Unidirectional, Intermittent Rotation of the Flagellum ofRhodobacter sphaeroides
JUDITH P. ARMITAGElt* AND ROBERT M. MACNAB2Department of Botany and Microbiology, University College London, London WCJE 6BT, United Kingdom,' and
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 065172
Received 11 July 1986/Accepted 23 October 1986
The single flagellum of the photosynthetic bacterium Rhodobacter sphaeroides was found to be mediallylocated on the cell body. Observation of free-swimming bacteria, and bacteria tethered by their flagellarfilaments, revealed that the flagellum could only rotate in the clockwise direction; switching of the direction ofrotation was never observed. Flagellar rotation stopped periodically, typically several times a minute for up toseveral seconds each. Reorientation of swimming cells appeared to be the result of Brownian rotation duringthe 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 motorstopped rotating, proceeding from the distal end towards the cell body. It is possible that both the relaxationprocess and the smaller frictional resistance after relaxation may act to enhance the rate of reorientation of thecell. The transition from the coiled to the normal form occurred when the motor restarted, proceeding fromthe proximal end outwards, which might further contribute to the reorientation of the cell before it reaches a
stable swimming geometry.
Bacteria swim by rotating semirigid helical flagellar fila-ments (see reference 12 for a review). Free-swimming bac-teria change direction every few seconds, altering the fre-quency of these directional changes when presented with a
change in the concentration of an attractant or repellent,thereby biassing their overall movement in a favorabledirection. There has been extensive investigation of themechanism of flagellar rotation and its tactic control over thepast 15 years (4, 12). All flagellated bacteria investigated sofar have been found to possess apparently the same basicmotility and tactic system. The flagella can rotate in either a
clockwise (CW) or a counterclockwise (CCW) direction,with each individual flagellum having its own inherentswitching frequency (8, 13). The switching frequency isaltered by an unknown signal from membrane-bound sen-sory receptors (methyl-accepting chemotaxis proteins[MCPs]) when a chemoeffector is encountered (3). Theswitching frequency can also be changed by an MCP-independent signal from the respiratory or photosyntheticelectron transport system (16).Although during propulsion the flagellar filament is a fairly
rigid helix, a change in the direction of rotation of theflagellum reverses the torsional stress on the filament andcan result in a discrete change in filament structure that iscaused by rearrangement of the constituent flagellin mono-mers (14). The helical waveform of a filament rotating CWmay therefore have a different amplitude, wavelength, andhandedness from that of the same filament rotating CCW.Interconversion of structures has also been demonstrated invitro (7). Thus, flagellar filaments display polymorphism(10).Evidence is presented here that the photosynthetic bacte-
* Corresponding author.t Present address: Microbiology Unit, Department of Biochemis-
try, Oxford University, Oxford OX1 3QU, United Kingdom.
nas sphaeroides) can swim and change direction by using a
single flagellum which rotates in the CW direction only, thechanges of direction being accomplished by briefly stoppingflagellar rotation and permitting Brownian motion to reorientthe cell. This stopping of rotation results in relaxation of theflagellar filament from the normal (small-amplitude, long-wavelength) helical polymorph used for propulsion to a
Growth media and conditions. R. sphaeroides wild-typestrains WS8 (gift from W. Sistrom), 8253, and 241 were
grown as described previously in either 25- or 100-ml flatbottles under low, continuous illumination at 25°C (6). Bac-teria were either examined directly or harvested and sus-
pended in 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.0).Antibody preparation. Flagellar filaments were sheared
from bacteria harvested in the early exponential growthphase, when the cells were maximally motile. Antibody topurified whole flagellar filaments (2) was produced in NewZealand white rabbits. Any somatic antigens present wereremoved by treatment with a nonflagellated R. sphaeroidesstrain, and the antibody was then stored at -15°C.
Observation of tethered bacteria. Suspensions of motilebacteria in 20 mM potassium phosphate buffer, pH 7.0, weremixed with dilute antifilament antibody and incubated at30°C for about 30 min. A drop of suspension was then addedto the slide, under a cover slip positioned on two fixed coverslips, and incubated upside down for 10 min to allowantibody and filaments to adhere to the cover slip surface.The whole slide mount was then turned right side up andexamined by phase-contrast microscopy. Cells were thusviewed looking from the tethered filament towards therotating cell body. Stopping and starting frequencies and
INTERMITTENT ROTATION OF R. SPHAEROIDES FLAGELLUM 515
helices of short wavelength and large amplitude (Fig. lb); thecircular appearance was therefore presumed to be a planview of a flatly coiled helix. A few examples were seen offilaments with a more conventional waveform for flagellatedbacteria (wavelength ca. 2.2 ,um, amplitude ca. 0.7 pLm; Fig.lc). These two forms will be referred to as coiled andnormal, respectively.
Observation of flagellated cells and detached flagella byelectron microscopy (Fig. 2) confirmed the existence of thecoiled form and revealed that the single flagellum arosemedially (i.e., from a point roughly midway along the longaxis of the cell) and not polarly, as is the case with manyother monoflagellate bacteria, e.g., Pseudomonas spp. Themedial origin of the flagellum could also be seen by dark-fieldlight microscopy (data not shown).
Behavior of free-swimming cells. Cells swam at speeds ofup to ca. 80 ,um/s, stopping intermittently (typically every 10s or so, but with wide variation from cell to cell and alsowithin any given cell) and restarting after a short interval(typically about 1 s, but again with wide variation). Duringthe stop, there was some angular motion of the cell, but itwas much less vigorous than is seen with peritrichousbacteria such as Salmonella typhimurium (14) and couldsimply have been the result of Brownian rotation.Swimming cells were always pushed by the flagellum, with
the counterrotating body oriented roughly perpendicular tothe swimming direction. The helical waveform was difficultto see on cells swimming at full speed (Fig. 3a), but on cellsswimming more slowly it appeared to be normal, i.e., similar
FIG. 1. Light micrographs of flagellar filaments from R.sphaeroides under high-intensity dark-field illumination. (a) Re-laxed, coiled form, which in plan view is seen as a circle (arrows).(b) Long filament in the coiled form, with its wavelength extendedby axial flow of fluid. (c) Filament trapped in the unrelaxed, normalform that is generated by rotation and used in propulsion. Bar, 5 Jim.
rotation rates were measured from video recordings of thetethered cells.
Observation of detached flagellar filaments and of filamentson free-swimming bacteria. All light-microscopic observa- fk rk I
tions of flagellar filaments were made as described previ-ously (11). Bacterial suspensions were illuminated by a450-W xenon short-arc lamp through an oil immersion dark- p -field condenser. Detached flagellar filaments and swimmingbacteria were recorded in U.S. format with a silicon inten-sifying target Vidicon camera (Dage/MTI model 650) and a1/2-in. (1.27-cm) reel-to-reel video recorder (Panasonic AC
NV-8030), and later transferred to European format foranalysis in the United Kingdom, using a Umaticvideorecorder (Sony 5800). Still photographs of single - iframes of-the video recordings were taken from an 800-line8-in. (20-cm) monitor (Panasonic).
Electron microscopy. Samples were either negatively istained with 2% potassium phosphotungstate or dried ontoFormvar-coated grids and shadowed with gold-palladium atan angle of 220 before examination in a Siemens Elmiskopelectron microscope.
RESULTS '-"' ____
Morphology of stationary filaments. Detached flagellar FIG. 2. Electron micrographs showing (a) the medial location offilaments, examined by high-intensity dark-field light micros- the flagellum on an intact cell of R. sphaeroides and (a, b, and c) thecopy (Fig. la), appeared as circles of constant diameter (ca. relaxed, coiled form exhibited both by attached and detached2.1 ,um) or occasionally, if the filament was very long, as flagellar filaments. Bar, 1 pm.
to those seen on other flagellated species and to occasionalimages of detached filaments of R. sphaeroides (Fig. lc); itcould be clearly differentiated from the coiled forms shownin Fig. la and 2.The axial length of the flagellar filament on different cells
varied from about 2 to 5 body lengths. On any given cell, thislength was the same when the filament was rotating (Fig. 3a)and immediately after stopping (Fig. 3b); there was noevidence of a polymorphic transition that might have oc-curred had there been a brief period of rotation in theopposite direction.
After flagellar rotation had stopped, the filament couldoften be seen to undergo a polymorphic transition, relaxingfrom the distal end into the coiled conformation seen indetached filkments (Fig. 4 and 5, i to iv). When rotationstarted again, the small-amplitude, long-wavelength normalhelix reformed from the cell body outwards (Fig. 5, v to vii)and therefore was presumed to be a consequence of thetorsional stress developed by motor rotation. Its waveform(unlike that of the relaxed coiled polymorph) made it func-tionally suited for propulsion.
Dividing bacteria were often observed with two flagella,one on each daughter cell, attached medially on opposingsides. When both flagella rotated, nontranslational spinningresulted. Often only one of the flagella on a pair of dividingcells would rotate, allowing translational motility.
Behavior of tethered cells. Bacteria tethered by theirflagellar filament to glass cover slips by antifilament antibodyrotated exclusively in the CCW direction as viewed, corre-sponding therefore to CW rotation of the flagellar filament ona free-swimming cell; this leads to the conclusion that thehelical form used during swimming must be right-handed.Several hundred cells of the three wild-type strains used,grown under various conditions and incubated both aerobi- -____cally and anaerobically, were monitored for many minuteseach; none were seen to rotate CW, even transiently.From cell to cell, there was a wide variation in speed and -...
stopping frequency (Fig. 6). However, even within an indi-vidual cell, both of these parameters fluctuated consider-ably.
DISCUSSIONR. sphaeroides rotates its flagellum in one direction only,
periodically stopping and restarting that rotation. (Rhizo-bium meliloti behaves similarly; R. Gotz and R. Schmitt,personal communication.) This form of behavior is quitedistinct from the CCW-CW switching that has been de- FIG. 4. Sequence of images of an R. sphaeroides cell taken fromscribed for other bacterial species. Three different wild-type a video recording, showing the relaxation of a stopped flagellarstrains of R. sphaeroides were examined, and all were found filament from its distal end. Total elapsed time, 3 s. Bar, 5 p.m.
to behave in the same manner. Reorientation of the cell byw:g^. Brownian motion (possibly augmented by other factors, see
below) during stops appeared to be responsible for thegeneration of a new swimming direction; this mechanismmay be regarded as a simplified version of the reorientationproduced actively by tumbling in peritrichous bacteria.
There is now a large body of evidence that flagellarrotation is driven by the proton electrochemical gradientacross the bacterial cytoplasmic membrane (15), Recentresults have suggested that the interaction of the protonsFIG. 3. Light micrographs of a swimming cell of R. sphaeroides wihtepoinofhelalarmordsntivleunder high-intensity dark-field illumination, showing that the axial wlth the proteins of the flagellar motor does not involve
length of the rotating flagellar filament (a) remained unchanged after covalent bonding, but simple acid-base interactions (1).the filament stopped (b). The apparent size of the cell body is However, in the case of the flagellar motor of R.exaggerated by scattering of the high-intensity light; the true cell sphaeroides, it appears that-in addition to the energy-size is indicated by the drawn outline. Bar. 5 ,um. transdticing components-there must be a control structure,
INTERMITTENT ROTATION OF R. SPHAEROIDES FLAGELLUM 517
lI) (m (m) W
_~~~~~~~~~~~~~O'0_ 0
(VIIH
FIG. 5. Cartoon illustrating an R. sphaeroides cell (i) whileswimming, (ii through iv) with flagellar rotation stopped and theflagellar filament progressively relaxing into the coiled form from itsdistal end, and (v through vii) with the flagellum once again rotatingand the filament reconverting from its proximal end to the normalform used for propulsion. Reorientation of the cell during the periodwhen the flagellum is not rotating is indicated in panel iv by thedouble-headed arrow.
perhaps similar to a gate control in the ion pores ofeucaryotes. Not only did flagella stop rotating periodically,but their speed while rotating could change by an order ofmagnitude (Fig. 6), even though the proton gradient, underconditions of photosynthetic saturation, was presumablyfairly constant. In this connection it is interesting thathydrophobic, anionic uncouplers appear to be able to inter-act directly with the flagellar motor of R. sphaeroides, butonly under certain conditions (6a): when the proton motiveforce is artificially reduced to zero in the presence of lowconcentrations of uncoupler, there is a delay in the reinitia-tion of motility after the proton motive force has beenrestored, yet the same concentration of uncoupler has noeffect on the swimming speed of cells that are fully energizedat the time of addition. It is therefore possible that the stateof the motor is different in the stopped and rotating states.
Variability of behavior was a pervasive characteristic ofthe motility of the organism. Observation of both filamentrotation and tethered-cell rotation showed that the speedvaried, even in saturating light, and that rotation could stopinstantly at both slow and fast rotation speeds. The durationof a stop also varied, from a fraction of a second to severalminutes. Individual bacteria within a field of observation allhad their own inherent average rotation speed and stoppingfrequency, a situation comparable to the different switchingfrequencies seen in motors of other bacterial flagella (8, 13).The coiled structure of detached and stopped filaments of
R. sphaeroides is not a common one (although similarstructures have been seen in isolated filaments of S.typhimurium at low pH [9], to a limited extent at the distalend of stationary filaments of Escherichia coli [14], and inisolated filaments of some spirochetes, in which the fila-ments are internalized between the inner and outer mem-branes of the bacterium [5]). We initially considered thepossibility that this coiled structure in R. sphaeroides, likethe tighter ("curly") helical form of filaments on Salmonellacells during tumbling (14), was caused by a brief period ofreverse motor rotation. Careful observation revealed, how-ever, that the coiled conformation always arose after theflagellum had stopped rotating. Furthermore, it developedby relaxation of the structure from the distal end of thefilament, not from the proximal end as would be expected ifthe change were being generated by motor rotation. Incontrast, the small-amplitude, long-wavelength, normal form
was only generated after reinitiation of rotation and devel-oped from the proximal end, suggesting that it was stableonly under the torsional stress generated by motor rotation.The normal waveform resembles that of other bacterial
flagellar filaments and is well suited for propulsion, whereasthe coiled form would be quite unsuited for this purpose.Does the coiled form serve any other function in the motilityof the cell? By drawing the filament close up to the cell body,it should reduce the resistance to Brownian rotation andhence increase the degree of reorientation that can occurwithin the time interval of a stop; dissipation of the storedconformational energy of the propulsive form after stoppingand the regeneration of that form from the coil after restart-ing may also actively contribute to the reorientation process.R. sphaeroides appears to lack the methylation-dependent
chemotaxis system found in other bacteria (R. Sockett and J.Armitage, unpublished data). Chemoeffectors for R.sphaeroides have been found to be those that interactdirectly with the electron transport pathways. Observationof tethered cells has shown that when stimulated by a changein chemical concentration, only a fraction of the populationresponds by changing their stopping frequency and that thisfraction can be increased by decreasing the baseline mem-brane potential of the population. This and the apparent lack
stopT2Hz
4Hz
spin(a) ~~~~~~~~stop
s|HZLL I I~~~~~~~~spin
(b)
stopI 2Hz
4Hz
Ispin
lOsecFIG. 6. Stopping and rotation behavior of three different R.
sphaeroides cells (a, b, and c) in the same microscopic field,tethered by antifilament antibody and observed under conditions ofsaturating illumination (150 microeinsteins/mm2). The duration of astop is indicated by the width of the corresponding square peak.While a cell is rotating, the rotational frequency is indicated by thevertical lines; the shorter the line, the higher the rotation frequency.Each cell showed different stopping frequencies and rotation rates;Cell a had short stops every few seconds, cell b had few stops but arelatively low average rotation frequency, and cell c rotated veryrapidly but also stopped frequently for periods of several seconds.
of MCPs suggest that R. sphaeroides only responds toenvironmental changes which interfere with the proton gra-dient, perhaps interacting directly with the flagellar motor.
ACKNOWLEDGMENTS
We thank May Kihara for her patience and technical help insetting up the experiments and M.C.W. Evans for critical reading ofthe manuscript.
This work was supported by a Lister Institute Research Fellow-ship and a travel grant from the Wellcome Trust (to J.P.A.) and byPublic Health Service grant AI-12202 from the National Institutes ofHealth (to R.M.M.).
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