記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

DOI： 10.2142/biophysico.bppb-v20.0033

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85175488429&origin=inward

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold Dictyostelium discoideum among eukaryotes. Dictyostelium cells undergo a transition from a unicellular to a multicellular state, ultimately forming a stalk and spores. While Dictyostelium is known to employ c-di-GMP to induce differentiation into stalk cells, there have been no reports of direct observation of c-di-GMP using fluorescent probes. In this study, we used a fluorescent probe used in bacteria to visualize its localization within Dictyostelium multicellular bodies. Cytosolic c-di-GMP concentrations were significantly higher at the tip of the multicellular body during stalk formation.

DOI： 10.3389/fcell.2023.1237778

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85166558089&origin=inward

担当区分：筆頭著者,　責任著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

The bacterial flagellum is driven by a rotational motor located at the base of the flagellum. The stator unit complex conducts cations such as protons (H+) and sodium ions (Na+) along the electrochemical potential across the cytoplasmic membrane and interacts with the rotor to generate the rotational force. Escherichia coli and Salmonella have the H+-type stator complex, which serves as a transmembrane H+ channel that couples H+ flow through an ion channel to torque generation whereas Vibrio and some Bacillus species have the Na+-type stator complex. In this chapter, we describe how to measure the ion conductivity of the transmembrane stator complex over-expressed in E. coli cells using fluorescent indicators. Intensity measurements of fluorescent indicators using either a fluorescence spectrophotometer or microscope allow quantitative detection of changes in the intracellular ion concentrations due to the ion channel activity of the transmembrane protein complex.

DOI： 10.1007/978-1-0716-3060-0_8

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85149053417&origin=inward

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

Bacteria employ the flagellar type III secretion system (fT3SS) to construct flagellum, which acts as a supramolecular motility machine. The fT3SS of Salmonella enterica serovar Typhimurium is composed of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. The transmembrane export gate complex is fueled by proton motive force across the cytoplasmic membrane and is divided into four distinct functional parts: a dual-fuel export engine; a polypeptide channel; a membrane voltage sensor; and a docking platform. ATP hydrolysis by the cytoplasmic ATPase complex converts the export gate complex into a highly efficient proton (H+)/ protein antiporter that couples inward-directed H+ flow with outward-directed protein export. When the ATPase ring complex does not work well in a given environment, the export gate complex will remain inactive. However, when the electric potential difference, which is defined as membrane voltage, rises above a certain threshold value, the export gate complex becomes an active H+ /protein antiporter to a considerable degree, suggesting that the export gate complex has a voltage-gated activation mechanism. Furthermore, the export gate complex also has a sodium ion (Na+) channel to couple Na+ influx with flagellar protein export. In this article, we review our current understanding of the activation mechanism of the dual-fuel protein export engine of the fT3SS. This review article is an extended version of a Japanese article, Membrane voltage-dependent activation of the transmembrane export gate complex in the bacterial flagellar type III secretion system, published in SEIBUTSU BUTSURI Vol. 62, p165–169 (2022).

DOI： 10.2142/biophysico.bppb-v19.0046

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85143436859&origin=inward

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

Calcium acts as a second messenger to regulate many cellular functions, including cell motility. In Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium waves propagate through the cell population during the early stages of development, including aggregation. In the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium dynamics during multicellular morphogenesis are still unclear. Here, live imaging of cytosolic calcium revealed that calcium wave propagation, depending on cAMP relay, disappeared at the onset of multicellular body (slug) formation. Later, other forms of occasional calcium bursts and their propagation were observed in both anterior and posterior regions of migrating slugs. This calcium signaling also occurred in response to mechanical stimuli. Two pathways—calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell—were involved in calcium signals induced by mechanical stimuli. These data suggest that calcium signaling is involved in mechanosensing in both the unicellular and multicellular phases of Dictyostelium development using different molecular mechanisms.

DOI： 10.1038/s41598-022-16774-3

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85134489418&origin=inward

記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）

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）

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and in functions such as virulence and biofilm formation, mainly via gene regulation. Here, we show that the swimming motility of the soil bacterium Leptospira kobayashii is rapidly modulated by light stimulation. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis identified the novel sensory gene, and its expression in Escherichia coli through codon optimization elucidated the light-dependent synthesis of cyclic adenosine monophosphate (cAMP). GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. These findings suggest a new role for cAMP in rapidly controlling the flagella-dependent motility of Leptospira and highlight the global distribution of the newly discovered photoactivated cyclase among diverse microbial species.

DOI： 10.1038/s41598-022-10556-7

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85128876265&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

FlgN, FliS, and FliT are flagellar export chaperones specific for FlgK/FlgL, FliC, and FliD, respectively, which are essential component proteins for filament formation. These chaperones facilitate the docking of their cognate substrates to a transmembrane export gate protein, FlhA, to facilitate their subsequent unfolding and export by the flagellar type III secretion system (fT3SS). Dynamic interactions of the chaperones with FlhA are thought to determine the substrate export order. To clarify the role of flagellar chaperones in filament assembly, we constructed cells lacking FlgN, FliS, and/or FliT. Removal of either FlgN, FliS, or FliT resulted in leakage of a large amount of unassembled FliC monomers into the culture media, indicating that these chaperones contribute to robust and efficient filament formation. The ∆flgN ∆fliS ∆fliT (∆NST) cells produced short filaments similarly to the ∆fliS mutant. Suppressor mutations of the ∆NST cells, which lengthened the filament, were all found in FliC and destabilized the folded structure of FliC monomer. Deletion of FliS inhibited FliC export and filament elongation only after FliC synthesis was complete. We propose that FliS is not involved in the transport of FliC upon onset of filament formation, but FliS-assisted unfolding of FliC by the fT3SS becomes essential for its rapid and efficient export to form a long filament when FliC becomes fully expressed in the cytoplasm.

DOI： 10.3389/fmicb.2021.756044

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85117502497&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

DOI： 10.1073/pnas.2026587118

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85106875656&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2021, The Author(s). The bacterial flagellar protein export machinery consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. The gate complex has two intrinsic and distinct H+-driven and Na+-driven engines to drive the export of flagellar structural proteins. Salmonella wild-type cells preferentially use the H+-driven engine under a variety of environmental conditions. To address how the Na+-driven engine is activated, we analyzed the fliJ(Δ13–24) fliH(Δ96–97) mutant and found that the interaction of the FlgN chaperone with FlhA activates the Na+-driven engine when the ATPase complex becomes non-functional. A similar activation can be observed with either of two single-residue substitutions in FlhA. Thus, it is likely that the FlgN-FlhA interaction generates a conformational change in FlhA that allows it to function as a Na+ channel. We propose that this type of activation would be useful for flagellar construction under conditions in which the proton motive force is severely restricted.

DOI： 10.1038/s42003-021-01865-0

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85102502357&origin=inward

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）

© 2021, Springer Nature Switzerland AG. One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

DOI： 10.1007/978-3-030-58971-4_8

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85097002900&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2020, The Author(s). Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of the rotational direction, which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150°. The flagellar helicity remained right-handed with a 1.3 μm pitch and 0.14 μm helix radius, which is consistent with the feature of a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.

DOI： 10.1038/s41598-020-72429-1

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85091647933&origin=inward

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）

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA-FliG interaction responsible for torque generation.

DOI： 10.3390/biom10091255

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85090181458&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2019 John Wiley & Sons Ltd The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.

DOI： 10.1111/mmi.14440

Kyutacar

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85077140959&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2020 The Author(s) Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.

DOI： 10.1016/j.bbrc.2020.02.060

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85079903105&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

Copyright © 2019 Sakai et al. The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.

DOI： 10.1128/mBio.00079-19

Kyutacar

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85064222930&origin=inward

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

Copyright © 2019 American Society for Microbiology. All Rights Reserved. The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella. The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-m beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-m beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly. IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella. We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

DOI： 10.1128/JB.00727-18

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85062186976&origin=inward

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2019, The Author(s). In Dictyostelium discoideum, a model organism for the study of collective cell migration, extracellular cyclic adenosine 3’,5’-monophosphate (cAMP) acts as a diffusible chemical guidance cue for cell aggregation, which has been thought to be important in multicellular morphogenesis. Here we revealed that the dynamics of cAMP-mediated signaling showed a transition from propagating waves to steady state during cell development. Live-cell imaging of cytosolic cAMP levels revealed that their oscillation and propagation in cell populations were obvious for cell aggregation and mound formation stages, but they gradually disappeared when multicellular slugs started to migrate. A similar transition of signaling dynamics occurred with phosphatidylinositol 3,4,5-trisphosphate signaling, which is upstream of the cAMP signal pathway. This transition was programmed with concomitant developmental progression. We propose a new model in which cAMP oscillation and propagation between cells, which are important at the unicellular stage, are unessential for collective cell migration at the multicellular stage.

DOI： 10.1038/s42003-018-0273-6

Kyutacar

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85071152501&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2018 The Author(s). We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca 2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 μm. In the absence of Ca 2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca 2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca 2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca 2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.

DOI： 10.1038/s41598-018-26337-0

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85047606949&origin=inward

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

DOI： 10.5772/intechopen.73277

記述言語：英語   掲載種別：研究論文（学術雑誌）

Copyright © 2018 The Authors. The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhA C ) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhA C (FlhA L ) is required not only for FlhA C ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhA L was required for cooperative ring formation of FlhA C . Alanine substitutions of residues involved in FlhA C ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhA C .

DOI： 10.1126/sciadv.aao7054

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85045963006&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© The Author(s) 2018. The FliI ATPase of the flagellar type III protein export apparatus forms the FliH2FliI complex along with its regulator FliH. The FliH2FliI complex is postulated to bring export substrates from the cytoplasm to the docking platform made of FlhA and FlhB although not essential for flagellar protein export. Here, to clarify the role of the FliH2FliI complex in flagellar assembly, we analysed the effect of FliH and FliI deletion on flagellar protein export and assembly. The hook length was not controlled properly in the ΔfliH-fliI flhB(P28T) mutant compared to wild-Type cells, whose hook length is controlled to about 55 nm within 10% error. The FlhA(F459A) mutation increased the export level of the hook protein FlgE and the ruler protein FliK by about 10-fold and 3-fold, respectively, and improved the hook length control in the absence of FliH and FliI. However, the ΔfliH-fliI flhB(P28T) flhA(F459A) mutant did not produce flagellar filaments efficiently, and a large amount of flagellin monomers were leaked out into the culture media. Neither the hook length control nor flagellin leakage was affected by the FlhB(P28T) and FlhA(F459A) mutations. We will discuss a hierarchical protein export mechanism of the bacterial flagellum.

DOI： 10.1038/s41598-018-20209-3

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85041224923&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

<p>The bacterial flagellum is a supramolecular motility machine consisting of the basal body as a rotary motor, the hook as a universal joint, and the filament as a helical propeller. Intact structures of the bacterial flagella have been observed for different bacterial species by electron cryotomography and subtomogram averaging. The core structures of the basal body consisting of the C ring, the MS ring, the rod and the protein export apparatus, and their organization are well conserved, but novel and divergent structures have also been visualized to surround the conserved structure of the basal body. This suggests that the flagellar motors have adapted to function in various environments where bacteria live and survive. In this review, we will summarize our current findings on the divergent structures of the bacterial flagellar motor.</p>

DOI： 10.2142/biophysico.14.0_191

CiNii Article

その他リンク： https://ci.nii.ac.jp/naid/130006252950

担当区分：責任著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（その他）

DOI： 10.2142/biophys.57.296

CiNii Article

その他リンク： http://ci.nii.ac.jp/naid/130006230285

担当区分：責任著者   記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

DOI： 10.21769/BioProtoc.2529

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2017 The AuthorsProtein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.

DOI： 10.1016/j.celrep.2017.04.030

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85019007573&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© The Author(s) 2017. The bacterial flagellar hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The hook protein FlgE shows considerable sequence and structural similarities to the distal rod protein FlgG; however, the hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the hook to suppress axial compression and extension of the protofilaments required for bending flexibility.

DOI： 10.1038/srep46723

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85026856088&origin=inward

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）

© Springer Science+Business Media LLC 2017. Fluorescence imaging techniques using green fluorescent protein (GFP) and related fluorescent proteins are utilized to monitor and analyze a wide range of biological processes in living cells. Stepwise photobleaching experiments can determine the stoichiometry of protein complexes. Fluorescence recovery after photobleaching (FRAP) experiments can reveal in vivo dynamics of biomolecules. In this chapter, we describe methods to detect the subcellular localization, stoichiometry, and turnovers of stator and rotor components of the Salmonella flagellar motor.

DOI： 10.1007/978-1-4939-6927-2_16

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85017329216&origin=inward

担当区分：責任著者   記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

DOI： 10.21769/BioProtoc.2093

担当区分：責任著者   記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）

DOI： 10.21769/BioProtoc.2092

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）

© 2016 Morimoto et al.Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+ flow to drive protein export. IMPORTANCE The flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed an in vivo pH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.

DOI： 10.1128/mBio.01911-16

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85007553998&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN–FlgK chaperone–substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN–FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly.

DOI： 10.1002/mbo3.340

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84974720864&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

DOI： 10.1371/journal.ppat.1005495

記述言語：英語   掲載種別：研究論文（学術雑誌）

© 2016 Nature America, Inc.Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.

DOI： 10.1038/nsmb.3172

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84959499452&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

DOI： 10.1016/j.bbrc.2015.09.004

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84927762705&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84923371148&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84923284551&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84896702974&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84902584400&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84891145792&origin=inward

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

その他リンク： http://ci.nii.ac.jp/naid/130003383787

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84889777894&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84878761389&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

DOI： 10.1093/jb/mvt011

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84873045159&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84871793328&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84868305790&origin=inward

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）

DOI： 10.5772/29968

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=80053415915&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=79955785935&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=79958063382&origin=inward

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

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=78649598805&origin=inward

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

Scopus

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

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

Scopus

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

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）

The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator-rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque-speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor-stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque-speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle. © 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2009.08.022

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=70349431086&origin=inward

記述言語：英語   掲載種別：研究論文（学術雑誌）

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (∞). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors. © 2009 Springer-Verlag.

DOI： 10.1007/s00359-008-0411-6

Scopus

その他リンク： https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=64149099402&origin=inward

開催期間： 2024年12月14日   記述言語：日本語  

開催期間： 2024年12月14日   記述言語：日本語  

開催期間： 2024年12月12日 - 2024年12月14日   記述言語：日本語  

開催期間： 2024年11月27日 - 2024年11月29日   記述言語：日本語  

開催期間： 2024年11月27日 - 2024年11月29日   記述言語：日本語  

開催期間： 2024年11月16日   記述言語：日本語  

開催期間： 2024年11月14日 - 2024年11月15日   記述言語：英語  

開催期間： 2024年10月13日 - 2024年10月16日   記述言語：英語  

開催期間： 2024年10月13日 - 2024年10月16日   記述言語：英語  

開催期間： 2024年10月05日 - 2024年10月06日   記述言語：日本語  

開催期間： 2024年10月05日 - 2024年10月06日   記述言語：日本語  

開催期間： 2024年10月05日 - 2024年10月06日   記述言語：日本語  

開催期間： 2024年09月04日 - 2024年09月06日   記述言語：日本語  

開催期間： 2024年06月24日 - 2024年06月28日   記述言語：英語  

開催期間： 2024年06月24日 - 2024年06月28日   記述言語：英語  

開催期間： 2024年06月24日 - 2024年06月28日   記述言語：英語  

開催期間： 2024年06月24日 - 2024年06月28日   記述言語：英語  

開催期間： 2024年06月24日 - 2024年06月28日   記述言語：英語  

開催期間： 2024年03月15日 - 2024年03月16日   記述言語：日本語  

開催期間： 2024年03月15日 - 2024年03月16日   記述言語：日本語  

開催期間： 2024年03月15日 - 2024年03月16日   記述言語：日本語  

開催期間： 2024年01月16日 - 2024年01月17日   記述言語：日本語  

開催期間： 2024年01月05日 - 2024年01月07日   記述言語：日本語  

開催期間： 2023年12月14日 - 2023年12月16日   記述言語：日本語  

開催期間： 2023年12月14日 - 2023年12月16日   記述言語：日本語  

開催期間： 2023年12月09日   記述言語：日本語  

開催期間： 2023年12月09日   記述言語：日本語  

開催期間： 2023年12月09日   記述言語：日本語  

開催期間： 2023年12月09日   記述言語：日本語  

開催期間： 2023年12月09日   記述言語：日本語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年11月14日 - 2023年11月16日   記述言語：英語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年10月21日 - 2023年10月22日   記述言語：日本語  

開催期間： 2023年09月10日 - 2023年09月15日   記述言語：英語   国名：大韓民国  

開催期間： 2023年09月10日 - 2023年09月15日   記述言語：英語   国名：大韓民国  

開催期間： 2023年09月10日 - 2023年09月15日   記述言語：英語   国名：大韓民国  

開催期間： 2023年09月07日 - 2023年09月08日   記述言語：日本語  

開催期間： 2023年03月06日 - 2023年03月07日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月17日   記述言語：日本語  

開催期間： 2022年12月03日   記述言語：日本語  

開催期間： 2022年10月08日 - 2022年10月09日   記述言語：日本語  

開催期間： 2022年10月08日 - 2022年10月09日   記述言語：日本語  

開催期間： 2022年10月08日 - 2022年10月09日   記述言語：日本語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年09月28日 - 2022年09月30日   記述言語：英語  

開催期間： 2022年03月29日 - 2022年03月31日   記述言語：日本語  

開催期間： 2022年03月09日 - 2022年03月10日   記述言語：日本語  

開催期間： 2022年01月07日 - 2022年01月09日   記述言語：日本語  

開催期間： 2021年11月26日   記述言語：英語  

開催期間： 2021年11月26日   記述言語：日本語  

開催期間： 2021年11月26日   記述言語：英語  

開催期間： 2021年10月23日   記述言語：日本語  

開催期間： 2021年10月23日   記述言語：日本語  

開催期間： 2021年06月14日 - 2021年06月16日   記述言語：日本語  

開催期間： 2021年06月14日 - 2021年06月16日   記述言語：日本語  

開催期間： 2021年06月14日 - 2021年06月16日   記述言語：日本語  

開催期間： 2020年12月09日 - 2020年12月11日   記述言語：日本語  

開催期間： 2020年11月14日   記述言語：日本語  

開催期間： 2020年11月14日   記述言語：日本語  

開催期間： 2020年11月14日   記述言語：日本語  

開催期間： 2020年09月16日 - 2020年09月18日   記述言語：英語  

開催期間： 2020年09月16日 - 2020年09月18日   記述言語：英語  

開催期間： 2020年09月16日 - 2020年09月18日   記述言語：英語  

開催期間： 2020年05月25日 - 2020年05月27日   記述言語：日本語  

開催期間： 2020年05月25日 - 2020年05月27日   記述言語：日本語  

開催期間： 2020年01月10日 - 2020年01月12日   記述言語：日本語  

開催期間： 2019年12月20日 - 2019年12月22日   記述言語：日本語  

開催期間： 2019年12月20日 - 2019年12月22日   記述言語：日本語  

開催期間： 2019年12月14日   記述言語：日本語  

開催期間： 2019年12月14日   記述言語：日本語  

開催期間： 2019年12月14日   記述言語：日本語  

開催期間： 2019年12月03日 - 2019年12月06日   記述言語：日本語  

開催期間： 2019年10月19日 - 2019年10月20日   記述言語：日本語  

開催期間： 2019年09月24日 - 2019年09月26日   記述言語：英語  

開催期間： 2019年01月04日 - 2019年01月06日   記述言語：日本語  

開催期間： 2018年12月26日 - 2018年12月28日   記述言語：日本語  

開催期間： 2018年12月08日   記述言語：日本語  

開催期間： 2018年12月06日 - 2018年12月08日   記述言語：日本語  

開催期間： 2018年10月20日 - 2018年10月21日   記述言語：日本語  

開催期間： 2018年09月15日 - 2018年09月17日   記述言語：英語  

開催期間： 2018年05月29日 - 2018年05月31日   記述言語：日本語  

開催期間： 2018年01月05日 - 2018年01月07日   記述言語：日本語  

開催期間： 2017年12月19日 - 2017年12月21日   記述言語：日本語  

開催期間： 2017年12月01日 - 2017年12月02日   記述言語：日本語  

開催期間： 2017年10月21日 - 2017年10月22日   記述言語：日本語  

開催期間： 2017年09月19日 - 2017年09月21日   記述言語：英語  

開催期間： 2017年09月19日 - 2017年09月21日   記述言語：英語  

開催期間： 2023年03月30日   発表言語：英語   講演種別：招待講演  

開催期間： 2021年11月25日   発表言語：英語   講演種別：招待講演  

開催期間： 2019年09月05日 - 2019年09月07日   発表言語：日本語   講演種別：招待講演  

開催期間： 2018年10月31日   発表言語：英語   講演種別：招待講演  

開催期間： 2017年09月19日 - 2017年09月21日   発表言語：英語   講演種別：招待講演  

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：大学院専門科目

科目区分：大学院専門科目

科目区分：大学院専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：大学院専門科目

科目区分：大学院専門科目

科目区分：大学院専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：大学院専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：学部専門科目

科目区分：大学院専門科目