Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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超好熱菌Aquifex aeolicusの運動解析と大腸菌を用いたAquifexモーター固定子の機能解析

本間道夫, 小嶋誠司, 竹川宜宏, 西山雅祥, 金関剛史, 金井保

日本細菌学雑誌 Vol. 70 ( 1 ) page： 147 - 147 2015.2

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

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

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

Web of Science

PubMed

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

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

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

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

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

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

Language：Japanese Publisher：The Crystallographic Society of Japan

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

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

Language：English Publisher：FRONTIERS RESEARCH FOUNDATION

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

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.

DOI： 10.1128/JB.01850-12

Web of Science

PubMed

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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Papers - KOJIMA, Seiji