2011.11 - 2013.10

  4. Nagoya University   Graduate School of Science Division of Biological Science   Assistant Professor

    2007.4 - 2011.10

  5. Nagoya University   Graduate School of Science Division of Biological Science   Assistant

    2007.3

  6. 独立行政法人 科学技術振興機構 ICORP超分子ナノマシンプロジェクト

    2004.3 - 2005.9

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    Country:Japan

  7. 日本学術振興会海外特別研究員

    2000.4 - 2002.3

  8. Postdoctoral research fellow, University of Utah

    1999.4 - 2004.2

  9. 日本学術振興会特別研究員DC2

    1997.4 - 1999.3

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    Country:Japan

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Education 2

  1. Nagoya University   Graduate School, Division of Natural Science

    - 1999.2

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    Country: Japan

  2. Nagoya University   Faculty of Science

    1990.4 - 1994.3

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    Country: Japan

Professional Memberships 5

  1. 日本生物物理学会

  2. 日本細菌学会

  3. 日本生化学会

  4. 日本分子生物学会

  5. Biophysical Society

Awards 2

  1. 小林六造記念賞

    2016.3   日本細菌学会   細菌べん毛の回転および本数制御機構に関する研究

    小嶋誠司

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    Award type:Award from Japanese society, conference, symposium, etc.  Country:Japan

  2. 小林六造記念賞

    2016.3   日本細菌学会  

    小嶋 誠司

 

Papers 213

  1. Interaction of FlhF, SRP-like GTPase with FliF, MS ring component assembling the initial structure of flagella in marine Vibrio

    Fukushima Yuria, Homma Michio, Kojima Seiji

    JOURNAL OF BIOCHEMISTRY   Vol. 174 ( 2 ) page: 125 - 130   2023.7

  2. 1. Function and structure of FlaK, a master regulator of the polar flagellar genes in marine Vibrio. Reviewed

    Homma M, Kobayakawa T, Hao Y, Nishikino T, Kojima S

    Journal of Bacteriology   Vol. 204 ( 11 ) page: e0032022   2022.11

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1128/jb.00320-22.

  3. The Periplasmic Domain of the Ion-Conducting Stator of Bacterial Flagella Regulates Force Generation

    Homma Michio, Kojima Seiji

    FRONTIERS IN MICROBIOLOGY   Vol. 13   2022.4

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    Language:Japanese  

    DOI: 10.3389/fmicb.2022.869187

    Web of Science

  4. Editorial: Flagellar Motors and Force Sensing in Bacteria

    Baker Matthew A. B., Kojima Seiji, Nord Ashley L., Partridge Jonathan D.

    FRONTIERS IN MICROBIOLOGY   Vol. 13   2022.2

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    Language:Japanese  

    DOI: 10.3389/fmicb.2022.833011

    Web of Science

  5. Probing the mechanism of bacterial flagella assembly by real-time fluorescent imaging

    Zhuang X. -Y., Zhao Z., Homma M., Kojima S., Bai F., Lo C. -J.

    EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS   Vol. 46   page: S297 - S297   2017.7

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  6. Sodium-Driven Energy Conversion for Flagellar Rotation of the Earliest Divergent Hyperthermophilic Bacterium

    Takekawa Norihiko, Nishiyama Masayoshi, Kaneseki Tsuyoshi, Kanai Tamotsu, Atomi Haruyuki, Kojima Seij, Homma Michio

    BIOPHYSICAL JOURNAL   Vol. 110 ( 3 ) page: 468A - 468A   2016.2

  7. Pressure-Speed Relationship of the Sodium-Driven Flagellar Motor of Vibrio Alginolyticus

    Nishiyama Masayoshi, Shimoda Yoshiki, Kimura Yoshifumi, Terazima Masahide, Homma Michio, Kojima Seiji

    BIOPHYSICAL JOURNAL   Vol. 106 ( 2 ) page: 578A - 578A   2014.1

  8. Structure and function of the peptidoglycan-binding (PGB) domain of bacterial flagellar motor component MotB using systematic mutagenesis and chimeric protein with Pal

    Hizukuri, Y; Morton, JF; Yakushi, T; Kojima, S; Homma, M

    GENES & GENETIC SYSTEMS   Vol. 83 ( 6 ) page: 488 - 488   2008.12

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  9. The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force

    Kojima S, Yamamoto K, Kawagishi I, Homma M

    JOURNAL OF BACTERIOLOGY   Vol. 181 ( 6 ) page: 1927 - 1930   1999.3

  10. Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor. International journal

    Yusuke Miyamura, Tatsuro Nishikino, Hiroaki Koiwa, Michio Homma, Seiji Kojima

    Genes to cells : devoted to molecular & cellular mechanisms     2024.2

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    Language:English   Publishing type:Research paper (scientific journal)  

    The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

    DOI: 10.1111/gtc.13102

    PubMed

  11. Changes in the hydrophobic network of the FliGMC domain induce rotational switching of the flagellar motor. International journal

    Tatsuro Nishikino, Atsushi Hijikata, Seiji Kojima, Tsuyoshi Shirai, Masatsune Kainosho, Michio Homma, Yohei Miyanoiri

    iScience   Vol. 26 ( 8 ) page: 107320 - 107320   2023.8

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    Language:English   Publishing type:Research paper (scientific journal)  

    The FliG protein plays a pivotal role in switching the rotational direction of the flagellar motor between clockwise and counterclockwise. Although we previously showed that mutations in the Gly-Gly linker of FliG induce a defect in switching rotational direction, the detailed molecular mechanism was not elucidated. Here, we studied the structural changes in the FliG fragment containing the middle and C-terminal regions, named FliGMC, and the switch-defective FliGMC-G215A, using nuclear magnetic resonance (NMR) and molecular dynamics simulations. NMR analysis revealed multiple conformations of FliGMC, and the exchange process between these conformations was suppressed by the G215A residue substitution. Furthermore, changes in the intradomain orientation of FliG were induced by changes in hydrophobic interaction networks throughout FliG. Our finding applies to FliG in a ring complex in the flagellar basal body, and clarifies the switching mechanism of the flagellar motor.

    DOI: 10.1016/j.isci.2023.107320

    PubMed

  12. Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo

    Hiroyuki Terashima, Michio Homma, Seiji Kojima

    Methods in Molecular Biology     page: 71 - 82   2023.2

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    Publishing type:Part of collection (book)   Publisher:Springer US  

    DOI: 10.1007/978-1-0716-3060-0_7

  13. Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane

    Kanji Takahashi, Tatsuro Nishikino, Hiroki Kajino, Seiji Kojima, Takayuki Uchihashi, Michio Homma

    BIOPHYSICS AND PHYSICOBIOLOGY   Vol. 20 ( 2 )   2023

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BIOPHYSICAL SOC JAPAN  

    The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor-stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the twotransmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF-fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.

    DOI: 10.2142/biophysico.bppb-v20.0028

    Web of Science

  14. Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor

    Hiroyuki Terashima, Kiyoshiro Hori, Kunio Ihara, Michio Homma, Seiji Kojima

    Scientific Reports   Vol. 12 ( 1 )   2022.12

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    Abstract

    The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction.

    DOI: 10.1038/s41598-022-06947-5

    Web of Science

    Other Link: https://www.nature.com/articles/s41598-022-06947-5

  15. Structure of MotA, a flagellar stator protein, from hyperthermophile

    Tatsuro Nishikino, Norihiro Takekawa, Duy Phuoc Tran, Jun-ichi Kishikawa, Mika Hirose, Sakura Onoe, Seiji Kojima, Michio Homma, Akio Kitao, Takayuki Kato, Katsumi Imada

    Biochemical and Biophysical Research Communications   Vol. 631   page: 78 - 85   2022.11

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.bbrc.2022.09.072

    Web of Science

  16. Function and Structure of FlaK, a Master Regulator of the Polar Flagellar Genes in Marine Vibrio. International journal

    Michio Homma, Tomoya Kobayakawa, Yuxi Hao, Tatsuro Nishikino, Seiji Kojima

    Journal of bacteriology   Vol. 204 ( 11 ) page: e0032022   2022.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    Vibrio alginolyticus has a flagellum at the cell pole, and the fla genes, involved in its formation, are hierarchically regulated in several classes. FlaK (also called FlrA) is an ortholog of Pseudomonas aeruginosa FleQ, an AAA+ ATPase that functions as a master regulator for all later fla genes. In this study, we conducted mutational analysis of FlaK to examine its ATPase activity, ability to form a multimeric structure, and function in flagellation. We cloned flaK and confirmed that its deletion caused a nonflagellated phenotype. We substituted amino acids at the ATP binding/hydrolysis site and at the putative subunit interfaces in a multimeric structure. Mutations in these sites abolished both ATPase activity and the ability of FlaK to induce downstream flagellar gene expression. The L371E mutation, at the putative subunit interface, abolished flagellar gene expression but retained ATPase activity, suggesting that ATP hydrolysis is not sufficient for flagellar gene expression. We also found that FlhG, a negative flagellar biogenesis regulator, suppressed the ATPase activity of FlaK. The 20 FlhG C-terminal residues are critical for reducing FlaK ATPase activity. Chemical cross-linking and size exclusion chromatography revealed that FlaK mostly exists as a dimer in solution and can form multimers, independent of ATP. However, ATP induced the interaction between FlhG and FlaK to form a large complex. The in vivo effects of FlhG on FlaK, such as multimer formation and/or DNA binding, are important for gene regulation. IMPORTANCE FlaK is an NtrC-type activator of the AAA+ ATPase subfamily of σ54-dependent promoters of flagellar genes. FlhG, a MinD-like ATPase, negatively regulates the polar flagellar number by collaborating with FlhF, an FtsY-like GTPase. We found that FlaK and FlhG interact in the presence of ATP to form a large complex. Mutational analysis revealed the importance of FlaK ATPase activity in flagellar gene expression and provided a model of the Vibrio molecular mechanism that regulates the flagellar number.

    DOI: 10.1128/jb.00320-22

    Web of Science

    PubMed

  17. Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus. International journal

    Michio Homma, Norihiro Takekawa, Kazushi Fujiwara, Yuxi Hao, Yasuhiro Onoue, Seiji Kojima

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 27 ( 9 ) page: 568 - 578   2022.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

    DOI: 10.1111/gtc.12975

    Web of Science

    PubMed

  18. Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control. Reviewed

    Homma M, Mizuno A, Hao Y, Kojima S

      Vol. 172 ( 2 ) page: 99 - 107   2022.6

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    Language:English  

    DOI: 10.1093/jb/mvac047

    Web of Science

  19. The Periplasmic Domain of the Ion-Conducting Stator of Bacterial Flagella Regulates Force Generation Reviewed

    Homma M, Kojima S

        2022.4

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  20. Roles of the second messenger c-di-GMP in bacteria: Focusing on the topics of flagellar regulation and Vibrio spp.

    Homma Michio, Kojima Seiji

    GENES TO CELLS   Vol. 27 ( 3 ) page: 157 - 172   2022.3

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    Language:Japanese  

    DOI: 10.1111/gtc.12921

    Web of Science

  21. Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor. International journal

    Tatsuro Nishikino, Yugo Sagara, Hiroyuki Terashima, Michio Homma, Seiji Kojima

    Journal of biochemistry   Vol. 171 ( 4 ) page: 443 - 450   2022.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

    DOI: 10.1093/jb/mvac001

    Web of Science

    PubMed

  22. Achievements in bacterial flagellar research with focus on Vibrio species

    Michio Homma, Tatsuro Nishikino, Seiji Kojima

    Microbiology and Immunology   Vol. 66 ( 2 ) page: 75 - 95   2022.1

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/1348-0421.12954

    Web of Science

    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12954

  23. ZomB is essential for chemotaxis of Vibrio alginolyticus by the rotational direction control of the polar flagellar motor. International journal

    Norihiro Takekawa, Tatsuro Nishikino, Kiyoshiro Hori, Seiji Kojima, Katsumi Imada, Michio Homma

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 26 ( 11 ) page: 927 - 937   2021.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from non-preferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY-Phosphate (CheY-P) to the C-ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp. have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for a clockwise-locked phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY-P. Purified ZomB proteins form multimers, suggesting that ZomB may function as a homo-oligomer. These observations imply that ZomB interacts with protein(s) involved in either flagellar motor rotation, chemotaxis or both. We provide the evidence that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus.

    DOI: 10.1111/gtc.12895

    Web of Science

    PubMed

  24. A slight bending of an α-helix in FliM creates a counterclockwise-locked structure of the flagellar motor in Vibrio. International journal

    Norihiro Takekawa, Tatsuro Nishikino, Toshiki Yamashita, Kiyoshiro Hori, Yasuhiro Onoue, Kunio Ihara, Seiji Kojima, Michio Homma, Katsumi Imada

    Journal of biochemistry   Vol. 170 ( 4 ) page: 531 - 538   2021.6

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    Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus, which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signaling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM-FliM interaction. The structural models of wild-type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring.

    DOI: 10.1093/jb/mvab074

    Web of Science

    PubMed

  25. Putative spanner function of the <i>Vibrio</i> PomB plug region in the stator rotation model for flagellar motor

    Michio Homma, Hiroyuki Terashima, Hiroaki Koiwa, Seiji Kojima

    Journal of Bacteriology   Vol. 203 ( 16 )   2021.6

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner.

    Importance

    The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

    DOI: 10.1128/jb.00159-21

    Web of Science

  26. Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring. Invited Reviewed

    mBio.     2021.3

  27. Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring

    Norihiro Takekawa, Akihiro Kawamoto, Mayuko Sakuma, Takayuki Kato, Seiji Kojima, Miki Kinoshita, Tohru Minamino, Keiichi Namba, Michio Homma, Katsumi Imada

    mBio   Vol. 12 ( 2 )   2021.3

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    Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    <title>ABSTRACT</title>
    The bacterial flagellum is a protein nanomachine essential for bacterial motility. The flagellar basal body contains several ring structures. The MS-ring is embedded in the cytoplasmic membrane and is formed at the earliest stage of flagellar formation to serve as the base for flagellar assembly as well as a housing for the flagellar protein export gate complex. The MS-ring is formed by FliF, which has two transmembrane helices and a large periplasmic region. A recent electron cryomicroscopy (cryoEM) study of the MS-ring formed by overexpressed FliF revealed a symmetry mismatch between the S-ring and inner part of the M-ring. However, the actual symmetry relation in the native MS-ring and positions of missing domains remain obscure. Here, we show the structure of the M-ring by combining cryoEM and X-ray crystallography. The crystal structure of the N-terminal half of the periplasmic region of FliF showed that it consists of two domains (D1 and D2) resembling PrgK D1/PrgH D2 and PrgK D2/PrgH D3 of the injectisome. CryoEM analysis revealed that the inner part of the M-ring shows a gear wheel-like density with the inner ring of C23 symmetry surrounded by cogs with C11 symmetry, to which 34 copies of FliF<sub>D1–D2</sub> fitted well. We propose that FliF<sub>D1–D2</sub> adopts two distinct orientations in the M-ring relative to the rest of FliF, with 23 chains forming the wheel and 11 chains forming the cogs, and the 34 chains come together to form the S-ring with C34 symmetry for multiple functions of the MS-ring.


    <bold>IMPORTANCE</bold> The bacterial flagellum is a motility organelle formed by tens of thousands of protein molecules. At the earliest stage of flagellar assembly, a transmembrane protein, FliF, forms the MS-ring in the cytoplasmic membrane as the base for flagellar assembly. Here, we solved the crystal structure of a FliF fragment. Electron cryomicroscopy (cryoEM) structural analysis of the MS-ring showed that the M-ring and S-ring have different rotational symmetries. By docking the crystal structure of the FliF fragment into the cryoEM density map of the entire MS-ring, we built a model of the whole periplasmic region of FliF and proposed that FliF adopts two distinct conformations to generate three distinct C11, C23, and C34 symmetries within the MS-ring for its multiple functions.

    DOI: 10.1128/mbio.03199-20

    Web of Science

  28. Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body. International journal

    Seiji Kojima, Hiroki Kajino, Keiichi Hirano, Yuna Inoue, Hiroyuki Terashima, Michio Homma

    Journal of bacteriology   Vol. 203 ( 9 )   2021.2

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    The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

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  29. Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor. International journal

    Hiroyuki Terashima, Seiji Kojima, Michio Homma

    Journal of bacteriology   Vol. 203 ( 9 )   2021.2

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    The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses, but have not been demonstrated biochemically. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.ImportanceThe bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo- and disulfide-crosslinking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

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  30. Stator Dynamics Depending on Sodium Concentration in Sodium-Driven Bacterial Flagellar Motors. International journal

    Tsai-Shun Lin, Seiji Kojima, Hajime Fukuoka, Akihiko Ishijima, Michio Homma, Chien-Jung Lo

    Frontiers in microbiology   Vol. 12   page: 765739 - 765739   2021

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    Bacterial flagellar motor (BFM) is a large membrane-spanning molecular rotary machine for swimming motility. Torque is generated by the interaction between the rotor and multiple stator units powered by ion-motive force (IMF). The number of bound stator units is dynamically changed in response to the external load and the IMF. However, the detailed dynamics of stator unit exchange process remains unclear. Here, we directly measured the speed changes of sodium-driven chimeric BFMs under fast perfusion of different sodium concentration conditions using computer-controlled, high-throughput microfluidic devices. We found the sodium-driven chimeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the number of functional stator units shows a rapidly excessive reduction and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.

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  31. The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. International journal

    Brittany L Carroll, Tatsuro Nishikino, Wangbiao Guo, Shiwei Zhu, Seiji Kojima, Michio Homma, Jun Liu

    eLife   Vol. 9   2020.9

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    The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

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  32. Live-cell fluorescence imaging reveals dynamic production and loss of bacterial flagella. International journal

    Xiang-Yu Zhuang, Shihao Guo, Zhuoran Li, Ziyi Zhao, Seiji Kojima, Michio Homma, Pengyuan Wang, Chien-Jung Lo, Fan Bai

    Molecular microbiology   Vol. 114 ( 2 ) page: 279 - 291   2020.8

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    Bacterial flagella are nanomachines that drive bacteria motility and taxis in response to environmental changes. Whether flagella are permanent cell structures and, if not, the circumstances and timing of their production and loss during the bacterial life cycle remain poorly understood. Here we used the single polar flagellum of Vibrio alginolyticus as our model and implementing in vivo fluorescence imaging revealed that the percentage of flagellated bacteria (PFB) in a population varies substantially across different growth phases. In the early-exponential phase, the PFB increases rapidly through the widespread production of flagella. In the mid-exponential phase, the PFB peaks at around 76% and the partitioning of flagella between the daughter cells are 1:1 and strictly at the old poles. After entering the stationary phase, the PFB starts to decline, mainly because daughter cells stop making new flagella after cell division. Interestingly, we observed that bacteria can actively abandon flagella after prolonged stationary culturing, though cell division has long been suspended. Further experimental investigations confirmed that flagella were ejected in V. alginolyticus, starting from breakage in the rod. Our results highlight the dynamic production and loss of flagella during the bacterial life cycle. IMPORTANCE: Flagella motility is critical for many bacterial species. The bacterial flagellum is made up of about 20 different types of proteins in its final structure and can be self-assembled. The current understanding of the lifetime and durability of bacterial flagella is very limited. In the present study, we monitored Vibrio alginolyticus flagellar assembly and loss by in vivo fluorescence labeling, and found that the percentage of flagellated bacteria varies substantially across different growth phases. The production of flagella was synchronized with cell growth but stopped when cells entered the stationary phase. Surprisingly, we observed that bacteria can actively abandon flagella after prolonged stationary culturing, as well as in the low glucose buffering medium. We then confirmed the ejection of flagella in V. alginolyticus started with breakage of the rod. Our results highlight the dynamic production and loss of flagella during the bacterial life cycle.

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  33. Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species. Reviewed International journal

    Hiroyuki Terashima, Keiichi Hirano, Yuna Inoue, Takaya Tokano, Akihiro Kawamoto, Takayuki Kato, Erika Yamaguchi, Keiichi Namba, Takayuki Uchihashi, Seiji Kojima, Michio Homma

    Journal of bacteriology   Vol. 202 ( 16 )   2020.6

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    The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

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  34. Regulation of the Single Polar Flagellar Biogenesis. International journal

    Seiji Kojima, Hiroyuki Terashima, Michio Homma

    Biomolecules   Vol. 10 ( 4 )   2020.4

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    Some bacterial species, such as the marine bacterium Vibrio alginolyticus, have a single polar flagellum that allows it to swim in liquid environments. Two regulators, FlhF and FlhG, function antagonistically to generate only one flagellum at the cell pole. FlhF, a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase, works as a positive regulator for flagellar biogenesis and determines the location of flagellar assembly at the pole, whereas FlhG, a MinD-type ATPase, works as a negative regulator that inhibits flagellar formation. FlhF intrinsically localizes at the cell pole, and guanosine triphosphate (GTP) binding to FlhF is critical for its polar localization and flagellation. FlhG also localizes at the cell pole via the polar landmark protein HubP to directly inhibit FlhF function at the cell pole, and this localization depends on ATP binding to FlhG. However, the detailed regulatory mechanisms involved, played by FlhF and FlhG as the major factors, remain largely unknown. This article reviews recent studies that highlight the post-translational regulation mechanism that allows the synthesis of only a single flagellum at the cell pole.

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  35. Characterization of the MinD/ParA-type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form. International journal

    Seiji Kojima, Yoshino Imura, Hikaru Hirata, Michio Homma

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 25 ( 4 ) page: 279 - 287   2020.4

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    FlhG is a MinD/ParA-type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here, we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP-binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild type, but ATP caused strong aggregation of the ATPase-stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution, and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably because of a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.

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  36. Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. International journal

    Tatsuro Nishikino, Hiroto Iwatsuki, Taira Mino, Seiji Kojima, Michio Homma

    Journal of biochemistry   Vol. 167 ( 4 ) page: 389 - 398   2020.4

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    The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2-3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1-2) and TM3 and TM4 (loop3-4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3-4 but hardly labelled Cys residues in loop1-2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41-120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1-2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1-2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

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  37. Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System. International journal

    Florencia Mengucci, Carolina Dardis, Elías J Mongiardini, María J Althabegoiti, Jonathan D Partridge, Seiji Kojima, Michio Homma, Juan I Quelas, Aníbal R Lodeiro

    Journal of bacteriology   Vol. 202 ( 5 )   2020.2

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    Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

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  38. In Situ Structure of the Vibrio Polar Flagellum Reveals a Distinct Outer Membrane Complex and Its Specific Interaction with the Stator. Reviewed International journal

    Shiwei Zhu, Tatsuro Nishikino, Norihiro Takekawa, Hiroyuki Terashima, Seiji Kojima, Katsumi Imada, Michio Homma, Jun Liu

    Journal of bacteriology   Vol. 202 ( 4 )   2020.1

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    The bacterial flagellum is a biological nanomachine that rotates to allow bacteria to swim. For flagellar rotation, torque is generated by interactions between a rotor and a stator. The stator, which is composed of MotA and MotB subunit proteins in the membrane, is thought to bind to the peptidoglycan (PG) layer, which anchors the stator around the rotor. Detailed information on the stator and its interactions with the rotor remains unclear. Here, we deployed cryo-electron tomography and genetic analysis to characterize in situ structure of the bacterial flagellar motor in Vibrio alginolyticus, which is best known for its polar sheathed flagellum and high-speed rotation. We determined in situ structure of the motor at unprecedented resolution and revealed the unique protein-protein interactions among Vibrio-specific features, namely the H ring and T ring. Specifically, the H ring is composed of 26 copies of FlgT and FlgO, and the T ring consists of 26 copies of a MotX-MotY heterodimer. We revealed for the first time a specific interaction between the T ring and the stator PomB subunit, providing direct evidence that the stator unit undergoes a large conformational change from a compact form to an extended form. The T ring facilitates the recruitment of the extended stator units for the high-speed motility in Vibrio species.IMPORTANCE The torque of flagellar rotation is generated by interactions between a rotor and a stator; however, detailed structural information is lacking. Here, we utilized cryo-electron tomography and advanced imaging analysis to obtain a high-resolution in situ flagellar basal body structure in Vibrio alginolyticus, which is a Gram-negative marine bacterium. Our high-resolution motor structure not only revealed detailed protein-protein interactions among unique Vibrio-specific features, the T ring and H ring, but also provided the first structural evidence that the T ring interacts directly with the periplasmic domain of the stator. Docking atomic structures of key components into the in situ motor map allowed us to visualize the pseudoatomic architecture of the polar sheathed flagellum in Vibrio spp. and provides novel insight into its assembly and function.

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  39. Tree of motility - A proposed history of motility systems in the tree of life. Reviewed International journal

    Makoto Miyata, Robert C Robinson, Taro Q P Uyeda, Yoshihiro Fukumori, Shun-Ichi Fukushima, Shin Haruta, Michio Homma, Kazuo Inaba, Masahiro Ito, Chikara Kaito, Kentaro Kato, Tsuyoshi Kenri, Yoshiaki Kinosita, Seiji Kojima, Tohru Minamino, Hiroyuki Mori, Shuichi Nakamura, Daisuke Nakane, Koji Nakayama, Masayoshi Nishiyama, Satoshi Shibata, Katsuya Shimabukuro, Masatada Tamakoshi, Azuma Taoka, Yosuke Tashiro, Isil Tulum, Hirofumi Wada, Ken-Ichi Wakabayashi

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 25 ( 1 ) page: 6 - 21   2020.1

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    Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

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  40. [Flagellar related genes and functions in Vibrio].

    Tatsuro Nishikino, Seiji Kojima, Michio Homma

    Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 75 ( 3 ) page: 195 - 214   2020

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    Bacteria can move or swim by flagella. On the other hand, the motile ability is not necessary to live at all. In laboratory, the flagella-deficient strains can grow just like the wild-type strains. The flagellum is assembled from more than 20 structural proteins and there are more than 50 genes including the structural genes to regulate or support the flagellar formation. The cost to construct the flagellum is so expensive. The fact that it evolved as a motor organ means even at such the large cost shows that the flagellum is essential for survival in natural condition. In this review, we would like to focus on the flagella-related researches conducted by the authors and the flagellar research on Vibrio spp.

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  41. Structure of the periplasmic domain of SflA involved in spatial regulation of the flagellar biogenesis of Vibrio reveals a TPR/SLR-like fold. Reviewed International journal

    Mayuko Sakuma, Shoji Nishikawa, Satoshi Inaba, Takehiko Nishigaki, Seiji Kojima, Michio Homma, Katsumi Imada

    Journal of biochemistry   Vol. 166 ( 2 ) page: 197 - 204   2019.8

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    Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein-protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function.

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  42. Effect of PlzD, a YcgR homologue of c-di-GMP-binding protein, on polar flagellar motility in Vibrio alginolyticus. Reviewed International journal

    Seiji Kojima, Takuro Yoneda, Wakako Morimoto, Michio Homma

    Journal of biochemistry   Vol. 166 ( 1 ) page: 77 - 88   2019.7

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    YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.

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  43. Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus. Reviewed

    Mino T, Nishikino T, Iwatsuki H, Kojima S, Homma M

    J. Biochem.   Vol. 166 ( 4 ) page: 331 - 341   2019.5

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  44. Structure of Vibrio FliL, a New Stomatin-like Protein That Assists the Bacterial Flagellar Motor Function. Reviewed International journal

    Norihiro Takekawa, Miyu Isumi, Hiroyuki Terashima, Shiwei Zhu, Yuuki Nishino, Mayuko Sakuma, Seiji Kojima, Michio Homma, Katsumi Imada

    mBio   Vol. 10 ( 2 ) page: e00292   2019.3

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    Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.

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  45. Rotational direction of flagellar motor from the conformation of FliG middle domain in marine Vibrio. Reviewed International journal

    Tatsuro Nishikino, Atsushi Hijikata, Yohei Miyanoiri, Yasuhiro Onoue, Seiji Kojima, Tsuyoshi Shirai, Michio Homma

    Scientific reports   Vol. 8 ( 1 ) page: 17793 - 17793   2018.12

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    FliG, which is composed of three distinctive domains, N-terminal (N), middle (M), and C-terminal (C), is an essential rotor component that generates torque and determines rotational direction. To determine the role of FliG in determining flagellar rotational direction, we prepared rotational biased mutants of fliG in Vibrio alginolyticus. The E144D mutant, whose residue is belonging to the EHPQR-motif in FliGM, exhibited an increased number of switching events. This phenotype generated a response similar to the phenol-repellent response in chemotaxis. To clarify the effect of E144D mutation on the rotational switching, we combined the mutation with other che mutations (G214S, G215A and A282T) in FliG. Two of the double mutants suppressed the rotational biased phenotype. To gain structural insight into the mutations, we performed molecular dynamic simulations of the FliGMC domain, based on the crystal structure of Thermotoga maritima FliG and nuclear magnetic resonance analysis. Furthermore, we examined the swimming behavior of the fliG mutants lacking CheY. The results suggested that the conformation of FliG in E144D mutant was similar to that in the wild type. However, that of G214S and G215A caused a steric hindrance in FliG. The conformational change in FliGM triggered by binding CheY may lead to a rapid change of direction and may occur in both directional states.

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  46. The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum. Reviewed International journal

    Shiwei Zhu, Tatsuro Nishikino, Seiji Kojima, Michio Homma, Jun Liu

    Journal of bacteriology   Vol. 200 ( 21 )   2018.11

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    The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, whereas spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here, we use cryo-electron tomography to visualize the polar sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio-specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes.IMPORTANCE Flagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella, such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

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  47. Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment. Reviewed International journal

    Tohru Minamino, Naoya Terahara, Seiji Kojima, Keiichi Namba

    Molecular microbiology   Vol. 109 ( 6 ) page: 723 - 734   2018.9

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    The bacterial flagellar motor is composed of a rotor and a transmembrane ion channel complex that acts as a stator unit. The ion channel complex consists of at least three structural parts: a cytoplasmic domain responsible for the interaction with the rotor, a transmembrane ion channel that forms a pathway for the transit of ions across the cytoplasmic membrane, and a peptidoglycan-binding (PGB) domain that anchors the stator unit to the peptidoglycan (PG) layer. A flexible linker connecting the ion channel and the PGB domain not only coordinates stator assembly with its ion channel activity but also controls the assembly of stator units to the motor in response to changes in the environment. When the ion channel complex encounters the rotor, the N-terminal portion of the PGB domain adopts a partially stretched conformation, allowing the PGB domain to reach and bind to the PG layer. The binding affinity of the PGB domain for the PG layer is affected by the force applied to its anchoring point and to the type of ionic energy source. In this review article, we will present current understanding of autonomous control mechanism of stator assembly in the bacterial flagellar motor.

    DOI: 10.1111/mmi.14092

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  48. Biochemical analysis of GTPase FlhF which controls the number and position of flagellar formation in marine Vibrio. Reviewed International journal

    Shota Kondo, Yoshino Imura, Akira Mizuno, Michio Homma, Seiji Kojima

    Scientific reports   Vol. 8 ( 1 ) page: 12115 - 12115   2018.8

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    FlhF controls the number and position of the polar flagellar formation of Vibrio species. FlhF, is a paralog of FtsY, a GTPase acting in the Sec membrane transport system of bacteria, and localizes at the cell pole. Mutations in the conserved GTPase motif of FlhF lost polar localization capability and flagellar formation. Vibrio FlhF has not, until now, been purified as soluble protein. Here, we report that addition of MgCl2 and GTP or GDP at the step of cell lysis greatly improved the solubility of FlhF, allowing us to purify it in homogeneity. Purified FlhF showed GTPase activity only in the presence of FlhG. Of twelve FlhF GTPase motif mutants showing reduced function, eleven were recovered as precipitate after the cell disruption. The E440K substitution could be purified and showed no GTPase activity even in the presence of FlhG. Interestingly an FlhF substitution in the putative catalytic residue for GTP hydrolysis, R334A, allowed normal flagellar formation although GTPase activity of FlhF was completely abolished. Furthermore, size exclusion chromatography of purified FlhF revealed that it forms dimers in the presence of GTP but exists as monomer in the presence of GDP. We speculate that the GTP binding allows FlhF to dimerize and localize at the pole where it initiates flagellar formation, and the GDP-bound form diffuses as monomer.

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  49. The role of conserved charged residues in the bidirectional rotation of the bacterial flagellar motor. Reviewed International journal

    Yasuhiro Onoue, Norihiro Takekawa, Tatsuro Nishikino, Seiji Kojima, Michio Homma

    MicrobiologyOpen   Vol. 7 ( 4 ) page: e00587   2018.8

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    Many bacteria rotate their flagella both counterclockwise (CCW) and clockwise (CW) to achieve swimming toward attractants or away from repellents. Highly conserved charged residues are important for that motility, which suggests that electrostatic interactions are crucial for the rotor-stator function. It remains unclear if those residues contribute equally to rotation in the CCW and CW directions. To address this uncertainty, in this study, we expressed chimeric rotors and stators from Vibrio alginolyticus and Escherichia coli in E. coli, and measured the rotational speed of each motor in both directions using a tethered-cell assay. In wild-type cells, the rotational speeds in both directions were equal, as demonstrated previously. Some charge-neutralizing residue replacements in the stator decreased the rotational speed in both directions to the same extent. However, mutations in two charged residues in the rotor decreased the rotational speed only in the CCW direction. Subsequent analysis and previous results suggest that these amino acid residues are involved in supporting the conformation of the rotor, which is important for proper torque generation in the CCW direction.

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  50. FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy. Reviewed International journal

    Tsai-Shun Lin, Shiwei Zhu, Seiji Kojima, Michio Homma, Chien-Jung Lo

    Scientific reports   Vol. 8 ( 1 ) page: 11172 - 11172   2018.7

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    Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.

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  51. The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx Reviewed

    Seiji Kojima, Masato Takao, Gaby Almira, Ikumi Kawahara, Mayuko Sakuma, Michio Homma, Chojiro Kojima, Katsumi Imada

    Structure   Vol. 26 ( 4 ) page: 590 - 598.e5   2018.4

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    The stator of the bacterial flagellar motor couples ion flow with torque generation. The ion-conducting stator channel opens only when incorporated into and anchored around the rotor via the peptidoglycan (PG) binding domain of the B subunit (MotBC). However, no direct evidence of PG binding coupled with channel activation has been presented. Here, we report the structural rearrangements of MotBC responsible for this coupling process. A MotBC fragment with the L119P replacement, which is known to cause channel activation, was able to bind PG. Nuclear magnetic resonance analysis of MotBC and the crystal structure of the MotBC-L119P dimer revealed major structural changes in helix α1. In vivo crosslinking results confirm that a major rearrangement occurs. Our results suggest that, upon stator incorporation into the motor, helix α1 of MotBC changes into an extended non-helical structure. We propose that this change allows the stator both to bind PG and to open its proton channel. Kojima et al. provide direct evidence for coupling between peptidoglycan (PG)-binding and activation of the flagellar stator protein. Using X-ray and NMR analyses they reveal that dynamic rearrangement of the N-terminal helix of the PG-binding domain of the stator protein MotB is key for its functional activation and energy conversion.

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  52. Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

    Liew C. W., Hynson R. M., Ganuelas L. A., Shah-Mohammadi N., Duff A. P., Kojima S., Homma M., Lee L. K.

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 495 ( 2 ) page: 1614-1619   2018.1

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    DOI: 10.1016/j.bbrc.2017.11.194

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  53. Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella Reviewed

    Yohei Miyanoiri, Atsushi Hijikata, Yuuki Nishino, Mizuki Gohara, Yasuhiro Onoue, Seiji Kojima, Chojiro Kojima, Tsuyoshi Shirai, Masatsune Kainosho, Michio Homma

    STRUCTURE   Vol. 25 ( 10 ) page: 1540 - +   2017.10

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    The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliG(C)). We have identified point mutations in FliG(C) from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliG(C) from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliG(C) are observed in the C alpha 1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliG(C) at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix alpha 1 in the C alpha 1-6 domain of FliG(C) contribute to ensure the precise regulation of the motor switching.

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  54. Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus Reviewed

    Shiwei Zhu, Tatsuro Nishikino, Bo Hu, Seiji Kojima, Michio Homma, Jun Liu

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 114 ( 41 ) page: 10966 - 10971   2017.10

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    Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use Vibrio alginolyticus as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in V. alginolyticus cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined a Vibrio-specific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring provides a static platform to recruit the PomA/B complexes, which are required to generate higher torques for rotation of the sheathed flagellum and fast motility of Vibrio cells. Furthermore, the Vibrio flagellar motor exhibits an intrinsic length variation between the inner and the outer membrane bound complexes, suggesting the outer membrane bound complex can shift slightly along the axial rod during flagellar rotation. Together, our detailed analyses of the polar flagella in intact cells provide insights into unique aspects of the sheathed flagellum and the distinct motility of Vibrio species.

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  55. Localization and domain characterization of the SflA regulator of flagellar formation in Vibrio alginolyticus Reviewed

    Satoshi Inaba, Takehiko Nishigaki, Norihiro Takekawa, Seiji Kojima, Michio Homma

    GENES TO CELLS   Vol. 22 ( 7 ) page: 619 - 627   2017.7

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    Many swimming bacteria use flagella as locomotive organelles. The spatial and numerical regulation of flagellar biosynthesis differs by bacterial species. The marine bacteria Vibrio alginolyticus use a single polar flagellum whose number is regulated positively by FlhF and negatively by FlhG. Cells lacking FlhF and FlhG have no flagellum. The motility defect in an flhFG deletion was suppressed by a mutation in the sflA gene that resulted in the production of multiple, peritrichous flagella. SflA is a Vibrio-specific protein. SlfA either facilitates flagellum growth at the cell pole or prevents flagellar formation on the cell body by an unknown mechanism. Fluorescent protein fusions to SflA localized to the cell pole in the presence of FlhF and FlhG, but exhibited both polar and lateral cell localization in Delta flhFG cells. Polar localization of SflA required the polar landmark protein HubP. Over-expression of the C-terminal region of SflA (SflA(C)) in Delta flhFG Delta sflA cells suppressed the lateral flagellar formation. Our results suggest that SflA localizes with the flagella and that SflA(C) represses the flagellar initiation in Delta flhFG strains. A model is presented where SflA inhibits lateral flagellar formation to facilitate single polar flagellum assembly in V. alginolyticus cells.

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  56. Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus Reviewed

    Ananthanarayanan Kumar, Miyu Isumi, Mayuko Sakuma, Shiwei Zhu, Yuuki Nishino, Yasuhiro Onoue, Seiji Kojima, Yohei Miyanoiri, Katsumi Imada, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 161 ( 4 ) page: 331 - 337   2017.4

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    The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (Delta TM FliL). Biochemical characterization and NMR analysis revealed that Delta TM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.

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  57. Mutational analysis and overproduction effects of MotX, an essential component for motor function of Na+-driven polar flagella of Vibrio. International journal

    Norihiro Takekawa, Seiji Kojima, Michio Homma

    Journal of biochemistry   Vol. 161 ( 2 ) page: 159 - 166   2017.2

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    The bacterial flagellar motor is a rotary motor complex composed of various proteins. The motor contains a central rod, multiple ring-like structures and stators. The Na+-driven polar flagellar motor of the marine bacterium Vibrio alginolyticus has a specific ring, called the ‘T-ring’, which consists of two periplasmic proteins, MotX and MotY. The T-ring is essential for assembly of the torque-generating unit, the PomA/PomB stator complex, into the motor. To investigate the role of the T-ring for motor function, we performed random mutagenesis of the motX gene on a plasmid. The isolated MotX mutants showed nonmotile, slow-motile, and up-motile phenotypes by the expression from the plasmid. Deletion analysis indicated that the C-terminal region and the signal peptide in MotX are not always essential for flagellar motor function. We also found that overproduction of MotX caused the delay of growth and aberrant cell shape. MotX might have unexpected roles not only in flagellar motor function but also in cell morphology control.

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  58. Structural and functional analysis of the C-terminal region of FliG, an essential motor component of Vibrio Na+-driven flagella. Reviewed

    Miyanoiri Y, Hijikata A, Nishino Y, Gohara M, Onoue Y, Kojima S, Kojima C, Shirai T, Kainosho M, Homma M.

    Structure   Vol. 25   page: 1-9   2017

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  59. Localization and domain characterization of the SflA regulator of flagellar formation in Vibrio alginolyticus. Reviewed

    Inaba S, Nishigaki T, Takekawa N, Kojima S, Homma M.

    Genes Cells   Vol. 7   page: 619-627   2017

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  60. Mechanism of Stator Assembly and Incorporation into the Flagellar Motor. Reviewed

    Kojima S

    Methods in molecular biology (Clifton, N.J.)   Vol. 1593   page: 147 - 159   2017

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    DOI: 10.1007/978-1-4939-6927-2_11

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  61. Analysis of the GTPase motif of FlhF in the control of the number and location of polar flagella in Vibrio alginolyticus. Reviewed

    Shota Kondo, Michio Homma, Seiji Kojima

    Biophysics and physicobiology   Vol. 14   page: 173 - 181   2017

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    Vibrio alginolyticus normally has a single polar flagellum whose number and placement are regulated positively by FlhF. FlhF is a GTPase and homolog of a signal recognition particle (SRP) protein called Ffh and SRP receptor FtsY. FlhF is located at the cell pole and directs formation of the flagellum. To study the mechanism of FlhF localization, we introduced random mutations into flhF by means of hydroxylamine and isolated mutants that could not generate the flagellum at the cell pole. The novel mutations were only mapped to the GTPase motif of FlhF. The mutant FlhF proteins showed reduced polar localization as compared to the wild type and still could associate with the membrane. These results support the assumption that the GTPase motif of FlhF plays a critical role in the polar localization of this protein during formation of the flagellum.

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  62. HubP, a Polar Landmark Protein, Regulates Flagellar Number by Assisting in the Proper Polar Localization of FlhG in Vibrio alginolyticus Reviewed

    Norihiro Takekawa, Soojin Kwon, Noriko Nishioka, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 198 ( 22 ) page: 3091 - 3098   2016.11

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    The marine bacterium Vibrio alginolyticus has a single polar flagellum, the number of which is regulated positively by FlhF and negatively by FlhG. FlhF is intrinsically localized at the cell pole, whereas FlhG is localized there through putative interactions with the polar landmark protein HubP. Here we focused on the role of HubP in the regulation of flagellar number in V. alginolyticus. Deletion of hubP increased the flagellar number and completely disrupted the polar localization of FlhG. It was thought that the flagellar number is determined primarily by the absolute amount of FlhF localized at the cell pole. Here we found that deletion of hubP increased the flagellar number although it did not increase the polar amount of FlhF. We also found that FlhG overproduction did not reduce the polar localization of FlhF. These results show that the absolute amount of FlhF is not always the determinant of flagellar number. We speculate that cytoplasmic FlhG works as a quantitative regulator, controlling the amount of FlhF localized at the pole, and HubP-anchored polar FlhG works as a qualitative regulator, directly inhibiting the activity of polar FlhF. This regulation by FlhF, FlhG, and HubP might contribute to achieving optimal flagellar biogenesis at the cell pole in V. alginolyticus.
    IMPORTANCE
    For regulation of the flagellar number in marine Vibrio, two proteins, FlhF and FlhG, work as positive and negative regulators, respectively. In this study, we found that the polar landmark protein HubP is involved in the regulation of flagellar biogenesis. Deletion of hubP increased the number of flagella without increasing the amount of pole-localizing FlhF, indicating that the number of flagella is not determined solely by the absolute amount of pole-localizing FlhF, which is inconsistent with the previous model. We propose that cytoplasmic FlhG and HubP-anchored polar FlhG negatively regulate flagellar formation through two independent schemes.

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  63. The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium Reviewed

    Norihiro Takekawa, Naoya Terahara, Takayuki Kato, Mizuki Gohara, Kouta Mayanagi, Atsushi Hijikata, Yasuhiro Onoue, Seiji Kojima, Tsuyoshi Shirai, Keiichi Namba, Michio Homma

    SCIENTIFIC REPORTS   Vol. 6   page: 31526   2016.8

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    Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four-and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator.

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  64. FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella Reviewed

    Tohru Minamino, Miki Kinoshita, Yumi Inoue, Yusuke V. Morimoto, Kunio Ihara, Satomi Koya, Noritaka Hara, Noriko Nishioka, Seiji Kojima, Michio Homma, Keiichi Namba

    MICROBIOLOGYOPEN   Vol. 5 ( 3 ) page: 424 - 435   2016.6

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    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 flhA mutant but not that of a fliH-fliI flhB(P28T) flhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the fliH-fliI flhB(P28T) flhA 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.

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  65. Serine suppresses the motor function of a periplasmic PomB mutation in the Vibrio flagella stator Reviewed

    Tatsuro Nishikino, Shiwei Zhu, Norihiro Takekawa, Seiji Kojima, Yasuhiro Onoue, Michio Homma

    GENES TO CELLS   Vol. 21 ( 5 ) page: 505 - 516   2016.5

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    The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross-linking experiments of double-Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double-Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)-biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che(-) mutants, in either a CCW-locked or clockwise (CW)-locked strain. In contrast, in a cheY (CCW-locked) strain, Vibrio (L160C/I186C) required serine to be rescued. We speculate that CheY affects stator conformation and motility restoration by serine is independent on the chemotaxis signaling pathway.

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  66. 細菌べん毛の回転および本数制御機構に関する研究 Invited

    小嶋誠司

    日本細菌学雑誌   Vol. 71 ( 3 ) page: 185-197   2016

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  67. Studies on the mechanism of bacterial flagellar rotation and the flagellar number regulation. Reviewed

    Seiji Kojima

    Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 71 ( 3 ) page: 185 - 97   2016

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    Many motile bacteria have the motility organ, the flagellum. It rotates by the rotary motor driven by the ion-motive force and is embedded in the cell surface at the base of each flagellar filament. Many researchers have been studying its rotary mechanism for years, but most of the energy conversion processes have been remained in mystery. We focused on the flagellar stator, which works at the core process of energy conversion, and found that the periplasmic region of the stator changes its conformation to be activated only when the stator units are incorporated into the motor and anchored at the cell wall. Meanwhile, the physiologically important supramolecular complex is localized in the cell at the right place and the right time with a proper amount. How the cell achieves such a proper localization is the fundamental question for life science, and we undertake this problem by analyzing the mechanism for biogenesis of a single polar flagellum of Vibrio alginolyticus. Here I describe the molecular mechanism of how the flagellum is generated at the specific place with a proper number, and also how the flagellar stator is incorporated into the motor to complete the functional motor assembly, based on our studies.

    DOI: 10.3412/jsb.71.185

    PubMed

  68. Effect of FliG three amino acids deletion in Vibrio polar-flagellar rotation and formation Reviewed

    Yasuhiro Onoue, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 158 ( 6 ) page: 523 - 529   2015.12

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    Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (Delta PSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(Delta PSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

    DOI: 10.1093/jb/mvv068

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  69. Dynamism and regulation of the stator, the energy conversion complex of the bacterial flagellar motor Reviewed

    Seiji Kojima

    CURRENT OPINION IN MICROBIOLOGY   Vol. 28   page: 66 - 71   2015.12

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    Many motile bacteria swim by rotating their motility organ, the flagellum. Rotation of the flagellum is driven by a motor at its base, and torque is generated by the rotor stator interaction coupled with the specific ion flow through the channel in the stator. Because the stator works as an energy-conversion complex in the motor, understanding the functional mechanism of the stator is critically important. But its characterization has been hampered due to the difficulty in isolating the functional stator complex from the membrane. Recently, successful new approaches for studying the stator have been reported that reveal its novel properties. Two of those, visualization of the in vivo behavior of stator units using fluorescently tagged proteins and structure-guided functional analyses of the soluble region in the stator, are summarized in this short review.

    DOI: 10.1016/j.mib.2015.07.015

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  70. FliL associates with the stator to support torque generation of the sodium-driven polar flagellar motor of Vibrio Reviewed

    Shiwei Zhu, Ananthanarayanan Kumar, Seiji Kojima, Michio Homma

    MOLECULAR MICROBIOLOGY   Vol. 98 ( 1 ) page: 101 - 110   2015.10

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    Flagellar motors generate torque to rotate flagellar filaments and drive bacterial cells. Each motor is composed of a rotor and many stators. The stator is a force-generating complex that converts ion flux into torque. Previous reports have suggested that the membrane protein FliL is located near the stator and is involved in torque generation. We investigated the role of FliL in the sodium-driven polar flagellar motor of Vibrio alginolyticus. Our results revealed that FliL is a cytoplasmic membrane protein and is located at the base of flagellum. The deletion of fliL did not affect the cell morphology or flagellation but resulted in a significant decrease of swimming speed, especially at a higher load thus suggesting that FliL is important for torque generation at high load conditions. Furthermore, the polar localization of the stator was decreased in a Delta fliL mutant, but the sodium-dependent assembly of the stator complex was still retained. The polar localization of FliL was lost in the absence of the stator complex, indicating that FliL interacts directly or indirectly with the stator. Our results suggest that FliL is localized along with the stator in order to support the motor functioning for swimming at high load conditions by maintaining the stator assembly.

    DOI: 10.1111/mmi.13103

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  71. The MinD homolog FlhG regulates the synthesis of the single polar flagellum of Vibrio alginolyticus Reviewed

    Hiroki Ono, Akari Takashima, Hikaru Hirata, Michio Homma, Seiji Kojima

    MOLECULAR MICROBIOLOGY   Vol. 98 ( 1 ) page: 130 - 141   2015.10

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    FlhG, a MinD homolog and an ATPase, is known to mediate the formation of the single polar flagellum of Vibrio alginolyticus together with FlhF. FlhG and FlhF work antagonistically, with FlhF promoting flagellar assembly and FlhG inhibiting it. Here, we demonstrate that purified FlhG exhibits a low basal ATPase activity. As with MinD, the basal ATPase activity of FlhG can be activated and the D171A residue substitution enhances its ATPase activity sevenfold. FlhG-D171A localizes strongly at the cell pole and severely inhibits motility and flagellation, whereas the FlhG K31A and K36Q mutants, which are defective in ATP binding, do not localize to the poles, cannot complement a flhG mutant and lead to hyperflagellation. A strong polar localization of FlhF is observed with the K36Q mutant FlhG but not with the wild-type or D171A mutant FlhG. Unexpectedly, an Ala substitution at the catalytic residue (D60A), which abolishes ATPase activity but still allows ATP binding, only slightly affects FlhG functions. These results suggest that the ATP-dependent polar localization of FlhG is crucial for its ability to downregulate the number of polar flagella. We speculate that ATP hydrolysis by FlhG is required for the fine tuning of the regulation.

    DOI: 10.1111/mmi.13109

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  72. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium Reviewed

    Norihiro Takekawa, Masayoshi Nishiyama, Tsuyoshi Kaneseki, Tamotsu Kanai, Haruyuki Atomi, Seiji Kojima, Michio Homma

    SCIENTIFIC REPORTS   Vol. 5   page: 12711   2015.8

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    Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 degrees C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 mu m s(-1) at 85 degrees C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.

    DOI: 10.1038/srep12711

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  73. Functional chimeras of flagellar stator proteins between E-coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E-coli Reviewed

    Yuuki Nishino, Yasuhiro Onoue, Seiji Kojima, Michio Homma

    MICROBIOLOGYOPEN   Vol. 4 ( 2 ) page: 323 - 331   2015.4

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    The bacterial flagellar motor has a stator and a rotor. The stator is composed of two membrane proteins, MotA and MotB in Escherichia coli and PomA and PomB in Vibrio alginolyticus. The Vibrio motor has a unique structure, the T ring, which is composed of MotX and MotY. Based on the structural information of PomB and MotB, we constructed three chimeric proteins between PomB and MotB, named PotB(91), PotB(129,) and PotB(138), with various chimeric junctions. When those chimeric proteins were produced with PomA in a motAB strain of E. coli or in pomAB and pomAB motX strains of Vibrio, all chimeras were functional in E. coli or Vibrio, either with or without the Tring, although the motilities were very weak in E. coli. Furthermore, we could isolate some suppressors in E. coli and identified the mutation sites on PomA or the chimeric B subunit. The weak function of chimeric PotBs in E. coli is derived mainly from the defect in the rotational switching of the flagellar motor. In addition, comparing the motilities of chimera strains in pomAB, PotB(138) had the highest motility. The difference between the origin of the 1 and 2 helices, E. coli MotB or Vibro PomB, seems to be important for motility in E. coli and especially in Vibrio.

    DOI: 10.1002/mbo3.240

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  74. Interaction of the C-Terminal Tail of FliF with FliG from the Na+-Driven Flagellar Motor of Vibrio alginolyticus Reviewed

    Ryo Ogawa, Rei Abe-Yoshizumi, Takaaki Kishi, Michio Homma, Seiji Kojima

    JOURNAL OF BACTERIOLOGY   Vol. 197 ( 1 ) page: 63 - 72   2015.1

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    Rotation of the polar flagellum of Vibrio alginolyticus is driven by a Na+-type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of the Vibrio FliF protein produced at high levels in Escherichia coli was found in the soluble fraction. Soluble FliF purifies as an oligomer of similar to 700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data from in vitro binding assays using the soluble FliF multimer correlate with data from in vivo functional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation in Vibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na+-driven flagellar motor, both in vivo and in vitro.

    DOI: 10.1128/JB.02271-14

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  75. The MinD homolog FlhG regulates the synthesis of the single polar flagellum of Vibrio alginolyticus. Reviewed

    Ono H, Takashima A, Hirata H, Homma M, Kojima S.

    Mol. Microbiol.   Vol. 98   page: 130-141   2015

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  76. FliL associates with the stator to support torque generation of the sodium-driven polar flagellar motor of Vibrio. Reviewed

    Zhu S, Kumar A, Kojima S, Homma M.

    Mol. Microbiol.   Vol. 98   page: 101-110   2015

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  77. Interaction of the C-terminal tail of FliF with FliG from the Na+-driven flagellar motor of Vibrio alginolyticus Reviewed

    Ogawa R, Abe-Yoshizumi R, Kishi T, Homma M, Kojima S.

    J. Bacteriol.   Vol. 197   page: 63-72   2015

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  78. Dynamism and regulation of the stator, the energy conversion complex of the bacterial flagellar motor. Invited Reviewed

    Seiji Kojima

    Curr. Opin. Microbiol.   Vol. 28   page: 66-71   2015

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  79. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria. Reviewed

    Ishii E, Chiba S, Hashimoto N, Kojima S, Homma M, Ito K, Akiyama Y, Mori H.

    Proc. Natl. Acad. Sci. USA   Vol. 112   page: E5513-E5522   2015

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  80. Structural Basis of the Assembly and Activation Mechanism of the Bacterial Flagellar Stator Complex

    IMADA Katsumi, KOJIMA Seiji

    X-RAYS   Vol. 57 ( 5 ) page: 291 - 296   2015

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    Bacteria swim in aqueous environment by rotating the flagellum driven by a reversible motor embedded in the cell membrane. The torque of the motor is generated by the rotor-stator interaction coupled with the ion flow through the stator. A dozen of stators assemble around the rotor and are fixed to the peptidoglycan layer. The ion-conductivity of the stator is coupled to its assembly, and proper anchoring of the stator is required for torque generation. We determined the crystal structures of C-terminal fragments of the B-subunit of the stator including the region responsible for anchoring the stator, and performed the functional analyses using the mutants designed based on the structure. These studies revealed that the conformational change of the N-terminal region of the fragment is essential to form a functional stator around the rotor.

    DOI: 10.5940/jcrsj.57.291

    CiNii Books

    Other Link: https://jlc.jst.go.jp/DN/JLC/20016787177?from=CiNii

  81. Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor Reviewed

    Shiwei Zhu, Masato Takao, Na Li, Mayuko Sakuma, Yuuki Nishino, Michio Homma, Seiji Kojima, Katsumi Imada

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 111 ( 37 ) page: 13523 - 13528   2014.9

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    The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomB(C)) at 2.0-angstrom resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121-153) and following the N-terminal two-thirds of alpha 1(154- 164) in PomBC changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.

    DOI: 10.1073/pnas.1324201111

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  82. Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na (+) -Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus Reviewed

    Norihiro Takekawa, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 196 ( 7 ) page: 1377 - 1385   2014.4

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    In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s)of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na (+)- driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na (+) -driven motor than in the MotA-FliG interactions in the H (+) -driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na (+) -driven motor seem to be more complex than those previously proposed in the H (+) -driven motor.

    DOI: 10.1128/JB.01392-13

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  83. Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor. Reviewed

    Onoue Y, Abe-Yoshizumi R, Gohara M, Kobayashi S, Nishioka N, Kojima S, Homma M.

    J. Biochem.   Vol. 155   page: 207-216   2014.3

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  84. Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor Reviewed

    Yasuhiro Onoue, Rei Abe-Yoshizumi, Mizuki Gohara, Shiori Kobayashi, Noriko Nishioka, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 155 ( 3 ) page: 207 - 216   2014.3

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    The membrane motor proteins, PomA (polar flagellar motility protein A) and PomB (polar flagellar motility protein B), of Vibrio alginolyticus form a stator complex that converts energy from the ion flow to mechanical work in bacterial flagellar motors. The cytoplasmic domain of PomA is believed to interact with the rotor protein FliG to make a torque. In this study, to investigate the function of the cytoplasmic domain of PomA, we constructed a series of fragments that flank the cytoplasmic loop of PomA between the second and third transmembrane (TM) domains (A-loop) and the C-terminal region, and expressed them in Escherichia coli together with PomA and PotB (a chimeric protein of PomB and MotB). We observed a dominant-negative effect of one PomA fragment on motility. We confirmed that these PomA fragments localized both in the membrane fraction and in the cytoplasmic fraction, and induced bacterial growth delay. Effect of additional point and deletion mutations into this fragment implies that the C-terminal region and TM domains used as a linker play a significant part in these observations. From these results, we conclude that the PomA fragments retain the structure important for functions. We expect that further constructions will provide a variety of experimental approaches to characterize the interaction between PomA and FliG.

    DOI: 10.1093/jb/mvt115

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  85. Biophysical characterization of the C-terminal region of FliG, an essential rotor component of the Na+-driven flagellar motor Reviewed

    Mizuki Gohara, Shiori Kobayashi, Rei Abe-Yoshizumi, Natsumi Nonoyama, Seiji Kojima, Yasuo Asami, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 155 ( 2 ) page: 83 - 89   2014.2

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    The bacterial flagellar motor generates a rotational force by the flow of ions through the membrane. The rotational force is generated by the interaction between the cytoplasmic regions of the rotor and the stator. FliG is directly involved in the torque generation of the rotor protein by its interaction. FliG is composed of three domains: the N-terminal, Middle and C-terminal domains, based on its structure. The C-terminal domain of FliG is assumed to be important for the interaction with the stator that generates torque. In this study, using CD spectra, gel filtration chromatography and DSC (differential scanning calorimetry), we characterized the physical properties of the C-terminal domain (G214-Stop) of wild-type (WT) FliG and its non-motile phenotype mutant derivatives (L259Q, L270R and L271P), which were derived from the sodium-driven motor of Vibrio. The CD spectra and gel filtration chromatography revealed a slight difference between the WT and the mutant FliG proteins, but the DSC results suggested a large difference in their stabilities. That structural difference was confirmed by differences in protease sensitivity. Based on these results, we conclude that mutations which confer the non-motile phenotype destabilize the C-terminal domain of FliG.

    DOI: 10.1093/jb/mvt100

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  86. Structure, gene regulation and environmental response of flagella in Vibrio Reviewed

    Shiwei Zhu, Seiji Kojima, Michio Homma

    FRONTIERS IN MICROBIOLOGY   Vol. 4 ( 410 ) page: 410   2013.12

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    Vibrio species are Gram-negative, rod-shaped bacteria that live in aqueous environments. Several species, such as V harveyi, V alginotyticus, and V splendidus, are associated with diseases in fish or shellfish. In addition, a few species, such as V cholerae and V parahaemolyticus, are risky for humans due to infections from eating raw shellfish infected with these bacteria or from exposure of wounds to the marine environment. Bacterial flagella are not essential to live in a culture medium. However, most Vibrio species are motile and have rotating flagella which allow them to move into favorable environments or to escape from unfavorable environments. This review summarizes recent studies about the flagellar structure, function, and regulation of Vibrio species, especially focused on the Na+-driven polar flagella that are principally responsible for motility and sensing the surrounding environment, and discusses the relationship between flagella and pathogenicity.

    DOI: 10.3389/fmicb.2013.00410

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  87. Mutation in the a-subunit of F1FO-ATPase causes an increased motility phenotype through the sodium-driven flagella of Vibrio Reviewed

    Hiroyuki Terashima, Takashi Terauchi, Kunio Ihara, Noriko Nishioka, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 154 ( 2 ) page: 177 - 184   2013.8

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    Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion as energy source and are composed of stator and rotor proteins. Vibrio alginolyticus has a Na+-driven motor and its stator is composed of PomA and PomB. Recently, we isolated increased motility strains (sp1-sp4) from the PomA-N194D/PomB-D24N mutant whose motility was quite weak. To detect the responsible mutation, we have used a next-generation sequencer and determined the entire genome sequences of the sp1 and sp2 strains. Candidate mutations were identified in the gene encoding the a-subunit of F1Fo-ATPase (uncB). To confirm this, we constructed a deletion strain, which gave the increased motility phenotype. The amount of membrane-bound ATPase was reduced in the sp2 and delta uncB mutants. From these results, we conclude that a mutation in the uncB gene causes the increased motility phenotype in V. alginolyticus. They confer faster motility in low concentrations of sodium than in the parental strain and this phenotype is suppressed in the presence of KCN. Those results may suggest that the proton gradient generated by the respiratory chain is increased by the uncB mutation, consequently the sodium motive force is increased and causes the increased motility phenotype.

    DOI: 10.1093/jb/mvt042

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  88. Fluorescence imaging of GFP-fused periplasmic components of Na+-driven flagellar motor using Tat pathway in Vibrio alginolyticus Reviewed

    Norihiro Takekawa, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 153 ( 6 ) page: 547 - 553   2013.6

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    The twin-arginine translocation (Tat) system works to export folded proteins across the cytoplasmic membrane via specific signal peptides harbouring a twin-arginine motif. In Escherichia coli, a functional GFP is exported to the periplasm through the Tat pathway by fusion of the signal peptide of TorA, which is one of the periplasmic proteins exported by the Tat pathway. In this study, we fused the signal peptide of Vibrio alginolyticus TorA (TorA(SP)) to GFP and demonstrate the export of functional GFP to the periplasm of V. alginolyticus. We also made fusions of TorA(SP)-GFP with MotX, MotY and FlgT, which are periplasmic components of the Na+-driven flagellar motor. Those fusion proteins were localized to the flagellar motor independent of the Na+ concentration in the environment.

    DOI: 10.1093/jb/mvt017

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  89. Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA Reviewed

    Norihiro Takekawa, Takashi Terauchi, Yusuke V. Morimoto, Tohru Minamino, Chien-Jung Lo, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 153 ( 5 ) page: 441 - 451   2013.5

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    PomA and PomB form the stator complex, which functions as a Na+ channel, in the Na+-driven flagellar motor of Vibrio alginolyticus. The plug region of PomB is thought to regulate the Na+ flow and to suppress massive ion influx through the stator channel. In this study, in order to measure the Na+ conductivity of the unplugged stator, we over-produced a plug-deleted stator of the Na+-driven flagellar motor in Escherichia coli. The over-production of the plug-deleted stator in E. coli cells caused more severe growth inhibition than in Vibrio cells and that growth inhibition depended on the Na+ concentration in the growth medium. Measurement of intracellular Na+ concentration by flame photometry and fluorescent analysis with a Na+ indicator, Sodium Green, revealed that over-production of the plug-deleted stator increased the Na+ concentration in cell. Some mutations in the channel region of PomB or in the cytoplasmic region of PomA suppressed both the growth inhibition and the increase in intracellular Na+ concentration. These results suggest that the level of growth inhibition correlates with the intracellular Na+ concentration, probably due to the Na+ conductivity through the stator due to the mutations.

    DOI: 10.1093/jb/mvt011

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  90. Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT Reviewed

    Hiroyuki Terashima, Na Li, Mayuko Sakuma, Masafumi Koike, Seiji Kojima, Michio Homma, Katsumi Imada

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 110 ( 15 ) page: 6133 - 6138   2013.4

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    Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus, is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coli. The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FlgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FlgT at 2.0-angstrom resolution. FlgT is composed of three domains, the N-terminal domain (FlgT-N), the middle domain (FlgT-M), and the C-terminal domain (FlgT-C). FlgT-M is similar to the N-terminal domain of TolB, and FlgT-C resembles the N-terminal domain of Flil and the alpha/beta subunits of F-1-ATPase. To elucidate the role of each domain, we prepared domain deletion mutants of FlgT and analyzed their effects on the basal-body ring formation. The results suggest that FlgT-N contributes to the construction of the H-ring structure, and FlgT-M mediates the T-ring association on the LP ring. FlgT-C is not essential but stabilizes the H-ring structure. On the basis of these results, we propose an assembly mechanism for the basal-body rings and the stator units of the sodium-driven flagellar motor.

    DOI: 10.1073/pnas.1222655110

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  91. A Novel dnaJ Family Gene, sflA, Encodes an Inhibitor of Flagellation in Marine Vibrio Species Reviewed

    Maya Kitaoka, Takehiko Nishigaki, Kunio Ihara, Noriko Nishioka, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 195 ( 4 ) page: 816 - 822   2013.2

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    The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The Delta flhF mutant makes no flagellum, whereas the Delta flhFG double-deletion mutant usually lacks a flagellum. However, the Delta flhFG mutant occasionally reverts to become motile by forming peritrichous flagella. We have isolated a suppressor pseudorevertant from the Delta flhFG strain (Delta flhFG-sup). The suppressor strain forms peritrichous flagella in the majority of cells. We identified candidate suppressor mutations by comparing the genome sequence of the parental strain, VIO5, with the genome sequences of the suppressor strains. Two mutations were mapped to a gene, named sflA (suppressor of Delta flhFG), at the VEA003730 locus of the Vibrio sp. strain EX25 genome. This gene is specific for Vibrio species and is predicted to encode a transmembrane protein with a DnaJ domain. When the wild-type gene was introduced into the suppressor strain, motility was impaired. Introducing a mutant version of the sflA gene into the Delta flhFG strain conferred the suppressor phenotype. Thus, we conclude that loss of the sflA gene is responsible for the suppressor phenotype and that the wild-type SflA protein plays a role in preventing polar-type flagella from forming on the lateral cell wall.

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  92. Mutation in the a-subunit of F1FO-ATPase causes an increased motility phenotype through the sodium-driven flagella of Vibrio. Reviewed

    Terashima H, Terauchi T, Ihara K, Nishioka N, Kojima S, Homma M.

    J. Biochem.   Vol. 154   page: 177-184   2013

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  93. Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT. Reviewed

    Terashima H, Li N, Sakuma M, Koike M, Kojima S, Homma M, Imada K.

    Proc. Natl. Acad. Sci. U. S. A.   Vol. 110   page: 6133-6138   2013

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  94. Fluorescence imaging of GFP-fused periplasmic components of Na+-driven flagellar motor using Tat pathway in Vibrio alginolyticus. Reviewed

    Takekawa N, Kojima S, Homma M.

    J. Biochem.   Vol. 153   page: 547-553   2013

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  95. Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor. Reviewed

    Abe-Yoshizumi R, Kobayashi S, Gohara M, Hayashi K, Kojima C, Kojima S, Sudo Y, Asami Y, Homma M.

    Biophysics   Vol. 9   page: 21-29   2013

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  96. Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA. Reviewed

    Takekawa N, Terauchi T, Morimoto YV, Minamino T, Lo CJ, Kojima S, Homma M.

    J. Biochem.   Vol. 153   page: 441-451   2013

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  97. A novel dnaJ family gene, sflA, encodes an inhibitor of flagellation in marine Vibrio species. Reviewed

    Kitaoka M, Nishigaki T, Ihara K, Nishioka N, Kojima S, Homma M.

    J. Bacteriol.   Vol. 195   page: 816-822   2013

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  98. Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor Reviewed

    Rei Abe-Yoshizumi, Shiori Kobayashi, Mizuki Gohara, Kokoro Hayashi, Chojiro Kojima, Seiji Kojima, Yuki Sudo, Yasuo Asami, Michio Homma

    Biophysics (Japan)   Vol. 9   page: 21 - 29   2013

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    Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function. © 2013 THE BIOPHYSICAL SOCIETY OF JAPAN.

    DOI: 10.2142/biophysics.9.21

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  99. Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB, a Chimeric Stator Protein of Sodium-Driven Flagella Reviewed

    Shiwei Zhu, Michio Homma, Seiji Kojima

    JOURNAL OF BACTERIOLOGY   Vol. 194 ( 24 ) page: 6728 - 6735   2012.12

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    The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H+ or Na+ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na+-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Delta 41-91], called PotB Delta L), which lacks the periplasmic linker region including the segment that works as a "plug" to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na+-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific alpha-helix that connects the transmembrane and peptidoglycan binding domains of PotB Delta L in the region of MotB. Overproduction of the PomA/PotB Delta L(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na+-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this alpha-helix optimizes PotB Delta L conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this alpha-helix plays a key role in assembly-coupled stator activation.

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  100. Bacterial Motility Measured by a Miniature Chamber for High-Pressure Microscopy Reviewed

    Masayoshi Nishiyama, Seiji Kojima

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   Vol. 13 ( 7 ) page: 9225 - 9239   2012.7

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    Hydrostatic pressure is one of the physical stimuli that characterize the environment of living matter. Many microorganisms thrive under high pressure and may even physically or geochemically require this extreme environmental condition. In contrast, application of pressure is detrimental to most life on Earth; especially to living organisms under ambient pressure conditions. To study the mechanism of how living things adapt to high-pressure conditions, it is necessary to monitor directly the organism of interest under various pressure conditions. Here, we report a miniature chamber for high-pressure microscopy. The chamber was equipped with a built-in separator, in which water pressure was properly transduced to that of the sample solution. The apparatus developed could apply pressure up to 150 MPa, and enabled us to acquire bright-field and epifluorescence images at various pressures and temperatures. We demonstrated that the application of pressure acted directly and reversibly on the swimming motility of Escherichia coli cells. The present technique should be applicable to a wide range of dynamic biological processes that depend on applied pressures.

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  101. Characterization of PomA Mutants Defective in the Functional Assembly of the Na+-Driven Flagellar Motor in Vibrio alginolyticus Reviewed

    Norihiro Takekawa, Na Li, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 194 ( 8 ) page: 1934 - 1939   2012.4

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    The polar flagellar motor of Vibrio alginolyticus rotates using Na+ influx through the stator, which is composed of 2 subunits, PomA and PomB. About a dozen stators dynamically assemble around the rotor, depending on the Na+ concentration in the surrounding environment. The motor torque is generated by the interaction between the cytoplasmic domain of PomA and the C-terminal region of FliG, a component of the rotor. We had shown previously that mutations of FliG affected the stator assembly around the rotor, which suggested that the PomA-FliG interaction is required for the assembly. In this study, we examined the effects of various mutations mainly in the cytoplasmic domain of PomA on that assembly. All mutant stators examined, which resulted in the loss of motor function, assembled at a lower level than did the wild-type PomA. A His tag pulldown assay showed that some mutations in PomA reduced the PomA-PomB interaction, but other mutations did not. Next, we examined the ion conductivity of the mutants using a mutant stator that lacks the plug domain, PomA/PomB(Delta L)(Delta 41-120), which impairs cell growth by overproduction, presumably because a large amount of Na+ is conducted into the cells. Some PomA mutations suppressed this growth inhibition, suggesting that such mutations reduce Na+ conductivity, so that the stators could not assemble around the rotor. Only the mutation H136Y did not impair the stator formation and ion conductivity through the stator. We speculate that this particular mutation may affect the PomA-FliG interaction and prevent activation of the stator assembly around the rotor.

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  102. Instantaneous and Quantitative Single Cells Viability Determination Using Dual Nanoprobe Inside ESEM Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Masaru Kojima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOTECHNOLOGY   Vol. 11 ( 2 ) page: 298 - 306   2012.3

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    We performed single pulses current measurement on single cells using dual nanoprobe through environmental scanning electron microscope nanomanipulator system. The ability to characterize the electrical property of single cells can be used as a novel method for cell viability detection in quantitative and instantaneous manners. The nanoprobe was successfully fabricated using focused ion beam tungsten deposition and etching processes. The characteristics of the nanoprobe were examined from the energy dispersion spectrometry and noise analyses. In this paper, for the first time, the electrical property of single cells under their native condition was presented. In order to apply this method for cell viability detection, two types of cells were used, i.e., dead cells and live cells. The results showed that there is a significant difference on the electrical measurement data between dead and live cells.

    DOI: 10.1109/TNANO.2011.2171989

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  103. Nanofork for Single Cells Adhesion Measurement via ESEM-Nanomanipulator System Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Masaru Kojima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOBIOSCIENCE   Vol. 11 ( 1 ) page: 70 - 78   2012.3

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    In this paper, single cells adhesion force was measured using a nanofork. The nanofork was used to pick up a single cell on a line array substrate inside an environmental scanning electron microscope (ESEM). The line array substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam(FIB) etching process while the line array substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line array substrate were used, i.e., 1 mu m and 2 mu m. Results showed that cells attached on the 1 mu m gap line array substrate required more force to be released as compared to the cells attached on the 1 mu m gap line array substrate.

    DOI: 10.1109/TNB.2011.2179809

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  104. ペリプラズム側構造から見たべん毛モーター構築とモーターの活性化機構 Reviewed

    小嶋誠司、今田勝巳

    生物物理   Vol. 52 ( 1 ) page: 18-21   2012.2

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  105. Assembly and Activation Mechanism of the Flagellar Stator Revealed by the Crystal Structure of Its Periplasmic Region

    KOJIMA Seiji, IMADA Katsumi

    Seibutsu Butsuri   Vol. 52 ( 1 ) page: 18 - 21   2012.1

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    Bacterial flagellar motor is an ion-driven supramolecular nanomachine embedded in the cell envelope. Rotor-stator interaction that couples to the specific ion translocation through the stator channel is the nature of torque generation. To produce fully functional motor, multiple stator complexes must be incorporated around the rotor at appropriate places. However, such stator assembly mechanism has not been investigated by the structural point of view. Here we describe stator assembly and activation mechanism revealed from the crystal structure of a motor component located in the periplasmic space, suggesting the dynamic conformational changes in the stator during its assembly-coupled activation.<br>

    DOI: 10.2142/biophys.52.018

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  106. Nanofork for single cells adhesion measurement via ESEM-nanomanipulator system Reviewed

    Ahmad MR, Nakajima M, Kojima M, Kojima S, Homma M, Fukuda T

    IEEE Trans Nanobioscience   Vol. 11   page: 70-78   2012

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  107. Characterization of PomA mutants defective in the functional assembly of the Na+-driven flagellar motor in Vibrio alginolyticus Reviewed

    Takekawa N, Li N, Kojima S, Homma M

    Journal of Bacteriology   Vol. 194   page: 1934-1939   2012

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  108. Bacterial motility measured by a miniature chamber for high-pressure microscopy. Reviewed

    Nishiyama M and Kojima S

    Int. J. Mol. Sci.   Vol. 13   page: 9225-9239   2012

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  109. Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella. Reviewed

    Zhu S, Homma M, Kojima S

    J. Bacteriol.   Vol. 194   page: 6728-6735   2012

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  110. Evaluation of the Single Yeast Cell's Adhesion to ITO Substrates With Various Surface Energies via ESEM Nanorobotic Manipulation System. Reviewed

    Shen Y, Ahmad MR, Nakajima M, Kojima S, Homma M, Fukuda T.

    IEEE Trans Nanobioscience   Vol. 10 ( 4 ) page: 217-224   2011.12

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  111. Evaluation of the Single Yeast Cell's Adhesion to ITO Substrates With Various Surface Energies via ESEM Nanorobotic Manipulation System Reviewed

    Yajing Shen, Mohd Ridzuan Ahmad, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOBIOSCIENCE   Vol. 10 ( 4 ) page: 217 - 224   2011.12

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    Cell-surface adhesion force is important for cell activities and the development of bio materials. In this paper, a method for in situ single cell (W303) adhesion force measurement was proposed based on nanorobotic manipulation system inside an environment scanning electron microscope (ESEM). An end effector was fabricated from a commercial atomic force microscope (AFM) cantilever by focused ion beam (FIB) etching. The spring constant of it was calibrated by nanomanipulation approach. Three kinds of hydrophilic and hydrophobic ITO plates were prepared by using VUV-irradiation and OTS coating techniques. The shear adhesion strength of the single yeast cell to each substrate was measured based on the deflection of the end effector. The results demonstrated that the cell adhesion force was larger under the wet condition in the ESEM environment than in the aqueous condition. It also showed that the cell adhesion force to hydrophilic surface was larger than that to the hydrophobic surface. Studies of single cell's adhesion on various plate surfaces and environments could give new insights into the tissue engineering and biological field.

    DOI: 10.1109/TNB.2011.2177099

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  112. Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na(+)-driven flagella of Vibrio alginolyticus. Reviewed

    Kojima S, Nonoyama N, Takekawa N, Fukuoka H, Homma M.

    J Mol Biol   Vol. 414 ( 1 ) page: 62-74   2011.11

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  113. Mutations Targeting the C-Terminal Domain of FliG Can Disrupt Motor Assembly in the Na+-Driven Flagella of Vibrio alginolyticus Reviewed

    Seiji Kojima, Natsumi Nonoyama, Norihiro Takekawa, Hajime Fukuoka, Michio Homma

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 414 ( 1 ) page: 62 - 74   2011.11

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    The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na+-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side. (C) 2011 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2011.09.019

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  114. Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter Reviewed

    Yajing Shen, Masahiro Nakajima, Seiji Kojima, Michio Homma, Masaru Kojima, Toshio Fukuda

    MEASUREMENT SCIENCE AND TECHNOLOGY   Vol. 22 ( 11 )   2011.11

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    Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

    DOI: 10.1088/0957-0233/22/11/115802

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  115. Sodium-driven motor of the polar flagellum in marine bacteria Vibrio Reviewed

    Na Li, Seiji Kojima, Michio Homma

    GENES TO CELLS   Vol. 16 ( 10 ) page: 985 - 999   2011.10

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    The Na+-driven bacterial flagellar motor is a molecular machine powered by an electrochemical potential gradient of sodium ions across the cytoplasmic membrane. The marine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim in liquid. The flagellar motor contains a basal body and a stator complexes, which are composed of several proteins. PomA, PomB, MotX, and MotY are thought to be essential components of the stator that are required to generate the torque of the rotation. Several mutations have been investigated to understand the characteristics and function of the ion channel in the stator and the mechanism of its assembly around the rotor to complete the motor. In this review, we summarize recent results of the Na+-driven motor in the polar flagellum of Vibrio.

    DOI: 10.1111/j.1365-2443.2011.01545.x

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  116. 細菌べん毛モーターエネルギー変換タンパク質の構造と機能 Invited Reviewed

    寺内尭史、小嶋誠司、本間道夫

    生化学   Vol. 83 ( 9 ) page: 822-833   2011.9

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  117. A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque Reviewed

    Takashi Terauchi, Hiroyuki Terashima, Seiji Kojima, Michio Homma

    MICROBIOLOGY-SGM   Vol. 157 ( Pt 8 ) page: 2422 - 2432   2011.8

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    Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

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  118. Characterization of the Periplasmic Region of PomB, a Na+-Driven Flagellar Stator Protein in Vibrio alginolyticus Reviewed

    Na Li, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 193 ( 15 ) page: 3773 - 3784   2011.8

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    The stator proteins PomA and PomB form a complex that couples Na+ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB(C)). To investigate the function of PomB(C), a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB(C) fragment consisting of residues 135 to 315 (PomB(C5)) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB(Delta L)) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na+ influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB(Delta L) stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal alpha-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a "plug" segment is important for the control of Na+ influx through the stator complex as well as for stator assembly.

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  119. Design and characterization of nanoknife with buffering beam for in situ single-cell cutting. Reviewed International journal

    Shen Y, Nakajima M, Yang Z, Kojima S, Homma M, Fukuda T

    Nanotechnology   Vol. 22 ( 30 ) page: 305701 - 305701   2011.7

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    A novel nanoknife with a buffering beam is proposed for single-cell cutting. The nanoknife was fabricated from a commercial atomic force microscopy (AFM) cantilever by focused-ion-beam (FIB) etching technique. The material identification of the nanoknife was determined using the energy dispersion spectrometry (EDS) method. It demonstrated that the gallium ion pollution of the nanoknife can be ignored during the etching processes. The buffering beam was used to measure the cutting force based on its deformation. The spring constant of the beam was calibrated based on a referenced cantilever by using a nanomanipulation approach. The tip of the nanoknife was designed with a small edge angle 5° to reduce the compression to the cell during the cutting procedure. For comparison, two other nanoknives with different edge angles, i.e. 25° and 45°, were also prepared. An in situ single-cell cutting experiment was performed using these three nanoknives inside an environmental scanning electron microscope (ESEM). The cutting force and the sample slice angle for each nanoknife were evaluated. It showed the compression to the cell can be reduced when using the nanoknife with a small edge angle 5°. Consequently, the nanoknife was capable for in situ single-cell cutting tasks.

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  120. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM Reviewed

    Yajing Shen, Masahiro Nakajima, Mohd Ridzuan Ahmad, Seiji Kojima, Michio Homma, Toshio Fukuda

    ULTRAMICROSCOPY   Vol. 111 ( 8 ) page: 1176 - 1183   2011.7

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    A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research. (C) 2011 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.ultramic.2011.02.008

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  121. Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker Reviewed

    Yajing Shen, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 409 ( 2 ) page: 160 - 165   2011.6

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    Cell's adhesion is important to cell's interaction and activates. In this paper, a novel method for cell-cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell-cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell-cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell-cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially. (C) 2011 Published by Elsevier Inc.

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  122. M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins Reviewed

    Yusuke V. Morimoto, Seiji Kojima, Keiichi Namba, Tohru Minamino

    PLOS ONE   Vol. 6 ( 5 ) page: e19598   2011.5

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    Background:. Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.
    Methodology/Principal Findings. Here, we we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.
    Conclusions/Significance: Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.

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  123. Buckling Nanoneedle for Characterizing Single Cells Mechanics Inside Environmental SEM Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOTECHNOLOGY   Vol. 10 ( 2 ) page: 226 - 236   2011.3

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    We propose a buckling nanoneedle as a force sensor for stiffness characterization of single cells. The buckling nanoneedle was easily fabricated by using focused ion beam etching from a commercialized atomic force microscope cantilever. There are notable advantages of using buckling nanoneedle for single cells stiffness characterizations. First, severe cell damage from an excessive indentation force could be prevented. Second, large variations in single cells stiffness property could be easily detected either from the dented mark on the cell surface after the indentation and/or by comparing the buckling length of the nanoneedle during the indentation. The calibrations of the buckling nanoneedle were done experimentally and numerically. The calibration results from both methods showed a good agreement. The calibration data show the relationship between the indentation force and the buckling length of the nanoneedle. This relationship was used for obtaining force data during a nanoindentation experiment between a buckling nanoneedle and single cells. We performed in situ measurements of mechanical properties of individual W303 wildtype yeast cells by using a buckling nanoneedle inside an integrated SEM (ESEM)-nanomanipulator system. Finer local stiffness property of single cells was compared at different pressure and different temperature ranges. This detection method of the stiffness variations of the single cells could be applied in the future fast disease diagnosis based on single-cell stiffness analysis.

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  124. Sodium-driven motor of the polar flagellum in marine bacteria Vibrio. Reviewed

    Li N, Kojima S, Homma M.

    Genes to Cells   Vol. 16 ( 10 ) page: 985-999   2011

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  125. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM. Reviewed

    Shen Y, Nakajima M, Ahmad MR, Kojima S, Homma M, Fukuda T.

    Ultramicroscopy   Vol. 111 ( 8 ) page: 1176-1183   2011

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  126. Design and characterization of nanoknife with buffering beam for in situ single-cell cutting. Reviewed

    Shen Y, Nakajima M, Yang Z, Kojima S, Homma M, Fukuda T.

    Nanotechnology   Vol. 22 ( 30 ) page: 305701   2011

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  127. A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque. Reviewed

    Terauchi T, Terashima H, Kojima S, Homma M.

    Microbiology   Vol. 157   page: 2422-2432   2011

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  128. Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus. Reviewed

    Li N, Kojima S, Homma M.

    J Bacteriol   Vol. 193 ( 15 ) page: 3773-3784   2011

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  129. Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker. Reviewed

    Shen Y, Nakajima M, Kojima S, Homma M, Fukuda T.

    Biochem Biophys Res Commun.   Vol. 409 ( 2 ) page: 160-165   2011

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  130. M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins Reviewed

    Morimoto YV, Kojima S, Namba K, Minamino T

    PLoS One   Vol. 6 ( 5 ) page: e19598   2011

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  131. Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the NA +-driven polar flagellum of Vibrio alginolyticus Reviewed

    Masafumi Koike, Noriko Nishioka, Seiji Kojima, Michio Homma

    Biophysics   Vol. 7   page: 59 - 67   2011

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    The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the Cterminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG. © 2011 THE BIOPHYSICAL SOCIETY OF JAPAN.

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  132. Fabrication and Application of Nanofork for Measuring Single Cells Adhesion Force inside ESEM Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Masaru Kojima, Seiji Kojima, Michio Homma, Toshio Fukuda

    ENABLING SCIENCE AND NANOTECHNOLOGY   Vol. 1341   page: 304 - +   2011

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    In this paper, single cell adhesion force was measured using a nanofork. The nanofork was used to pick-up a single cell on a line-patterned substrate inside ESEM. The line-patterned substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick-up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line-patterned substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line-patterned substrate were used, i.e. 1 mu m and 2 mu m. Results showed that cells attached on the 1 mu m gap line-patterned substrate required more force to be released as compared to the cells attached on the 2 mu m gap line-patterned substrate.

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  133. Structure and function of energy transduction protein complex of bacterial flagellar motor Reviewed

    Takashi Terauchi, Seiji Kojima, Michio Homma

    Seikagaku   Vol. 83 ( 9 ) page: 822 - 833   2011

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  134. The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor Reviewed

    Hiroyuki Terashima, Masafumi Koike, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 192 ( 21 ) page: 5609 - 5615   2010.11

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    In Vibrio alginolyticus, the flagellar motor can rotate at a remarkably high speed, ca. three to four times faster than the Escherichia coli or Salmonella motor. Here, we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla(-) and Mot(-) phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of Delta flgT cells revealed a smaller LP ring structure compared to the wild type, and most of the T ring was lost. His(6)-tagged FlgT could be coisolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.

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  135. Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor Reviewed

    Yohei Hizukuri, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 148 ( 3 ) page: 309 - 318   2010.9

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    The flagellar motor is composed of the stator and the rotor, and the interaction between the stator and the rotor at the cytoplasmic region is believed to produce mechanical force for the rotation of flagella. The periplasmic region of the stator has been proposed to play an important role in assembly around and incorporation into the motor. In this study, we provide evidence suggesting that the periplasmic region of the stator component MotB interacts with the P-ring component FlgI, which functions as a bearing for the rotor along with the L-ring protein FlgH, from a site-directed disulphide cross-linking approach. First, we prepared four FlgI and three MotB cysteine-substituted mutant proteins and co-expressed them in various combinations in Escherichia coli. We detected cross-linked combinations of FlgI G11C and MotB S248C when treated with the oxidant Cu-phenanthroline or bismaleimide cross-linkers. Furthermore, we performed Cys-scanning mutagenesis around these two residues and found additional combinations of cross-linked residues. Treatment with a protonophore CCCP significantly reduced the cross-linking efficiency between FlgI and MotB in flagellated cells, but not in non-flagellated cells. These results suggest a direct contact between MotB and FlgI upon assembly of the stator into a motor.

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  136. Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor Reviewed

    Hiroyuki Terashima, Seiji Kojima, Michio Homma

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 397 ( 3 ) page: 689 - 696   2010.4

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    Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H(+) or Na(+)) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na(+) across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na(+)-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na(+)-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex. (c) 2010 Elsevier Ltd. All rights reserved.

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  137. Nanoindentation Methods to Measure Viscoelastic Properties of Single Cells Using Sharp, Flat, and Buckling Tips Inside ESEM Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOBIOSCIENCE   Vol. 9 ( 1 ) page: 12 - 23   2010.3

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    In this paper, methods to measure viscoelastic properties of time-dependent materials are proposed using sharp, flat, and buckling tips inside an environmental SEM. Single W303 yeast cells were employed in this study. Each of the tips was used to indent single cells in a nanoindentation test. Three loading histories were used: 1) a ramp loading history, in which a sharp indenter was used; 2) a step loading history, in which a flat indenter was implemented; and 3) a fast unloading history, in which a buckling nanoneedle was applied. Analysis of the viscoelastic properties of single cells was performed for each of the loading histories by choosing an appropriate theory between the correspondence principle and the functional equation. Results from each of the tests show good agreement, from which strong conclusion can be drawn.

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  138. Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus Reviewed

    Masafumi Koike, Hiroyuki Terashima, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 192 ( 1 ) page: 375 - 378   2010.1

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    To investigate the Na+-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.

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  139. The flagellar basal body-associated protein FlgT is essential for a novel ring structure in the sodium-driven Vibrio motor. Reviewed

    Terashima H., Koike M., Kojima S. & Homma M.

    Journal of Bacteriology   Vol. 192   page: 5609-5615   2010

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  140. Isolation of basal bodies with C-ring components from the Na+-driven flagellar motor of Vibrio alginolyticus. Reviewed

    Koike M., Terashima H., Kojima S. & Homma M.

    Journal of Bacteriology   Vol. 192   page: 375-378   2010

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  141. Functional transfer of an essential aspartate for the ion-binding site in the stator proteins of the bacterial flagellar motor. Reviewed

    Terashima, H., Kojima, S & Homma M.

    Journal of Molecular Biology   Vol. 397   page: 689-696   2010

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  142. Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor. Reviewed

    Hizukuri Y., Kojima, S. & Homma, M.

    Journal of Biochemistry (Tokyo)   Vol. 148   page: 309-318   2010

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  143. Interaction between Na+ Ion and Carboxylates of the PomA-PomB Stator Unit Studied by ATR-FTIR Spectroscopy Reviewed

    Yuki Sudo, Yuya Kitade, Yuji Furutani, Masaru Kojima, Seiji Kojima, Michio Homma, Hideki Kandori

    BIOCHEMISTRY   Vol. 48 ( 49 ) page: 11699 - 11705   2009.12

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    Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na+ influx to torque generation in this supramolecular motor, but little is known about how Na+ associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na+ to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na+ affinity of Asp24 is estimated to be similar to 85 mM, close to the apparent K-m value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na+, but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na+ interaction sites in the PomA-PomB complex. This work reveals features of the Na+ pathway in the PomA-PomB Na+ channel by using vibrational spectroscopy.

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  144. Rotational Speed Control of Na+-Driven Flagellar Motor by Dual Pipettes Reviewed

    Kousuke Nogawa, Masaru Kojima, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOBIOSCIENCE   Vol. 8 ( 4 ) page: 341 - 348   2009.12

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    Single cell analysis has attracted much attention to reveal the detailed and localized biological information. Local environmental control technique is desired to analyze the detailed and localized properties of single cells. In this paper, we propose the local environmental control system with nano/micro dual pipettes to control the local reagent concentration dynamically and arbitrarily. Local environmental control by dual pipettes is applied to the rotational speed control of bacterial flagellar motor, which is a rotary molecular machine. We demonstrate quick response and iterative rotational speed control of Na+-driven flagellar motor in both accelerating and relaxing directions by switching the local spout between Na+-containing and Na+-free solutions with dual pipettes. It is shown that the rotational speedmight be controllable by changing the spouting velocity of Na+-containing solution with multiplying the applied dc voltage.

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  145. Mutational Analysis of the GTP-binding Motif of FlhF which Regulates the Number and Placement of the Polar Flagellum in Vibrio alginolyticus Reviewed

    Akiko Kusumoto, Noriko Nishioka, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 146 ( 5 ) page: 643 - 650   2009.11

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    Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.

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  146. Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB Reviewed

    Seiji Kojima, Katsumi Imada, Mayuko Sakuma, Yuki Sudo, Chojiro Kojima, Tohru Minamino, Michio Homma, Keiichi Namba

    MOLECULAR MICROBIOLOGY   Vol. 73 ( 4 ) page: 710 - 718   2009.8

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    Torque generation in the Salmonella flagellar motor is coupled to translocation of H(+) ions through the proton-conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan-binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C-terminal periplasmic fragment of MotB (MotBC) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton-conducting channel. Drastic conformational changes in the N-terminal portion of MotBC are required both for PG binding and the proton channel activation.

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  147. The peptidoglycan-binding (PGB) Domain of the Escherichia coli Pal Protein can also Function as the PGB Domain in E. coli Flagellar Motor Protein MotB Reviewed

    Yohei Hizukuri, John Frederick Morton, Toshiharu Yakushi, Seiji Kojima, Michio Hommay

    JOURNAL OF BIOCHEMISTRY   Vol. 146 ( 2 ) page: 219 - 229   2009.8

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    The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a motB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys mutations in the crystal structure of Escherichia coli Pal. We found that the MotB mutations that affected motility nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that, although the functions of MotB and Pal are very different, the PGB region of Pal is interchangeable with the PGB region of MotB.

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  148. Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors Reviewed

    Hajime Fukuoka, Tomoyuki Wada, Seiji Kojima, Akihiko Ishijima, Michio Homma

    MOLECULAR MICROBIOLOGY   Vol. 71 ( 4 ) page: 825 - 835   2009.2

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    The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H(+) or Na(+). The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na(+) motor of Vibrio alginolyticus, is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na(+)-binding site. Furthermore, we demonstrated that the coupling ion, Na(+), is required for stator assembly and that phenamil (an inhibitor of the Na(+)-driven motor) inhibited the assembly. Carbonyl cyanide m-chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na(+) influx through the channel, including Na(+) binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.

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  149. Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors. Reviewed

    Fukuoka, H., Wada, T., Kojima, S., Ishijima, A. & Homma, M.

    Molecular Microbiology   Vol. 71   page: 825-835   2009

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  150. Mutational analysis of the GTP-binding motif of FlhF which regulates the number and placement of the polar flagellum in Vibrio alginolyticus. Reviewed

    Kusumoto, A., Nishioka, N., Kojima, S. & Homma, M.

    Journal of Biochemistry (Tokyo)   Vol. 146   page: 643-650   2009

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  151. *Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB. Reviewed

    Kojima, S., Imada, K., Sakuma, M., Sudo, Y., Kojima, C., Minamino, T., Homma, M. & Namba, K.

    Molecular Microbiology   Vol. 73   page: 710-718   2009

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  152. Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors. Reviewed

    Sudo, Y., Terashima, H., Abe-Yoshizumi, R., Kojima, S. & Homma, M.

    Biophysics   Vol. 5   page: 45-52   2009

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  153. The peptidoglycan-binding (PGB) domain of the Escherichia coli Pal protein can also function as the PGB domain in E. coli flagellar motor protein MotB. Reviewed

    Hizukuri, Y., Morton, J.F., Yakushi, T., Kojima, S. & Homma, M.

    Journal of Biochemistry (Tokyo)   Vol. 146   page: 219-229   2009

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  154. Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors Reviewed

    Yuki Sudo, Hiroyuki Terashima, Rei Abe-Yoshizumi, Seiji Kojima, Michio Homma

    Biophysics   Vol. 5   page: 45 - 52   2009

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    Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively. © 2009.

    DOI: 10.2142/biophysics.5.45

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  155. Rotational Speed Control of Na+-driven Flagellar Motor by Nano/Micro Dual Pipettes

    Nogawa Kousuke, Kojima Masaru, Nakajima Masahiro, Kojima Seiji, Homma Michio, Fukuda Toshio

    2009 9TH IEEE CONFERENCE ON NANOTECHNOLOGY (IEEE-NANO)     page: 522 - 525   2009

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  156. Cell-free Synthesis of the Torque-Generating Membrane Proteins, PomA and PomB, of the Na+-driven Flagellar Motor in Vibrio alginolyticus Reviewed

    Hiroyuki Terashima, Rei Abe-Yoshizumi, Seiji Kojima, Michio Homma

    JOURNAL OF BIOCHEMISTRY   Vol. 144 ( 5 ) page: 635 - 642   2008.11

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    Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na+-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na+ channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.

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  157. Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation Reviewed

    Yong-Suk Che, Shuichi Nakamura, Seiji Kojima, Nobunori Kami-ike, Keiichi Namba, Tohru Minamino

    JOURNAL OF BACTERIOLOGY   Vol. 190 ( 20 ) page: 6660 - 6667   2008.10

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    MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/ deprotonation of Glu-33 but not maximum proton conductivity.

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  158. In-situ Single Cell Mechanics Characterization of Yeast Cells using Nanoneedles inside Environmental SEM

    AHMAD M. R.

    IEEE Trans. on Nanotech.   Vol. 7 ( 5 ) page: 607 - 616   2008.9

  159. The effects of cell sizes, environmental conditions, and growth phases on the strength of individual W303 yeast cells inside ESEM Reviewed

    Mohd Ridzuan Ahmad, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

    IEEE TRANSACTIONS ON NANOBIOSCIENCE   Vol. 7 ( 3 ) page: 185 - 193   2008.9

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    We performed in situ measurements of mechanical properties of individual W303 wild-type yeast cells by using an integrated environmental scanning electron microscope (ESEM)-nanomanipulator system. Compression experiments to penetrate the cell walls of single cells of different cell sizes (about 3-6 mu m diameter), environmental conditions (600 Pa and 3 mPa), and growth phases (early log, mid log, late log and saturation) were conducted. The compression experiments were performed inside ESEM, embedded with a 7 DOF nanomanipulator with a sharp pyramidal end effector and a cooling stage, i.e., a temperature controller. ESEM itself can control the chamber pressure. Data clearly show an increment in penetration force, i.e., 96 +/- 2, 124 +/- 10, 163 +/- 1, and 234 +/- 14 nN at 3, 4, 5, and 6 mu m cell diameters, respectively. Whereas, 20-fold increase in penetration forces was recorded at different environmental conditions for 5 mu m cell diameter, i.e., 163 +/- 1 nN and 2.95 +/- 0.23 mu N at 600 Pa (ESEM mode) and 3 mPa (HV mode), respectively. This was further confirmed from quantitative estimation of average cell rigidity through the Hertz model, i.e., ESEM mode (3.31 +/- 0.11 MPa) and HV mode (26.02 +/- 3.66 MPa) for 5 mu m cell diameter. Finally, the penetration forces at different cell growth phases also show the increment pattern from log (early, mid, and late) to saturation phases, i.e., 161 +/- 25, 216 +/- 15, 255 +/- 21, and 408 +/- 41 nN, respectively.

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  160. Insights into the stator assembly of the Vibrio flagellar motor from the crystal structure of MotY Reviewed

    Seiji Kojima, Akari Shinohara, Hiroyuki Terashima, Toshiharu Yakushi, Mayuko Sakuma, Michio Homma, Keiichi Namba, Katsumi Imada

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 105 ( 22 ) page: 7696 - 7701   2008.6

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    Rotation of the sodium-driven polar flagellum of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX, and MotY. PomA and PomB form a sodium-ion channel in the cytoplasmic membrane that functions as a stator complex to couple sodium-ion flux with torque generation. MotX and MotY are components of the T-ring, which is located beneath the P-ring of the polar flagellar basal body and is involved in incorporation of-the PomA/PomB complex into the motor. Here, we describe,the determination of the crystal structure of MotY at 2.9 angstrom resolution. The structure shows two distinct domains: an N-terminal domain (MotY-N) and a C-terminal domain (MotY-C). MotY-N has a unique structure. MotY-C contains a putative peptidoglycan-binding motif that is remarkably similar to those of peptidoglycan-binding proteins, such as Pal and RmpM, but this region is disordered in MotY. Motility assay of cells producing either of the MotY-N and MotY-C fragments and subsequent biochemical analyses indicate that MotY-N is essential for association of the stator units around the rotor, whereas MotY-C stabilizes the association by binding to the peptidoglycan layer. Based on these observations, we propose a model for the mechanism of stator assembly around the rotor.

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  161. Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor Reviewed

    Seiji Kojima, Yukio Furukawa, Hideyuki Matsunarni, Tohru Minamino, Kelichi Narnba

    JOURNAL OF BACTERIOLOGY   Vol. 190 ( 9 ) page: 3314 - 3322   2008.5

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    MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton How with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta 197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C) (Delta 197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.

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  162. Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus Reviewed

    Akiko Kusumoto, Akari Shinohara, Hiroyuki Terashima, Seiji Kojima, Toshiharu Yakushi, Michio Homma

    MICROBIOLOGY-SGM   Vol. 154 ( Pt 5 ) page: 1390 - 1399   2008.5

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    Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number of polar flagella is positively regulated by FlhF and negatively regulated by FIhG. We now show that Delta flhF cells are non-flagellated as are most Delta flhFG cells; however, some of the Delta flhFG cells have several flagella at lateral positions. We found that FlhF-GFP was localized at the flagellated pole, and its polar localization was seen more intensely in Delta flhFG cells. On the other hand, most of the FlhG-GFP was diffused throughout the cytoplasm, although some was localized at the pole. To investigate the FlhF-FlhG interaction, immunoprecipitation was performed by using an anti-FlhF antibody, and FIhG co-precipitated with FlhF. From these results we propose a model in which FlhF localization at the pole determines polar location and production of a flagellum, FIhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FIhG negatively regulates flagellar number in V alginolyticus cells.

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  163. Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor Reviewed

    Madoka Obara, Toshiharu Yakushi, Seiji Kojima, Michio Homma

    JOURNAL OF BACTERIOLOGY   Vol. 190 ( 10 ) page: 3565 - 3571   2008.5

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    Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na+-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na+-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na+-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na+-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.

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  164. Systematic Cys mutagenesis of FIgI, the flagellar P-ring component of Escherichia coli Reviewed

    Yohei Hizukuri, Seiji Kojima, Toshiharu Yakushi, Ikuro Kawagishi, Michio Homma

    MICROBIOLOGY-SGM   Vol. 154 ( Pt 3 ) page: 810 - 817   2008.3

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    The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli P-ring protein FIgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FIgI. Immunoblot analysis against FIgI protein revealed that the cellular amounts of five FIgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FIgI G21C, completely disrupted FIgI function. The five residues that impaired motility of the cells were localized in the N terminus of FIgI. To demonstrate which residue(s) of FIgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FIgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FIgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FIgI proteins, the highly conserved region in the N terminus, residues 1-120, of FIgI is speculated to play important roles in the stabilization of FIgI structure and the formation of the P ring by interacting with FIgI molecules and/or other flagellar components.

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  165. 極毛単べん毛のビブリオ菌から周毛多べん毛の変異体の単離とその解析

    西岡 典子, 楠本 晃子, 小嶋 誠司, 本間 道夫, 小嶋 勝

    日本細菌学雑誌   Vol. 63 ( 1 ) page: 100 - 100   2008.2

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  166. Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli. Reviewed

    Hizukuri Y, Kojima S, Yakushi T, Kawagishi I, Homma M.

    Microbiology   Vol. 154   page: 810-817   2008

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  167. Flagellar motility in bacteria: Structure and function of flagellar motor Invited Reviewed

    Terashima H, Kojima S, Homma M

    International review of cell and molecular biology   Vol. 270   page: 39-85   2008

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  168. Cell-free synthesis of the torque-generating membrane proteins, PomA and PomB, of the Na+-driven flagellar motor in Vibrio alginolyticus. Reviewed

    Terashima H, Yoshizumi R, Kojima S, Homma M.

    Journal of Biochemistry (Tokyo)   Vol. 144   page: 635-642   2008

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  169. Flagellar motility in bacteria structure and function of flagellar motor. Invited Reviewed

    Terashima, H., Kojima, S. & Homma, M.

    International Review of Cell and Molecular Biology   Vol. 270   page: 39-85   2008

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  170. Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation. Reviewed

    Che YS, Nakamura S, Kojima S, Kami-ike N, Namba K, Minamino T.

    J. Bacteriol.   Vol. 190   page: 6660-6667   2008

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  171. The effects of cell sizes, environmental conditions, and growth phases on the strength of individual W303 yeast cells inside ESEM. Reviewed

    Ahmad MR, Nakajima M, Kojima S, Homma M, Fukuda T.

    IEEE Trans Nanobioscience   Vol. 7   page: 185-193   2008

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  172. *Insights into the stator assembly of the Vibrio flagellar motor from the crystal structure of MotY. Reviewed

    Kojima S, Shinohara A, Terashima H, Yakushi T, Sakuma M, Homma M, Namba K, Imada K.

    Proc. Natl. Acad. Sci. USA   Vol. 105   page: 7696-7701   2008

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  173. *Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor. Reviewed

    Kojima S, Furukawa Y, Matsunami H, Minamino T, Namba K.

    Journal of Bacteriology   Vol. 190   page: 3314-3322   2008

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  174. Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus. Reviewed

    Kusumoto A, Shinohara A, Terashima H, Kojima S, Yakushi T, Homma M.

    Microbiology   Vol. 154   page: 1390-1399   2008

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  175. Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor. Reviewed

    Obara M, Yakushi T, Kojima S, Homma M.

    Journal of Bacteriology   Vol. 190   page: 3565-3571   2008

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  176. FLAGELLAR MOTILITY IN BACTERIA: STRUCTURE AND FUNCTION OF FLAGELLAR MOTOR Reviewed

    Hiroyuki Terashima, Seiji Kojima, Michio Homma

    INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY, VOL 270   Vol. 270   page: 39 - 85   2008

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    Bacterial flagella are filamentous organelles that drive cell locomotion. They thrust cells in liquids (swimming) or on surfaces (swarming) so that cells can move toward favorable environments. At the base of each flagellum, a reversible rotary motor, which is powered by the proton- or the sodium-motive force, is embedded in the cell envelope. The motor consists of two parts: the rotating part, or rotor, that is connected to the hook and the filament, and the nonrotating part, or stator, that conducts coupling ion and is responsible for energy conversion. Intensive genetic and biochemical studies of the flagellum have been conducted in Salmonella typhimurium and Escherichia coli, and more than 50 gene products are known to be involved in flagellar assembly and function. The energy-coupling mechanism, however, is still not known. In this chapter, we survey our current knowledge of the flagellar system, based mostly on studies from Salmonella, E. coli, and marine species Vibrio alginolyticus, supplemented with distinct aspects of other bacterial species revealed by recent studies.

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  177. Single evaluation of C. Elegans inside environmental scanning electron microscope Reviewed

    Masahiro Nakajima, Mohd Ridzuan Ahmad, Seiji Kojima, Michio Homma, Toshio Fukuda

    2008 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2008     page: 83 - 88   2008

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    This paper presents the single evaluation of Caenorhabditìs Elegans (C. Elegans) by the nanorobotic manipulation system inside an Environmental-Scanning Electron Microscope (E-SEM). C. Elegans has complex outer and inner structures constructed by approximately one thousand cells. Their fine structures were observed by E-SEM directly
    without any drying or dyeing processes. For example, the lateral alae are the surface mark of seam cell body. The observation environments are controlled under different E-SEM chamber pressures for clear observation of C. Elegans. The local stiffness evaluation method of C. Elegans was proposed by buckling nanoprobe. The Silicon nanoprobe was fabricated by Focus Ion Beam (FIB) etching at the tip of Atomic Force Microscope (AFM) cantilever. The measurement position can arbitrarily be controlled by the nanorobotic manipulator inside the E-SEM. The local stiffness on or around the lateral alae evaluated from the experimental results. This local stiffness measurement technique can readily be applied to reveal unknown biological local stiffness, cell health conditions and novel cell diagnosis. © 2008 IEEE.

    DOI: 10.1109/MHS.2008.4752427

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  178. Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane Reviewed

    Hajime Fukuoka, Yoshiyuki Sowa, Seiji Kojima, Akihiko Ishijima, Michio Homma

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 367 ( 3 ) page: 692 - 701   2007.3

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    The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a Delta fliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble. (c) 2007 Elsevier Ltd. All rights reserved.

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  179. Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane Reviewed

    Fukuoka H, Sowa Y, Kojima S, Ishijima A and Homma M

    Journal of Molecular Biology   Vol. 367 ( 3 ) page: 692-701   2007.3

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  180. Crystallization and preliminary X-ray analysis of MotY, a stator component of the Vibrio alginolyticus polar flagellar motor Reviewed

    Shinohara A, Sakuma M, Yakushi T, Kojima S, Namba K Homma M and Imada K

    Acta Crystallograph Sect F   Vol. 63   page: 89-92   2007.2

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  181. Crystallization and preliminary X-ray analysis of MotY, a stator component of the Vibrio alginolyticus polar flagellar motor Reviewed

    Akari Shinohara, Mayuko Sakuma, Toshiharu Yakushi, Seiji Kojima, Keiichi Namba, Michio Homma, Katsumi Imada

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   Vol. 63 ( Pt 2 ) page: 89 - 92   2007.2

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    The polar flagellum of Vibrio alginolyticus is rotated by the sodium motor. The stator unit of the sodium motor consists of four different proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium motor, form the T-ring structure attached to the LP ring in the periplasmic space. MotY has a putative peptidoglycan-binding motif in its C-terminal region and MotX is suggested to interact with PomB. Thus, MotX and MotY are thought to be required for incorporation and stabilization of the PomA/B complex. In this study, mature MotY composed of 272 amino-acid residues and its SeMet derivative were expressed with a C-terminal hexahistidine-tag sequence, purified and crystallized. Native crystals were grown in the hexagonal space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 104.1, c = 132.6 angstrom. SeMet-derivative crystals belonged to the same space group with the same unit-cell parameters as the native crystals. Anomalous difference Patterson maps of the SeMet derivative showed significant peaks in their Harker sections, indicating that the derivatives are suitable for structure determination.

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  182. The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation Reviewed

    Terashima H, Fukuoka H, Yakushi T, Kojima S and Homma M

    Molecular Microbiology   Vol. 62 ( 4 ) page: 1170-1180   2006.11

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  183. The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na+-driven flagella and required for stator formation Reviewed

    Hiroyuki Terashima, Hajime Fukuoka, Toshiharu Yakushi, Seiji Kojima, Michio Homma

    MOLECULAR MICROBIOLOGY   Vol. 62 ( 4 ) page: 1170 - 1180   2006.11

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    The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na+-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a Delta motX or Delta motY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.

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  184. [Topology of bacterial cells focused on polar-localized proteins]. Reviewed

    Kusumoto A, Kojima S, Homma M

    Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 61 ( 3 ) page: 325 - 337   2006.8

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  185. Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli Reviewed

    TF Braun, LQ Al-Mawsawi, S Kojima, DF Blair

    BIOCHEMISTRY   Vol. 43 ( 1 ) page: 35 - 45   2004.1

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    The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

    DOI: 10.1021/bi035406d

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    PubMed

  186. Solubilization and purification of the MotA/MotB complex of Escherichia coli Reviewed

    S Kojima, DF Blair

    BIOCHEMISTRY   Vol. 43 ( 1 ) page: 26 - 34   2004.1

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    Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of similar to300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. S-35-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.

    DOI: 10.1021/bi0354051

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  187. The bacterial flagellar motor: Structure and function of a complex molecular machine. Invited

    Kojima, S., & Blair, D.F.

    International Review of Cytology   Vol. 233   page: 93-134   2004

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  188. Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli Reviewed

    Braun, T.F., Al-Mawsawi, L.Q., Kojima, S., & Blair, D.F.

    Biochemistry   Vol. 43   page: 35-45   2004

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  189. Solubilization and purification of the MotA/MotB complex of Escherichia coli Reviewed

    Kojima, S., and Blair, D.F.

    Biochemistry   Vol. 43   page: 26-35   2004

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  190. The bacterial flagellar motor: Structure and function of a complex molecular machine Reviewed

    S Kojima, DF Blair

    INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL. 233   Vol. 233   page: 93 - 134   2004

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    DOI: 10.1016/S0074-7696(04)33003-2

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    PubMed

  191. Rotary motors (bacterial). Invited

    Kojima, S., & Blair, D.F.

    McGraw-Hill Yearbook of Science & Technology 2003     page: 363-366   2003

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  192. Conformational change in the stator of the bacterial flagellar motor Reviewed

    S Kojima, DF Blair

    BIOCHEMISTRY   Vol. 40 ( 43 ) page: 13041 - 13050   2001.10

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    MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.

    Web of Science

  193. *Conformational change in the stator of the bacterial flagellar motor. Reviewed

    Kojima, S., & Blair, D.F.

    Biochemistry   Vol. 40   page: 13041-13050   2001

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  194. A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor Reviewed

    S Kojima, T Shoji, Y Asai, Kawagishi, I, M Homma

    JOURNAL OF BACTERIOLOGY   Vol. 182 ( 11 ) page: 3314 - 3318   2000.6

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    PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio alginolyticus. We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.

    Web of Science

  195. A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor

    Kojima S, Shoji T, Asai Y, Kawagishi I, Homma M

    JOURNAL OF BACTERIOLOGY   Vol. 182 ( 11 ) page: 3314 - 3318   2000.6

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    Web of Science

  196. A slow-motility phenotype caused by substitutions at residue Asp31 in the PomA channel component of a sodium-driven flagellar motor.

    Kojima, S., Shoji, T., Asai, Y., Kawagishi, I & Homma, M.

    Journal of Bacteriology   Vol. 182   page: 3314-3318   2000

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  197. Random mutagenesis of the pomA gene encoding a putative channel component of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. International journal

    Seiji Kojima, Mitsuyo Kuroda, Ikuro Kawagishi, Michio Homma

    Microbiology (Reading, England)   Vol. 145   page: 1759 - 1767   1999.7

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    PomA and PomB are integral membrane proteins and are essential for the rotation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H(+)-driven motor, they are thought to form the Na(+)-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.

    DOI: 10.1099/13500872-145-7-1759

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    PubMed

  198. The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force Reviewed

    S Kojima, K Yamamoto, Kawagishi, I, M Homma

    JOURNAL OF BACTERIOLOGY   Vol. 181 ( 6 ) page: 1927 - 1930   1999.3

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    Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated, We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.

    Web of Science

  199. Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components Reviewed

    S Kojima, Y Asai, T Atsumi, Kawagishi, I, M Homma

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 285 ( 4 ) page: 1537 - 1547   1999.1

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    The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpa(r) ((m) under bar otility (r) under bar esistant to (p) under bar hen (a) under bar mil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpa(r) strains, NMB205 and NM8201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Go-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpa(r) phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex.(C) 1999 Academic Press.

    DOI: 10.1006/jmbi.1998.2377

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  200. *Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations of putative channel components. Reviewed

    Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I. & Homma, M.

    Journal of Molecular Biology   Vol. 285   page: 1537-1547   1999

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  201. The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force. Reviewed

    Kojima, S., Yamamoto, K., Kawagishi, I. & Homma, M.

    Journal of Bacteriology   Vol. 181   page: 1927-1930   1999

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  202. Random mutagenesis of the pomA gene encoding a putative channel component of the Na+-driven polar flagellar motor of Vibrio alginolyticus. Reviewed

    Kojima, S., Kuroda, M., Kawagishi, I. & Homma, M.

    Microbiology   Vol. 145   page: 1759-1767   1999

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  203. Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium Reviewed

    Y Asai, S Kojima, H Kato, N Nishioka, Kawagishi, I, M Homma

    JOURNAL OF BACTERIOLOGY   Vol. 179 ( 16 ) page: 5104 - 5110   1997.8

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    The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions, Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel, MotX Has proposed to be a component of a sodium channel, Were we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions, These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene. suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors mag have similar structure and function as proton driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.

    Web of Science

  204. Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium

    Asai Y, Kojima S, Kato H, Nishioka N, Kawagishi I, Homma M

    JOURNAL OF BACTERIOLOGY   Vol. 179 ( 16 ) page: 5104 - 5110   1997.8

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    Web of Science

  205. Characterization of polar-flagellar-length mutants in Vibrio alginolyticus Reviewed

    M Furuno, T Atsumi, T Yamada, S Kojima, N Nishioka, Kawagishi, I, M Homma

    MICROBIOLOGY-UK   Vol. 143 ( 5 ) page: 1615 - 1621   1997.5

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    Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof(+) Laf(-)), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5 +/- 0.9 mu m, 2.5 +/- 0.6 mu m and 11.2 +/- 3.6 mu m, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. in the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 mu m and 0.062 mu m, respectively, whereas those of the wild-type flagella were 1.5 mu m and 0.16 mu m. The relative amounts of the various molecular components of the long Pof were different from those of the wildtype or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.

    DOI: 10.1099/00221287-143-5-1615

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  206. Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor.

    Kojima, S, Atsumi, T, Muramoto, K, Kudo, S, Kawagishi, I, Homma, M

    Journal of Molecular Biology   Vol. 265 ( 3 ) page: 310 - 318   1997.1

  207. Characterization of the polar-flagellar length mutants in Vibrio alginolitycus. Reviewed

    Furuno, M., Atsumi, T., Kojima, S., Nishioka, N., Kawagishi, I. & Homma, M.

    Microbiology   Vol. 143   page: 1615-1621   1997

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  208. Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor. Reviewed

    Kojima, S., Atsumi, T., Muramoto, K., Kudo, S., Kawagishi, I. & Homma, M.

    Journal of Molecular Biology   Vol. 265   page: 310-318   1997

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  209. Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium. Reviewed

    Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi, I. & Homma, M.

    Journal of Bacteriology   Vol. 179   page: 5104-5110   1997

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  210. Bacterial Sodium-driven Flagellar Motor

    KOJIMA Seiji, KAWAGISHI Ikuro, HONMA Michio

    KAGAKU TO SEIBUTSU   Vol. 34 ( 11 ) page: 730 - 737   1996.11

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    Language:Japanese   Publisher:Japan Society for Bioscience, Biotechnology, and Agrochemistry  

    DOI: 10.1271/kagakutoseibutsu1962.34.730

    CiNii Books

    Other Link: https://jlc.jst.go.jp/DN/JALC/00083125607?from=CiNii

  211. Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus Reviewed

    M Homma, H Oota, S Kojima, Kawagishi, I, Y Imae

    MICROBIOLOGY-UK   Vol. 142   page: 2777 - 2783   1996.10

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    Chemotactic responses in Vibrio alginolyticus, which has lateral and polar flagellar systems in one cell, were investigated. A lateral-flagella-defective (Pof(+) Laf(-)) mutant, which has only a polar flagellum, usually swam forward by the pushing action of its flagellum and occasionally changed direction by backward swimming. When the repellent phenol was added, Pof(+) Laf(-) cells moved frequently forward and backward (tumbling state). The tumbling was derived from the frequent changing between counter-clockwise and clockwise (CW) rotation of the flagellar motor, as was confirmed by the tethered-cell method. Furthermore, we found that the tumbling cells did not adapt to the phenol stimulus. When the attractant serine was added, the phenol-treated cells ceased tumbling and swam smoothly, adapting to the attractant stimulus after several minutes. We isolated chemotaxis-defective (Che(-)) mutants from the Pof(+) Laf(-) mutant; the tumbling mutants were not isolated. One interesting mutant swam backwards continuously, with its flagellum leading the cell and its flagellar motor rotating CW continuously. A polar-flagella-defective mutant (Pof(-) Laf(+)) stopped swimming after phenol addition and then recovered swimming ability within 10 min, indicating that lateral flagella can adapt to the repellent stimulus. This may represent a functional difference between the two flagellar systems in Vibrio cells, and between the chemotaxis systems affecting the two types of flagella.

    Web of Science

  212. Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus

    Homma M, Oota H, Kojima S, Kawagishi I, Imae Y

    MICROBIOLOGY-UK   Vol. 142   page: 2777 - 2783   1996.10

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    Web of Science

  213. Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus.

    Homma, M., Oota, H., Kojima, S., Kawagishi, I. & Imae, Y.

    Microbiology   Vol. 142   page: 2777-2783   1996

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MISC 15

  1. Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. International journal

    Tatsuro Nishikino, Hiroto Iwatsuki, Taira Mino, Seiji Kojima, Michio Homma

    Journal of biochemistry   Vol. 167 ( 4 ) page: 389 - 398   2020.4

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    The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2-3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1-2) and TM3 and TM4 (loop3-4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3-4 but hardly labelled Cys residues in loop1-2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41-120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1-2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1-2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

    DOI: 10.1093/jb/mvz102

    PubMed

  2. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria (vol 112, pg E5513, 2015)

    Eiji Ishii, Shinobu Chiba, Narimasa Hashimoto, Seiji Kojima, Michio Homma, Koreaki Ito, Yoshinori Akiyama, Hiroyuki Mori

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 112 ( 51 ) page: E7157 - E7157   2015.12

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    DOI: 10.1073/pnas.1522482112

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  3. 超好熱菌Aquifex aeolicusのべん毛運動とモーター固定子タンパク質の機能解析・精製

    竹川宜宏, 西山雅祥, 土方敦司, 金関剛史, 郷原瑞樹, 真柳浩太, 小嶋誠司, 金井保, 白井剛, 本間道夫

    日本生体エネルギー研究会討論会講演要旨集   Vol. 40th   page: 76 - 77   2014.12

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    J-GLOBAL

  4. ビブリオ菌のタンパク質分泌マシナリー:SecDFパラログの発現制御機構

    MORI HIROYUKI, HASHIMOTO NARIMASA, KOJIMA SEIJI, HONMA MICHIO, AKIYAMA YOSHINORI

    日本生化学会大会(Web)   Vol. 85th   page: 2S15-6 (WEB ONLY)   2012

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    J-GLOBAL

  5. ビブリオ菌SecDF パラログの生理機能の解明に向けて Reviewed

    橋本成祐, 森博幸, 秋山芳展, 本間道夫, 小嶋誠司

    第9 回21世紀大腸菌研究会、長浜、2012年6月21日- 22日     2012

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  6. Evaluation of nanoknife's edge angle for single cell cutting by using nanorobotic manipulators inside ESEM

    Yajing Shen, Masahiro Nakajimat, Zhan Yang, Seiji Kojima, Michio Homma, Masaru Kojima, Toshio Fukuda

    Proceedings of the IEEE Conference on Nanotechnology     page: 155 - 160   2011

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    Cell cutting is an important step in cell analysis processes. To address the single cell cutting at nano scale, Three types of nanoknives are designed in this paper. The first type of nanoknive was fabricated from a tungsten and the second type was fabricated from an atomic force microscopy(AFM)cantilever. In order to protect the nanoknife tip during the cell cutting, a novel nanoknife with a buffering beam is proposed. This kind of nanoknife was fabricated from a commercial AFM cantilever by focused ion beam (FIB) etching technique. The buffering beam can be also used to measure the cutting force based on its deformation. The spring constant of the beam was calibrated based on a referenced cantilever by using a nanomanipulation approach. The tip of the nanoknife was designed with a small edge angle 5 to reduce the compression to the cell during the cutting procedure. For comparison, two other nanoknives with different edge angles, i.e. 25° and 45°, were also prepared. An in-situ single cell cutting experiment was performed using these three nanoknives inside an environmental scanning electron microscope (ESEM). The cutting force and the sample slice angle for each nanoknife were evaluated. It showed the compression to the cell can be reduced when using the nanoknife with a small edge angle 5°. Consequently, the nanoknife was capable for in- situ single cell cutting tasks. © 2011 IEEE.

    DOI: 10.1109/NANO.2011.6144510

    Scopus

  7. 周毛性ビブリオ菌となったflhFG二重欠失株の抑圧変異体の性質と変異同定の試み

    西岡典子, 小嶋勝, 小嶋誠司, 本間道夫

    日本細菌学雑誌   Vol. 65 ( 1 )   2010

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  8. 完全な状態での大腸菌べん毛モーターの単離を視野に入 れた固定子と軸受け間のジスルフィド架橋 Reviewed

    檜作洋平, 小嶋誠司, 本間道夫

    第6 回21 世紀大腸菌研究会, 熱海,2009 年6 月11 日-12 日     2009

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    Language:Japanese  

  9. 細菌べん毛モーターの再構成に向けた固定子と軸受け間 のジスルフィド架橋 Reviewed

    檜作洋平, 小嶋誠司, 本間道夫

    第82 回日本生化学会大会、神戸、2009 年10 月21 日 -24 日     2009

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    Language:Japanese  

  10. Vibrio alginolyticus極べん毛形成位置と本数制御に対するFlhFのGTP結合モチーフの関与

    楠本晃子, 西岡典子, 小嶋誠司, 本間道夫

    日本細菌学雑誌   Vol. 64 ( 1 )   2009

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  11. FlhGがFlhFの極局在を調節することで Vibrio alginolyticus 極べん毛は1本に制御されている

    楠本 晃子, 小嶋 誠司, 本間 道夫

    日本細菌学雑誌   Vol. 62 ( 1 ) page: 83 - 83   2007.2

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    Language:Japanese  

    CiNii Books

  12. Vibrio alginolyticusの極べん毛数制御因子FlhFとFlhGの相互作用解析

    楠本晃子, 薬師寿治, 小嶋誠司, 本間道夫

    日本細菌学雑誌   Vol. 61 ( 1 )   2006

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  13. Random mutagenesis of the pomA gene encoding a putative channel component of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. International journal

    Seiji Kojima, Mitsuyo Kuroda, Ikuro Kawagishi, Michio Homma

    Microbiology (Reading, England)   Vol. 145 ( Pt 7)   page: 1759 - 1767   1999.7

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    Language:English  

    PomA and PomB are integral membrane proteins and are essential for the rotation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H(+)-driven motor, they are thought to form the Na(+)-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.

    DOI: 10.1099/13500872-145-7-1759

    PubMed

  14. Rotation rate of the Na^+-driven flagellar motor under the expression contorl of the torque-generating component, PomA.

    Kojima S., Kudo S., Kawagishi I., Homma M.

    Biophysics   Vol. 38 ( 2 ) page: S72   1998.9

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    Language:Japanese   Publisher:The Biophysical Society of Japan General Incorporated Association  

    CiNii Books

  15. Analysis of the slow motile pomA mutants in the Na^+-driven flagellar motor

    Shoji T., Kojima S., Kawagishi I., Homma M.

    Biophysics   Vol. 38 ( 2 ) page: S72   1998.9

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    Language:Japanese   Publisher:The Biophysical Society of Japan General Incorporated Association  

    CiNii Books

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Presentations 7

  1. Cell-free synthesis of the torque generating bacterial flagellar motor proteins. International conference

    BIT Life Sciences' 3rd Annual Protein and Peptide Conference (PepCon-2010) 

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    Event date: 2010.3

    Language:English   Presentation type:Oral presentation (invited, special)  

  2. Structural insight into active flagellar motor formation through the periplasmic region of MotB International conference

    BLAST X 

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    Event date: 2009.1

    Language:English   Presentation type:Oral presentation (general)  

  3. Crystal structure of MotY, an essential component for the torque generation of sodium-driven polar flagellar motor of Vibrio alginolyticus International conference

    BLAST IX 

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    Event date: 2007.1

    Language:English   Presentation type:Oral presentation (general)  

  4. Conformational changes in the MotA/MotB stator complex of the bacterial flagellar motor International conference

    Bacterial locomotion and signal transduction VII meeting 

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    Event date: 2003

    Language:English   Presentation type:Oral presentation (general)  

  5. henamil-resistant mutations in the sodium-driven flagellar motor of Vibrio alginolyticus International conference

    Bacterial locomotion and signal transduction V meeting 

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    Event date: 1999

    Language:English   Presentation type:Oral presentation (general)  

  6. トルク発生ユニット構成タンパク質 PomA の発現制御下におけるNa+ 駆動型べん毛モーターの回転速度の解析

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    Event date: 1998

    Language:Japanese   Presentation type:Oral presentation (general)  

    Country:Japan  

  7. Na+駆動型鞭毛モーター回転阻害剤(フェナミル)耐性株の単離と解析

    第32回 日本生物物理学会年会 

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    Event date: 1994

    Language:Japanese   Presentation type:Oral presentation (general)  

    Country:Japan  

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KAKENHI (Grants-in-Aid for Scientific Research) 18

  1. 活性化型固定子から迫る細菌べん毛モーターエネルギー変換装置の機能発現メカニズム

    Grant number:23H02447  2023.4 - 2027.3

    科学研究費助成事業  基盤研究(B)

    小嶋 誠司

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    Authorship:Principal investigator 

    Grant amount:\18330000 ( Direct Cost: \14100000 、 Indirect Cost:\4230000 )

    全ての生物は、細胞膜を介したイオンの電気化学勾配エネルギー(イオン駆動力)を膜蛋白質により変換して、多様な生命現象に利用している。我々は、細菌がイオン駆動力を利用して運動器官のべん毛を回転させる仕組みの解明を目指している。べん毛は基部に存在するモーターにより回転し、そのエネルギー変換装置である固定子の回転がギアのように回転子に伝搬してモーターを駆動する「固定子回転モデル」が提唱されている。しかしモーターに組込まれて活性化し機能する固定子の実態は未だ明らかではない。本研究では、活性化固定子の性質、特にイオン透過特性や活性制御の仕組み、及び活性化後の固定子構造安定化の解明に挑む。

  2. 細菌べん毛本数を厳密に制御する分子機構

    2014.4 - 2016.3

    科学研究費補助金  新学術領域研究

    小嶋誠司

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    Authorship:Principal investigator 

  3. 細菌べん毛を1本に制御する仕組み

    2012.4 - 2014.3

    科学研究費補助金  新学術領域研究

    小嶋誠司

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    細胞の機能を担う超分子複合体は、細胞内の指定された場所に必要な数の分子が自己集合して機能している。この現象は高等生物だけでなく構造の単純な細菌でも共通に見られる。細菌は効率良く運動するために、べん毛の形成位置と本数を厳密に制御している。ビブリオ菌は細胞の極に1本だけべん毛を形成するため、細胞における超分子の位置と数の制御機構を解析するにおいて優れたモデル系である。我々は以前同一オペロン上に存在するGTPaseのFlhFとATP結合モチーフを持つFlhGがべん毛の本数と形成位置を制御していることを見いだした。FlhF自身は極に局在しべん毛数を正に制御し、FlhGはFlhFに作用し極局在を阻害することで負に制御しているが、なぜ極に1本だけべん毛を形成できるのかその詳細はまだ分かっていない。本研究ではまずFlhFとFlhGの生化学的活性が、本数・位置を制御するのか詳細に調べる。続いてFlhF/FlhGと結合する因子の免疫沈降と質量分析による同定、及び欠失変異体からの抑圧変異の取得を行うことで、FlhFとFlhGの関わるネットワークを調べる。そして、蛍光標識したFlhFの細胞内動態観察を本領域で開発される少数分子可視化技術を用いて行い、何分子のFlhFが極に集合することで1個の基部体形成を開始するのかを明らかにしたい。FlhFは分泌蛋白質の膜へのターゲティングを担うFtsY/Ffhとホモロジーがあり、同じGTPaseファミリーに属していることから、べん毛の本数・位置の制御が蛋白質輸送系と共通の機構で行われる可能性がある。高等生物にも一般化できる知見が得られると期待している。

  4. エネルギー変換ユニットの活性を制御する動的構造変換

    2012.4 - 2014.3

    科学研究費補助金 

    小嶋誠司

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    細菌べん毛モーターは、エネルギー変換を担う固定子が回転子周囲の適切な場所に十数個配置された状態で機能する。驚くべきことに、固定子はモーターへ集合と解離を繰り返す動的な性質を持つ。ビブリオ菌PomA/PomB固定子複合体は、モーターに集合するとPomBのペリプラズム領域に大きな構造変化を誘起して固定され、イオン透過能を活性化すると考えられているが、本当にPomBに機能に必須な構造変化が生じているのかは不明である。本研究では、精製固定子をリポソーム上に再構成し、PomBの構造変化を結晶構造から予測される構造変化部位に、局所環境変化により蛍光強度を変化させるプローブを導入して、構造変化の検出を試みる。

  5. 自己集合する細菌べん毛モーター固定子の結晶化

    2008.4 - 2010.3

    科学研究費補助金  特定領域研究

    小嶋 誠司

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    Authorship:Principal investigator 

  6. 細菌べん毛モーターの回転子複合体と固定子複合体の結晶化

    2006.4 - 2008.3

    科学研究費補助金  特定領域研究

    小嶋誠司

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    Authorship:Principal investigator 

  7. ナトリウムイオン駆動型細菌べん毛モーターの固定子における生化学的解析

    2006.4 - 2009.3

    科学研究費補助金  若手研究(B)

    小嶋誠司

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  8. Detection of the physical interaction between rotor and stator essential for torque generation in the bacterial flagellar motor

    Grant number:18K19293  2018.6 - 2020.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Kojima Seiji

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    Grant amount:\6240000 ( Direct Cost: \4800000 、 Indirect Cost:\1440000 )

    Bacterial flagellar motor is a rotary nanomachine driven by the ion-motive force across the cytoplasmic membrane. Rotational force is generated by the rotor-stator interaction that couples to ion conduction through the stator complex. Since the rotor-stator interaction occurs very transient and rapidly, it was not detected physically or biochemically in protein level. Here we employed the in vivo photo-crosslink method to capture this rapid interaction. In this approach, the photo-reactive but non-natural amino acid (pBPA) was site-directly incorporated into the rotor or stator protein respectively (FliG or MotA/PomA) in Eschericha coli cell. After the UV flash irradiation to the cell, the photo-crosslinked protein was detected by the SDS-PAGE followed by the immunoblotting. Using this method, we could detect for the first time FliG-MotA or FliG-PomA interactions between the residues reported genetically for the electrostatic interactions.

  9. Functional mechanism of the GTP/ATP-binding proteins that regulate bacterial flagellar number.

    Grant number:16H04774  2016.4 - 2020.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Kojima Seiji

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    Authorship:Principal investigator 

    Grant amount:\17290000 ( Direct Cost: \13300000 、 Indirect Cost:\3990000 )

    Vibrio alginolyticus has a single polar flagellum used for swimming motility. We found that FlhG, the negative regulator for flagellar number and ATPase, exists as a monomer in the presence of ATP and do not form dimer as seen for its paralog MinD. We suggest that an ATP-bound FlhG localizes at cell pole via the membrane protein HubP, and then directly inhibits FlhF, the positive regulator for flagellar number. On the other hand, FlhF intrinsically localizes at cell pole. We found that a GTP-bound FlhF forms dimer and its GTPase activity was stimulated by the N-terminal region of FlhG. Our current model proposes that the nucleotide-bound active states of FlhF and FlhG function at cell pole, and their regulatory activities to determine flagellar number is strictly balanced at pole, so as to generate only a single polar flagellum.

  10. 細菌べん毛本数を厳密に制御する分子機構

    Grant number:26115705  2014.4 - 2016.3

    日本学術振興会  科学研究費助成事業  新学術領域研究(研究領域提案型)

    小嶋 誠司

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    Authorship:Principal investigator 

    Grant amount:\9750000 ( Direct Cost: \7500000 、 Indirect Cost:\2250000 )

    ビブリオ菌は細胞の極に1本だけべん毛を形成する。極べん毛の位置と本数は、GTPaseのFlhFが正に、同一オペロン上のATPaseであるFlhGが負に制御することがわかっている。昨年、ATPase活性の高いFlhG変異体はべん毛本数を強く負に制御するが、ATPase活性を失った触媒部位の変異体でも本数を負に制御する活性が維持されていることを見出し、FlhGのATP結合能が機能に必須であることを論文にまとめて発表した。ATPの加水分解は、べん毛形成を1本のみに厳密に制御するためのファインチューニングの役割を果たしていると考えている。本年度は、FlhGのATP結合能を生化学的に検証することを目的として、精製したFlhGがアイソトープで標識されたATPに結合するかどうか検証を試みた。ところが、FlhG自身の凝集しやすい性質が解析を阻み、ATP結合を評価できなかった。次にFlhGが凝集する条件を検討したところ、ATPase活性が高くべん毛本数を強く負に制御するD171A変異体は野生型に比べて凝集しやすく、ATPを加えるとその凝集性が増すことがわかった。ATPase活性と凝集性には構造を介した相関があると考えられる。精製したFlhGの動的光散乱解析、及び細胞破砕液を分画して得た可溶性画分のゲルろ過クロマトグラフィーの結果から、同じファミリーに属するMinDとは異なり、FlhGは細胞質においてATP存在下でも単量体として存在する可能性が示唆された。FlhGはATP依存的に極に移行すると構造変化し、べん毛本数を負に制御する機能を発現している可能性を考えている。また、FlhFの細胞極における分子数の計測を、昨年度作成したFlhF-Venus融合タンパク質発現株を材料として、共同研究先の一分子観察が可能な顕微システムを用いて行った。10数個のFlhFが極に存在する様子が見え始めている。
    27年度が最終年度であるため、記入しない。
    27年度が最終年度であるため、記入しない。

  11. Generalization of harmonized supramolecular motility machinery and its diversity

    Grant number:24117001  2012.6 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    MIYATA Makoto, MORI Hiroyuki, UYEDA Q. Taro, KOJIMA Seiji, KATAYAMA Eisaku, KODERA Noriyuki, TAOKA Azuma, KAWAKAMI Masaru, KOUYAMA Tsutomu, ISHIWATA Shin'ichi, KITA Kiyoshi, SASAKAWA Chihiro, NAMBA Kiichi

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    Authorship:Collaborating Investigator(s) (not designated on Grant-in-Aid) 

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), “Harmonized supramolecular motility machinery and its diversity” is aimed at promoting "Studies on motility supramolecular motility machinery” at each stage and eventually clarifying them at atomic level. The general team of this area seriously received the significance of the existence of the research expenses category "Grant-in-Aid for Scientific Research on Innovative Areas New academic area" and aimed to give synergy effects to the whole area that cannot be obtained with other research funds. We performed the activities difficult for individual research groups, (1) activation of discussion, (2) development and provision of technology necessary for the development of the area, and (3) outreach activities.

  12. Dynamic conformational changes that regulate the energy-converting unit

    Grant number:24657087  2012.4 - 2014.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    KOJIMA Seiji, HOMMA Michio

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    Authorship:Principal investigator 

    Grant amount:\3640000 ( Direct Cost: \2800000 、 Indirect Cost:\840000 )

    The PomA/PomB complex, that functions as an energy-converting stator unit in the bacterial flagellar motor, becomes functional when installed into the appropriate place around the rotor. In the functional stator, it is believed that a conformational change in the periplasmic region of PomB is induced to anchor it and simultaneously to activate ion conduction activity of the stator. This study aims to detect such a conformational change using the fluorescent probe and the purified PomA/B complex reconstituted into the liposome. We are still on the way to establish the analyzing system, but we greatly improved the overproduction and purification procedure for the PomA/B complex, so that we could systematically screen conditions to reconstitute the functional stator complex to the liposome. We also succeeded in narrowing down the region where the conformational change occurs, by using the systematic disulfide bridge formation analysis.

  13. 細菌べん毛形成を1本に制御する仕組み

    Grant number:24115506  2012.4 - 2014.3

    日本学術振興会  科学研究費助成事業  新学術領域研究(研究領域提案型)

    小嶋 誠司

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    Grant amount:\7280000 ( Direct Cost: \5600000 、 Indirect Cost:\1680000 )

    海洋性細菌であるビブリオ菌の運動器官「べん毛」は、細胞の極に1本だけ形成するため、位置・数の制御機構の解析において優れた研究対象である。べん毛形成位置と数の制御には、GTPaseのFlhFとATP結合モチーフを持つFlhGが関与している。FlhFは極に局在しべん毛数を正に制御する一方、FlhGはFlhFに作用し極局在を阻害することで負に制御している。本研究では、なぜ極に1本だけべん毛を形成できるのかを明らかにするため、FlhFとFlhGの生化学的性質、FlhFとFlhGと作用する因子のネットワーク、細胞の極に局在するFlhFの分子数とべん毛本数の関係に着目した。残念ながらFlhFの分子数計測やネットワークの解析は2年間で進まなかったが、FlhGの生化学的性質とべん毛形成制御との関連について、明らかにすることが出来た。前年度に、変異体を用いた解析からFlhGのATPaseモチーフは機能に重要であり、FlhGの極局在がべん毛形成阻害に関与することが明らかとなっていたので、今年度はFlhGを精製し、実際にATPase活性を測定して、べん毛数制御との関係を探った。FlhGを大腸菌内で大量発現させると凝集が起こり、半数が沈殿してしまった。様々な条件を試して、可溶性画分に残ったものを、グリセロールを含み比較的高い塩濃度の緩衝液を用いることで精製に成功した。精製したFlhGは4ºCで3日間は安定に保たれ、2 mg/ml以上濃縮すると沈殿してしまう。精製後すぐにATPase活性を測定することで、確かにFlhGはATP加水分解活性を示し、べん毛数を減少させる変異体(D171A)ではその活性が非常に高いことが明らかとなった。従って、FlhGのATPase活性を適切に制御することで、極にあるFlhFの分子数が制御され、その結果べん毛の形成が1本に制御されていると考えられる。
    25年度が最終年度であるため、記入しない。
    25年度が最終年度であるため、記入しない。

  14. 自己集合する細菌べん毛モーター固定子の結晶化

    Grant number:20051009  2008 - 2009

    日本学術振興会  科学研究費助成事業  特定領域研究

    小嶋 誠司, 本間 道夫

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    Authorship:Principal investigator 

    Grant amount:\6400000 ( Direct Cost: \6400000 )

    細菌のべん毛モーターは、細胞表層に自己集合し、エネルギー変換を担う超分子複合体である。モーターの回転力は、エネルギー源となる共役イオンが固定子中を流れる際に生じる、固定子-回転子間相互作用により発生すると考えられている。回転力発生のメカニズムを明らかにするためには、モーターの心臓部である固定子の構造情報が不可欠である。本研究では、ビブリオ菌Na^+駆動型モーターの固定子PomA/PomB複合体の結晶化を試みた。今年度は、これまで困難であった複合体の膜からの抽出の際に、界面活性剤Cymal-5を用いることで可溶化と精製度の向上が見られたので、大量精製し結晶化のスクリーニングを行ったが、現在のところ結晶はまだ得られていない。
    我々は部分構造の結晶化も同時に行い、H^+駆動型のサルモネラ菌固定子蛋白質MotBのC末端ペリプラズム側断片(MotB_C)の結晶構造を分解能1.75Aで決定することが出来た。固定子はこの領域に存在する推定ペプチドグリカン結合(PGB)ドメインを介してPG層に固定されていると考えられている。またイオンの透過は固定子がモーターに設置されて始めて活性化される。本研究で用いたMotB_CにはPGBドメインだけでなく、ペリプラズム側において運動に必須な部分がすべて含まれている。MotB_Cは1つのドメインで構成され、二量体を形成していた。MotB_Cが予想以上にコンパクトな構造であるため、ペプチドグリカン層に作用し固定するためには、MotB_Cにおいて大きな構造変化が起こらなければならない。構造情報をもとに行った機能解析により、PGBドメインでの二量体形成が固定子のチャネル部分を形成する膜貫通ヘリックスの適切な配置に重要であること、MotB_CのN末端部分の大きな構造変化がPG結合とプロトンチャネルの活性化に必要であることが明らかとなった。

  15. Study on the mechanism of force generation in the sodium-driven flagellar motor

    Grant number:18074003  2006 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research on Priority Areas

    HOMMA Michio, KOJIMA Seiji, KOJIMA Seiji, KAKINUMA Yohimi, MURATA Takeshi, KOJIMA Seiji

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    Authorship:Coinvestigator(s) 

    The bacterial flagellar motor is a molecular machine powered by an electrochemical potential gradient of ions across the cytoplasmic membrane. The marine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim in liquid by Na^+ ions. Until this study, the ion flux pathway in the stator complex is almost unknown. We experimentally showed that Na^+ ions bind to PomB-24 by ATR-FTIR. Furthermore, the ion pathway was inferred by the mutations of the transmembrane regions of stator proteins. Next, we investigated the localization of the GFP-fused stator complex and we found that the stator is assembled into a functional motor around the rotor only in the presence of Na+ ions. Furthermore, we determined the crystal structure of a C-terminal periplasmic fragment of a stator protein and we could suggest that drastic conformational changes in the N-terminal portion of the stator protein are required both for PG binding and the ion channel activation.

  16. Study on the structure and funciotion of supramolecular flagellar motor

    Grant number:18207011  2006 - 2009

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (A)

    HOMMA Michio, KOJIMA Seiji

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    Authorship:Coinvestigator(s) 

    The proteins, PomA, PomB, MotX and MotY have been identified as the motor proteins that are necessary for the flagella rotation. The T ring newly found by us is necessary for the assembly. The structure of the component protein, MotY, of the T ring is determined with a 2.9 Å resolution. We proposed the molecule dynamic model of the flagella motor proteins to assemble as a functional motor. We showed that the C ring structure was easily dissociated from the flagella base body. Furthermore we found that FlgT was the basal body component to construct a ring outside of the LP ring. The ring was named an H ring. The new ring may contribute on the Vibro motor function as a strong shaft to support the high-speed rotation of the motor.

  17. Biochemical analysis of the stator in the sodium-driven bacterial flagellar motor

    Grant number:18770129  2006 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Young Scientists (B)

    KOJIMA Seiji

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    Authorship:Principal investigator 

    Grant amount:\3840000 ( Direct Cost: \3600000 、 Indirect Cost:\240000 )

  18. 細菌べん毛モーターの回転子複合体と固定子複合体の結晶化

    Grant number:18054010  2006 - 2007

    日本学術振興会  科学研究費助成事業  特定領域研究

    小嶋 誠司, 本間 道夫

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    Authorship:Principal investigator 

    Grant amount:\6500000 ( Direct Cost: \6500000 )

    1)海洋性ビブリオ菌極べん毛モーター固定子複合体PomA/PomBの結晶化へ向けた大量発現と精製
    細菌べん毛モーターのエネルギー変換機構を理解するためには、共役イオンの流入に共役したトルク発生を担う、固定子複合体の立体構造を解明することが必須である。我々はNa^+駆動型のビブリオ菌を材料に用い、PomA/PomB固定子複合体の結晶化に向けて、ヒスチジンタグを融合した複合体の大腸菌での大量発現系を構築した。現在、膜画分からの抽出に最適な界面活性剤のスクリーニングを行っている。
    2)サルモネラ菌固定子蛋白質MotBのペリプラズム断片の結晶化
    我々は固定子複合体だけでなく、可溶性断片による部分構造の結晶化も進めてきた。特に固定子蛋白質MotBは、そのC末端側に固定に関わるペプチドグリカン結合モチーフを持ち、部分構造としても興味深い。サルモネラ菌を材料に、このモチーフを含む可溶性のMotB断片(MotB_c)を作成し解析した。MotB_cは野生株の運動能を低下させることから、野生型MotBとヘテロダイマーを形成し固定子複合体の集合を阻害するか、モーター中の固定子結合部位に作用していると予想され、またペリプラズムにおいて安定な二量体を形成していた(文献1)。MotB_cの精製は容易で、1リットル培養あたり25mg程度の精製標品が得られる。結晶化が難航したため、MotB_cのNMRスペクトル解析を行ったところ、N末端側とC末端側の構造が不安定であることが予想された。そこで、これら両末端を削った複数種のMotB_c断片を作成し結晶化条件を探索したところ、N末端を10残基、C末端を18残基欠失させた断片MotB_<c6>において、実験室でのX線回折実験で高い分解能の回折像を得ることができた。現在Spring-8での回折実験を目標とし、MotB_<c6>のセレノメチオニン置換体結晶を準備している。

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