Binding of the Chemotaxis Response Regulator CheY to the Isolated, Intact Switch Complex of the Bacterial Flagellar 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.

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Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor

Nature Communications ◽

10.1038/s41467-021-25774-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jyot D. Antani ◽

Rachit Gupta ◽

Annie H. Lee ◽

Kathy Y. Rhee ◽

Michael D. Manson ◽

...

Keyword(s):

Adaptive Response ◽

Mechanical Load ◽

Response Regulator ◽

Mechanical Stimulus ◽

Viscous Resistance ◽

Mechanical Forces ◽

Mechanical Stimuli ◽

Flagellar Motor ◽

Neuromuscular Systems ◽

Bacterial Flagellar Motor

AbstractReversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

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Robustness in an Ultrasensitive Motor

mBio ◽

10.1128/mbio.03050-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

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.

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Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor

10.21203/rs.3.rs-437908/v1 ◽

2021 ◽

Author(s):

Jyot Antani ◽

Rachit Gupta ◽

Annie Lee ◽

Kathy Rhee ◽

Michael Manson ◽

...

Keyword(s):

Adaptive Response ◽

Mechanical Load ◽

Response Regulator ◽

Mechanical Stimulus ◽

Viscous Resistance ◽

Mechanical Forces ◽

Mechanical Stimuli ◽

Flagellar Motor ◽

Neuromuscular Systems ◽

Bacterial Flagellar Motor

Abstract Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

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Motile ghosts of the halophilic archaeon, Haloferax volcanii

10.1101/2020.01.08.899351 ◽

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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