High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P

Steps in the bacterial flagellar motor

The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps [Sowa et al. (2005) Nature 437, 916--919]. Here we propose a simple physical model that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties, and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. Our model also predicts a sublinear torque-speed relationship at low torque, and a peak in rotor diffusion as a function of torque

Steps and bumps: precision extraction of discrete states of molecular machines using physically-based, high-throughput time series analysis

We report new statistical time-series analysis tools providing significant improvements in the rapid, precision extraction of discrete state dynamics from large databases of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we demonstrate new techniques for recovering discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of step-like rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, symmetries and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a major advance in the drive towards automated, precision, highthroughput studies of molecular machine dynamics. Modular, open-source software that implements these techniques is provided at http://www.eng.ox.ac.uk/samp/members/max/software

Microscopic Analysis of Bacterial Motility at High Pressure

Freely available on the publisher's website.The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment

Flagellar polymorphism-dependent bacterial swimming motility in a structured environment

Most motile bacteria use supramolecular motility machinery called bacterial flagellum, which converts the chemical energy gained from ion flux into mechanical rotation. Bacterial cells sense their external environment through a two-component regulatory system consisting of a histidine kinase and response regulator. Combining these systems allows the cells to move toward favorable environments and away from their repellents. A representative example of flagellar motility is run-and-tumble swimming in Escherichia coli, where the counter-clockwise (CCW) rotation of a flagellar bundle propels the cell forward, and the clockwise (CW) rotation undergoes cell re-orientation (tumbling) upon switching the direction of flagellar motor rotation from CCW to CW. In this mini review, we focus on several types of chemotactic behaviors that respond to changes in flagellar shape and direction of rotation. Moreover, our single-cell analysis demonstrated back-and-forth swimming motility of an original E. coli strain. We propose that polymorphic flagellar changes are required to enhance bacterial movement in a structured environment as a colony spread on an agar plate

Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20

The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force; however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli &Delta;motAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis &Delta;motAB&Delta;motPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation