Papers - KOJIMA, Seiji

Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species. Reviewed International journal

Hiroyuki Terashima, Keiichi Hirano, Yuna Inoue, Takaya Tokano, Akihiro Kawamoto, Takayuki Kato, Erika Yamaguchi, Keiichi Namba, Takayuki Uchihashi, Seiji Kojima, Michio Homma

Journal of bacteriology   Vol. 202 ( 16 )   2020.6

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The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

DOI： 10.1128/JB.00236-20

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Regulation of the Single Polar Flagellar Biogenesis. International journal

Seiji Kojima, Hiroyuki Terashima, Michio Homma

Biomolecules   Vol. 10 ( 4 )   2020.4

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Some bacterial species, such as the marine bacterium Vibrio alginolyticus, have a single polar flagellum that allows it to swim in liquid environments. Two regulators, FlhF and FlhG, function antagonistically to generate only one flagellum at the cell pole. FlhF, a signal recognition particle (SRP)-type guanosine triphosphate (GTP)ase, works as a positive regulator for flagellar biogenesis and determines the location of flagellar assembly at the pole, whereas FlhG, a MinD-type ATPase, works as a negative regulator that inhibits flagellar formation. FlhF intrinsically localizes at the cell pole, and guanosine triphosphate (GTP) binding to FlhF is critical for its polar localization and flagellation. FlhG also localizes at the cell pole via the polar landmark protein HubP to directly inhibit FlhF function at the cell pole, and this localization depends on ATP binding to FlhG. However, the detailed regulatory mechanisms involved, played by FlhF and FlhG as the major factors, remain largely unknown. This article reviews recent studies that highlight the post-translational regulation mechanism that allows the synthesis of only a single flagellum at the cell pole.

DOI： 10.3390/biom10040533

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Characterization of the MinD/ParA-type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form. International journal

Seiji Kojima, Yoshino Imura, Hikaru Hirata, Michio Homma

Genes to cells : devoted to molecular & cellular mechanisms   Vol. 25 ( 4 ) page： 279 - 287   2020.4

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FlhG is a MinD/ParA-type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here, we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP-binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild type, but ATP caused strong aggregation of the ATPase-stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution, and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably because of a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.

DOI： 10.1111/gtc.12754

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Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. International journal

Tatsuro Nishikino, Hiroto Iwatsuki, Taira Mino, Seiji Kojima, Michio Homma

Journal of biochemistry   Vol. 167 ( 4 ) page： 389 - 398   2020.4

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The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2-3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1-2) and TM3 and TM4 (loop3-4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3-4 but hardly labelled Cys residues in loop1-2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41-120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1-2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1-2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

DOI： 10.1093/jb/mvz102

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Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System. International journal

Florencia Mengucci, Carolina Dardis, Elías J Mongiardini, María J Althabegoiti, Jonathan D Partridge, Seiji Kojima, Michio Homma, Juan I Quelas, Aníbal R Lodeiro

Journal of bacteriology   Vol. 202 ( 5 )   2020.2

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Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

DOI： 10.1128/JB.00708-19

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In Situ Structure of the Vibrio Polar Flagellum Reveals a Distinct Outer Membrane Complex and Its Specific Interaction with the Stator. Reviewed International journal

Shiwei Zhu, Tatsuro Nishikino, Norihiro Takekawa, Hiroyuki Terashima, Seiji Kojima, Katsumi Imada, Michio Homma, Jun Liu

Journal of bacteriology   Vol. 202 ( 4 )   2020.1

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The bacterial flagellum is a biological nanomachine that rotates to allow bacteria to swim. For flagellar rotation, torque is generated by interactions between a rotor and a stator. The stator, which is composed of MotA and MotB subunit proteins in the membrane, is thought to bind to the peptidoglycan (PG) layer, which anchors the stator around the rotor. Detailed information on the stator and its interactions with the rotor remains unclear. Here, we deployed cryo-electron tomography and genetic analysis to characterize in situ structure of the bacterial flagellar motor in Vibrio alginolyticus, which is best known for its polar sheathed flagellum and high-speed rotation. We determined in situ structure of the motor at unprecedented resolution and revealed the unique protein-protein interactions among Vibrio-specific features, namely the H ring and T ring. Specifically, the H ring is composed of 26 copies of FlgT and FlgO, and the T ring consists of 26 copies of a MotX-MotY heterodimer. We revealed for the first time a specific interaction between the T ring and the stator PomB subunit, providing direct evidence that the stator unit undergoes a large conformational change from a compact form to an extended form. The T ring facilitates the recruitment of the extended stator units for the high-speed motility in Vibrio species.IMPORTANCE The torque of flagellar rotation is generated by interactions between a rotor and a stator; however, detailed structural information is lacking. Here, we utilized cryo-electron tomography and advanced imaging analysis to obtain a high-resolution in situ flagellar basal body structure in Vibrio alginolyticus, which is a Gram-negative marine bacterium. Our high-resolution motor structure not only revealed detailed protein-protein interactions among unique Vibrio-specific features, the T ring and H ring, but also provided the first structural evidence that the T ring interacts directly with the periplasmic domain of the stator. Docking atomic structures of key components into the in situ motor map allowed us to visualize the pseudoatomic architecture of the polar sheathed flagellum in Vibrio spp. and provides novel insight into its assembly and function.

DOI： 10.1128/JB.00592-19

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Tree of motility - A proposed history of motility systems in the tree of life. Reviewed International journal

Makoto Miyata, Robert C Robinson, Taro Q P Uyeda, Yoshihiro Fukumori, Shun-Ichi Fukushima, Shin Haruta, Michio Homma, Kazuo Inaba, Masahiro Ito, Chikara Kaito, Kentaro Kato, Tsuyoshi Kenri, Yoshiaki Kinosita, Seiji Kojima, Tohru Minamino, Hiroyuki Mori, Shuichi Nakamura, Daisuke Nakane, Koji Nakayama, Masayoshi Nishiyama, Satoshi Shibata, Katsuya Shimabukuro, Masatada Tamakoshi, Azuma Taoka, Yosuke Tashiro, Isil Tulum, Hirofumi Wada, Ken-Ichi Wakabayashi

Genes to cells : devoted to molecular & cellular mechanisms   Vol. 25 ( 1 ) page： 6 - 21   2020.1

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Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

DOI： 10.1111/gtc.12737

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[Flagellar related genes and functions in Vibrio].

Tatsuro Nishikino, Seiji Kojima, Michio Homma

Nihon saikingaku zasshi. Japanese journal of bacteriology   Vol. 75 ( 3 ) page： 195 - 214   2020

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

Bacteria can move or swim by flagella. On the other hand, the motile ability is not necessary to live at all. In laboratory, the flagella-deficient strains can grow just like the wild-type strains. The flagellum is assembled from more than 20 structural proteins and there are more than 50 genes including the structural genes to regulate or support the flagellar formation. The cost to construct the flagellum is so expensive. The fact that it evolved as a motor organ means even at such the large cost shows that the flagellum is essential for survival in natural condition. In this review, we would like to focus on the flagella-related researches conducted by the authors and the flagellar research on Vibrio spp.

DOI： 10.3412/jsb.75.195

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Structure of the periplasmic domain of SflA involved in spatial regulation of the flagellar biogenesis of Vibrio reveals a TPR/SLR-like fold. Reviewed International journal

Mayuko Sakuma, Shoji Nishikawa, Satoshi Inaba, Takehiko Nishigaki, Seiji Kojima, Michio Homma, Katsumi Imada

Journal of biochemistry   Vol. 166 ( 2 ) page： 197 - 204   2019.8

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Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein-protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function.

DOI： 10.1093/jb/mvz027

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Effect of PlzD, a YcgR homologue of c-di-GMP-binding protein, on polar flagellar motility in Vibrio alginolyticus. Reviewed International journal

Seiji Kojima, Takuro Yoneda, Wakako Morimoto, Michio Homma

Journal of biochemistry   Vol. 166 ( 1 ) page： 77 - 88   2019.7

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YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.

DOI： 10.1093/jb/mvz014

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Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus. Reviewed

Mino T, Nishikino T, Iwatsuki H, Kojima S, Homma M

J. Biochem.   Vol. 166 ( 4 ) page： 331 - 341   2019.5

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DOI： 10.1093/jb/mvz040

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Structure of Vibrio FliL, a New Stomatin-like Protein That Assists the Bacterial Flagellar Motor Function. Reviewed International journal

Norihiro Takekawa, Miyu Isumi, Hiroyuki Terashima, Shiwei Zhu, Yuuki Nishino, Mayuko Sakuma, Seiji Kojima, Michio Homma, Katsumi Imada

mBio   Vol. 10 ( 2 ) page： e00292   2019.3

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Many motile bacteria swim or swarm using a filamentous rotating organelle, the flagellum. FliL, a component protein of the flagellar motor, is known to enhance the motor performance under high-load conditions in some bacteria. Here we determined the structure of the periplasmic region of FliL (FliLPeri) of the polar flagellum of Vibrio alginolyticus FliLPeri shows a remarkable structural similarity to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of stomatin family proteins, some of which are involved in modulation of ion channel activities in various organisms. FliLPeri forms a ring assembly in the crystal with an inner diameter of around 8 nm, which is comparable to the size of the stator unit. Mutational analyses suggest that the FliL ring forms a complex with the stator unit and that the length of the periplasmic linkers of FliL and the stator B-subunit is essential for the complex formation. We propose a model of the FliL-stator complex to discuss how Vibrio FliL modulates stator function in the bacterial flagellar motor under conditions of high viscosity.IMPORTANCE Some flagellated bacteria regulate motor torque in response to the external load change. This behavior is critical for survival, but the mechanism has remained unknown. Here, we focused on a key protein, FliL of Vibrio alginolyticus, and solved the crystal structure of its periplasmic region (FliLPeri). FliLPeri reveals striking structural similarity to a conserved domain of stomatin, which is involved in ion channel regulation in some organisms, including mammals. FliLPeri forms a ring with an inner diameter that is comparable in size to the stator unit. The mutational analyses suggested that the presence of the ring-like assembly of FliL around the stator unit enhances the surface swarming of Vibrio cells. Our study data also imply that the structural element for the ion channel regulation is conserved from bacteria to mammals.

DOI： 10.1128/mBio.00292-19

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Rotational direction of flagellar motor from the conformation of FliG middle domain in marine Vibrio. Reviewed International journal

Tatsuro Nishikino, Atsushi Hijikata, Yohei Miyanoiri, Yasuhiro Onoue, Seiji Kojima, Tsuyoshi Shirai, Michio Homma

Scientific reports   Vol. 8 ( 1 ) page： 17793 - 17793   2018.12

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FliG, which is composed of three distinctive domains, N-terminal (N), middle (M), and C-terminal (C), is an essential rotor component that generates torque and determines rotational direction. To determine the role of FliG in determining flagellar rotational direction, we prepared rotational biased mutants of fliG in Vibrio alginolyticus. The E144D mutant, whose residue is belonging to the EHPQR-motif in FliGM, exhibited an increased number of switching events. This phenotype generated a response similar to the phenol-repellent response in chemotaxis. To clarify the effect of E144D mutation on the rotational switching, we combined the mutation with other che mutations (G214S, G215A and A282T) in FliG. Two of the double mutants suppressed the rotational biased phenotype. To gain structural insight into the mutations, we performed molecular dynamic simulations of the FliGMC domain, based on the crystal structure of Thermotoga maritima FliG and nuclear magnetic resonance analysis. Furthermore, we examined the swimming behavior of the fliG mutants lacking CheY. The results suggested that the conformation of FliG in E144D mutant was similar to that in the wild type. However, that of G214S and G215A caused a steric hindrance in FliG. The conformational change in FliGM triggered by binding CheY may lead to a rapid change of direction and may occur in both directional states.

DOI： 10.1038/s41598-018-35902-6

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The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum. Reviewed International journal

Shiwei Zhu, Tatsuro Nishikino, Seiji Kojima, Michio Homma, Jun Liu

Journal of bacteriology   Vol. 200 ( 21 )   2018.11

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The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, whereas spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here, we use cryo-electron tomography to visualize the polar sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio-specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes.IMPORTANCE Flagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella, such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

DOI： 10.1128/JB.00387-18

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Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment. Reviewed International journal

Tohru Minamino, Naoya Terahara, Seiji Kojima, Keiichi Namba

Molecular microbiology   Vol. 109 ( 6 ) page： 723 - 734   2018.9

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The bacterial flagellar motor is composed of a rotor and a transmembrane ion channel complex that acts as a stator unit. The ion channel complex consists of at least three structural parts: a cytoplasmic domain responsible for the interaction with the rotor, a transmembrane ion channel that forms a pathway for the transit of ions across the cytoplasmic membrane, and a peptidoglycan-binding (PGB) domain that anchors the stator unit to the peptidoglycan (PG) layer. A flexible linker connecting the ion channel and the PGB domain not only coordinates stator assembly with its ion channel activity but also controls the assembly of stator units to the motor in response to changes in the environment. When the ion channel complex encounters the rotor, the N-terminal portion of the PGB domain adopts a partially stretched conformation, allowing the PGB domain to reach and bind to the PG layer. The binding affinity of the PGB domain for the PG layer is affected by the force applied to its anchoring point and to the type of ionic energy source. In this review article, we will present current understanding of autonomous control mechanism of stator assembly in the bacterial flagellar motor.

DOI： 10.1111/mmi.14092

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Biochemical analysis of GTPase FlhF which controls the number and position of flagellar formation in marine Vibrio. Reviewed International journal

Shota Kondo, Yoshino Imura, Akira Mizuno, Michio Homma, Seiji Kojima

Scientific reports   Vol. 8 ( 1 ) page： 12115 - 12115   2018.8

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FlhF controls the number and position of the polar flagellar formation of Vibrio species. FlhF, is a paralog of FtsY, a GTPase acting in the Sec membrane transport system of bacteria, and localizes at the cell pole. Mutations in the conserved GTPase motif of FlhF lost polar localization capability and flagellar formation. Vibrio FlhF has not, until now, been purified as soluble protein. Here, we report that addition of MgCl2 and GTP or GDP at the step of cell lysis greatly improved the solubility of FlhF, allowing us to purify it in homogeneity. Purified FlhF showed GTPase activity only in the presence of FlhG. Of twelve FlhF GTPase motif mutants showing reduced function, eleven were recovered as precipitate after the cell disruption. The E440K substitution could be purified and showed no GTPase activity even in the presence of FlhG. Interestingly an FlhF substitution in the putative catalytic residue for GTP hydrolysis, R334A, allowed normal flagellar formation although GTPase activity of FlhF was completely abolished. Furthermore, size exclusion chromatography of purified FlhF revealed that it forms dimers in the presence of GTP but exists as monomer in the presence of GDP. We speculate that the GTP binding allows FlhF to dimerize and localize at the pole where it initiates flagellar formation, and the GDP-bound form diffuses as monomer.

DOI： 10.1038/s41598-018-30531-5

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The role of conserved charged residues in the bidirectional rotation of the bacterial flagellar motor. Reviewed International journal

Yasuhiro Onoue, Norihiro Takekawa, Tatsuro Nishikino, Seiji Kojima, Michio Homma

MicrobiologyOpen   Vol. 7 ( 4 ) page： e00587   2018.8

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Many bacteria rotate their flagella both counterclockwise (CCW) and clockwise (CW) to achieve swimming toward attractants or away from repellents. Highly conserved charged residues are important for that motility, which suggests that electrostatic interactions are crucial for the rotor-stator function. It remains unclear if those residues contribute equally to rotation in the CCW and CW directions. To address this uncertainty, in this study, we expressed chimeric rotors and stators from Vibrio alginolyticus and Escherichia coli in E. coli, and measured the rotational speed of each motor in both directions using a tethered-cell assay. In wild-type cells, the rotational speeds in both directions were equal, as demonstrated previously. Some charge-neutralizing residue replacements in the stator decreased the rotational speed in both directions to the same extent. However, mutations in two charged residues in the rotor decreased the rotational speed only in the CCW direction. Subsequent analysis and previous results suggest that these amino acid residues are involved in supporting the conformation of the rotor, which is important for proper torque generation in the CCW direction.

DOI： 10.1002/mbo3.587

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FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy. Reviewed International journal

Tsai-Shun Lin, Shiwei Zhu, Seiji Kojima, Michio Homma, Chien-Jung Lo

Scientific reports   Vol. 8 ( 1 ) page： 11172 - 11172   2018.7

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Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.

DOI： 10.1038/s41598-018-29447-x

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The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx Reviewed

Seiji Kojima, Masato Takao, Gaby Almira, Ikumi Kawahara, Mayuko Sakuma, Michio Homma, Chojiro Kojima, Katsumi Imada

Structure   Vol. 26 ( 4 ) page： 590 - 598.e5   2018.4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

The stator of the bacterial flagellar motor couples ion flow with torque generation. The ion-conducting stator channel opens only when incorporated into and anchored around the rotor via the peptidoglycan (PG) binding domain of the B subunit (MotBC). However, no direct evidence of PG binding coupled with channel activation has been presented. Here, we report the structural rearrangements of MotBC responsible for this coupling process. A MotBC fragment with the L119P replacement, which is known to cause channel activation, was able to bind PG. Nuclear magnetic resonance analysis of MotBC and the crystal structure of the MotBC-L119P dimer revealed major structural changes in helix α1. In vivo crosslinking results confirm that a major rearrangement occurs. Our results suggest that, upon stator incorporation into the motor, helix α1 of MotBC changes into an extended non-helical structure. We propose that this change allows the stator both to bind PG and to open its proton channel. Kojima et al. provide direct evidence for coupling between peptidoglycan (PG)-binding and activation of the flagellar stator protein. Using X-ray and NMR analyses they reveal that dynamic rearrangement of the N-terminal helix of the PG-binding domain of the stator protein MotB is key for its functional activation and energy conversion.

DOI： 10.1016/j.str.2018.02.016

Web of Science

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PubMed

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella Reviewed

Yohei Miyanoiri, Atsushi Hijikata, Yuuki Nishino, Mizuki Gohara, Yasuhiro Onoue, Seiji Kojima, Chojiro Kojima, Tsuyoshi Shirai, Masatsune Kainosho, Michio Homma

STRUCTURE   Vol. 25 ( 10 ) page： 1540 - +   2017.10

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

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliG(C)). We have identified point mutations in FliG(C) from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliG(C) from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliG(C) are observed in the C alpha 1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliG(C) at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix alpha 1 in the C alpha 1-6 domain of FliG(C) contribute to ensure the precise regulation of the motor switching.

DOI： 10.1016/j.str.2017.08.010

Web of Science

PubMed