Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar 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.

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

Journal of Bacteriology ◽

10.1128/jb.00016-21 ◽

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.

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Structure of the molecular bushing of the bacterial flagellar motor

Nature Communications ◽

10.1038/s41467-021-24715-3 ◽

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.

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Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H+-Driven and Na+-Driven Motors in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.188.4.1466-1472.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1466-1472 ◽

Cited By ~ 57

Author(s):

Toshiharu Yakushi ◽

Junghoon Yang ◽

Hajime Fukuoka ◽

Michio Homma ◽

David F. Blair

Keyword(s):

Escherichia Coli ◽

Comparative Study ◽

Electrostatic Interactions ◽

General Feature ◽

Flagellar Motor ◽

Motor Patterns ◽

E Coli ◽

Flagellar Motors ◽

Charged Residues ◽

Flagellar Rotation

ABSTRACT In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713655114 ◽

2017 ◽

Vol 114 (47) ◽

pp. 12478-12482 ◽

Cited By ~ 10

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.

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Catch bond drives stator mechanosensitivity in the bacterial flagellar motor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716002114 ◽

2017 ◽

Vol 114 (49) ◽

pp. 12952-12957 ◽

Cited By ~ 30

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143110 ◽

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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