Dataset Information

Dynamics of the bacterial flagellar motor: the effects of stator compliance, back steps, temperature, and rotational asymmetry.

ABSTRACT: The rotation of a bacterial flagellar motor (BFM) is driven by multiple stators tethered to the cell wall. Here, we extend a recently proposed power-stroke model to study the BFM dynamics under different biophysical conditions. Our model explains several key experimental observations and reveals their underlying mechanisms. 1), The observed independence of the speed at low load on the number of stators is explained by a force-dependent stepping mechanism that is independent of the strength of the stator tethering spring. Conversely, without force-dependent stepping, an unrealistically weak stator spring is required. 2), Our model with back-stepping naturally explains the observed absence of a barrier to backward rotation. Using the same set of parameters, it also explains BFM behaviors in the high-speed negative-torque regime. 3), From the measured temperature dependence of the maximum speed, our model shows that stator-stepping is a thermally activated process with an energy barrier. 4), The recently observed asymmetry in the torque-speed curve between counterclockwise- and clockwise-rotating BFMs can be quantitatively explained by the asymmetry in the stator-rotor interaction potentials, i.e., a quasilinear form for the counterclockwise motor and a quadratic form for the clockwise motor.

SUBMITTER: Meacci G

PROVIDER: S-EPMC3077703 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Dynamics of the bacterial flagellar motor: the effects of stator compliance, back steps, temperature, and rotational asymmetry.

Meacci Giovanni G Lan Ganhui G Tu Yuhai Y

Biophysical journal 20110401 8

The rotation of a bacterial flagellar motor (BFM) is driven by multiple stators tethered to the cell wall. Here, we extend a recently proposed power-stroke model to study the BFM dynamics under different biophysical conditions. Our model explains several key experimental observations and reveals their underlying mechanisms. 1), The observed independence of the speed at low load on the number of stators is explained by a force-dependent stepping mechanism that is independent of the strength of th ...[more]

PMID: 21504735

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Project description:The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(?PAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(?PEV)). The FliG(?PAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(?PAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(?PEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.

Project description: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- 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 in PomA 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.ImportanceThe 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- and disulfide-crosslinking 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.

Dataset Information

Dynamics of the bacterial flagellar motor: the effects of stator compliance, back steps, temperature, and rotational asymmetry.

Publications

Dynamics of the bacterial flagellar motor: the effects of stator compliance, back steps, temperature, and rotational asymmetry.

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