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 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 Ligand Sensing Enhances Bacterial Flagellar Motor Output via Stator Recruitment

2020 ◽  
Author(s):  
Farha Naaz ◽  
Megha Agrawal ◽  
Soumyadeep Chakraborty ◽  
Mahesh S. Tirumkudulu ◽  
K.V. Venkatesh
Keyword(s):  
Escherichia Coli ◽  
Population Level ◽  
Combined Effect ◽  
Flagellar Motor ◽  
Chemical Concentration ◽  
Motor Speed ◽  
The Past ◽  
Mutant Strains ◽  
Bacterial Flagellar Motor

AbstractThe phenomenon of chemotaxis in bacteria, where the cells migrate towards or away from chemicals, has been extensively studied in the past. For flagellated bacteria such as Escherichia coli, a change in chemical concentration in its environment is sensed by a chemoreceptor and communicated via a well-characterised signalling pathway to the flagellar motor. It has been widely accepted that the signals change the rotation bias of the motor without influencing the motor speed. Here, we present results to the contrary and show that the bacteria is also capable of modulating motor speed on merely sensing a ligand. Step changes in concentration of non-metabolisable ligand cause temporary recruitment of stators leading to a momentary increase in motor speeds. For metabolisable ligand, the combined effect of sensing and metabolism leads to higher motor speeds for longer durations. Swimming speeds measured at the population level corroborate the observations. Experiments performed with mutant strains delineate the role of metabolism and sensing in the modulation of motor speed and show how speed changes along with changes in bias can significantly enhance bacteria’s response to changes in its environment.

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 Speed of the bacterial flagellar motor near zero load depends on the number of stator units

2017 ◽  
Vol 114 (44) ◽  
pp. 11603-11608 ◽  
Author(s):  
Ashley L. Nord ◽  
Yoshiyuki Sowa ◽  
Bradley C. Steel ◽  
Chien-Jung Lo ◽  
Richard M. Berry
Keyword(s):  
Escherichia Coli ◽  
Motive Force ◽  
Flagellar Motor ◽  
Single Curve ◽  
Ion Gradient ◽  
Membrane Bound ◽  
Bacterial Flagellar Motor ◽  
Ion Conducting ◽  
Relationship Of

The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque–speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+-driven motors. We measure the full torque–speed relationship of one and two H+units inEscherichia coliby selecting the number of H+units and controlling the number of Na+units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque–speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque–speed curves to be collapsed onto a single curve by simple rescaling.

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 Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

2019 ◽  
Vol 116 (10) ◽  
pp. 1952-1959 ◽  
Author(s):  
Kento Sato ◽  
Shuichi Nakamura ◽  
Seishi Kudo ◽  
Shoichi Toyabe
Keyword(s):  
Dynamic Load ◽  
Load Control ◽  
Duty Ratio ◽  
Flagellar Motor ◽  
Bacterial Flagellar Motor

scholarly journals Robustness in an Ultrasensitive Motor

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Guangzhe Liu ◽  
Antai Tao ◽  
Rongjing Zhang ◽  
Junhua Yuan
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Motor Output ◽  
Flagellar Motor ◽  
Content Type ◽  
P Concentration ◽  
Bacterial Flagellar Motor ◽  
Cell Variation

ABSTRACT In Escherichia coli, the chemotaxis response regulator CheY-P binds to FliM, a component of the switch complex at the base of the bacterial flagellar motor, to modulate the direction of motor rotation. The bacterial flagellar motor is ultrasensitive to the concentration of unbound CheY-P in the cytoplasm. CheY-P binds to FliM molecules both in the cytoplasm and on the motor. As the concentration of FliM unavoidably varies from cell to cell, leading to a variation of unbound CheY-P concentration in the cytoplasm, this raises the question whether the flagellar motor is robust against this variation, that is, whether the rotational bias of the motor is more or less constant as the concentration of FliM varies. Here, we showed that the motor is robust against variations of the concentration of FliM. We identified adaptive remodeling of the motor as the mechanism for this robustness. As the level of FliM molecules changes, resulting in different amounts of the unbound CheY-P molecules, the motor adaptively changes the composition of its switch complex to compensate for this effect. IMPORTANCE The bacterial flagellar motor is an ultrasensitive motor. Its output, the probability of the motor turning clockwise, depends sensitively on the occupancy of the protein FliM (a component on the switch complex of the motor) by the input CheY-P molecules. With a limited cellular pool of CheY-P molecules, cell-to-cell variation of the FliM level would lead to large unwanted variation of the motor output if not compensated. Here, we showed that the motor output is robust against the variation of FliM level and identified the adaptive remodeling of the motor switch complex as the mechanism for this robustness.

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 A Slow-Motility Phenotype Caused by Substitutions at Residue Asp31 in the PomA Channel Component of a Sodium-Driven Flagellar Motor

2000 ◽  
Vol 182 (11) ◽  
pp. 3314-3318 ◽  
Author(s):  
Seiji Kojima ◽  
Tomokazu Shoji ◽  
Yukako Asai ◽  
Ikuro Kawagishi ◽  
Michio Homma
Keyword(s):  
Negative Charge ◽  
Dark Field ◽  
Sodium Ion ◽  
Flagellar Motor ◽  
Wild Type ◽  
Or Efficiency ◽  
Motor Complex ◽  
Dark Field Microscopy ◽  
Nacl Concentration ◽  
High Concentrations

ABSTRACT PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio alginolyticus. We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.

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