Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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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

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

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

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

Biophysics Vol. 5 page： 45 - 52 2009

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

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

Open Access

Scopus

PubMed

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

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

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

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.

DOI： 10.1093/jb/mvn110

Web of Science

PubMed

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

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

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

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.

DOI： 10.1128/JB.00503-08

Web of Science

PubMed

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

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

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

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.

DOI： 10.1109/TNB.2008.2002281

Web of Science

PubMed

Insights into the stator assembly of the Vibrio flagellar motor from the crystal structure of MotY Reviewed Open Access

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

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

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.

DOI： 10.1073/pnas.0800308105

Open Access

Web of Science

PubMed

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

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

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

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

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.

DOI： 10.1128/JB.01710-07

Web of Science

PubMed

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

Madoka Obara, Toshiharu Yakushi, Seiji Kojima, Michio Homma

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

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

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.

DOI： 10.1128/JB.00849-07

Web of Science

PubMed

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

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

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

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

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.

DOI： 10.1099/mic.0.2007/012641-0

Web of Science

PubMed

Systematic Cys mutagenesis of FIgI, the flagellar P-ring component of Escherichia coli Reviewed Open Access

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

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

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

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.

DOI： 10.1099/mic.0.2007/013854-0

Open Access

Web of Science

PubMed

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

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

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

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

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

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

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

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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Papers - KOJIMA, Seiji