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The spirochete Leptospira has a coiled cell body and two periplasmic flagella (PFs) that reside beneath the outer sheath. PFs extend from each end of the cell body and are attached to the right-handed spiral protoplasmic cylinder (PC) via a connection with the flagellar motor embedded in the inner membrane. PFs bend each end of the cell body into left-handed spiral (S) or planar hook (H) shapes, allowing leptospiral cells to swim using combined anterior S-end and posterior H-end gyrations with PC rotations. As a plausible mechanism for motility, S- and H-end gyrations by PFs and PC rotations by PF countertorque imply mutual influences among the three parts. Here we show a correlation between H-end gyration and PC rotation from the time records of rotation rates and rotational directions of individual swimming cells. We then qualitatively explain the observed correlation using a simple rotation model based on the measurements of motility and intracellular arrangements of PFs revealed by cryo-electron microscopy and electron cryotomography.

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.

Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H(+) is used in Escherichia coli, and Na(+) is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H(+) is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na(+), though it vigorously moved even under Na(+)-free conditions. These results suggest that H(+) is preferentially used and that Na(+) is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment.

The deformation of a flagellum of Vibrio alginolyticus, single-flagellate bacteria, is analyzed theoretically assuming the shape of the flagellum to be a circular helix. The viscous force exerted on the flagellum in aqueous fluid is estimated applying the resistive-force theory based on the Stokes flow. The moment of force in the flagellum are described in analytical expressions and also the curvature and the torsion of the deformed flagellum are expressed analytically according to the Kirchhoff rod model. The deformation of the flagellum is obtained numerically solving evolution equations which determine a space curve from the curvature and the torsion. Comparing variations of the pitch of helical flagella between the numerical solutions and the results of measurement, the flexural rigidity or the elastic bending coefficient for the flagellum of Vibrio alginolyticus is estimated.

The forward and backward swimming speeds and periods of a Vibrio alginolyticus strain that has a single polar flagellum were measured. The backward swimming speeds were 1.5 times greater than the forward ones on average and the average period of backward swimming was shorter than forward swimming. However, the swimming speed and period were not correlated. Similar results were obtained for a mutant that has a 1.6 times longer flagellum on average.

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 (σω) of 9% of ω. When ω was decreased to about 200 r.p.s. by the presence of 1.5 mM amiloride, σωincreased 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 σωup 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.

This paper gives an overview of consecutive studies on the asymmetrical motion of Vibrio alginolyticus cells, which possess a single polar flagellum. Inertial forces are negligible because of the cell size and the motion is expected to be symmetrical. However, asymmetrical characteristics between forward and backward motions were observed. The asymmetry observed in trajectory, swimming speed, and residence time appears only when a cell swims close to a surface. In backward motion, a cell traces circular path, while in forward motion the cell moves in a straight line. The backward swimming speed is faster than the forward speed. Backward swimming cells tend to stay close to a surface longer than forward swimming cells do. An explanation for these asymmetrical characteristics is given based on the results of boundary element analyses of creeping flow around a cell model that consists of a cell body and a rotating flagellum. According to the explanation, the attitude of a cell relative to a surface produces the asymmetry. The studies presented here indicate that the fluid-dynamic interaction between bacterial cells and a surface produces the unexpected asymmetrical motion. This asymmetry may help cells search for preferable states on a surface or to attach to the surface.

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.

BACTERIA such as Escherichia coli and Salmonella typhlmurium swim by rotating their flagella1,2, each of which consists of an external helical filament and a rotary motor embedded in the cell surface (see ref. 3 for a review). The function of the flagellar motor has been examined mainly by tethering the flagellar filament to a glass slide and observing the resultant rotation of the cell body2. But under these conditions the motor operates at a very low speed (about lOr.p.s.) owing to the unnaturally high load conditions inherent in this technique. Lowe et al.4 analysed the frequency of light scattered from swimming cells to estimate the average rotation speed of flagellar bundles of E. coli as about 270 r.p.s. To analyse motor function in more detail, however, measurement of high-speed rotation of a single flagellum (at low load) with a temporal resolution better than 1 ms is needed. We have now developed a new method—laser dark-field microscopy—which fulfils these requirements. We find that although the average rotation speed of S. typhimurium flagella is rather stable, there are occasional abrupt slowdowns, pauses and reversals (accomplished within 1 ms). These changes were frequently observed in mutants defective in one of the motor components (called the switch complex), suggesting that this component is important not only in switching rotational direction but also in torque generation or regulation.

Many bacteria swim by rotating their helical flagellar filaments which are driven by flagellar motors embedded in the cell membranes. In mechanical engineering, bacterial swimming is an interesting subtopic of robotics and nano-mechanics since countless nano-machines made of bio-molecules are packed into 1 µm cells. In this paper, we present three exceptionally interesting facts about swimming bacteria, which have been known for the past decade. First, a flagellar motor rotates extremely fast (the maximum recorded is 1,700 rps). This information produces many new questions regarding, for example, the torque generation mechanism and the wear. The second fact concerns the flagellar filament as an intelligent material. It is sufficiently rigid for a use as a propeller and yet can change its helical form to relax the stress when an excessive force acts on it. The mechanism is now being explored at an atomic level. The last fact is that bacterial cells sometimes swim well in viscous environments. This phenomenon contradicts common knowledge but could be explained by a new hypothesis in which the effect of the polymer network on the bacterial motion was expressed mathematically. We were impressed by the acumen of bacteria. (Review)

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.

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.

Many kind of bacteria swim in aqueous solution by rotating their helical flagellar filaments. Each filament is driven by a rotary motor (flagellar motor) embedded in the cytoplasmic membrane and can rotate very fast. The rotation speed reaches from 100 to more than 1000 r/sec. The possible deformation of such fast rotating flagellar filaments has not yet been observed because of difficulties mainly caused by their extremely small size. We tried to observe the deformation of the flagellar helices of swimming Vibrio alginolyticus cells with laser dark-field microscope. The laser dark-field image was recorded by using an image intensified CCD camera equipped with the electrical shutter. The change in the helical pitch was observed to be rather small, around 10%.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.

Bacterial cells possess ultra-small motors on their surfaces with which to rotate their flagellar filaments. The motor utilizes the electrochemical energy stored in the proton gradient across the cytoplasmic membrane, and can rotate at more than 200 r.p.s. without a load. It can rotate in both clockwise and counterclockwise directions and switch the rotational direction in 1 msec. Its rotator is made of about 10 kinds of proteins and is about 30 nm in diameter. To analyze the motor function in detail, the authors have developed a laser dark-field microscopy technique by which high-speed rotation of a single flagellum can be measured. They have also succeeded in controlling the rotation speed by applying an external electric pulse to a bacterial cell that is held at the tip of a micropipette. >