Asynchronous switching of flagellar motors on a single bacterial cell

Salmonella possesses several flagella, each capable of counterclockwise and clockwise rotation. Counterclockwise rotation produces swimming, clockwise rotation produces tumbling. Switching between senses occurs stochastically. The rotational sense of individual flagella on a single cell could be monitored under special conditions (partially de-energized cells of cheC and cheZ mutants). Switching was totally asynchronous, indicating that the stochastic process operates at the level of the individual organelle. Coordinated rotation in the flagellar bundle during swimming may therefore derive simply from a high counterclockwise probability enhanced by mechanical interactions, and not from a synchronizing switch mechanism. Different flagella on a given cell had different switching probabilities, on a time scale (greater than 2 min) spanning many switching events. This heterogeneity may reflect permanent structural differences, or slow fluctuations in some regulatory process.     

Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise.

Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise. Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes.     

Morphology, function and isolation of halobacterial flagella

Halobacterium halobium has right-handed helical flagella. During the logarithmic phase of growth, cells are predominantly monopolar, whereas in the stationary phase they are mostly bipolarly flagellated. The flagellar bundle consists of several filaments. Halobacteria swim forward by clockwise and backwards by counterclockwise rotation of their flagella. The flagellar bundle does not fly apart when the sense of rotation changes. In addition to the flagella attached to the cells, large amounts of loose flagella, which aggregate into thick super-flagella, can be observed at all phases of growth. During stationary phase, the production of these super-flagella, which are generally 10 to 20 times longer than the cell body, is significantly higher. Dissociation and association by high temperature and differential centrifugation allow the isolation of pure flagella. Three different protein bands, of 23,500, 26,500 and 31,500 apparent molecular weights, are seen on sodium dodecyl sulphate/polyacrylamide gels. Antibodies against halobacterial flagella were produced in chicken; these antibodies interact with the flagella even in 4 M-NaCl. Rotation of tethered cells demonstrates that Halobacteria move due to the rotation of the flagella.     

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.     

Caulobacter crescentus flagellar filament has a right-handed helical form

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.     

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.     

Coordination of flagella on filamentous cells of Escherichia coli.

Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli. Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis. Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies. The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass. The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously. The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time. Variations in bias were correlated, provided that the motors were within a few micrometers of one another. Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are. This signal appears to have a limited range.     

In vitro Approach to Bacterial Chemotaxis

An in vitro approach to study bacterial motility and chemotaxis is described. The approach is based on a preparation of flagellated cell envelopes. The envelopes are prepared from bacteria by a penicillin treatment and subsequent osmotic lysis. When the envelopes are energized, their flagella rotate. The direction of rotation in wild type envelopes is counterclockwise. Inclusion of the CheY protein within the envelopes may restore clockwise rotation. The advantages and disadvantages of this approach are pointed out.     

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.     

Throwing the switch in bacterial chemotaxis

In Escherichia coli chemotaxis, the switch from counterclockwise to clockwise rotation of the flagella occurs as a result of binding of the phosphorylated CheY protein to the base of the flagellum. Analysis of CheY variants has provided a picture of the surface of CheY that undergoes conformational shifts, as a result of phosphorylation, to interact directly with the flagellum. Whether phospho-CheY binding and flagellar switching are sequential steps or can occur in a concerted fashion has yet to be determined.     

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.     

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.     

Torque and switching in the bacterial flagellar motor. An electrostatic model.

A model is presented for the rotary motor that drives bacterial flagella, using the electrochemical gradient of protons across the cytoplasmic membrane. The model unifies several concepts present in previous models. Torque is generated by proton-conducting particles around the perimeter of the rotor at the base of the flagellum. Protons in channels formed by these particles interact electrostatically with tilted lines of charges on the rotor, providing "loose coupling" between proton flux and rotation of the flagellum. Computer simulations of the model correctly predict the experimentally observed dynamic properties of the motor. Unlike previous models, the motor presented here may rotate either way for a given direction of the protonmotive force. The direction of rotation only depends on the level of occupancy of the proton channels. This suggests a novel and simple mechanism for the switching between clockwise and counterclockwise rotation that is the basis of bacterial chemotaxis.     

Reversal of flagellar rotation is important in initial attachment of Escherichia coli to glass in a dynamic system with high- and low-ionic-strength buffers

The attachment rates of wild-type, smooth-swimming, tumbly, and paralyzed Escherichia coli to glass was measured at fluid velocities of 0.0044 and 0.044 cms(-1) (corresponding to shear rates of 0.34 and 3.4 s(-1), respectively), in 0.02 and 0.2 M buffer solutions. At the highest ionic strength, we did not observe a significant difference in the attachment rate of wild-type and paralyzed cells at either fluid velocity. However, when the ionic strength was reduced, paralyzed bacteria attached at rates 4 and 10 times lower than that of the wild type under fluid velocities of 0.0044 and 0.044 cms(-1), respectively. This suggested that the rotation of the flagella assisted in attachment. We then compared the attachment rates of smooth-swimming (counterclockwise rotation only) and tumbly (clockwise rotation only) cells to the wild type to determine whether the direction of rotation was important to cell attachment. At 0.0044 cms(-1), the smooth-swimming cells attached at rates similar to that of the wild type in both buffer solutions but significantly less at the higher fluid velocity. Tumbly cells attached at much lower rates under all conditions. Thus, the combination of clockwise and counterclockwise flagellar rotation and their coupling appeared to be important in cell attachment. We considered a number of hypotheses to interpret these observations, including a residence time analysis and a comparison of traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to soft-particle theory.     

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.     

Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor.

FliG, FliM, and FliN are three proteins of Salmonella typhimurium that affect the rotation and switching of direction of the flagellar motor. An analysis of mutant alleles of FliM has been described recently (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992). We have now analyzed a large number of mutations in the fliG and fliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively). The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate. However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly. Missense mutations in fliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene. CW-bias and CCW-bias mutations tended to segregate into separate subclusters. The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes. Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias. The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.     

Changing the direction of flagellar rotation in bacteria by modulating the ratio between the rotational states of the switch protein FliM

One of the major questions in bacterial chemotaxis is how the switch, which controls the direction of flagellar rotation, functions. It is well established that binding of the signaling molecule CheY to the switch protein FliM shifts the rotation from the default direction, counterclockwise, to clockwise. How this shift is done is still a mystery. Our aim in this study was to determine the correlation between the fraction of FliM molecules in the clockwise state (i.e. occupied by CheY) and the probability of clockwise rotation. For this purpose we gradually expressed, from a plasmid, a clockwise FliM mutant protein in cells that express, from the chromosome, wild-type FliM but no chemotaxis proteins. We verified that plasmid-borne FliM exchanges chromosomal FliM in the switch. Surprisingly, a substantial clockwise probability was not obtained before the large majority of the FliM molecules in the switch were clockwise molecules. Thereafter, the rise in clockwise probability was very steep. These results suggest that an increase in the clockwise probability requires a high level of FliM occupancy by CheY approximately P. They further suggest that the steep increase in clockwise rotation upon increasing CheY levels, reported in several studies, is due, at least in part, to cooperativity of post-binding interactions within the switch. We also carried out the inverse experiment, in which wild-type FliM was gradually expressed in a background of a clockwise fliM mutant. In this case, the level of the clockwise mutant protein, required for establishing a certain clockwise probability, was lower than in the original experiment. If our system (in which the ratio between the rotational states of FliM in the switch is established by slow exchange) and the native system (in which the ratio is established by fast changes in FliM occupancy) are comparable, the results suggest that hysteresis is involved in the switch function. Such a situation might reflect a damping mechanism, which prevents a situation in which fluctuations in the phosphorylation level of CheY throw the switch from one direction of rotation to the other.     

Reversal of Flagellar Rotation Is Important in Initial Attachment of Escherichia coli to Glass in a Dynamic System with High- and Low-Ionic-Strength Buffers

The attachment rates of wild-type, smooth-swimming, tumbly, and paralyzed Escherichia coli to glass was measured at fluid velocities of 0.0044 and 0.044 cms⁻¹ (corresponding to shear rates of 0.34 and 3.4 s⁻¹, respectively), in 0.02 and 0.2 M buffer solutions. At the highest ionic strength, we did not observe a significant difference in the attachment rate of wild-type and paralyzed cells at either fluid velocity. However, when the ionic strength was reduced, paralyzed bacteria attached at rates 4 and 10 times lower than that of the wild type under fluid velocities of 0.0044 and 0.044 cms⁻¹, respectively. This suggested that the rotation of the flagella assisted in attachment. We then compared the attachment rates of smooth-swimming (counterclockwise rotation only) and tumbly (clockwise rotation only) cells to the wild type to determine whether the direction of rotation was important to cell attachment. At 0.0044 cms⁻¹, the smooth-swimming cells attached at rates similar to that of the wild type in both buffer solutions but significantly less at the higher fluid velocity. Tumbly cells attached at much lower rates under all conditions. Thus, the combination of clockwise and counterclockwise flagellar rotation and their coupling appeared to be important in cell attachment. We considered a number of hypotheses to interpret these observations, including a residence time analysis and a comparison of traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to soft-particle theory.     

Signal formation in the halobacterial photophobic response mediated by a fourth retinal protein (P480)

Halobacterial cells swim forward by clockwise, and backward by counterclockwise, rotation of their flagella. The changes of direction of rotation occur statistically and can be quantitatively described by a four-state model of the motor. Stimulation of the cells with blue light induces the formation of a signal that causes the motor to switch the direction of rotation. The results of step-up and flash experiments led to a kinetic equation that describes the signal formation as a photocatalytic process. The stimulating blue light is sensed either by sensory rhodopsin in the presence of green background light or by protein P480, which has a maximum in the action spectrum around 480 nm. P480, but not sensory rhodopsin, is synthesized by the cells constitutively, and both pigments together allow the cells to find optimal conditions during aerobic and phototrophic growth. The work presented here was reported at the U.S.-Israel Binational Science Foundation Meeting in Jerusalem, March 1986.