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.     

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.     

Bacterial Flagellar Motor Switch in Response to CheY-P Regulation and Motor Structural Alterations

The bacterial flagellar motor (BFM) is a molecular machine that rotates the helical filaments and propels the bacteria swimming toward favorable conditions. In our previous works, we built a stochastic conformational spread model to explain the dynamic and cooperative behavior of BFM switching. Here, we extended this model to test whether it can explain the latest experimental observations regarding CheY-P regulation and motor structural adaptivity. We show that our model predicts a strong correlation between rotational direction and the number of CheY-Ps bound to the switch complex, in agreement with the latest finding from Fukuoka et al. It also predicts that the switching sensitivity of the BFM can be fine-tuned by incorporating additional units into the switch complex, as recently demonstrated by Yuan et al., who showed that stoichiometry of FliM undergoes dynamic change to maintain ultrasensitivity in the motor switching response. In addition, by locking some rotor switching units on the switch complex into the stable clockwise-only conformation, our model has accurately simulated recent experiments expressing clockwise-locked FliG(ΔPAA) into the switch complex and reproduced the increased switching rate of the motor.     

Specificity of motor components in the dual flagellar system of Shewanella putrefaciens CN-32

Bacterial flagellar motors are intricate nanomachines in which the stator units and rotor component FliM may be dynamically exchanged during function. Similar to other bacterial species, the gammaproteobacterium Shewanella putrefaciens CN-32 possesses a complete secondary flagellar system along with a corresponding stator unit. Expression of the secondary system occurs during planktonic growth in complex media and leads to the formation of a subpopulation with one or more additional flagella at random positions in addition to the primary polar system. We used physiological and phenotypic characterizations of defined mutants in concert with fluorescent microscopy on labelled components of the two different systems, the stator proteins PomB and MotB, the rotor components FliM(1) and FliM(2), and the auxiliary motor components MotX and MotY, to determine localization, function and dynamics of the proteins in the flagellar motors. The results demonstrate that the polar flagellum is driven by a Na(+)-dependent FliM(1)/PomAB/MotX/MotY flagellar motor while the secondary system is rotated by a H(+)-dependent FliM(2)/MotAB motor. The components were highly specific for their corresponding motor and are unlikely to be extensively swapped or shared between the two flagellar systems under planktonic conditions. The results have implications for both specificity and dynamics of flagellar motor components.     

Conformational spread in the flagellar motor switch: a model study

The reliable response to weak biological signals requires that they be amplified with fidelity. In E. coli, the flagellar motors that control swimming can switch direction in response to very small changes in the concentration of the signaling protein CheY-P, but how this works is not well understood. A recently proposed allosteric model based on cooperative conformational spread in a ring of identical protomers seems promising as it is able to qualitatively reproduce switching, locked state behavior and Hill coefficient values measured for the rotary motor. In this paper we undertook a comprehensive simulation study to analyze the behavior of this model in detail and made predictions on three experimentally observable quantities: switch time distribution, locked state interval distribution, Hill coefficient of the switch response. We parameterized the model using experimental measurements, finding excellent agreement with published data on motor behavior. Analysis of the simulated switching dynamics revealed a mechanism for chemotactic ultrasensitivity, in which cooperativity is indispensable for realizing both coherent switching and effective amplification. These results showed how cells can combine elements of analog and digital control to produce switches that are simultaneously sensitive and reliable.     

Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.     

Torque and rotation rate of the bacterial flagellar motor.

This paper describes an analysis of microscopic models for the coupling between ion flow and rotation of bacterial flagella. In model I it is assumed that intersecting half-channels exist on the rotor and the stator and that the driving ion is constrained to move together with the intersection site. Model II is based on the assumption that ion flow drives a cycle of conformational transitions in a channel-like stator subunit that are coupled to the motion of the rotor. Analysis of both mechanisms yields closed expressions relating the torque M generated by the flagellar motor to the rotation rate v. Model I (and also, under certain assumptions, model II) accounts for the experimentally observed linear relationship between M and v. The theoretical equations lead to predictions on the relationship between rotation rate and driving force which can be tested experimentally.     

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.     

A quantitative model of the switch cycle of an archaeal flagellar motor and its sensory control

By reverse-engineering we have detected eight kinetic phases of the symmetric switch cycle of the Halobacterium salinarum flagellar motor assembly and identified those steps in the switch cycle that are controlled by sensory rhodopsins during phototaxis. Upon switching the rotational sense, the flagellar motor assembly passes through a stop state from which all subunits synchronously resume rotation in the reverse direction. The assembly then synchronously proceeds through three subsequent functional states of the switch: Refractory, Competent, and Active, from which the rotational sense is switched again. Sensory control of the symmetric switch cycle occurs at two steps in each rotational sense by inversely regulating the probabilities for a change from the Refractory to the Competent and from Competent to the Active rotational mode. We provide a mathematical model for flagellar motor switching and its sensory control, which is able to explain all tested experimental results on spontaneous and light-controlled motor switching, and give a mechanistic explanation based on synchronous conformational transitions of the subunits of the switch complex after reversible dissociation and binding of a response regulator (CheYP). We conclude that the kinetic mechanism of flagellar motor switching and its sensory control is fundamentally different in the archaeon H. salinarum and the bacterium Escherichia coli.     

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.     

Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H⁺ or Na⁺. The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na⁺ motor of Vibrio alginolyticus , is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na⁺-binding site. Furthermore, we demonstrated that the coupling ion, Na⁺, is required for stator assembly and that phenamil (an inhibitor of the Na⁺-driven motor) inhibited the assembly. Carbonyl cyanide m -chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na⁺ influx through the channel, including Na⁺ binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.     

Exchange of rotor components in functioning bacterial flagellar motor

The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s⁻¹, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

A Molecular Mechanism of Bacterial Flagellar Motor Switching

The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliG_C) from FliM, modulating the FliG_C-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis.     

Control of speed modulation (chemokinesis) in the unidirectional rotary motor of Sinorhizobium meliloti

Swimming cells of Sinorhizobium meliloti are driven by flagella that rotate only clockwise. They can modulate rotary speed (achieve chemokinesis) and reorient the swimming path by slowing flagellar rotation. The flagellar motor is energized by proton motive force, and torque is generated by electrostatic interactions at the rotor/stator (FliG/MotA-MotB) interface. Like the Escherichia coli flagellar motor that switches between counterclockwise and clockwise rotation, the S. meliloti rotary motor depends on electrostatic interactions between conserved charged residues, namely, Arg294 and Glu302 (FliG) and Arg90, Glu98 and Glu150 (MotA). Unlike in E. coli, however, Glu150 is essential for torque generation, whereas residues Arg90 and Glu98 are crucial for the chemotaxis-controlled variation of rotary speed. Substitutions of either Arg90 or Glu98 by charge-neutralizing residues or even by their smaller, charge-maintaining isologues, lysine and aspartate, resulted in top-speed flagellar rotation and decreased potential to slow down in response to tactic signalling (chemokinesis-defective mutants). The data infer a novel mechanism of flagellar speed control by electrostatic forces acting at the rotor/stator interface. These features have been integrated into a working model of the speed-modulating rotary motor.     

Flagellar movement driven by proton translocation

The bacterial flagellar motor couples ion flow to rotary motion at high speed and with apparently fixed stoichiometry. The functional properties of the motor are quite well understood, but its molecular mechanism remains unknown. Recent studies of motor physiology, coupled with mutational and biochemical studies of the components, put significant constraints on the mechanism. Rotation is probably driven by conformational changes in membrane-protein complexes that form the stator. These conformational changes occur as protons move on and off a critical Asp residue in the stator protein MotB, and the resulting forces are applied to the rotor protein FliG.     

Forced rotation of Na+-driven flagellar motor in a coupling ion-free environment

Rotational characteristics of Na+-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na+, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na+, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.     

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.