Putative spanner function of the Vibrio PomB plug region in the stator rotation model for flagellar motor

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. Importance The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

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Function of the Vibrio PomB plug region based on the stator rotation model for bacterial flagellar motor

10.1101/2021.03.16.435749 ◽

2021 ◽

Author(s):

Michio Homma ◽

Hiroyuki Terashima ◽

Hiroaki Koiwa ◽

Seiji Kojima

Keyword(s):

Cross Linking ◽

Ion Flow ◽

Bacterial Flagella ◽

B Subunit ◽

Loop Region ◽

Flagellar Motors ◽

Flagellar Filament ◽

A Subunit ◽

Biological World ◽

Protein Motor

AbstractBacterial flagella are the only real rotational motor organs in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. The base of the flagellar filament has a protein motor consisting of a stator and a rotor embedded in the membrane. The motor part has stators composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters coupled to the ion flow that assemble around the rotor. Recently, structures of the stator, in which two molecules of MotB stuck in the center of the MotA ring made of five molecules, were reported and a model in which the MotA ring rotates with respect to MotB, which is coupled to the influx of ions, was proposed. We focused on the Vibrio PomB plug region, which has been reported to control the activation of flagellar motors. We searched for the plug region, which is the interacting region, through site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that it interacts with the extracellular short loop region of PomA, which is between transmembrane 3 and 4. Although the motor halted following cross-linking, its function was recovered with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion inflow by stopping the rotation of the rotor.ImportanceThe flagellar biological motor resembles a mechanical motor, which is composed of stator and rotor and where the rotational force is transmitted by gear-like movements. We hypothesized that the flagellar the rotation of stator that the pentamer of A subunits revolves around the axis of the B subunit dimer with ion flow. The plug region of the B subunit has been shown to regulate the ion flow. Herein, we demonstrated that the ion flow was terminated by the crosslinking between the plug region and the A subunit. These finding support the rotation hypothesis and explain the role of the plug region in terminating the rotation.

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Structural insights into flagellar stator–rotor interactions

eLife ◽

10.7554/elife.48979 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 14

Author(s):

Yunjie Chang ◽

Ki Hwan Moon ◽

Xiaowei Zhao ◽

Steven J Norris ◽

MD A Motaleb ◽

...

Keyword(s):

Conformational Change ◽

High Speed ◽

Electron Tomography ◽

Deletion Mutants ◽

Flagellar Motor ◽

Wild Type ◽

Cryo Electron Tomography ◽

Flagellar Filament ◽

Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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

Structure ◽

10.1016/j.str.2018.02.016 ◽

2018 ◽

Vol 26 (4) ◽

pp. 590-598.e5 ◽

Cited By ~ 42

Author(s):

Seiji Kojima ◽

Masato Takao ◽

Gaby Almira ◽

Ikumi Kawahara ◽

Mayuko Sakuma ◽

...

Keyword(s):

B Subunit ◽

Ion Influx ◽

Periplasmic Domain

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Bacterial flagella grow through an injection-diffusion mechanism

eLife ◽

10.7554/elife.23136 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 39

Author(s):

Thibaud T Renault ◽

Anthony O Abraham ◽

Tobias Bergmiller ◽

Guillaume Paradis ◽

Simon Rainville ◽

...

Keyword(s):

Fundamental Problem ◽

Diffusion Mechanism ◽

Chain Mechanism ◽

Bacterial Flagellum ◽

Bacterial Flagella ◽

Self Assembling ◽

Single Protein ◽

Flagellar Filament ◽

Dynamic Assembly

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

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Mechanics of Fibrous Biological Materials With Hierarchical Chirality

Journal of Applied Mechanics ◽

10.1115/1.4034225 ◽

2016 ◽

Vol 83 (10) ◽

Cited By ~ 5

Author(s):

Huijuan Zhu ◽

Takahiro Shimada ◽

Jianshan Wang ◽

Takayuki Kitamura ◽

Xiqiao Feng

Keyword(s):

Biological Materials ◽

Bottom Up ◽

Chirality Transfer ◽

Bacterial Flagella ◽

Physical Mechanisms ◽

Mechanics Model ◽

Chiral Structures ◽

Biological World ◽

Constituent Elements ◽

Structural Levels

Chirality simultaneously exists at different length scales in many biological materials, e.g., climbing tendrils and bacterial flagella. It can transfer from lower structural levels to higher structural levels, which is tightly associated with the growth and assembly of biological materials. In this paper, a continuum mechanics model is presented for understanding the bottom–up transfer of chirality in fibrous biological materials. Basic physical mechanisms underlying the chirality transfer in biological world are revealed. It is demonstrated that the chirality of constituent elements at the microscale can induce the twisting of higher-level structures, which may further transfer into the macroscopic morphology in different manners, rendering the formation of hierarchically chiral structures in tissues or organs. The bottom–up transfer mechanism of chirality may provide a limit to the macroscopic size of biological materials through the accumulative contribution of twisting.

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Flagella-Driven Motility of Bacteria

Biomolecules ◽

10.3390/biom9070279 ◽

2019 ◽

Vol 9 (7) ◽

pp. 279 ◽

Cited By ~ 50

Author(s):

Shuichi Nakamura ◽

Tohru Minamino

Keyword(s):

Cell Body ◽

Molecular Mechanisms ◽

Bacterial Species ◽

Structural Diversity ◽

Flagellar Motor ◽

Bacterial Flagellum ◽

Structure And Dynamics ◽

Ring Complex ◽

Flagellar Filament ◽

Experimental And Theoretical Studies

The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.

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In situstructures of periplasmic flagella inBorreliareveals conformational changes essential for flagellar rotation

10.1101/553289 ◽

2019 ◽

Author(s):

Yunjie Chang ◽

Kihwan Moon ◽

Xiaowei Zhao ◽

J. Norris Steven ◽

Md A. Motaleb ◽

...

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

High Speed ◽

Electron Tomography ◽

Proton Translocation ◽

Three Dimensional ◽

Dimensional Structure ◽

Flagellar Motor ◽

Flagellar Filament ◽

Flagellar Rotation

SUMMARYThe bacterial flagellar motor is a molecular machine that rotates the flagellar filament at high speed. Torque is generated by the stator-rotor interaction coupled to an ion flux through the torque-generating stator. Here, we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirocheteBorrelia burgdorferi. By analysis of the motor structures of wild-type and stator mutants, we localize the torque-generating units precisely and determine three-dimensional structure of the stator and its interactions with the rotor. Our study shows that the cytoplasmic domains of the stator are packed regularly around the circumference of the flagellar C-ring. The stator-rotor interaction induces a profound conformational change in the C-ring. Analysis of the motors of a less motilemotB-D24E mutant and a non-motilemotB-D24N mutant, in which the proton translocation is reduced and blocked, respectively, provides evidence that the conformational change of the C-ring is essential for flagellar rotation.

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Molecular structure of the intact bacterial flagellar basal body

10.1101/2020.12.05.413195 ◽

2020 ◽

Author(s):

Steven Johnson ◽

Emily J. Furlong ◽

Justin C. Deme ◽

Ashley L. Nord ◽

Joseph Caesar ◽

...

Keyword(s):

Basal Body ◽

Drive Shaft ◽

Inner Membrane ◽

Bacterial Flagella ◽

Cryo Electron Microscopy ◽

Different Types ◽

Flagellar Filament ◽

A Cell ◽

Insight Into

AbstractBacterial flagella self-assemble a strong, multi-component drive shaft that couples rotation in the inner membrane to the microns-long flagellar filament that powers bacterial swimming in viscous fluids. We here present structures of the intact Salmonella flagellar basal body, solved using cryo-electron microscopy to resolutions between 2.2 and 3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the inner membrane rotor component, and at the other end passes through a molecular bearing that is anchored in the outer membrane via interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insight into the assembly process of this molecular machine.

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