scholarly journals Internal and external components of the bacterial flagellar motor rotate as a unit

2016 ◽  
Vol 113 (17) ◽  
pp. 4783-4787 ◽  
Author(s):  
Basarab G. Hosu ◽  
Vedavalli S. J. Nathan ◽  
Howard C. Berg
Keyword(s):  
Polarized Light ◽  
Sodium Ion ◽  
Reasonable Assumption ◽  
Flagellar Motor ◽  
Electrochemical Gradient ◽  
Flexible Coupling ◽  
Latex Beads ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor ◽  
Peptidoglycan Layer

Most bacteria that swim, includingEscherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.

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 Motile ghosts of the halophilic archaeon, Haloferax volcanii

2020 ◽  
Vol 117 (43) ◽  
pp. 26766-26772
Author(s):  
Yoshiaki Kinosita ◽  
Nagisa Mikami ◽  
Zhengqun Li ◽  
Frank Braun ◽  
Tessa E. F. Quax ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Atp Hydrolysis ◽  
Response Regulator ◽  
Model System ◽  
Flagellar Motor ◽  
Domains Of Life ◽  
Bacterial Flagellar Motor ◽  
Archaeal Flagellum ◽  
Triton Model ◽  
Rotary Motor

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5′-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma. We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque–speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

scholarly journals Bacterial flagellar motor

2008 ◽  
Vol 41 (2) ◽  
pp. 103-132 ◽  
Author(s):  
Yoshiyuki Sowa ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Cell Envelope ◽  
Molecular Motor ◽  
Motive Force ◽  
Flagellar Motor ◽  
Large Size ◽  
Structural Details ◽  
Bacterial Flagellar Motor ◽  
And Function ◽  
Rotary Motor

AbstractThe bacterial flagellar motor is a reversible rotary nano-machine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+or Na+ions across the cytoplasmic membrane driven by an electrochemical gradient, the proton-motive force or the sodium-motive force. Each motor rotates a helical filament at several hundreds of revolutions per second (hertz). In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signalling proteins. The bacterial flagellar motor was confirmed as a rotary motor in the early 1970s, the first direct observation of the function of a single molecular motor. However, because of the large size and complexity of the motor, much remains to be discovered, in particular, the structural details of the torque-generating mechanism. This review outlines what has been learned about the structure and function of the motor using a combination of genetics, single-molecule and biophysical techniques, with a focus on recent results and single-molecule techniques.

scholarly journals Limiting (zero-load) speed of the rotary motor of Escherichia coli is independent of the number of torque-generating units

2017 ◽  
Vol 114 (47) ◽  
pp. 12478-12482 ◽  
Author(s):  
Bin Wang ◽  
Rongjing Zhang ◽  
Junhua Yuan
Keyword(s):  
Escherichia Coli ◽  
Optical Trapping ◽  
Dark Field ◽  
Duty Ratio ◽  
Flagellar Motor ◽  
The Past ◽  
Dark Field Microscopy ◽  
Bacterial Flagellar Motor ◽  
Mechanochemical Cycle ◽  
Rotary Motor

Rotation of the bacterial flagellar motor is driven by multiple torque-generating units (stator elements). The torque-generating dynamics can be understood in terms of the “duty ratio” of the stator elements, that is, the fraction of time a stator element engages with the rotor during its mechanochemical cycle. The dependence of the limiting speed (zero-load speed) of the motor on the number of stator elements is the determining test of the duty ratio, which has been controversial experimentally and theoretically over the past decade. Here, we developed a method combining laser dark-field microscopy and optical trapping to resolve this controversy. We found that the zero-load speed is independent of the number of stator elements for the bacterial flagellar motor in Escherichia coli, demonstrating that these elements have a duty ratio close to 1.

scholarly journals A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

2017 ◽  
Author(s):  
AL Nord ◽  
E Gachon ◽  
R Perez-Carrasco ◽  
JA Nirody ◽  
A Barducci ◽  
...  
Keyword(s):  
Dissociation Rate ◽  
Flagellar Motor ◽  
Motor Speed ◽  
Adsorption Model ◽  
Catch Bond ◽  
Before And After ◽  
Sequential Adsorption ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor ◽  
Kinetics Of

AbstractThe bacterial flagellar motor (BFM) is the rotary motor which powers 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 mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

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

2019 ◽  
Vol 167 (4) ◽  
pp. 389-398
Author(s):  
Tatsuro Nishikino ◽  
Hiroto Iwatsuki ◽  
Taira Mino ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Ion Channel ◽  
Energy Transduction ◽  
Sodium Ion ◽  
Flagellar Motor ◽  
Ion Flow ◽  
Cytoplasmic Region ◽  
Marine Vibrio ◽  
Bacterial Flagellar Motor

Abstract 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.

scholarly journals Theories of rotary motors

2000 ◽  
Vol 355 (1396) ◽  
pp. 503-509 ◽  
Author(s):  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Structural Data ◽  
Geometric Constraints ◽  
Flagellar Motor ◽  
Motor Behaviour ◽  
Electrochemical Gradient ◽  
Rotary Motors ◽  
Bacterial Flagellar Motor ◽  
Near Future ◽  
Turbine Model

The bacterial flagellar motor and the ATP–hydrolysing F 1 portion of the F 1 F o –ATPase are known to be rotary motors, and it seems highly probable that the H + –translocating F o portion rotates too. The energy source in the case of F o and the flagellar motor is the flow of ions, either H + (protons) or Na + , down an electrochemical gradient across a membrane. The fact that ions flow in a particular direction through a well–defined structure in these motors invites the possibility of a type of mechanism based on geometric constraints between the rotor position and the paths of ions flowing through the motor. The two beststudied examples of such a mechanism are the ‘turnstile’ model of Khan and Berg and the ‘proton turbine’ model of Lauger or Berry. Models such as these are typically represented by a small number of kinetic states and certain allowed transitions between them. This allows the calculation of predictions of motor behaviour and establishes a dialogue between models and experimental results. In the near future structural data and observations of single–molecule events should help to determine the nature of the mechanism of rotary motors, while motor models must be developed that can adequately explain the measured relationships between torque and speed in the flagellar motor.

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.

scholarly journals Motile ghosts of the halophilic archaeon, Haloferax volcanii

2020 ◽  
Author(s):  
Yoshiaki Kinosita ◽  
Nagisa Mikami ◽  
Zhengqun Li ◽  
Frank Braun ◽  
Tessa EF. Quax ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Atp Hydrolysis ◽  
Response Regulator ◽  
Model System ◽  
Flagellar Motor ◽  
Domains Of Life ◽  
Bacterial Flagellar Motor ◽  
Archaeal Flagellum ◽  
Triton Model ◽  
Rotary Motor

SummaryMotility is seen across all domains of life1. Prokaryotes exhibit various types of motilities, such as gliding, swimming, and twitching, driven by supramolecular motility machinery composed of multiple different proteins2. In archaea only swimming motility is reported, driven by the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament which acts as a propeller3,4. Unlike the bacterial flagellar motor (BFM), adenosine triphosphate (ATP) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum5,6. However, direct evidence is still lacking due to the lack of a versatile model system. Here we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella7 and gliding Mycoplasma8,9. We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation, which is the opposite of a previous report10. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life11,12, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

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