研究者詳細 - 森本　雄祐

論文 - 森本　雄祐

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A lactose fermentation product produced bylactococcus lactis subsp Lactis acetate inhibitsthe motility of flagellated pathogenic bacteria 査読有り

Nakamura S., Morimoto Y., Kudo S.

Microbiology (United Kingdom) 161 ( 4 ) 701 - 707 2015年04月

記述言語：英語掲載種別：研究論文（学術雑誌）

© 2015 The Authors.Many strains of lactic acid bacteria have been used for the production of probiotics. Some metabolites produced by lactic acid bacteria impair the motilities of pathogenic bacteria. Because bacterial motility is strongly associated with virulence, the metabolic activities of lactic acid bacteria are effective for suppressing bacterial infections. Here we show that lactose fermentation by Lactococcus lactis subsp. lactis inhibits the motility of Salmonella enterica serovar Typhimurium. A single-cell tracking and rotation assay for a single flagellum showed that the swimming behaviour of Salmonella was severely but transiently impaired through disruption of flagellar rotation on exposure to media cultivated with Lac. lactis. Using a pH-sensitive fluorescent protein, we observed that the intracellular pH of Salmonella was decreased because of some fermentation products of Lac. lactis. We identified acetate as the lactose fermentation product of Lac. lactis triggering the paralysis of Salmonella flagella. The motilities of Pseudomonas, Vibrio and Leptospira strains were also severely disrupted by lactose utilization by Lac. lactis. These results highlight the potential use of Lac. lactis for preventing infections by multiple bacterial species.

DOI： 10.1099/mic.0.000031

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The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis 査読有り

Minamino T., Morimoto Y., Morimoto Y., Kinoshita M., Aldridge P., Namba K., Namba K.

Scientific Reports 4 2014年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI6 FliJ complex is structurally similar to the α3β 3γ complex of FO F1- -ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI 6 FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI 6 FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

DOI： 10.1038/srep07579

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Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus 査読有り

Bai F., Bai F., Morimoto Y., Morimoto Y., Yoshimura S., Hara N., Kami-Ike N., Namba K., Namba K., Minamino T.

Scientific Reports 4 2014年10月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

© 2014, Nature Publishing Group. All rights reserved.For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assembly-disassembly cycle of FliI during flagellar assembly. We propose that the FliH2FliI complex and FliI6 ring function as a dynamic substrate carrier and a static substrate loader, respectively.

DOI： 10.1038/srep06528

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Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body 査読有り国際誌

Morimoto Y., Morimoto Y., Ito M., Hiraoka K., Che Y., Bai F., Bai F., Kami-ike N., Namba K., Namba K., Minamino T.

Molecular Microbiology 91 ( 6 ) 1214 - 1226 2014年03月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

Summary: The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane-embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C-terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C-termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF-YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA-CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA-YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co-ordinated manner. © 2014 John Wiley & Sons Ltd.

DOI： 10.1111/mmi.12529

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Structure and function of the bi-directional bacterial flagellar motor 査読有り国際誌

Morimoto Y., Morimoto Y., Minamino T.

Biomolecules ( Biomolecules ) 4 ( 1 ) 217 - 234 2014年02月

担当区分：筆頭著者記述言語：英語掲載種別：記事・総説・解説・論説等（学術雑誌）

© 2014 by the authors; licensee MDPI, Basel, Switzerland.The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor-stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor.

DOI： 10.3390/biom4010217

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Load-sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor 査読有り国際誌

Che Y., Che Y., Nakamura S., Nakamura S., Morimoto Y., Morimoto Y., Kami-ike N., Namba K., Namba K., Minamino T.

Molecular Microbiology 91 ( 1 ) 175 - 184 2014年01月

記述言語：英語掲載種別：研究論文（学術雑誌）

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their 'unplugged' forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation. © 2013 John Wiley & Sons Ltd.

DOI： 10.1111/mmi.12453

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The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor 査読有り国際誌

Castillo David J., Nakamura Shuichi, Morimoto Yusuke V., Che Yong-Suk, Kami-ike Nobunori, Kudo Seishi, Minamino Tohru, Namba Keiichi

BIOPHYSICS ( The Biophysical Society of Japan ) 9 ( 0 ) 173 - 181 2013年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

The bacterial flagellar motor is made of a rotor and stators. In <i>Salmonella</i> it is thought that about a dozen MotA/B complexes are anchored to the peptidoglycan layer around the motor through the C-terminal peptidoglycan-binding domain of MotB to become active stators as well as proton channels. MotB consists of 309 residues, forming a single transmembrane helix (30-50), a stalk (51-100) and a C-terminal peptidoglycan-binding domain (101-309). Although the stalk is dispensable for torque generation by the motor, it is required for efficient motor performance. Residues 51 to 72 prevent premature proton leakage through the proton channel prior to stator assembly into the motor. However, the role of residues 72-100 remains unknown. Here, we analyzed the torque-speed relationship of the MotB(Δ72-100) motor. At a low speed near stall, this mutant motor produced torque at the wild-type level. Unlike the wild-type motor, however, torque dropped off drastically by slight decrease in external load and then showed a slow exponential decay over a wide range of load by its further reduction. Since it is known that the stator is a mechanosensor and that the number of active stators changes in a load-dependent manner, we interpreted this unusual torque-speed relationship as anomaly in load-dependent control of the number of active stators. The results suggest that residues 72-100 of MotB is required for proper load-dependent control of the number of active stators around the rotor.<br>

DOI： 10.2142/biophysics.9.173

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Common and distinct structural features of Salmonella injectisome and flagellar basal body 査読有り

Kawamoto A., Kawamoto A., Morimoto Y., Morimoto Y., Miyata T., Minamino T., Hughes K., Kato T., Namba K., Namba K.

Scientific Reports 3 2013年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

DOI： 10.1038/srep03369

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Role of the Dc domain of the bacterial hook protein FlgE in hook assembly and function 査読有り国際誌

Moriya N., Minamino T., Ferris H., Morimoto Y., Morimoto Y., Ashihara M., Kato T., Namba K., Namba K.

Biophysics (Japan) 9 63 - 72 2013年06月

記述言語：英語掲載種別：研究論文（学術雑誌）

The bacterial flagellar hook acts as a universal joint to smoothly transmit torque produced by the motor to the filament. The hook protein FlgE assembles into a 55 nm tubular structure with the help of the hook cap (FlgD). FlgE consists of four domains, D0, Dc, D1 and D2, arranged from the inner to the outer part of the tubular structure of the hook. The Dc domain contributes to the structural stability of the hook, but it is unclear how this Dc domain is responsible for the universal joint mechanism. Here, we carried out a deletion analysis of the FlgE Dc domain. FlgEΔ4/5 with deletion of residues 30 to 49 was not secreted into the culture media. FlgEΔ5 and FlgEΔ6 with deletions of residues 40 to 49 and 50 to 59, respectively, still formed hooks, allowing the export apparatus to export the hook-filament junction proteins FlgK and FlgL and flagellin FliC. However, these deletions inhibited the replacement of the FlgD hook cap by FlgK at the hook tip, thereby abolishing filament formation. Deletion of residues 50 to 59 significantly affected hook morphology. These results suggest that the Dc domain is responsible not only for hook assembly but also for FlgE export, the interaction with FlgK, and the polymorphic supercoiling mechanism of the hook. © 2013 THE BIOPHYSICAL SOCIETY OF JAPAN.

DOI： 10.2142/biophysics.9.63

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Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA 査読有り国際誌

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

Journal of Biochemistry 153 441 - 451 2013年05月

Distinct roles of highly conserved charged residues at the MotA-FliG interface in bacterial flagellar motor rotation 査読有り国際誌

Morimoto Y., Morimoto Y., Nakamura S., Hiraoka K., Namba K., Namba K., Minamino T.

Journal of Bacteriology 195 ( 3 ) 474 - 481 2013年02月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

Electrostatic interactions between the stator protein MotA and the rotor protein FliG are important for bacterial flagellar motor rotation. Arg90 and Glu98 of MotA are required not only for torque generation but also for stator assembly around the rotor, but their actual roles remain unknown. Here we analyzed the roles of functionally important charged residues at the MotA-FliG interface in motor performance. About 75% of the motA(R90E) cells and 45% of the motA(E98K) cells showed no fluorescent spots of green fluorescent protein (GFP)-MotB, indicating reduced efficiency of stator assembly around the rotor. The FliG(D289K) and FliG(R281V) mutations, which restore the motility of the motA(R90E) and motA(E98K) mutants, respectively, showed reduced numbers and intensity of GFP-MotB spots as well. The FliG(D289K) mutation significantly recovered the localization of GFP-MotB to the motor in the motA(R90E) mutant, whereas the FliG(R281V) mutation did not recover the GFP-MotB localization in the motA(E98K) mutant. These results suggest that the MotA-Arg90-FliG-Asp289 interaction is critical for the proper positioning of the stators around the rotor, whereas the MotA-Glu98-FliG-Arg281 interaction is more important for torque generation. © 2013, American Society for Microbiology.

DOI： 10.1128/JB.01971-12

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Isolation of Salmonella Mutants Resistant to the Inhibitory Effect of Salicylidene acylhydrazides on Flagella-Mediated Motility 査読有り国際誌

Martinez-Argudo I., Martinez-Argudo I., Veenendaal A., Veenendaal A., Liu X., Roehrich A., Ronessen M., Franzoni G., van Rietschoten K., Morimoto Y., Saijo-Hamano Y., Avison M., Studholme D., Namba K., Minamino T., Minamino T., Blocker A., Blocker A.

PLoS ONE 8 ( 1 ) 2013年01月

記述言語：英語掲載種別：研究論文（学術雑誌）

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB(P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB(P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification. © 2013 Martinez-Argudo et al.

DOI： 10.1371/journal.pone.0052179

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Interaction of the extreme N-terminal region of flih with FlhA is required for efficient bacterial flagellar protein export 査読有り

Hara N., Morimoto Y., Morimoto Y., Kawamoto A., Kawamoto A., Namba K., Namba K., Minamino T., Minamino T.

Journal of Bacteriology 194 ( 19 ) 5353 - 5360 2012年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliHEN) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliHEN with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export. © 2012, American Society for Microbiology.

DOI： 10.1128/JB.01028-12

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Assembly and activation of the MotA/B proton channel complex of the proton-driven flagellar motor of Salmonella enterica 査読有り国際誌

Yusuke V. Morimoto and Tohru Minamino

Salmonella - Distribution, Adaptation, Control Measures and Molecular Technologies ( InTech ) 391 - 404 2012年06月

An energy transduction mechanism used in bacterial flagellar type III protein export 査読有り

Minamino T., Minamino T., Morimoto Y., Hara N., Namba K.

Nature Communications 2 ( 1 ) 2011年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton - protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH - FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus. © 2011 Macmillan Publishers Limited. All rights reserved.

DOI： 10.1038/ncomms1488

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M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins 査読有り

Morimoto Y., Kojima S., Namba K., Minamino T., Minamino T.

PLoS ONE 6 ( 5 ) 2011年05月

記述言語：英語掲載種別：研究論文（学術雑誌）

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 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 a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins. © 2011 Morimoto et al.

DOI： 10.1371/journal.pone.0019598

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Structural insight into the rotational switching mechanism of the bacterial flagellar motor 査読有り

Minamino T., Minamino T., Imada K., Imada K., Kinoshita M., Nakamura S., Nakamura S., Morimoto Y., Namba K.

PLoS Biology 9 ( 5 ) 2011年05月

記述言語：英語掲載種別：研究論文（学術雑誌）

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliGN, FliGM, and FliGC, and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliGMC domains of Thermotoga maritima, which consist of the FliGM and FliGC domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliGMC domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliGMC(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliGC relative to FliGM in a single molecule was different among the three crystals, a conserved FliGM-FliGC unit was observed in all three of them. We suggest that the conserved FliGM-FliGC unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliGM and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor. © 2011 Minamino et al.

DOI： 10.1371/journal.pbio.1000616

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Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor 査読有り国際誌

Morimoto Y., Nakamura S., Kami-Ike N., Namba K., Minamino T., Minamino T.

Molecular Microbiology 78 ( 5 ) 1117 - 1129 2010年12月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotAC), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotAC with FliG is required for the efficient assembly of the stators around the rotor. © 2010 Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2958.2010.07391.x

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Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella 査読有り国際誌

Morimoto Y., Che Y., Minamino T., Minamino T., Namba K., Namba K.

FEBS Letters 584 ( 6 ) 1268 - 1272 2010年03月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

MotA and MotB form the proton-channel complex of the proton-driven bacterial flagellar motor. A plug segment of Escherichia coli MotB suppresses proton leakage through the MotA/B complex when it is not assembled into the motor. Using a ratiometric pH indicator protein, pHluorin, we show that the proton-conductivity of a Salmonella MotA/B complex not incorporated into the motor is two orders of magnitude lower than that of a complex that is incorporated and activated. This leakage is, however, significant enough to change the cytoplasmic pH to a level at which the chemotaxis signal transduction system responds. © 2010 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2010.02.051

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=77950369485&origin=inward

Roles of the extreme N-terminal region of FliH for efficient localization of the FliH-FliI complex to the bacterial flagellar type III export apparatus 査読有り

Minamino T., Yoshimura S., Morimoto Y., González-Pedrajo B., Kami-Ike N., Namba K.

Molecular Microbiology 74 ( 6 ) 1471 - 1483 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self-assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH-FliI complex also binds to the C ring of the basal body through a FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI-YFP by fluorescence microscopy. FliI-YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI-YFP formed a complex with FliHΔ1 (missing residues 2-10) but the complex did not show any localization. FliHΔ1 did not interact with FliN, and alanine-scanning mutagenesis revealed that only Trp-7 and Trp-10 of FliH are essential for the interaction with FliN. Overproduction of the FliH-FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)-FliI nor FliH(W10A)-FliI complexes did, suggesting that Trp-7 and Trp-10 of FliH are also required for efficient localization of the FliH-FliI complex to the export gate. © 2009 The Authors.

DOI： 10.1111/j.1365-2958.2009.06946.x

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=72049109167&origin=inward

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