Unidirectional, intermittent rotation of the flagellum of Rhodobacter sphaeroides.

The single flagellum of the photosynthetic bacterium Rhodobacter sphaeroides was found to be medially located on the cell body. Observation of free-swimming bacteria, and bacteria tethered by their flagellar filaments, revealed that the flagellum could only rotate in the clockwise direction; switching of the direction of rotation was never observed. Flagellar rotation stopped periodically, typically several times a minute for up to several seconds each. Reorientation of swimming cells appeared to be the result of Brownian rotation during the stop periods. The flagellar filament displayed polymorphism; detached and nonrotating filaments were usually seen as large-amplitude helices of such short wavelength that they appeared as flat coils or circles, whereas the filaments on swimming cells showed a normal (small-amplitude, long-wavelength) helical form. With attached filaments, the transition from the normal to the coiled form occurred when the flagellar motor stopped rotating, proceeding from the distal end towards the cell body. It is possible that both the relaxation process and the smaller frictional resistance after relaxation may act to enhance the rate of reorientation of the cell. The transition from the coiled to the normal form occurred when the motor restarted, proceeding from the proximal end outwards, which might further contribute to the reorientation of the cell before it reaches a stable swimming geometry.     

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.     

Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.     

Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling.     

Quantitative measurements of proton motive force and motility in Bacillus subtilis.

The protein motive force of metabolizing Bacillus subtilis cells was only slightly affected by changes in the external pH between 5 and 8, although the electrical component and the chemical component of the proton motive force contributed differently at different external pH. The electrical component of the proton motive force was very small at pH 5, and the chemical component was almost negligible at pH 7.5. At external pH values between 6 and 7.7, swimming speed of the cells stayed constant. Thus, either the electrical component or the chemical component of the proton motive force could drive the flagellar motor. When the proton motive force of valinomycin-treated cells was quantitatively decreased by increasing the external K+ concentration, the swimming speed of the cells changed in a unique way: the swimming speed was not affected until about--100 mV, then decreased linearly with further decrease in the proton motive force, and was almost zero at about--30 mV. The rotation rate of a flagellum, measured by a tethered cell, showed essentially the same characteristics. Thus, there are a threshold proton motive force and a saturating proton motive force for the rotation of the B. subtilis flagellar motor.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.     

Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.     

The lifetime of photosensory signals in Halobacterium halobium and its dependence on protein methylation

Halobacteria spontaneously reverse their swimming direction about every 10 s. This behavioral pattern is transiently disturbed upon stimulation through sensory photosystems of different spectral sensitivity. As a result of stimulation, a single swimming interval is either prolonged (attractant response) or shortened (repellent response). Thereafter the cell returns to its autonomous reversal rhythm, i.e., it quickly adapts. Method are presented to determine the lifetime of repellent as well as of attractant cellular signals at the site of signal integration, using particular stimulation programs. Independent of the photosystem through which the signals were generated, the total lifetime of a repellent signal was 1.3 s. The decay of the signal was rapid during the first 100 ms and slow thereafter. The lifetime of an attractant signal was about 4 s and likewise did not depend on the photosystems. The degree of methylation of membrane proteins was increased by attractant stimuli and decreased by repellent stimuli. Inhibition of protein methylation by homocysteine was accompanied by a slowdown of the decay of both the repellent and attractant signal. A mutant strain with an increased demethylation also gave increased signal lifetimes. A lowered Ca2+ concentration, which activates methylation in vivo, led to shortened signal lifetimes. Methylation is proposed to be the mechanism which limits the signal lifetime and thereby allows the cells to adapt.     

On torque and tumbling in swimming Escherichia coli

Bacteria swim by rotating long thin helical filaments, each driven at its base by a reversible rotary motor. When the motors of peritrichous cells turn counterclockwise (CCW), their filaments form bundles that drive the cells forward. We imaged fluorescently labeled cells of Escherichia coli with a high-speed charge-coupled-device camera (500 frames/s) and measured swimming speeds, rotation rates of cell bodies, and rotation rates of flagellar bundles. Using cells stuck to glass, we studied individual filaments, stopping their rotation by exposing the cells to high-intensity light. From these measurements we calculated approximate values for bundle torque and thrust and body torque and drag, and we estimated the filament stiffness. For both immobilized and swimming cells, the motor torque, as estimated using resistive force theory, was significantly lower than the motor torque reported previously. Also, a bundle of several flagella produced little more torque than a single flagellum produced. Motors driving individual filaments frequently changed directions of rotation. Usually, but not always, this led to a change in the handedness of the filament, which went through a sequence of polymorphic transformations, from normal to semicoiled to curly 1 and then, when the motor again spun CCW, back to normal. Motor reversals were necessary, although not always sufficient, to cause changes in filament chirality. Polymorphic transformations among helices having the same handedness occurred without changes in the sign of the applied torque.     

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.     

Transformations in flagellar structure of Rhodobacter sphaeroides and possible relationship to changes in swimming speed

Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops. Free-swimming R. sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions. DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations. However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix. The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament. In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form. This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation. This form was always significantly longer than the coiled or helical form from which it was derived. The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix. Examination of the three-dimensional swimming pattern showed that R. sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops. The rate of acceleration out of stops was also variable. The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells. The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed.     

Study of the torque of the bacterial flagellar motor using a rotating electric field.

Bacterial flagella are driven by a rotary motor that is energized by an electrochemical ion gradient across the cell membrane. In this study the torque generated by the flagellar motor was measured in tethered cells of a smooth-swimming Escherichia coli strain by using rotating electric fields to determine the relationship between the torque and speed over a wide range. By measuring the electric current applied to the sample cell and combining the data obtained at different viscosities, the torque of the flagellar motor was estimated up to 55 Hz, and also at negative rotation rates. By this method we have found that the torque of the flagellar motor linearly decreases with rotation rate from negative through positive rate of rotation. In addition, the dependence of torque upon temperature was also investigated. We showed that torque at the high speeds encountered in swimming cells had a much steeper dependence on temperature that at the low speeds encountered in tethered cells. From these results, the activation energy of the proton transfer reaction in the torque-generating unit was calculated to be about 7.0 x 10(-20) J.     

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.     

Functional roles of the hook in a rotating tethered cell

A bacterial cell tethered through a flagellum on a glass slide rotates its cell body in either counter-clockwise or clockwise at around 10 Hz. To analyze the detailed manner of rotation, we have constructed and expressed yellow fluorescent protein (YFP)-FliN fusion protein in a fliN deletion mutant, resulting in the recovery of motility of the Fla^- mutant cells. The tethered cells that incorporated the fusion protein in the flagellar motor rotate around one of the fluorescent spots. Tracing the center spot of a rotating cell, we have found that the rotating circles of the tethered cells were often distorted, and that the cell has seldom rotated smoothly but gyrated around the center point. The radii of the gyrating circles were 100-200 nm for the wild-type cells, and 50 nm for the cells carrying short hooks, suggesting that the flexibility of the hook is responsible for asymmetrical rotation. These observations indicate that tethered cells almost always interact with the glass surface in one cycle of rotation, where the length and flexibility of the hook have an important role.     

How dinoflagellates swim

Dinoflagellates possess two flagella; usually these are directed perpendicular to one another constituting a transversal flagellum and a longitudinal, trailing flagellum, respectively. The transversal flagellum causes the cell to rotate around its length axis. The trailing flagellum is responsible for the translation of the cell; due to its asymmetric insertion it also causes a rotation of the cell around an axis perpendicular to the longitudinal axis. Together, these two rotational components result in a helical swimming path. Cells can vary the two rotational components independently as well as the translational velocity. With these three degrees of freedom, cells can vary the parameters of their helical swimming paths for steering. Dinoflagellates use this mechanism for orientation in chemical concentration gradients (“helical klinotaxis”).     

Functions of the flagellar modes of rotation in bacterial motility and chemotaxis

Bacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a three-state device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these findings.     

Adaptation kinetics in bacterial chemotaxis.

Cells of Escherichia coli, tethered to glass by a single flagellum, were subjected to constant flow of a medium containing the attractant alpha-methyl-DL-aspartate. The concentration of this chemical was varied with a programmable mixing apparatus over a range spanning the dissociation constant of the chemoreceptor at rates comparable to those experienced by cells swimming in spatial gradients. When an exponentially increasing ramp was turned on (a ramp that increases the chemoreceptor occupancy linearly), the rotational bias of the cells (the fraction of time spent spinning counterclockwise) changed rapidly to a higher stable level, which persisted for the duration of the ramp. The change in bias increased with ramp rate, i.e., with the time rate of change of chemoreceptor occupancy. This behavior can be accounted for by a model for adaptation involving proportional control, in which the flagellar motors respond to an error signal proportional to the difference between the current occupancy and the occupancy averaged over the recent past. Distributions of clockwise and counterclockwise rotation intervals were found to be exponential. This result cannot be explained by a response regular model in which transitions between rotational states are generated by threshold crossings of a regular subject to statistical fluctuation; this mechanism generates distributions with far too many long events. However, the data can be fit by a model in which transitions between rotational states are governed by first-order rate constants. The error signal acts as a bias regulator, controlling the values of these constants.