Papers - NISHIKINO Tatsuro
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Low-temperature FTIR spectroscopy of the L/Q switch of proteorhodopsin. Reviewed International journal
Tatsuro Nishikino, Teppei Sugimoto, Hideki Kandori
Physical chemistry chemical physics : PCCP 26 ( 35 ) 22959 - 22967 2024.09
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)
Rhodopsins are photoreceptive membrane proteins containing a retinal chromophore, and the color tuning mechanism in rhodopsins is one of the important topics. Color switch is a color-determining residue at the same position, where replacement of red- and blue-shifting amino acids in two wild-type rhodopsins causes spectral blue- and red-shifts, respectively. The first and most famous color switch in microbial rhodopsins is the L/Q switch in proteorhodopsins (PRs). Green- or blue-absorbing PR (GPR or BPR) contains Leu and Gln at position 105 of the C-helix (TM3), respectively, and their replacement converted absorbing colors. The L/Q switch enables bacteria to absorb green or blue light in shallow or deep ocean waters, respectively. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, a comprehensive mutation study of position 105 in GPR revealed that the λmax correlated with the volume of residues, not the hydropathy index. To gain structural insights into the mechanism, we applied low-temperature FTIR spectroscopy of L105Q GPR, and the obtained spectra were compared with those of GPR and BPR. The difference FTIR spectra of L105Q GPR were similar to those of BPR, not GPR, implying that the L/Q switch converts the GPR structure into a BPR structure in terms of the local environments of the retinal chromophore. It includes retinal skeletal vibration, hydrogen-bonding strength of the protonated Schiff base, amide-A vibration (peptide backbone), and protein-bound water molecules. Consequently color is switched accompanying such structural alterations, and known as the L/Q switch.
DOI： 10.1039/d4cp02248c
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Changes in the hydrophobic network of the FliGMC domain induce rotational switching of the flagellar motor. Reviewed International journal
Tatsuro Nishikino, Atsushi Hijikata, Seiji Kojima, Tsuyoshi Shirai, Masatsune Kainosho, Michio Homma, Yohei Miyanoiri
iScience 26 ( 8 ) 107320 - 107320 2023.08
Language：English Publishing type：Research paper (scientific journal)
The FliG protein plays a pivotal role in switching the rotational direction of the flagellar motor between clockwise and counterclockwise. Although we previously showed that mutations in the Gly-Gly linker of FliG induce a defect in switching rotational direction, the detailed molecular mechanism was not elucidated. Here, we studied the structural changes in the FliG fragment containing the middle and C-terminal regions, named FliGMC, and the switch-defective FliGMC-G215A, using nuclear magnetic resonance (NMR) and molecular dynamics simulations. NMR analysis revealed multiple conformations of FliGMC, and the exchange process between these conformations was suppressed by the G215A residue substitution. Furthermore, changes in the intradomain orientation of FliG were induced by changes in hydrophobic interaction networks throughout FliG. Our finding applies to FliG in a ring complex in the flagellar basal body, and clarifies the switching mechanism of the flagellar motor.
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Site-Specific Isotope Labeling of FliG for Studying Structural Dynamics Using Nuclear Magnetic Resonance Spectroscopy Reviewed
Tatsuro Nishikino, Yohei Miyanoiri
Methods in Molecular Biology 57 - 70 2023.02
Publishing type：Part of collection (book) Publisher：Springer US
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The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. Reviewed International journal
Brittany L Carroll, Tatsuro Nishikino, Wangbiao Guo, Shiwei Zhu, Seiji Kojima, Michio Homma, Jun Liu
eLife 9 2020.09
Language：English Publishing type：Research paper (scientific journal)
The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.
DOI： 10.7554/eLife.61446
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Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. Reviewed International journal
Tatsuro Nishikino, Hiroto Iwatsuki, Taira Mino, Seiji Kojima, Michio Homma
Journal of biochemistry 167 ( 4 ) 389 - 398 2020.04
Language：English
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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Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus. Reviewed International journal
Shiwei Zhu, Tatsuro Nishikino, Bo Hu, Seiji Kojima, Michio Homma, Jun Liu
Proceedings of the National Academy of Sciences of the United States of America 114 ( 41 ) 10966 - 10971 2017.10
Language：English
Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use Vibrio alginolyticus as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in V. alginolyticus cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined a Vibrio-specific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring provides a static platform to recruit the PomA/B complexes, which are required to generate higher torques for rotation of the sheathed flagellum and fast motility of Vibrio cells. Furthermore, the Vibrio flagellar motor exhibits an intrinsic length variation between the inner and the outer membrane bound complexes, suggesting the outer membrane bound complex can shift slightly along the axial rod during flagellar rotation. Together, our detailed analyses of the polar flagella in intact cells provide insights into unique aspects of the sheathed flagellum and the distinct motility of Vibrio species.
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Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor. Reviewed International journal
Norihiro Takekawa, Tatsuro Nishikino, Jun-Ichi Kishikawa, Mika Hirose, Miki Kinoshita, Seiji Kojima, Tohru Minamino, Takayuki Uchihashi, Takayuki Kato, Katsumi Imada, Michio Homma
mBio e0126124 2024.09
Language：English Publishing type：Research paper (scientific journal)
The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.
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Structural insight into sodium ion pathway in the bacterial flagellar stator from marine<i>Vibrio</i>
Tatsuro Nishikino, Norihiro Takekawa, Jun-ichi Kishikawa, Mika Hirose, Seiji Kojima, Michio Homma, Takayuki Kato, Katsumi Imada
2024.07
Publisher：Cold Spring Harbor Laboratory
Abstract
Many bacteria swim in liquid or swarm on surface using the flagellum rotated by a motor driven by specific ion flow. The motor consists of the rotor and stator, and the stator converts the energy of ion flow to mechanical rotation. However, the ion pathway and the mechanism of stator rotation coupled with specific ion flow are still obscure. Here, we determined the structures of the Na<sup>+</sup>-driven stator ofVibrio, namely PomAB, in the presence and absence of sodium ions and the structure with its specific inhibitor, phenamil, by cryo-electron microscopy. The structures and following functional analysis revealed the sodium ion pathway, the mechanism of ion selectivity, and the inhibition mechanism by phenamil. We propose a model of sodium ion flow coupled with the stator rotation based on the structures. This work provides insights into the molecular mechanisms of ion specificity and conversion of the electrochemical potential into mechanical functions. -
Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor. Reviewed International journal
Yusuke Miyamura, Tatsuro Nishikino, Hiroaki Koiwa, Michio Homma, Seiji Kojima
Genes to cells : devoted to molecular & cellular mechanisms 2024.02
Language：English Publishing type：Research paper (scientific journal)
The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.
DOI： 10.1111/gtc.13102
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高圧NMR測定による細菌べん毛モータータンパク質FliGの構造変化がモーターの回転方向を決定する分子機構の解明
Tatsuro NISHIKINO, Yohei MIYANOIRI
The Review of High Pressure Science and Technology 33 ( 2 ) 83 - 90 2023.06
Publishing type：Research paper (scientific journal) Publisher：The Japan Society of High Pressure Science and Technology
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Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane Reviewed
Kanji Takahashi, Tatsuro Nishikino, Hiroki Kajino, Seiji Kojima, Takayuki Uchihashi, Michio Homma
BIOPHYSICS AND PHYSICOBIOLOGY 20 ( 2 ) 2023
Language：English Publishing type：Research paper (scientific journal) Publisher：BIOPHYSICAL SOC JAPAN
The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor-stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the twotransmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF-fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.
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Structure of MotA, a flagellar stator protein, from hyperthermophile. Reviewed International journal
Tatsuro Nishikino, Norihiro Takekawa, Duy Phuoc Tran, Jun-Ichi Kishikawa, Mika Hirose, Sakura Onoe, Seiji Kojima, Michio Homma, Akio Kitao, Takayuki Kato, Katsumi Imada
Biochemical and biophysical research communications 631 78 - 85 2022.11
Authorship：Lead author, Corresponding author Language：English Publishing type：Research paper (scientific journal)
Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA5MotB2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA.
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Function and Structure of FlaK, a Master Regulator of the Polar Flagellar Genes in Marine Vibrio. Reviewed International journal
Michio Homma, Tomoya Kobayakawa, Yuxi Hao, Tatsuro Nishikino, Seiji Kojima
Journal of bacteriology e0032022 2022.10
Language：English Publishing type：Research paper (scientific journal)
Vibrio alginolyticus has a flagellum at the cell pole, and the fla genes, involved in its formation, are hierarchically regulated in several classes. FlaK (also called FlrA) is an ortholog of Pseudomonas aeruginosa FleQ, an AAA+ ATPase that functions as a master regulator for all later fla genes. In this study, we conducted mutational analysis of FlaK to examine its ATPase activity, ability to form a multimeric structure, and function in flagellation. We cloned flaK and confirmed that its deletion caused a nonflagellated phenotype. We substituted amino acids at the ATP binding/hydrolysis site and at the putative subunit interfaces in a multimeric structure. Mutations in these sites abolished both ATPase activity and the ability of FlaK to induce downstream flagellar gene expression. The L371E mutation, at the putative subunit interface, abolished flagellar gene expression but retained ATPase activity, suggesting that ATP hydrolysis is not sufficient for flagellar gene expression. We also found that FlhG, a negative flagellar biogenesis regulator, suppressed the ATPase activity of FlaK. The 20 FlhG C-terminal residues are critical for reducing FlaK ATPase activity. Chemical cross-linking and size exclusion chromatography revealed that FlaK mostly exists as a dimer in solution and can form multimers, independent of ATP. However, ATP induced the interaction between FlhG and FlaK to form a large complex. The in vivo effects of FlhG on FlaK, such as multimer formation and/or DNA binding, are important for gene regulation. IMPORTANCE FlaK is an NtrC-type activator of the AAA+ ATPase subfamily of σ54-dependent promoters of flagellar genes. FlhG, a MinD-like ATPase, negatively regulates the polar flagellar number by collaborating with FlhF, an FtsY-like GTPase. We found that FlaK and FlhG interact in the presence of ATP to form a large complex. Mutational analysis revealed the importance of FlaK ATPase activity in flagellar gene expression and provided a model of the Vibrio molecular mechanism that regulates the flagellar number.
DOI： 10.1128/jb.00320-22
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Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor. Reviewed International journal
Tatsuro Nishikino, Yugo Sagara, Hiroyuki Terashima, Michio Homma, Seiji Kojima
Journal of biochemistry 2022.01
Language：English Publishing type：Research paper (scientific journal)
Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.
DOI： 10.1093/jb/mvac001
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ZomB is essential for chemotaxis of Vibrio alginolyticus by the rotational direction control of the polar flagellar motor. Reviewed International journal
Norihiro Takekawa, Tatsuro Nishikino, Kiyoshiro Hori, Seiji Kojima, Katsumi Imada, Michio Homma
Genes to cells : devoted to molecular & cellular mechanisms 2021.09
Language：English Publishing type：Research paper (scientific journal)
Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from non-preferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY-Phosphate (CheY-P) to the C-ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp. have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for a clockwise-locked phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY-P. Purified ZomB proteins form multimers, suggesting that ZomB may function as a homo-oligomer. These observations imply that ZomB interacts with protein(s) involved in either flagellar motor rotation, chemotaxis or both. We provide the evidence that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus.
DOI： 10.1111/gtc.12895
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A slight bending of an α-helix in FliM creates a counterclockwise-locked structure of the flagellar motor in Vibrio. Reviewed International journal
Norihiro Takekawa, Tatsuro Nishikino, Toshiki Yamashita, Kiyoshiro Hori, Yasuhiro Onoue, Kunio Ihara, Seiji Kojima, Michio Homma, Katsumi Imada
Journal of biochemistry 2021.06
Language：English Publishing type：Research paper (scientific journal)
Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus, which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signaling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM-FliM interaction. The structural models of wild-type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring.
DOI： 10.1093/jb/mvab074
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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 202 ( 4 ) 2020.01
Language：English
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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Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus. Reviewed International journal
Taira Mino, Tatsuro Nishikino, Hiroto Iwatsuki, Seiji Kojima, Michio Homma
Journal of biochemistry 2019.05
Language：English
The sodium driven flagellar stator of Vibrio alginolyticus is a hetero-hexamer membrane complex composed of PomA and PomB, and acts as a sodium ion channel. The conformational change in the cytoplasmic region of PomA for the flagellar torque generation, which interacts directly with a rotor protein, FliG, remains a mystery. In this study, we introduced cysteine mutations into cytoplasmic charged residues of PomA, which are highly conserved and interact with FliG, to detect the conformational change by the reactivity of biotin maleimide. In vivo labeling experiments of the PomA mutants revealed that the accessibility of biotin maleimide at position of E96 was reduced with sodium ions. Such a reduction was also seen in the D24N and the plug deletion mutants of PomB, and the phenomenon was independent in the presence of FliG. This sodium ions specific reduction was also detected in Escherichia coli that produced PomA and PomB from a plasmid, but not in the purified stator complex. These results demonstrated that sodium ions cause a conformational change around the E96 residues of loop2-3 in the biological membrane.
DOI： 10.1093/jb/mvz040
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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 8 ( 1 ) 17793 - 17793 2018.12
Language：English
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.
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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 200 ( 21 ) 2018.11
Language：English
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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