J. Mol. Biol. (1996) 259, 687–695

Rotational Fluctuation of the Sodium-driven Flagellar
Motor of Vibrio alginolyticus Induced by Bindingof Inhibitors
Kazumasa Muramoto1, Yukio Magariyama2, Michio Homma1
Ikuro Kawagishi1, Shigeru Sugiyama2, Yasuo Imae1 andSeishi Kudo2,3*1

Department of Molecular
Biology, Faculty of Science
Nagoya University
Chikusa-Ku Nagoya 464-01
Japan
2

Tsukuba ResearchLaboratory, Yaskawa Electric
Corporation, 5-9-10 Tokodai
Tsukuba 300-26, Japan
3

PRESTO (Precursory
Research for Embryonic
Science and Technology)
JRDC (Research Development
Corporation of Japan)
5-9-10 Tokodai, Tsukuba
300-26, Japan*Corresponding author

Rotation of the Na+-driven flagellar motor of Vibrio alginolyticus wasinvestigated under the influence of inhibitors specific to the motor,
amiloride and phenamil. The rotation rate of a single flagellum on a cellstuck to a glass slide was examined using laser dark-field microscopy. In
the presence of 50 mM NaCl, the average rotation rate (v̄) was about
600 r.p.s. with a standard deviation (sv ) of 9% of v̄. When v̄ was decreased
to about 200 r.p.s. by the presence of 1.5 mM amiloride, sv increased to
15% of v̄. On the other hand, when v̄ was decreased to about 200 r.p.s. by
the addition of 0.6 mM phenamil, a large increase in sv up to 50% of v̄, wasobserved. Similarly large fluctuations were observed at other concentrations of phenamil. These observations suggest that dissociation ofphenamil 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 moleculeof phenamil strongly inhibits the torque generation in the flagellar motor.
7 1996 Academic Press Limited

Keywords: flagella; laser dark-field microscopy; amiloride; phenamil;
VibrioIntroduction
Flagellar motors embedded in the cytoplasmic
membrane of bacteria convert the electrochemical
potential gradient of specific ions into torque(reviewed by Imae & Atsumi, 1989; Blair, 1990).
Proton is used as the coupling ion of the motorsof neutrophilic bacteria such as Bacillus subtilis,
Streptococcus sp. and Escherichia coli (Matsuura et al.,
1977; Manson et al., 1977). Sodium ion is usedin alkaliphilic Bacillus spp. and some Vibrio
spp. (Hirota et al., 1981; Chernyak et al., 1983;
Atsumi et al., 1992b).
The components of the motor, ‘‘force-generating
Professor Yasuo Imae died of a cerebral
haemorrhage on July 2nd, 1993. This article isdedicated to him.
Present address: K. Muramoto, Department of
Molecular Biophysics and Biochemistry, Yale University,
New Haven, CT 06520-8114, USA.
Abbreviations used: LDM, laser dark-fieldmicroscopy; 6-IA, 6-iodoamiloride; r.p.s., revolutions
per second.
0022–2836/96/240687–09 $18.00/0

units’’ have the ability to translocate the coupling
ion and generate torque through their interaction
with some rotor elements (reviewed by Jones &Aizawa, 1991; Khan, 1993). Previous studies haveshown that a single motor has multiple independently functioning force-generating units (Block &Berg, 1984; Blair & Berg, 1988; Muramoto et al.,1994). In the H+-driven motor of E. coli, the presenceof eight units in a fully functioning motor was
suggested by experiments involving successive
incorporation of subunits of the motor proteins,
MotA and MotB (Blair & Berg, 1988). In the
Na+-driven motor of Bacillus firmus, the presence offive to nine units was suggested by irreversible
inactivation by a photoreactive amiloride analog,
6-iodoamiloride (6-IA: Muramoto et al., 1994).
Electron microscopic studies also suggested that
there are several ring particles, which might
correspond to the force-generating units, in thecytoplasmic membranes of E. coli, Salmonella
typhimurium, B. firmus, B. subtilis, Aquaspirillum
serpens and Streptococcus spp. (Khan, 1993).
Amiloride (Figure 1A), an inhibitor of Na+
transport in Na+ channels, Na+/H+ exchangers and
7 1996 Academic Press Limited

688

Na+-driven flagellar motor in V. alginolyticus

rotation of V. alginolyticus in the presence of
amiloride and phenamil. We have found thatphenamil causes very large fluctuations in rotation
rate, distinct from those caused by rotational
Brownian motion. The processes of association anddissociation of inhibitors at sites on the force-generating units were considered to play a major role in
the fluctuation.

Results
Changes in rotational fluctuation caused
by inhibitors

Figure 1. The structure of amiloride and phenamil. A,
Amiloride. The values of IC50 of amiloride are 0.2 mM for
Na+ channel, 84 mM for Na+/H+ exchange and 1100 mM
for Na+/Ca2+ exchange (Cragoe et al., 1992). B, Phenamil.
One of the hydrogen atoms of the terminal amino groupsof the guanidino moiety in amiloride is substituted by aphenyl group. The potencies of phenamil relative toamiloride are 17 for Na+ channel, 0.1 for Na+/H+
exchange and 6 for Na+/Ca2+ exchange (Cragoe et al.,1992).

Na+/Ca2+ exchangers (Kleyman & Cragoe, 1988;
Cragoe et al., 1992), specifically and reversibly
inhibits rotation of the Na+-driven motors ofalkaliphilic Bacillus spp. and Vibrio spp. (Sugiyama
et al., 1988; Atsumi et al., 1992a,b; Kawagishi et al.,1995). Some amiloride analogs also inhibit the
Na+-driven motor (Sugiyama et al., 1988). Phenamil(Figure 1B), the most potent inhibitor known atpresent, completely inhibits the motility of B. firmus
and Vibrio alginolyticus at a concentration of muchless than 1 mM. Amiloride and 6-IA were suggested
to interact with the Na+-binding site at the externalsurface of the force-generating unit (Sugiyama et al.,
1988; Atsumi et al., 1990; Muramoto et al., 1994), andthereby block the Na+ influx. Analyses of theinhibitory mechanisms of these amiloride analogs
should be useful in characterizing the coupling
between ion translocation and force generation in
the Na+-driven motor.
Laser dark-field microscopy (LDM) has beendeveloped to measure flagellar rotation with hightemporal resolution (Kudo et al., 1990). In thismethod, the rotating flagellum is irradiated by athin laser beam and the rotation rate is measured
from the intensity change of the scattered light.
Recently, we analyzed the Na+-driven polar flagellar
rotation of V. alginolyticus using LDM and found thatthe rotation rate was very fast, up to 1700 r.p.s.
at 35°C (Magariyama et al., 1994). Rotational fluctuation was also examined, with results thatsuggested that the fluctuation was mainly causedby rotational Brownian motion (Muramoto et al.,1995). In other words, it appeared that the motor
mechanism involved free diffusion.
In the present study, we have used LDM withhigh temporal resolution to measure flagellar

We examined the inhibitory effect of amiloride
and phenamil on the Na+-driven flagellar motor of
V. alginolyticus using LDM. Figure 2A, C and E show
the intensity change of the scattered light from therotating flagellum. Figure 2B, D and F show therotation rates (v) as a function of time and their
histogram. Each value of v was determined as thereciprocal of the peak to peak interval of theintensity change (see equation (2)). Figure 2A andB show the flagellar rotation in the absence of
inhibitors (in HG medium (pH 7.0) supplemented
with 50 mM NaCl and 250 mM KCl). The flagellum
rotated fast and smoothly. The average rotation rate(v̄) for one second was about 600 r.p.s., and thestandard deviation of v (sv ) was about 8% of v̄.
When 1.5 mM of amiloride was added, v̄ decreased to about 200 r.p.s. (Figure 2C and D). The
value of sv was about 10% of v̄. When 0.6 mM
phenamil was added, v̄ decreased to about 200 r.p.s.
(Figure 2E and F), and sv increased substantially. In
Figure 2F, for example, v decreased rapidly andmarkedly from about 200 to 50 r.p.s. at 180 ms andincreased from 50 to 200 r.p.s. at 250 ms. The valueof sv for one second was about 43% of v̄. This value
was much larger than those in the absence of
inhibitors or in the presence of amiloride.
Figure 3 shows the changes in v caused byphenamil for five consecutive seconds including theone second period shown in Figure 2F. A large
fluctuation in v was seen in every period. The
values of v̄ for each one second interval in Figure
3A to E were 229, 212, 148, 188 and 232 r.p.s.; andthose of sv were 27, 27, 43, 35 and 32% of v̄,respectively. These differences in v̄ and sv indicatethat one second is rather short for statisticalprocessing of the data. On the contrary, one second
is sufficiently long for statistical processing for the
inhibition by amiloride, because the values of v̄ andsv for any of the one second periods in themeasurement were almost the same as those for theperiod shown in Figure 2D (data not shown). Thatis, the changes in v induced by phenamil occur
more slowly than those induced by amiloride.
Rotational fluctuation as a function of theinhibitor concentration
Figure 4 shows the effects of amiloride andphenamil on v̄ and sv /v̄ (for five seconds) asNa+-driven flagellar motor in V. alginolyticus

689

Figure 2. Change of rotation rate in the presence of amiloride and phenamil. YM3 cells were prepared in HG medium
containing 50 mM NaCl and 250 mM KCl. A and B, In the absence of inhibitor; C and D, in the presence of 1.5 mM
amiloride; E and F, in the presence of 0.6 mM phenamil. The laser beam was focused on a single freely rotating flagellum
on a cell whose body was stuck to a glass surface. A, C and E, The intensity changes of the scattered light from therotating flagellum. B, D and F, The values of v over a one second interval and their histogram. The resolution was limitedby the sampling interval of the light intensity change (200 ms), and is seen as the discrete feature of obtained v values.

functions of their concentrations. As shown in
Figure 4A, v̄ monotonically decreased with increasing concentration of amiloride, with 2 mM amiloride causing more than a 90% decrease in v̄. The
value of IC50 , which is the concentration of an
inhibitor required for decreasing v̄ by 50%, wasestimated as about 1 mM for amiloride. The valueof sv /v̄ was about 0.09 in the absence of amiloride,and only slightly increased with increasing concentration of amiloride (Figure 4B). Both v̄ and sv /v̄
varied rather widely from cell to cell, and the
distributions seemed to be independent of thepresence of the inhibitors.
In the case of phenamil (Figure 4C), the decreasein v̄ with increasing concentration of the inhibitor
was steeper than that seen with amiloride. Only
2.5 mM phenamil was sufficient to cause more than
a 90% decline of v̄. The value of IC50 of phenamil
was estimated to be about 0.25 mM, which was 4000
times smaller than that of amiloride. For swimming
speed, similar values of IC50 were obtained for bothamiloride and phenamil (data not shown).
Phenamil also caused an increase in sv /v̄. The
values of sv /v̄ increased steeply from 0.09 to about
0.4 with increasing concentration of phenamilwithin the range 0 to 1 mM (Figure 4D). Further
addition of phenamil caused a gradual increase in
sv /v̄, reaching up to 0.6 in the presence of 5 mM
phenamil. Thus, the presence of phenamil caused adecrease in v̄ and an increase in the fluctuation in
the rotation of flagellar motor, with the magnitudeof the fluctuation depending on the concentration ofphenamil.

The difference of fluctuation between amiloride
and phenamil seems to be caused by a difference
between the rates of association and/or dissociationof the inhibitors.
Rotational fluctuation after rapid application of
the inhibitors
To test whether the fluctuation in v directlyreflected the association and dissociation of theinhibitor molecule to the motor, transient effects of
the inhibitors on v were examined. A small amountof 3 mM amiloride or 10 mM phenamil was rapidly
applied to a stuck cell using a microinjector and thechanges in v were measured.
Figure 5A shows the change in v upon applying
amiloride. In the absence of amiloride, the flagellum
rotated at about 420 r.p.s. Within one second after
the application of amiloride, the value of v started
to decrease, continued to decrease for about 200 ms,
and reached a value of about 160 r.p.s. The fluctuation of flagellar rotation during the decreasing
process and after reaching the lower level were
almost the same as that at the initial level. The
duration of the decrease in v probably corresponds
to the period required for increasing the concentration of amiloride around the cell after theapplication. It should be noted that the inhibitor was
estimated to arrive at the cell within 0.5 to one
second after the injection.
When phenamil was applied (Figure 5B), a
sudden decrease in v (to about 250 r.p.s. on average)
was observed within one second after the appli-

690

Na+-driven flagellar motor in V. alginolyticus

Figure 5. Transitional change of rotation rate upon
rapid application of (A) 3 mM amiloride and (B) 10 mM
phenamil in HG medium by a microinjector.

Figure 3. Rotation rate over an extended period in thepresence of phenamil. Rotation rate changes for fiveconsecutive seconds in the presence of 0.6 mM ofphenamil. The values of v̄ and sv for each second were:A, 229 2 62 r.p.s.; B, 212 2 57 r.p.s.; C, 148 2 63 r.p.s.;
D, 188 2 65 r.p.s.; and E, 232 2 74 r.p.s.

cation. At the same time, the rotational fluctuation
started to increase. This result suggests that thelarge fluctuation is due to the direct interaction
between phenamil and the motor. Since theconcentration of phenamil around the cell presumably increased gradually, as in the case of

Figure 4. Dependence of rotation rate and its
fluctuation on amiloride and phenamil concentrations.
The values of v̄ and sv /v̄ for five seconds are shown forseveral cells at each concentration of amiloride (A and B)
and phenamil (C and D). Each point indicates the dataobtained from an individual cell.

amiloride, the large decrease in v and the increasein the fluctuation upon application of phenamilmust have occurred at quite low concentrations.
This interpretation is consistent with the steep
decrease in v̄ and increase in sv /v̄ seen at low
concentration of phenamil (Figure 4C and D).
Both with amiloride and with phenamil, v̄ andthe fluctuation completely recovered to the initiallevel after the inhibitors were removed (data notshown).
Rotational fluctuation at low concentrationof inhibitor
If a cell is placed in a very dilute solution of an
inhibitor (IC50 ), so that there is only slightinhibition of flagellar rotation, it might be possible
to observe events induced by a single inhibitor
molecule. As shown in Figure 6A, v and its
fluctuation in the presence of 0.2 mM amiloride(corresponding to 20% of IC50 ) were almost equal tothose in the absence of amiloride (Figure 2B).
Figure 6B to D shows the changes in v in thepresence of 0.04 mM phenamil (corresponding to
16% of IC50 ). The values of v and its fluctuation
were generally similar to those observed in theabsence of inhibitors. However, rapid and large
decreases in v with short duration (50 to 300 ms)
were occasionally observed (Figure 6B, at 350 ms;
C, at 700 ms; D, at 50, 500, and 800 ms). The
durations of periods of decreasing v were different
from one another and were considered to directlyreflect the binding durations of phenamil to themotor. Their values suggest that dissociation of theinhibitor from the motor is slow.
Since the concentration of phenamil was very low
and there were rather long periods in which v was
not affected, the decreases in v in Figure 6B to D

691

Na+-driven flagellar motor in V. alginolyticus

Figure 6. Changes of rotation rate in the presence of low
concentration of amiloride and phenamil. Stuck YM3 cells
were prepared in HG medium containing 50 mM NaCl,
250 mM KCl and 0.2 mM amiloride (A) or 0.04 mM
phenamil (B to D).

were considered to be induced by a single phenamilmolecule. However, the decreases in v were
unexpectedly large, frequently more than 70% (forexample, v suddenly dropped from 450 r.p.s. to
150 r.p.s. at 350 ms in Figure 6B).
Cell growth in the presence of the inhibitor
To preclude the possibility that the changes inrotation rate and its fluctuation are secondary
effects of cell damage, we examined cell growth in
the presence of phenamil. Although the motility of
YM3 cells was completely inhibited by 100 mM
phenamil, cell growth was unaffected (data notshown). It has been reported that 2 mM amiloride,
which almost completely inhibits the motility of
V. alginolyticus, does not affect membrane potential
and inhibits cell growth only slightly (Atsumi et al.,1992a). We conclude that, at the concentrations used,phenamil did not affect cellular physiology andamiloride did not severely affect it, and that thechanges in v and its fluctuation were not secondary
effects but the results of direct interaction of theinhibitors with the motor. The facts that thedecreases in v were observed immediately after
inhibitor addition (Figure 5) and that phenamilaffected v even at very low concentrations (Figure 6) were consistent with the idea.
Computer simulation of the inhibition process
To analyze the relation between the inhibition of
the motor rotation and its fluctuation, we carried out

Figure 7. Examples of the results of the simulation.
The simulations were carried out based on equations (5a)and (6) to (9) using the following parameters: Nu = 6(Muramoto et al., 1994), f = 2.2 × 10−22 Nms (Muramoto
et al., 1995), T0 = 1.4 × 10−19 Nm (Magariyama et al., 1995);
A to C, IC50 = 1.0 × 10−3 M, k+ = 108 M−1 s−1, k− = 105 s−1 andI = 1.6 mM (corresponding to the inhibition by amiloride);
D to F, IC50 = 2.5 × 10−7 M, k+ = 108 M−1 s−1, k− = 25 s−1 andI = 0.5 mM (corresponding to the inhibition by phenamil).
Data were calculated for time intervals Dt of 1 ms. The
values of Tm (t) and cos u(t) averaged over 200 suchintervals are shown. The values of v̄(k) and sv(k) for one
second were: A to C, 300 2 14 r.p.s.; D to F,
258 2 113 r.p.s.

computer simulation of the kinetics of inhibition of
the motor (for details, see Materials and Methods).
We made the following assumptions: (1) The motor
has multiple force-generating units, each of whichfunctions independently, and the torque generated
by a motor is the sum of the torque generated byeach unit (Block & Berg, 1984; Blair & Berg, 1988;
Muramoto et al., 1994). (2) An inhibitor binds to aforce-generating unit with intrinsic rate constants ofassociation (k+ ) and dissociation (k− : Sugiyama et al.,
1988; Atsumi et al., 1990, 1992b). (3) The force-generating unit bound by an inhibitor molecule
generates no torque (Sugiyama et al., 1988; Atsumi
et al., 1990, 1992b).
The simulation was carried out for several pairsof k+ and k− values, which are related to each otherthrough IC50 within the framework of the present
model as follows:k− = k+ IC50

(1)

For the inhibition by amiloride and phenamil, IC50
of 1 mM and 0.25 mM were used, respectively.
Figure 7A to C shows an example of the calculated results of inhibition by amiloride (IC50 = 1 mM,
k+ = 108 M−1 s−1, k− = 105 s−1, and I = 1.6 mM). Bothbinding and release of amiloride occurred withinmuch shorter times than the rotation period, which
resulted in frequent changes of instantaneous
torque, Tm (t) (Figure 7A). Consequently, the changes
in Tm (t) were averaged in each revolution. That is,changes in v(k) determined from the peak intervalsof cosu in Figure 7B were rather small (Figure 7C).

692

Figure 8. Simulated rotation rate and its fluctuation
as functions of inhibitor concentrations. Results of thesimulation are summarized. Parameters used were: A
and B, IC50 = 1.0 × 10−3 M; open circle, k+ = 107 M−1 s−1,
k− = 104 s−1, filled triangle, k+ = 108 M−1 s−1, k− = 105 s−1. C
and D, IC50 = 2.5 × 10−7 M; open circle, k+ = 108 M−1 s−1,
k− = 25 s−1, filled triangle, k+ = 109 M−1 s−1, k− = 250 s−1.
Values of v̄ and sv /v̄ were calculated for one second(A and B) and for five seconds (C and D).

Figure 7D to F shows calculated results forphenamil (IC50 = 0.25 mM, k+ = 108 M−1 s−1, k− = 25 s−1,
and I = 0.5 mM). In this case, the frequency ofbinding and release of the inhibitor was much lower
than that for amiloride (Figure 7D), and the change
of Tm (t) appeared as a large fluctuation in v(k)(Figure 7F). The basic features of calculated changesof v(k) for amiloride and phenamil were consistent
with those of the experimental results shown in
Figure 2D and F.
As summarized in Figure 8, the results ofsimulation showed features of v̄ and sv /v̄
essentially similar to those obtained experimentally(Figure 4: for details, see Discussion). Since
calculated values of v̄ were independent of thevalues of k+ and k− , appropriate values of k+ and k−
were evaluated by comparing calculated values ofsv /v̄ with the experimental ones. In the case ofamiloride, appropriate values of k+ and k− were
estimated to be 107 to 108 M−1 s−1 and 104 to 105 s−1,
respectively. In the case of phenamil, they were 108
to 109 M−1 s−1 and 25 to 250 s−1, respectively.

Discussion
Here, we used LDM to examine the inhibition of
the Na+-driven motor in V. alginolyticus by amiloride
and phenamil. The values of IC50 of amiloride andphenamil were determined to be 1 mM and
0.25 mM, respectively (Figure 4A and C), and so thepotency of phenamil is 4000 times larger than thatof amiloride. The fluctuation in rotation rateinduced by phenamil was much larger than thatinduced by amiloride (Figure 4B and D). Much
slower dissociation of phenamil than amiloride wassuggested to cause the differences.
The effects of inhibitors on the motor rotation

Na+-driven flagellar motor in V. alginolyticus

were analyzed by a simple kinetic model. Results ofcomputer simulation using this model could explain
the essential features of the experimental results.
Values of association and dissociation constants ofinhibitors to the motor were estimated as: k+ = 107 to
108 M−1 s−1 and k− = 104 to 105 s−1 for amiloride; andk+ = 108 to 109 M−1 s−1 and k− = 25 to 250 s−1 forphenamil, respectively. The smaller IC50 value forphenamil was reflected as a smaller k− value, i.e.
slower dissociation, compared with that foramiloride. With millisecond temporal resolution of
LDM, the dissociation of phenamil was slow
enough that it could be observed, whereas that ofamiloride could not.
The simulation did not include the effects ofother fluctuations observed in the absence of
inhibitors, which are thought to be caused mainly
by rotational Brownian motion of the flagellar
filament (Muramoto et al., 1995). In the case ofamiloride, the experimental values of sv /v̄ were
two to four times larger than the calculated one andslightly increased with increasing inhibitor concentration, while the calculated values remained
almost constant (Figures 4B and 8B). If thecontribution of the Brownian motion together with
the instrumental uncertainty (Muramoto et al., 1995)
are added to the calculated sv /v̄, the magnitude
and slight increase of sv /v̄ obtained experimentally
can be well explained. In the case of phenamil, themagnitude of fluctuation induced by the inhibitor issufficiently large that the contribution of otherfluctuations can be neglected in the present
analysis.
Although the basic features of the experimental
results could be explained by the present model, thedetailed dependence of sv /v̄ on the concentrationof phenamil is different between Figures 4D and
8D. Furthermore, in the presence of low concentrations of phenamil, the rotation rate slowed
remarkably (Figure 6). The binding of a single
phenamil molecule might decrease v much morethan is predicted by the model, or it might benecessary to consider co-operative interaction
between phenamil and the motor. In the former
case, binding of phenamil to a force-generating unit
may cause a block of the Na+ influx through theunit and other effects, such as inactivation of otherunits or additional drag torque in the motor.
If inactivation of other units occurs, the large
slowdown at low concentrations of phenamil would
be observed also under high-load conditions. On
the contrary, if phenamil induces only the additionaldrag torque in the motor, the large slowdown would
not be observed under such conditions, because v
is mainly determined by the larger external dragtorque. Hence analysis of the slowdown underhigh-load conditions, e.g., observation of tetheredcell rotation with high temporal resolution, isdesired to further understand the inhibitory
mechanism of phenamil.
In a previous study, we used the photoreactive
amiloride analog, 6-IA, to successively inactivate theforce-generating units of the Na+-driven motor in693

Na+-driven flagellar motor in V. alginolyticus

B. firmus (Muramoto et al., 1994). Without UV
irradiation, the inhibitory effect of 6-IA was similar
to that of amiloride. Photoactivation of 6-IA caused
an irreversible and stepwise decrease of the rotation
rate of tethered cells. In this case, the sizes of speed
drops were roughly equal in each step and thenumber of steps was estimated to be from five tonine. These results suggest that irreversible bindingof 6-IA merely causes a block of the Na+ influx
through the unit or the increase in drag caused bybinding of 6-IA, if any, is much smaller than thedrag on the rotating cell body.
The features of rotational fluctuation induced byamiloride and phenamil were remarkably different.
The estimated dissociation constants had different
orders of magnitude. Phenamil is more hydrophobic
than amiloride, and hence might strongly interact
with the hydrophobic region in the force-generating unit, or more easily permeate through thecytoplasmic membrane and interact with internal
Na+-binding sites of the force-generating units,
which were suggested by Yoshida et al. (1990).
Previous studies reported that the inhibition byamiloride is competitive with external Na+, while
inhibition by phenamil is non-competitive(Sugiyama et al., 1988; Atsumi et al., 1990). Theinteraction between amiloride analogs and proteins,
such as various Na+-transporters and anti-amiloride
antibody, has been studied (Cragoe et al., 1992;
Lin et al., 1994). Negulyaev and Vedernikova (1994)
showed, by using single patch measurements, thatthe blocking mechanisms of amiloride and its
derivative ethylisopropilamiloride are different. The
Na+ channels in epithelial cells have been proposed
to have one or more drug-binding sites (Lane et al.,1991).
Recently, in V. parahaemolyticus, two genes
encoding components of the polar flagellar motor,
which might be the force-generating unit, have been
identified (McCarter 1994a, 1994b). In V. alginolyticus, a procedure for electroporation was established(Kawagishi et al., 1994), and genes for motility
have been cloned (Okunishi et al., 1996). Becauseamiloride and phenamil inhibited the motor
rotation in distinct manners, it might be possible toisolate the mutants whose sensitivities to amiloride
and phenamil are different. The analyses of suchmutants would provide important clues to theidentification and characterization of the proteinsthat play important roles in the force-generating
units, and hence the force-generation mechanism of
the flagellar motor.

Materials and Methods
Bacterial strain and media
A smooth-swimming mutant of V. alginolyticus, YM3,
was used (Muramoto et al., 1995). Heart Infusion (HI)broth contained 25 g of heart infusion broth (Difco) and
15 g of NaCl per liter of distilled water. HG medium
contained 50 mM Hepes-KOH (pH 7.0), 5 mM glucose
and 5 mM MgCl2 . Amiloride (Sigma Chemical Co.,

St Louis, MO) and phenamil (provided by T. Atsumi
of Suzuka College of Technology) were dissolved indimethylsulfoxide and stored at −20°C.
Preparation of stuck cells and measurement ofrotation rate of a single flagellum
YM3 cells were grown at 30°C with shaking in HI brothand harvested during the late logarithmic phase of
growth. Cells from 1 ml of culture were washed bycentrifugation in HG medium supplemented with 50 mM
NaCl and 250 mM KCl, and resuspended in 5 ml of thesame medium.
‘‘Stuck cells’’ whose bodies were fixed to a glass surface
and whose flagella rotated freely were prepared as
described (Muramoto et al., 1995). Rotation rate of a single
flagellum on a cell attached to the coverslip was measured
by LDM according to Kudo et al. (1990) and Muramoto
et al. (1995). The rotation period (t(k)) for the kth
revolution was determined from the peak to peak intervalof the LDM data, and the rotation rate (v(k)) for the kth
revolution was calculated as:v(k) =

1
t(k)

(2)

All measurements were made at room temperature.
Analysis of rotational fluctuation
The statistical values used in this study were calculated
as follows:Ns v(k)t(k)

v̄ =

k=1
N

=

s t(k)

N
t0

(3)

k=1

N

s 4v(k) − v̄52t(k)

sv =

k=1
N

s t(k)=CNs v(k)
k=1

t0

− v̄2

(4)

k=1

Here, t0 is the duration of the measurement, v̄ is theaverage of the rotation rate (v) for the duration, N is thetotal number of revolutions, and sv is the standard
deviation of v.
Rapid application of inhibitor around a cell
Stuck YM3 cells were prepared in HG medium
containing 50 mM NaCl and 250 mM KCl. A thin glass
capillary was filled with the same medium supplemented
with 3 mM amiloride or 10 mM phenamil. The capillary
was inserted into the space between the glass slide andthe cover slip, where the stuck cell was prepared. The
capillary was placed with its tip near the stuck cell, on theside opposite from the flagellum, and was oriented
parallel with the flagellum. The inhibitor was flushed
toward the stuck cell with a microinjector (CIJ-1;
Shimadzu, Tokyo, Japan). The HG medium around thecell was flowed slowly parallel with the direction of
injection during the measurement.

694

Na+-driven flagellar motor in V. alginolyticus

Computer simulation of the inhibition of the motor
We assumed that the motor has independently
functioning force-generating units, each of which isinactivated by binding of inhibitors. The torque generated
by the motor at time t, Tm (t), is given by summing thetorque generated by each unit as follows:
Tm (t) = 4Nu − nu (t)5T0(5a)Here, Nu is total number of the force-generating units ina motor, nu (t) is the number of the inactivatedforce-generating units at time t, and T0 is the torquegenerated by a functioning unit. In equation (5a), torquegenerated by the motor was assumed to be independentof the rotation rate (Washizu et al., 1993; Berg & Turner,
1993). It should be noted that other experiments indicatedthe torque to linearly decrease with the rotation rate
(Lowe et al., 1987; Iwazawa et al., 1993). The motor torqueis expressed as:
Tm (t) = 4Nu − nu (t)5T0 −

T0
v
v0

(5b)

Here T0 is the zero-rotation-rate torque generated by afunctioning unit and v0 is the zero-torque rotation rate.
However, the results of simulation using equation (5b) isessentially equivalent to those using equation (5a). Hence
we adopted simpler torque characteristic (equation (5a))
in the present study.
To determine the value of nu (t) of equation (5a) in thesimulation, we considered the transition probabilities for
a force-generating unit from active to inactive state (P+ )and inactive to active state (P− ) in Dt:
P+ = k+ IDt(6)

P− = k− Dt(7)Here, I is a concentration of an inhibitor in the medium,and k+ and k− are rate constants of association anddissociation of inhibitors to the force-generating units,
respectively. In the simulation, a random number from 0
to 1 was generated by a computer for each force-generating unit. If a force-generating unit was active and therandom number was smaller than P+ , the state of the unit
was changed from active to inactive. On the contrary, if aunit was inactive and the number was smaller than P− , thestate was changed from inactive to active. The value ofnu (t) was determined as the resultant number of inactive
units. The value was determined on every time-step Dt,
where the value of Dt was set as 10−6 second.
The rotation angle of the motor at time t, u(t), and theaverage rotation rate for the kth revolution, v(k), are givenby the following equations:u(t + Dt) = u(t) +v(k) =Tm (t)
Dtf

1
tu=2kp − tu=2(k − 1)p

(8)
(9)

Here, f is the rotational frictional drag coefficient of theflagellar filament (Holwill & Burge, 1963; Magariyama
et al., 1995), and tu=2kp is time when u(t) is 2kp (seeFigure 7).

Acknowledgements
We thank F. Oosawa and H. Hotani for support anddiscussion, T. Atsumi for providing reagent, and R. M.
Macnab for critical reading of the manuscript. This work

was partly supported by grants-in-aid for scientificresearches (to K.M., I.K., and M.H.) from the Ministry of
Education, Science and Culture of Japan.

References
Atsumi, T., Sugiyama, S., Cragoe, E. J., Jr & Imae, Y.
(1990). Specific inhibition of the Na+-driven flagellar
motors of alkalophilic Bacillus strains by theamiloride analog phenamil. J. Bacteriol. 172, 1634–
1639.
Atsumi, T., Maekawa, Y., Tokuda, H. & Imae, Y. (1992a).
Amiloride at pH 7.0 inhibits the Na+-driven flagellar
motors of Vibrio alginolyticus but allows the cell
growth. FEBS Letters, 314, 114–116.
Atsumi, T., McCarter, L. & Imae, Y. (1992b). Polar andlateral flagellar motors of marine Vibrio are driven bydifferent ion-motive forces. Nature, 355, 182–184.
Berg, H. C. & Turner, L. (1993). Torque generated by theflagellar motor of Escherichia coli. Biophys. J. 65,
2201–2216.
Blair, D. F. & Berg, H. C. (1988). Restoration of torque in
defective flagellar motors. Science, 242, 1678–1681.
Blair, D. F. (1990). The bacterial flagellar motor. Semi. Cell
Biol. 1, 75–85.
Block, S. M. & Berg, H. C. (1984). Successive incorporationof force-generating units in the bacterial rotary
motor. Nature, 309, 470–472.
Chernyak, B. V., Dibrov, P. A., Glagolev, A. N., Sherman,
M. Yu. & Skulachev, V. P. (1983). A novel type ofenergetics in a marine alkali-tolerant bacterium.
Dm̃Na+-driven motility and sodium cycle. FEBS
Letters, 164, 38–42.
Cragoe, E. J., Jr, Kleyman, T. R. & Simchowitz, L. (1992).
Amiloride and its Analogs. Unique Cation Transport
Inhibitors, VCH Publishers, Inc., New York.
Hirota, N., Kitada, M. & Imae, Y. (1981). Flagellar motorsof alkalophilic Bacillus are powered by an electrochemical potential gradient of Na+. FEBS Letters, 132,
278–280.
Holwill, M. E. J. & Burge, R. E. (1963). A hydrodynamic
study of the motility of flagellated bacteria. Arch.
Biochem. Biophys. 101, 249–260.
Imae, Y. & Atsumi, T. (1989). Na+-driven bacterial flagellar
motors. J. Bioenerg. Biomembr. 21, 705–716.
Iwazawa, J., Imae, Y. & Kobayashi, S. (1993). Study of thetorque of the bacterial flagellar motor using a rotating
electric field. Biophys. J. 64, 925–933.
Jones, C. J. & Aizawa, S.-I. (1991). The bacterial flagellum
and flagellar motor: structure, assembly and function. Advan. Microb. Phys. 32, 110–172.
Kawagishi, I., Okunishi, I., Homma, M. & Imae, Y. (1994).
Removal of the periplasmic DNase before electroporation enhances efficiency of transformation in themarine bacterium Vibrio alginolyticus. Microbiology,
140, 2355–2361.
Kawagishi, I., Maekawa, Y., Atsumi, T., Homma, M. &Imae, Y. (1995). Isolation of the polar and lateral
flagellum-defective mutants in Vibrio alginolyticus
and identification of their flagellar driving energy
sources. J. Bacteriol. 177, 5158–5160.
Khan, S. (1993). Gene to ultrastructure: the case of theflagellar basal body. J. Bacteriol. 175, 2169-2174.
Kleyman, T. R. & Cragoe, E. J., Jr (1988). Amiloride andits analogs as tool in the study of ion transport.
J. Membr. Biol. 105, 1–21.
Kudo, S., Magariyama, Y. & Aizawa, S.-I. (1990). Abruptchanges in flagellar rotation observed by laser
dark-field microscopy. Nature, 346, 677–680.

Na+-driven flagellar motor in V. alginolyticus

Lane, J. W., McBride, D. W. & Hamill, O. P. (1991).
Amiloride block of mechanosensitive cation channel
in Xenopus oocytes. J. Physiol. 441, 347–366.
Lin, C., Kieber-Emmons, T., Villalobos, A. P., Foster,M. H., Wahlgren, C. & Kleyman T. R. (1994).
Topology of an amiloride-binding protein. J. Biol.
Chem. 269, 2805–2813.
Lowe, G., Meister, M. & Berg, H. C. (1987). Rapid rotationof flagellar bundles in swimming bacteria. Nature,
325, 637–640.
Magariyama, Y., Sugiyama, S., Muramoto, K., Maekawa,
Y., Kawagishi, I., Imae, Y. & Kudo, S. (1994). Very fast
flagellar rotation. Nature, 371, 752.
Magariyama, Y., Sugiyama, S., Muramoto, K., Kawagishi,
I., Imae, Y. & Kudo, S. (1995). Simultaneous
measurement of bacterial flagellar rotation rate andswimming speed. Biophys. J. 61, 2154–2162.
Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M. &van der Drift, C. (1977). A protonmotive force drives
bacterial flagella. Proc. Natl Acad. Sci. USA, 74,
3060–3064.
Matsuura, S., Shioi, J.-I. & Imae, Y. (1977). Motility in
Bacillus subtilis driven by an artificial protonmotive
force. FEBS Letters, 82, 187–190.
McCarter, L. L. (1994a). MotY, a component of thesodium-type flagellar motor. J. Bacteriol. 176, 4219–
4225.
McCarter, L. L. (1994b). MotX, the channel component of
the sodium-type flagellar motor. J. Bacteriol. 176,
5988–5998.

695
Muramoto, K., Sugiyama, S., Cragoe, E. J., Jr & Imae, Y.
(1994). Successive inactivation of the force-generating
units of sodium-driven bacterial flagellar motors bya photoreactive amiloride analog. J. Biol. Chem. 269,
3374–3380.
Muramoto, K., Kawagishi, I., Kudo, S., Magariyama, Y.,
Imae, Y. & Homma, M. (1995). High-speed rotationand speed stability of sodium-driven flagellar motorin Vibrio alginolyticus. J. Mol. Biol. 251, 50–58.
Negulyaev, Y. A. & Vedernikova, E. A. (1994). Sodium-selective channels in membranes of rat macrophages.
J. Membr. Biol. 138, 37–45.
Okunishi, I., Kawagishi, I. & Homma, M. (1996). Cloning
and characterization of motY, a gene coding for acomponent of the sodium-driven flagellar motor in
Vibrio alginolyticus. J. Bacteriol. 178, 2409–2415.
Sugiyama, S., Cragoe, E. J., Jr & Imae, Y. (1988). Amiloride,a specific inhibitor for the Na+-driven flagellar
motors of alkalophilic Bacillus. J. Biol. Chem. 263,
8215–8219.
Yoshida, S., Sugiyama, S., Hojo, Y., Tokuda, H. & Imae, Y.
(1990). Intracellular Na+ kinetically interfereswith the rotation of Na+-driven flagellar motorsof Vibrio alginolyticus. J. Biol. Chem. 265, 20346–
20350.
Washizu, M., Kurahashi, Y., Iochi, H., Kurosawa, O.,
Aizawa, S.-I., Kudo, S., Magariyama, Y. & Hotani, H.
(1993). Dielectrophoretic measurement of bacterial
motor characteristics. IEEE Trans. Indust. Appl. 29,
286–294.

Edited by J. Karn
(Received 12 January 1996; received in revised form 28 March 1996; accepted 2 April 1996)