Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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

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

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

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

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

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

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

Language：English Publishing type：Part of collection (book) Publisher：ELSEVIER ACADEMIC PRESS INC

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.

DOI： 10.1016/S1937-6448(08)01402-0

Web of Science

PubMed

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

Language：English Publishing type：Research paper (international conference proceedings)

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

Scopus

Na+駆動型べん毛モーターにおける遊泳速度の圧力応答(Pressure-velocity relationship of sodium-driven flagellar motor of Vibrio alginolyticus)

下田義樹, 西山雅祥, 小嶋誠司, 本間道夫, 木村佳文, 寺嶋正秀

生物物理 Vol. 47 ( Suppl.1 ) page： S247 - S247 2007.11

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

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

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.

DOI： 10.1016/j.jmb.2007.01.015

Web of Science

PubMed

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

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

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

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

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

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

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.

DOI： 10.1107/S1744309106055850

Web of Science

PubMed

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

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

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

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

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

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.

DOI： 10.1111/j.1365-2958.2006.05435.x

Web of Science

PubMed

[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

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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

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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Papers - KOJIMA, Seiji