scholarly journals Structure of the molecular bushing of the bacterial flagellar motor

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Fumiaki Makino ◽  
Tomoko Miyata ◽  
Tohru Minamino ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
Rotational Symmetry ◽  
Ring Structure ◽  
Flagellar Motor ◽  
Electron Cryomicroscopy ◽  
Bacterial Flagellum ◽  
Inner Surface ◽  
Intersubunit Interactions ◽  
Rotary Motor ◽  
Stable Rotation ◽  
Initial Assembly

AbstractThe basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.

scholarly journals Structure of the molecular bushing of the bacterial flagellar motor

2020 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Fumiaki Makino ◽  
Tomoko Miyata ◽  
Tohru Minamino ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
Basal Body ◽  
High Speed ◽  
Rotational Symmetry ◽  
Ring Structure ◽  
Repulsive Force ◽  
Bacterial Flagellum ◽  
Ring Assembly ◽  
High Speed Rotation ◽  
Speed Rotation ◽  
Intersubunit Interactions

AbstractThe bacterial flagellum is a motility organelle, consisting of the basal body acting as a rotary motor, the filament as a helical propeller and the hook connecting these two as a universal joint1,2. The basal body contains three rings: the MS ring as the transmembrane core of the rotor; the C ring essential for torque generation and switching regulation; and the LP ring as a bushing supporting the distal rod for its rapid, stable rotation without much friction. The negatively charged surface of the distal rod suggested electrostatic repulsive force in supporting high-speed rotation of the rod as a drive shaft3, but the LP ring structure was needed to see the actual mechanisms of its bushing function and assembly against the repulsive force. Here we report the LP ring structure by electron cryomicroscopy at 3.5 Å resolution, showing 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners that explains the structural stability. The inner surface is charged both positively and negatively, and positive charges on the P ring presumably play important roles in its initial assembly around the rod in the peptidoglycan layer followed by the L ring assembly in the outer membrane.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of FljB and FliC

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 246 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Shoko Toma ◽  
Naoya Terahara ◽  
Tomoko Miyata ◽  
Masamichi Ashihara ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
High Viscosity ◽  
Filament Formation ◽  
Electron Cryomicroscopy ◽  
Bacterial Flagellum ◽  
Serovar Typhimurium ◽  
Flagellar Filament ◽  
Position And Orientation ◽  
Rotary Motor

The bacterial flagellum is a motility organelle consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation and autonomously switches their expression at a frequency of 10−3–10−4 per cell per generation. We report here differences in their structures and motility functions under high-viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than one expressing FliC under high viscosity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure was nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.

scholarly journals Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of FljB and FliC

2019 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Shoko Toma ◽  
Naoya Terahara ◽  
Tomoko Miyata ◽  
Masamichi Ashihara ◽  
...  
Keyword(s):  
Salmonella Enterica Serovar Typhimurium ◽  
High Viscosity ◽  
Filament Formation ◽  
Electron Cryomicroscopy ◽  
Bacterial Flagellum ◽  
Serovar Typhimurium ◽  
Flagellar Filament ◽  
Position And Orientation ◽  
The One ◽  
Rotary Motor

The bacterial flagellum is a motility organelle, consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation and autonomously switches their expression at a frequency of 10-3–10-4 per cell per generation. We report here differences in their structures and motility functions under high viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than the one expressing FliC under high viscousity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure is nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.

Three-dimensional reconstruction of the bacterial basal body/switch complex by electron cryomicroscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 514-515
Author(s):  
Noreen R. Francis ◽  
Gina E. Sosinsky ◽  
Dennis Thomas ◽  
David J. DeRosier
Keyword(s):  
Basal Body ◽  
Three Dimensional ◽  
Structural Proteins ◽  
Three Dimensional Reconstruction ◽  
Electron Cryomicroscopy ◽  
Clockwise Rotation ◽  
Bacterial Flagellum ◽  
Dimensional Reconstruction ◽  
Rotary Motor

The bacterial flagellum is unique among Nature's motors in that it posesses a reversible, rotary motor and a propeller that converts torque into thrust The basal body, that part of the flagellum isolated from cells in attempts to purify the motor, contains eight different structural proteins. In Salmonella typhimurimi, the basal body consists of four rings (denoted M, S, L, and P) threaded on a coaxial rod. The M-S, L and Prings are each composed of a different protein, FliF, FlgH, and Flgl, each of which is present in ∽26 copies. The rod contains four different proteins, FlgB, FlgC, FlgF, and FlgG. Also present is FliE. These all are present in ∽6 copies except for FlgG present in ∽26 copies. The proteins important for motor rotation, however, are missing in standard basal body preparations. These missing proteins include the three “switch” proteins, FliG, FliM, and FliN, which control motor reversal (clockwise and counter-clockwise rotation) and may correspond to the rotor and gearbox of the motor. FliG has recently been shown to be localized at the M ring.

scholarly journals Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor

2021 ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Cysteine Residue ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Disulfide Crosslinking ◽  
Bacterial Flagellum ◽  
Rotary Motor

The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are embedded in the cytoplasmic membrane. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been suggested by genetic analyses, but have not been demonstrated directly. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.

scholarly journals Structure of Salmonella Flagellar Hook Reveals Intermolecular Domain Interactions for the Universal Joint Function

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 462 ◽  
Author(s):  
Péter Horváth ◽  
Takayuki Kato ◽  
Tomoko Miyata ◽  
Keiichi Namba
Keyword(s):  
Self Assembly ◽  
Bacterial Species ◽  
Atomic Model ◽  
Universal Joint ◽  
Electron Cryomicroscopy ◽  
Joint Function ◽  
Bacterial Flagellum ◽  
Domain Interactions ◽  
Density Map ◽  
Rotary Motor

The bacterial flagellum is a motility organelle consisting of a rotary motor and a long helical filament as a propeller. The flagellar hook is a flexible universal joint that transmits motor torque to the filament in its various orientations that change dynamically between swimming and tumbling of the cell upon switching the motor rotation for chemotaxis. Although the structures of the hook and hook protein FlgE from different bacterial species have been studied, the structure of Salmonella hook, which has been studied most over the years, has not been solved at a high enough resolution to allow building an atomic model of entire FlgE for understanding the mechanisms of self-assembly, stability and the universal joint function. Here we report the structure of Salmonella polyhook at 4.1 Å resolution by electron cryomicroscopy and helical image analysis. The density map clearly revealed folding of the entire FlgE chain forming the three domains D0, D1 and D2 and allowed us to build an atomic model. The model includes domain Dc with a long β-hairpin structure that connects domains D0 and D1 and contributes to the structural stability of the hook while allowing the flexible bending of the hook as a molecular universal joint.

scholarly journals Catch bond drives stator mechanosensitivity in the bacterial flagellar motor

2017 ◽  
Vol 114 (49) ◽  
pp. 12952-12957 ◽  
Author(s):  
Ashley L Nord ◽  
Emilie Gachon ◽  
Ruben Perez-Carrasco ◽  
Jasmine A. Nirody ◽  
Alessandro Barducci ◽  
...  
Keyword(s):  
Flagellar Motor ◽  
Motor Speed ◽  
Adsorption Model ◽  
Catch Bond ◽  
Bacterial Flagellum ◽  
Kinetic Rates ◽  
Surface Sensing ◽  
Bacterial Flagellar Motor ◽  
Sense And Respond ◽  
Rotary Motor

The bacterial flagellar motor (BFM) is the rotary motor that rotates each bacterial flagellum, powering the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force-powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechanosensitive, with the number of engaged units dependent on the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechanosensitivity is unknown. Here, we directly measure the kinetics of arrival and departure of the stator units in individual motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. The kinetic rates obtained, robust with respect to the details of the applied adsorption model, indicate that the lifetime of an assembled stator unit increases when a higher force is applied to its anchoring point in the cell wall. This provides strong evidence that a catch bond (a bond strengthened instead of weakened by force) drives mechanosensitivity of the flagellar motor complex. These results add the BFM to a short, but growing, list of systems demonstrating catch bonds, suggesting that this “molecular strategy” is a widespread mechanism to sense and respond to mechanical stress. We propose that force-enhanced stator adhesion allows the cell to adapt to a heterogeneous environmental viscosity and may ultimately play a role in surface-sensing during swarming and biofilm formation.

The three-dimensional structure of frozen-hydrated left- and right-handed forms of bacterial flagellar filaments from an identical serotype

1986 ◽  
Vol 44 ◽  
pp. 298-299
Author(s):  
S. Trachtenberg ◽  
D. J. DeRosier
Keyword(s):  
Ionic Strength ◽  
Basal Body ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Protein Species ◽  
Liquid Environment ◽  
Bacterial Flagellum ◽  
Left And Right ◽  
Rotary Motor ◽  
Helical Parameters

The bacterial cell is propelled through the liquid environment by means of one or more rotating flagella. The bacterial flagellum is composed of a basal body (rotary motor), hook (universal coupler), and filament (propellor). The filament is a rigid helical assembly of only one protein species — flagellin. The filament can adopt different morphologies and change, reversibly, its helical parameters (pitch and hand) as a function of mechanical stress and chemical changes (pH, ionic strength) in the environment.

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