Constraints on models for the flagellar rotary motor

Most bacteria that swim are propelled by flagellar filaments, each driven at its base by a rotary motor embedded in the cell wall and cytoplasmic membrane. A motor is about 45 nm in diameter and made up of about 20 different kinds of parts. It is assembled from the inside out. It is powered by a proton (or in some species, a sodium-ion) flux. It steps at least 400 times per revolution. At low speeds and high torques, about 1000 protons are required per revolution, speed is proportional to protonmotive force, and torque varies little with temperature or hydrogen isotope. At high speeds and low torques, torque increases with temperature and is sensitive to hydrogen isotope. At room temperature, torque varies remarkably little with speed from about -100 Hz (the present limit of measurement) to about 200 Hz, and then it declines rapidly reaching zero at about 300 Hz. These are facts that motor models should explain. None of the existing models for the flagellar rotary motor completely do so.     

FliZ induces a kinetic switch in flagellar gene expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.     

The switching dynamics of the bacterial flagellar motor

Many bacteria are propelled by flagellar motors that stochastically switch between the clockwise and counterclockwise rotation direction. Although the switching dynamics is one of their most important characteristics, the mechanisms that control it are poorly understood. We present a statistical–mechanical model of the bacterial flagellar motor. At its heart is the assumption that the rotor protein complex, which is connected to the flagellum, can exist in two conformational states and that switching between these states depends on the interactions with the stator proteins, which drive the rotor. This couples switching to rotation, making the switch sensitive to torque and speed. Another key element is that after a switch, it takes time for the load to build up, due to conformational transitions of the flagellum. This slow relaxation dynamics of the filament leads, in combination with the load dependence of the switching frequency, to a characteristic switching time, as recently observed. Hence, our model predicts that the switching dynamics is not only controlled by the chemotaxis‐signaling network, but also by mechanical feedback of the flagellum.     

Temperature-induced switching of the bacterial flagellar motor.

Chemotaxis signaling proteins normally control the direction of rotation of the flagellar motor of Escherichia coli. In their absence, a wild-type motor spins exclusively counterclockwise. Although the signaling pathway is well defined, relatively little is known about switching, the mechanism that enables the motor to change direction. We found that switching occurs in the absence of signaling proteins when cells are cooled to temperatures below about 10 degrees C. The forward rate constant (for counterclockwise to clockwise, CCW to CW, switching) increases and the reverse rate constant (for CW to CCW switching) decreases as the temperature is lowered. At about -2 degrees C, most motors spin exclusively CW. At temperatures for which reversals are frequent enough to generate a sizable data set, both CCW and CW interval distributions appear to be exponential. From the rate constants we computed equilibrium constants and standard free energy changes, and from the temperature dependence of the standard free energy changes we determined standard enthalpy and entropy changes. Using transition-state theory, we also calculated the activation free energy, enthalpy, and entropy. We conclude that the CW state is preferred at very low temperatures and that it is relatively more highly bonded and restricted than the CCW state.     

Helix rotation model of the flagellar rotary motor

A new model of the flagellar motor is proposed that is based on established dynamics of the KcsA potassium ion channel and on known genetic, biochemical, and biophysical facts, which accounts for the mechanics of torque generation, force transmission, and reversals of motor rotation. It predicts that proton (or in some species sodium ion) flow generates short, reversible helix rotations of the MotA-MotB channel complex (the stator) that are transmitted by Coulomb forces to the FliG segments at the rotor surface. Channels are arranged as symmetric pairs, S and T, that swing back and forth in synchrony. S and T alternate in attaching to the rotor, so that force transmission proceeds in steps. The sense of motor rotation can be readily reversed by conformationally switching the position of charged groups on the rotor so that they interact with the stator during the reverse rather than forward strokes. An elastic device accounts for the observed smoothness of rotation and a prolonged attachment of the torque generators to the rotor, i.e., a high duty ratio of each torque-generating unit.     

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.     

Energy transduction in the bacterial flagellar motor. Effects of load and pH.

The effect of load and pH on the relation between proton potential and flagellar rotation has been studied in cells of a smooth-swimming Streptococcus strain. The driving potential, speeds of free-swimming bacteria, and rotation rates of bacteria tethered to glass by a single flagellum were measured. The relation between rotation rate of tethered bacteria and potential was remarkably linear up to nearly -200 mV. The relation between swimming speed and potential exhibited both saturation and threshold, as previously observed in other species. The form of these relations depended on pH. The equivalence of the electrical and chemical potential components of the proton potential in enabling swimming depended on the voltage. Our observations may be most simply accommodated by a kinetic scheme that links transmembrane proton transits to a tightly coupled work cycle. The properties of this scheme were elucidated by computer simulations of the experimental plots. These simulations indicated that the protonable groups that participate in the rate limiting reactions have a fractional electrical distance between three-fourths to all of the way toward the cytoplasm with a corresponding mean proton binding affinity of 10(-7.3)-10(-7.0) M, respectively.     

The rate of protein secretion dictates the temporal dynamics of flagellar gene expression

Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.     

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.     

The proton flux through the bacterial flagellar motor

Bacterial flagella are driven by a rotary motor that utilizes the free energy stored in the electrochemical proton gradient across the cytoplasmic membrane to do mechanical work. The flux of protons coupled to motor rotation was measured in Streptococcus and found to be directly proportional to motor speed. This supports the hypothesis that the movement of protons through the motor is tightly coupled to the rotation of its flagellar filament. Under this assumption the efficiency of energy conversion is close to unity at the low speeds encountered in tethered cells but only a few percent at the high speeds encountered in swimming cells. This difference appears to be due to dissipation by processes internal to the motor. The efficiency at high speeds exhibits a steep temperature dependence and a sizable deuterium solvent isotope effect.     

Na(+)-driven flagellar motor of Vibrio

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the coupling ion: namely the H(+)-driven motor and the Na(+)-driven motor. Analysis of the flagellar motor at the molecular level is far more advanced in the H(+)-driven motor than in the Na(+)-driven motor. Recently, the genes of the Na(+)-driven motor have been cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In this review, we summarize recent studies of the Na(+)-driven flagellar motor.