Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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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

Language：English Publishing type：Research paper (international conference proceedings) Publisher：AMER INST PHYSICS

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.

DOI： 10.1063/1.3587007

Web of Science

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

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

Hiroyuki Terashima, Masafumi Koike, Seiji Kojima, Michio Homma

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

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.00720-10

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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.

DOI： 10.1093/jb/mvq067

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

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.

DOI： 10.1016/j.jmb.2010.01.050

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

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.

DOI： 10.1109/TNB.2009.2034849

Web of Science

PubMed

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

Masafumi Koike, Hiroyuki Terashima, Seiji Kojima, Michio Homma

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

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.01121-09

Web of Science

PubMed

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

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

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

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

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

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

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.

DOI： 10.1021/bi901517n

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

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.

DOI： 10.1109/TNB.2009.2035281

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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.

DOI： 10.1093/jb/mvp109

Web of Science

PubMed

Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB Reviewed Open Access

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

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

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.

DOI： 10.1111/j.1365-2958.2009.06802.x

Web of Science

PubMed

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

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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.

DOI： 10.1093/jb/mvp061

Web of Science

PubMed

Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors Reviewed Open Access

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

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

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

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.

DOI： 10.1111/j.1365-2958.2008.06569.x

Web of Science

PubMed

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

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

Molecular Microbiology Vol. 71 page： 825-835 2009

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

*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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Papers - KOJIMA, Seiji