Switching of Bacterial Flagellar Motors Is Triggered by Mutant FliG

Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliG^CW). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CW_bias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CW_bias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors—in motors containing identical fractions of mutant FliM^CW in otherwise wild-type motors, the CW_bias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors.     

A Computational Model of the Fluid Dynamics of Undulatory and Flagellar Swimming

We present a mathematical model and numerical method designedto study the fluid dynamics of swimming organisms. The fullNavier— Stokes equations are solved in a domain of fluidwithin which an organism undergoing time—dependent motionsis immersed. Of interest are both the dynamics of a single organismand the relationship of its morphology to its motility properties,as well as the collective hydrodynamic interactions of groupsof swimmers with each other and their environment. Biologicalapplications include spermatozoa motility in the reproductivetract, swimming of non-smooth filaments, and collective swimmingof algal cells.     

Serological Study of Bacterial Flagellar Hooks

Bacterial hooks were partially purified from flagella isolated from Salmonella SJ25, by treatment with heat to depolymerize flagellar filaments and with n-butanol and calcium chloride to remove membranes. Antihook serum was obtained from a rabbit inoculated with a preparation of hooks. The serum contained antibodies directed against the flagellar filament and cell membrane. These antibodies could be removed from the serum by absorption with purified flagellar filaments and cells of a nonflagellated mutant strain. It was shown by electron microscopy that anti-SJ25-hook antibody reacts with hooks from a number of strains of Salmonella which differed from SJ25 in H and O antigens, flagellar shape, and motility. Hooks possessed by various strains of Salmonella have a common antigenicity. In addition, anti-SJ25-hook cross-reacted with hooks from Escherichia coli W3110 but did not react at all which those from strains of Serratia, Proteus, Aerobacter, and Klebsiella. It is well known that bacteria stop moving upon addition of antiflagella serum to the medium. However, the addition of purified antihook was found to have little effect on motility. At physiological ionic strength and pH, flagellin (Salmonella) can polymerize into flagellar filaments only in the presence of seeds. It was shown that a crude preparation of hooks was able to initiate in vitro polymerization of flagellin.     

Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.     

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.     

Gains of Bacterial Flagellar Motility in a Fungal World

The maintenance of energetically costly flagella by bacteria in non-water-saturated media, such as soil, still presents an evolutionary conundrum. Potential explanations have focused on rare flooding events allowing dispersal. Such scenarios, however, overlook bacterial dispersal along mycelia as a possible transport mechanism in soils. The hypothesis tested in this study is that dispersal along fungal hyphae may lead to an increase in the fitness of flagellated bacteria and thus offer an alternative explanation for the maintenance of flagella even in unsaturated soils. Dispersal along fungal hyphae was shown for a diverse array of motile bacteria. To measure the fitness effect of dispersal, additional experiments were conducted in a model system mimicking limited dispersal, using Pseudomonas putida KT2440 and its nonflagellated (ΔfliM) isogenic mutant in the absence or presence of Morchella crassipes mycelia. In the absence of the fungus, flagellar motility was beneficial solely under conditions of water saturation allowing dispersal, while under conditions limiting dispersal, the nonflagellated mutant exhibited a higher level of fitness than the wild-type strain. In contrast, in the presence of a mycelial network under conditions limiting dispersal, the flagellated strain was able to disperse using the mycelial network and had a higher level of fitness than the mutant. On the basis of these results, we propose that the benefit of mycelium-associated dispersal helps explain the persistence of flagellar motility in non-water-saturated environments.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

A Mechanochemical Model of Flagellar Activity

A theory is presented which quantitatively links the physical properties of a flagellum with parameters which characterize the chemical reactions responsible for deforming the flagellum. Realistic values for the wave parameters are predicted when order-of-magnitude values for the appropriate constants are used. The model may be useful in other fields where mechanochemical coupling occurs.     

A Non-Poissonian Flagellar Motor Switch Increases Bacterial Chemotactic Potential

We investigate bacterial chemotactic strategies using run-tumble and run-reverse-flick motility patterns. The former is typically observed in enteric bacteria such as Escherichia coli and Salmonella and the latter was recently observed in the marine bacteria Vibrio alginolyticus and is possibly exhibited by other polar flagellated species. It is shown that although the three-step motility pattern helps the bacterium to localize near hot spots, an exploitative behavior, its exploratory potential in short times can be significantly enhanced by employing a non-Poissonian regulation scheme for its flagellar motor switches.     

Dynamics of Bacterial Swarming

When vegetative bacteria that can swim are grown in a rich medium on an agar surface, they become multinucleate, elongate, synthesize large numbers of flagella, produce wetting agents, and move across the surface in coordinated packs: they swarm. We examined the motion of swarming Escherichia coli, comparing the motion of individual cells to their motion during swimming. Swarming cells' speeds are comparable to bulk swimming speeds, but very broadly distributed. Their speeds and orientations are correlated over a short distance (several cell lengths), but this correlation is not isotropic. We observe the swirling that is conspicuous in many swarming systems, probably due to increasingly long-lived correlations among cells that associate into groups. The normal run-tumble behavior seen in swimming chemotaxis is largely suppressed, instead, cells are continually reoriented by random jostling by their neighbors, randomizing their directions in a few tenths of a second. At the edge of the swarm, cells often pause, then swim back toward the center of the swarm or along its edge. Local alignment among cells, a necessary condition of many flocking theories, is accomplished by cell body collisions and/or short-range hydrodynamic interactions.     

Effect of Intracellular pH on the Torque-Speed Relationship of Bacterial Proton-Driven Flagellar Motor

Bacterial flagella responsible for motility are driven by rotary motors powered by the electrochemical potential difference of specific ions across the cytoplasmic membrane. The stator of proton-driven flagellar motor converts proton influx into mechanical work. However, the energy conversion mechanism remains unclear. Here, we show that the motor is sensitive to intracellular proton concentration for high-speed rotation at low load, which was considerably impaired by lowering intracellular pH, while zero-speed torque was not affected. The change in extracellular pH did not show any effect. These results suggest that a high intracellular proton concentration decreases the rate of proton translocation and therefore that of the mechanochemical reaction cycle of the motor but not the actual torque generation step within the cycle by the stator-rotor interactions.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

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.     

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.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.