Populational Heterogeneity vs. Temporal Fluctuation in Escherichia coli Flagellar Motor Switching

The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations.     

Populational Heterogeneity vs. Temporal Fluctuation in Escherichia coli Flagellar Motor Switching

The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations.     

Distinct in situ structures of the Borrelia flagellar motor

Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.     

Microscopic analysis of bacterial motility at high pressure

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.     

Rotation and switching of the flagellar motor assembly in Halobacterium halobium.

Halobacterium halobium swims with a polarly inserted motor-driven flagellar bundle. The swimming direction of the cell can be reserved by switching the rotational sense of the bundle. The switch is under the control of photoreceptor and chemoreceptor proteins that act through a branched signal chain. The swimming behavior of the cells and the switching process of the flagellar bundle were investigated with a computer-assisted motion analysis system. The cells were shown to swim faster by clockwise than by counterclockwise rotation of the flagellar bundle. From the small magnitude of speed fluctuations, it is concluded that the majority, if not all, of the individual flagellar motors of a cell rotate in the same direction at any given time. After stimulation with light (blue light pulse or orange light step-down), the cells continued swimming with almost constant speed but then slowed before they reversed direction. The cells passed through a pausing state during the change of the rotational sense of the flagellar bundle and then exhibited a transient acceleration. Both the average length of the pausing period and the transient acceleration were independent of the stimulus size and thus represent intrinsic properties of the flagellar motor assembly. The average length of the pausing period of individual cells, however, was not constant. The time course of the probability for spontaneous motor switching was calculated from frequency distribution and shown to be independent of the rotational sense. The time course further characterizes spontaneous switching as a stochastic rather than an oscillator-triggered event.     

The bidirectional polar and unidirectional lateral flagellar motors of Vibrio alginolyticus are controlled by a single CheY species

The bacterial flagellar motor is an elaborate molecular machine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is its swift switching of the rotational direction or speed upon binding of the response regulator phospho-CheY, which causes the changes in swimming that achieve chemotaxis. Vibrio alginolyticus has dual flagellar systems: the Na(+)-driven polar flagellum (Pof) and the H(+)-driven lateral flagella (Laf), which are used for swimming in liquid and swarming over surfaces respectively. Here we show that both swimming and surface-swarming of V. alginolyticus involve chemotaxis and are regulated by a single CheY species. Some of the substitutions of CheY residues conserved in various bacteria have different effects on the Pof and Laf motors, implying that CheY interacts with the two motors differently. Furthermore, analyses of tethered cells revealed that their switching modes are different: the Laf motor rotates exclusively counterclockwise and is slowed down by CheY, whereas the Pof motor turns both counterclockwise and clockwise, and CheY controls its rotational direction.     

Measurement of the Internal Frictional Drag of the Bacterial Flagellar Motor by Fluctuation Analysis

The bacterial flagellar motor generates the torque that drives the rotation of bacterial flagellar filaments. The torque it generates depends sensitively on the frictional viscous drag on the motor, which includes the frictional viscous drag on the filaments (external load) and the internal frictional viscous drag on the rotor (internal load). The internal load was roughly estimated previously by modeling it as a sphere of a radius of 20 nm rotating in a lipid of viscosity of 100 cp but was never measured experimentally. Here, we measured the internal load by fluctuation analysis of the motor velocity traces. A similar approach should be applicable to other molecular motors.     

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB_C2) and Vibrio alginolyticus (PomB_C5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotB_C2, the dimeric conformation of the PomB_C5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Quantification of flagellar motor stator dynamics through in vivo proton‐motive force control

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane‐bound stator complexes. We used the light‐driven proton pump proteorhodopsin (pR) to control the proton‐motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s^?1. Using GFP‐tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s^?1.     

Phosphorylation-independent bacterial chemoresponses correlate with changes in the cytoplasmic level of fumarate.

Bacterial chemotaxis is based on modulation of the probability to switch the direction of flagellar rotation. Responses to many stimuli are transduced by a two-component system via reversible phosphorylation of CheY, a small cytoplasmic protein that directly interacts with the switch complex at the flagellar motor. We found that the chemorepellents indole and benzoate induce motor switching in Escherichia coli cells with a disabled phosphorylation cascade. This phosphorylation-independent chemoresponse is explained by reversible inhibition of fumarase by indole or benzoate which leads to an increased level of cellular fumarate, a compound involved in motor switching for bacteria and archaea. Genetic deletion of fumarase increased the intracellular concentration of fumarate and enhanced the switching frequency of the flagellar motors irrespective of the presence or absence of the phosphorylation cascade. These correlations provide evidence for fumarate-dependent metabolic signal transduction in bacterial chemosensing.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.     

Rotational Fluctuation of the Sodium-driven Flagellar Motor ofVibrio alginolyticusInduced by Binding of Inhibitors

Rotation of the Na⁺-driven flagellar motor ofVibrio alginolyticuswas investigated under the influence of inhibitors specific to the motor, amiloride and phenamil. The rotation rate of a single flagellum on a cell stuck to a glass slide was examined using laser dark-field microscopy. In the presence of 50 mM NaCl, the average rotation rate (ω) was about 600 r.p.s. with a standard deviation (σ_ω) of 9% of ω. When ω was decreased to about 200 r.p.s. by the presence of 1.5 mM amiloride, σ_ωincreased to 15% of ω. On the other hand, when ω was decreased to about 200 r.p.s. by the addition of 0.6 μM phenamil, a large increase in σ_ωup to 50% of ω, was observed. Similarly large fluctuations were observed at other concen trations of phenamil. These observations suggest that dissociation of phenamil from the motor was much slower than that of amiloride. A very low concentration of phenamil caused a transient but substantial reduction in rotation rate. This might suggest that binding of only a single molecule of phenamil strongly inhibits the torque generation in the flagellar motor.     

The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding

Swimming speed (v) and flagellar-bundle rotation rate (f) of Salmonella typhimurium, which has peritrichous flagella, were simultaneously measured by laser dark-field microscopy (LDM). Clear periodic changes in the LDM signals from a rotating bundle indicated in-phase rotation of the flagella in the bundle. A roughly linear relation between v and f was observed, though the data points were widely distributed. The ratio of v to f (v-f ratio), which indicates the propulsive distance during one flagellar rotation, was 0.27 microm (11% of the flagellar pitch) on average. The experimental v-f ratio was twice as large as the calculated one on the assumption that a cell had a single flagellum. A flagellar bundle was considered to propel a cell more efficiently than a single flagellum.     

Mechanical limits of bacterial flagellar motors probed by electrorotation.

We used the technique of electrorotation to apply steadily increasing external torque to tethered cells of the bacterium Escherichia coli while continuously recording the speed of cell rotation. We found that the bacterial flagellar motor generates constant torque when rotating forward at low speeds and constant but considerably higher torque when rotating backward. At intermediate torques, the motor stalls. The torque-speed relationship is the same in both directional modes of switching motors. Motors forced backward usually break, either suddenly and irreversibly or progressively. Motors broken progressively rotate predominantly at integral multiples of a unitary speed during the course of both breaking and subsequent recovery, as expected if progressive breaking affects individual torque-generating units. Torque is reduced by the same factor at all speeds in partially broken motors, implying that the torque-speed relationship is a property of the individual torque-generating units.     

The bacterial flagellar motor

The bacterial flagellar motor is a remarkable molecular machine that converts chemical energy into work. Knowledge of the structure, genetics, and dynamics of the motor has expanded steadily. Recent progress is reviewed, with an emphasis on the dynamics of flagellar rotation. Previous results with tethered cells, which rotate slowly, are contrasted with recent work on swimming cells, whose motors turn very rapidly. Genetic evidence delineates a small set of proteins that are likely to participate directly in the process of torque generation. An explicit hypothesis for torque generation is described, in which roles are envisaged for each of these proteins.     

A chimeric N-terminal Escherichia coli--C-terminal Rhodobacter sphaeroides FliG rotor protein supports bidirectional E. coli flagellar rotation and chemotaxis

Flagellate bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium typically express 5 to 12 flagellar filaments over their cell surface that rotate in clockwise (CW) and counterclockwise directions. These bacteria modulate their swimming direction towards favorable environments by biasing the direction of flagellar rotation in response to various stimuli. In contrast, Rhodobacter sphaeroides expresses a single subpolar flagellum that rotates only CW and responds tactically by a series of biased stops and starts. Rotor protein FliG transiently links the MotAB stators to the rotor, to power rotation and also has an essential function in flagellar export. In this study, we sought to determine whether the FliG protein confers directionality on flagellar motors by testing the functional properties of R. sphaeroides FliG and a chimeric FliG protein, EcRsFliG (N-terminal and central domains of E. coli FliG fused to an R. sphaeroides FliG C terminus), in an E. coli FliG null background. The EcRsFliG chimera supported flagellar synthesis and bidirectional rotation; bacteria swam and tumbled in a manner qualitatively similar to that of the wild type and showed chemotaxis to amino acids. Thus, the FliG C terminus alone does not confer the unidirectional stop-start character of the R. sphaeroides flagellar motor, and its conformation continues to support tactic, switch-protein interactions in a bidirectional motor, despite its evolutionary history in a bacterium with a unidirectional motor.     

Torque generated by the bacterial flagellar motor close to stall.

In earlier work in which electrorotation was used to apply external torque to tethered cells of the bacterium Escherichia coli, it was found that the torque required to force flagellar motors backward was considerably larger than the torque required to stop them. That is, there appeared to be substantial barrier to backward rotation. Here, we show that in most, possibly all, cases this barrier is an artifact due to angular variation of the torque applied by electrorotation, of the motor torque, or both; the motor torque appears to be independent to speed or to vary linearly with speed up to speeds of tens of Hertz, in either direction. However, motors often break catastrophically when driven backward, so backward rotation is not equivalent to forward rotation. Also, cells can rotate backward while stalled, either in randomly timed jumps of 180 degrees or very slowly and smoothly. When cells rotate slowly and smoothly backward, the motor takes several seconds to recover after electrorotation is stopped, suggesting that some form of reversible damage has occurred. These findings do not affect the interpretation of electrorotation experiments in which motors are driven rapidly forward.     

Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error‐prone PCR to introduce single point mutations into the sodium‐powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium‐channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C‐terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion‐channel blockers.     

Coordinated reversal of flagellar motors on a single Escherichia coli cell

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.