J. Mol. Biol. (1997) 265, 310–318

Vibrio alginolyticus Mutants Resistant to Phenamil,a Specific Inhibitor of the Sodium-driven
Flagellar Motor
Seiji Kojima1, Tatsuo Atsumi2, Kazumasa Muramoto1, Seishi Kudo3
Ikuro Kawagishi1 and Michio Homma1*
1

Division of Biological
Science Graduate School of
Science Nagoya University
Chikusa-ku, Nagoya 464-01
Japan2

Department of Physics
Suzuka College ofTechnology, Shiroko-cho
Suzuka, Mie 510-02, Japan
3

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

The polar flagella of Vibrio alginolyticus are driven by sodium motive force
and those motors are specifically and strongly inhibited by phenamil, an
amiloride analog that is thought to interact with a sodium channel of theflagellar motor. To study the sodium ion coupling site, we isolated motility
mutants resistant to phenamil and named the phenotype Mpar for motility
resistant to phenamil. The motility of the wild-type (Mpas) was inhibitedby 50 mM phenamil, whereas Mpar strains were still motile in the presenceof 200 mM phenamil. The Ki value for phenamil in the Mpar strain was
estimated to be five times larger than that in the Mpas strain. However, thesensitivities to amiloride or benzamil, another amiloride analog, were notdistinctly changed in the Mpar strain. The rotation rate of the wild-type
Na+-driven motor fluctuates greatly in the presence of phenamil, which canbe explained in terms of a relatively slow dissociation rate of phenamilfrom the motor. We therefore studied the stability of the rotation of the
Mpar and Mpas motors by phenamil. The speed fluctuations of the Mpar
motors were distinctly reduced relative to the Mpas motors. The steadierrotation of the Mpar motors can be explained by an increase in thephenamil dissociation rate from a sodium channel of the motor, whichsuggests that a phenamil-specific binding site of the motor is mutated in
the Mpar strain.
7 1997 Academic Press Limited*Corresponding author

Keywords: flagella; amiloride; Vibrio; motor; sodium-motive force

Introduction
Bacterial flagellar motors are molecular machines
powered by the electrochemical potential gradientof specific ions across the membrane (Blair, 1995;
Imae & Atsumi, 1989). The flagellar motors can beclassified by coupling ion: some are H+-driven andsome are Na+-driven. The motor contains several
independently functioning force-generating units,
which should interact with coupling ion at theexternal surface of the cytoplasmic membrane andfunction as ion channels. It has been estimated thatthe H+-driven motor of Escherichia coli and the Na+driven motor of an alkalophilic Bacillus have eight
Present address: K. Muramoto, Department of
Molecular Biophysics and Biochemistry, Yale
University, New Haven, CT 06520-8114, USA.
Abbreviations used: Pof, polar flagellum; Laf, lateral
flagellum; Mpar, motility resistant to phenamil; LDM,
laser-dark field microscopy.
0022–2836/97/030310–09 $25.00/0/mb960732

and five to nine force-generating units, respectively(Block & Berg, 1984; Blair & Berg, 1988; Muramoto
et al., 1994). For studies of the flagellar motor, theNa+-driven type presents certain advantages:
the sodium motive force can be easily changed by
the Na+ concentration of the medium and the motor
rotation can be specifically and competitively
inhibited by amiloride (Sugiyama et al., 1988),
which are potent inhibitors of Na+ channel, Na+/H+
exchangers, and Na+/Ca2+ exchangers (Cragoe
et al., 1992). An amiloride analog, phenamil, whose
inhibition is non-competitive with Na+, is morepotent than amiloride without affecting cell growthof alkalophiles (Atsumi et al., 1990; Sugiyama et al.,1988). The inhibitors are thought to interact withthe force-generating units of the motors (Sugiyama
et al., 1988; Atsumi et al., 1990; Muramoto et al.,
1994, 1995).
Some marine Vibrio species have both types ofmotors in one cell. A single polar flagellum (Pof)rotates by a Na+-driven motor and multiple lateral
7 1997 Academic Press Limited

311

Phenamil-resistant V. alginolyticus Mutants

flagella (Laf) rotate by H+-driven motors (Atsumi
et al., 1992a; Kawagishi et al., 1995). The polar
flagella work better for swimming in a low-viscosity environment than the lateral flagella (Atsumi
et al., 1996). The restriction of the function of thepolar flagellum appears to trigger the expression oflateral flagella (Kawagishi et al., 1996; McCarter
et al., 1988). The polar flagella are covered with asheath that is contiguous with the outer membrane(Follett & Gordon, 1963; Allen & Baumann, 1971)
and the single filament is easily observed under the
dark-field microscope.
The high-speed rotation of flagella has been
analyzed by laser-dark field microscopy (LDM;
Kudo et al., 1990; Kami-ike et al., 1991). Using thissystem, it was shown that the rotation of polar
flagella was very fast in V. alginolyticus; the speedsof 600 r.p.s. were observed in the presence of
50 mM NaCl at room temperature, and surprisingly, the speeds increase up to 1700 r.p.s. in thepresence of 300 mM NaCl at 35°C (Magariyama
et al., 1994, 1995; Muramoto et al., 1995). It was also
shown that the rotation of this motor was stable athigh speed (Muramoto et al., 1995). The standard
deviation of the rotation period was only 7% to 16%of the average rotation period in polar flagella of V.
alginolyticus. When the rotation rate was reduced
by decreasing external concentration of NaClor adding amiloride or carbonylcyanide mchlorophenylhydrazone (CCCP), the motor rotatedsmoothly. On the other hand, we found that thepolar flagella showed remarkably larger speed
fluctuations when the rotation rate was reduced byphenamil (Muramoto et al., 1996). The speed
fluctuations induced by phenamil can be explainedby the slow dissociation rate of the inhibitor fromthe force-generating unit.
To identify the force-generating units andinvestigate the sodium ion coupling site of the
motor, it would be useful to identify the target ofamiloride and its analogs in the flagellar motor. In
this work, we synthesized phenamil, which had notbeen commercially available, isolated V. alginolyticus motility mutants resistant to phenamil andcharacterized the mutants with respect to the effectof phenamil on the rotation of the motor.

It has been reported that the cell growth ofalkalophiles and V. parahaemolyticus is not affectedby phenamil, even at concentrations that fully blockrotation of the flagellar motors (Atsumi et al., 1990,
1992b; Kawagishi et al., 1996). V. alginolyticus cells
grew normally in VC medium containing a highconcentration (500 mM) of phenamil, while motilityof the wild-type cells was completely inhibited(data not shown). This indicates that phenamilworks as a specific inhibitor of the Na+-motors of V.
alginolyticus, without affecting cell growth.
V. alginolyticus VIO5 (Pof+ Laf− Rifr ) cells weretreated with ethylmethane sulfonate (EMS) andstreaked onto the surface of 0.25% agar VD plates
supplemented with 500 mM phenamil, and theplates were incubated. Cells that spread out fromthe point of inoculation on the plate were isolated,
and two isolates were named NMB201 and
NMB205. We confirmed the resistant phenotype ina 0.25% agar VG plate containing 200 mM phenamil(Figure 1). Because these mutants and the parent
showed very tumble-biased swimming in thepresence of inhibitors, it was quite difficult tomeasure their swimming speed. We therefore
isolated smooth swimming (che) mutants, NMB136(from VIO5), NMB157 (from NMB201), and
NMB160 (from NMB205). These che mutants swam
at the same swimming speed as the parent strains,and showed the same inhibition profiles against thephenamil concentrations (data not shown). Thus,
the motor function seems not to be affected in theche mutants. NMB136 (phenamil-sensitive) and
NMB157 (phenamil-resistant) were then characterized more closely. We named the phenotype Mpar
for motility resistant to phenamil. Accordingly, thewild-type phenotype was designated as Mpas.
Effect of specific inhibitors on the mutants
We first examined whether the motility of Mpar
cells (NMB157) and Mpas cells (NMB136) showed

Results
Isolation of motility mutants resistantto phenamil
We tried to isolate mutants whose flagellar
motors could function in the presence of amiloride
or its more potent analogs, phenamil and benzamil.
Experiments using amiloride or benzamil, which
are commercially available, were not successful
because their effects seem to be weak in the growth
medium of V. alginolyticus whose ionic strength andNa+ concentration are high (Atsumi et al., 1992b).
Phenamil, which is known to be the most potent
inhibitor of Na+-driven motors was then synthesized by Cragoe’s methods (Cragoe et al., 1967).

Figure 1. Swarming abilities of the mutants in thepresence of phenamil. A VG semi-solid plate containing
200 mM phenamil was incubated at 30°C for 20 hours.
1, the Mpas parent strain (VIO5); 2 and 3, the Mpar
mutants (NMB201 and NMB205).

312

Phenamil-resistant V. alginolyticus Mutants

affects mainly the phenamil interaction site in the
Na+-driven motor.
To quantify the effect of the mutation on
phenamil binding, we estimated the inhibition
constant (Ki ) in both strains. We measured theirswimming speeds in the presence of 6.25, 25, and100 mM NaCl under various concentrations ofphenamil. Figure 4 is a Dixon plot of the phenamilconcentration and the Na+ influx estimated fromthe swimming speed by a kinetic analysis described
previously (Sugiyama et al., 1988). This shows thatthe inhibition profile of phenamil in a Mpar strainchanges into a rather competitive manner with Na+.
The Ki values of the Mpas and Mpar strains were0.14 mM and 0.74 mM, respectively.
Fluctuation of the rotation rate in thephenamil-resistant mutant

Figure 2. Relationship between Na+ concentration ofmedia and swimming speed of Mpar cells. Cells from the
Mpar strain (W) or the Mpas strain (w) were harvested
at late logarithmic phase and suspended in TNM
medium (pH 7.5). The cell suspension was diluted about
100-fold into the TNM medium containing various
concentrations of NaCl. Swimming speed was measured
within one minute as described in Materials andMethods.

similar dependence on the external Na+ concentration. Their motility profiles were indistinguishable, although Mpar cells swam a little slower thanMpas cells (Figure 2). This suggests that the affinity
for Na+ is not affected by the mutation and the Na+binding region is essentially unchanged in Mpar
cells.
Next, we examined the swimming speed of Mpar
cells in various concentrations of phenamil,
amiloride or benzamil (Figure 3). The motility of
Mpas cells was completely inhibited by 50 mM
phenamil. In contrast, Mpar cells still swam at
12 mm/second and were stopped only by 500 mM
phenamil. When amiloride was used, differences
between Mpar and Mpas cells were less pronounced. Mpar cells swam 20 or 30% faster thanMpas cells in the range of 0.5 mM to 2.0 mM
amiloride. Difference in swimming speed wereobserved when Na+ concentration was varied in thepresence of 1 mM amiloride. At lower Na+
concentrations, Mpar cells swam faster than Mpas
cells (data not shown). This difference indicates thatthe mutation affects the amiloride sensitivity of the
motor. However, we could not detect any
difference between the swarming abilities of Mpar
and Mpas strains in a semi-solid plate containing
1 mM amiloride. The inhibition profile by benzamil,
whose potency is intermediate between phenamiland amiloride (Sugiyama et al., 1988; Imae., 1991),
showed no difference between the Mpar and Mpas
strain. These results suggest that the mutation

It has been shown that the rotation rate becomesvery unstable in the presence of phenamil but notin the presence of amiloride or CCCP, even when
the inhibitors are used at concentrations that reduce
the rotation rate by the same amount (Muramoto
et al., 1996). We investigated the stability of Mpar
flagellar rotation at 50 mM NaCl in the presence ofphenamil and amiloride. The Mpar and Mpas cellbodies were fixed on the glass surface, and the freerotation of a single flagellum was measured byusing laser-dark field microscopy (LDM). Figure 5
shows the periodic change in photon counts
observed under each condition. When the rotation
rate of a Mpas flagellum was decreased to about
200 r.p.s. by 1 mM phenamil, large fluctuations inrotation rate were observed (Figure 5(A)). In thecase of a Mpar cell, when the rotation rate wasdecreased to about 200 r.p.s. by 5 mM phenamil, thespeed fluctuations were significantly smaller thanin the Mpas strain (Figure 5(B)). On the other hand,when the rotation rate was decreased to about
200 r.p.s. by 0.5 mM amiloride, both the Mpar andMpas flagella rotated very smoothly and there waslittle change of the rotation period, peak-to-peakinterval (Figure 5(C) and 5(D)). The fluctuations
seen with phenamil in the Mpar strain were stilllarger than those seen with amiloride.
Rotation rates in the presence of phenamil or
amiloride were calculated from the rotation periods
and their distribution plotted on histograms (Figure 6). When the rotation rate was reduced byphenamil, the rotation rate of the Mpas flagellafluctuated between 50 and 500 r.p.s., and the slow
rotation sometimes persisted for relatively longtimes (Figure 6(A)). The variation in the rotation
period of Mpar flagella was smaller, and periods ofslow rotation were shorter (Figure 6(B)). In thepresence of amiloride, the rotation rates of both
Mpar and Mpas flagella were distributed sharply
around the average rotation rate (Figure 6(C) and(D)).
To evaluate this difference in various cells, we
obtained the standard deviation of the rotation rate(sv ) and expressed the fluctuation as (sv /v̄) ×Phenamil-resistant V. alginolyticus Mutants

313

Figure 3. Motility inhibition of
Mpar cells by specific inhibitors.
Cells of the Mpar strain (W) or the
Mpas strain (w) were suspended
in TNM medium containing various
concentrations of inhibitors, andthe swimming speed was measured
as described in Materials andMethods.

100 (%). The data obtained from four cells are
summarized in Table 1. In the presence ofphenamil, the speed fluctuations of Mpas cells
increased to 40%, and in Mpar cells 24%. When weused 0.5 mM amiloride as a control, both Mpas andMpar strains showed the same fluctuation, about
16%. In the absence of phenamil or amiloride, themotors of Mpas and Mpar strains rotated at 640 and
550 r.p.s., respectively, with fluctuations of 15%(data not shown). These results indicate that thefluctuations are significantly reduced in the Mparstrain, relative to the wild-type.

Discussion
To study the Na+-driven motor, we isolated
motility mutants resistant to phenamil (Mpar ).
Phenamil is the most potent specific inhibitor of a
Na+-driven motor and does not severely affect cell
growth or physiology. The growth and Na+-dependent motility of the Mpar mutant were the same as
those of the parent Mpas strain, which is wild-type
as far as the polar flagellum is concerned. The
mutant was significantly more resistant tophenamil, exhibiting an inhibition constant about
five times larger than that of the Mpas strain. Thissuggests that the Mpar mutation affects theinteraction between phenamil and the motor.
The sensitivity to benzamil was not changed in
the mutant and the sensitivity to amiloride wasonly slightly reduced. These results suggest that the
mutation affects mainly the phenamil interaction

site in the flagellar motor, and to a lesser extent theamiloride interaction site. In previous work,
motility mutants resistant to phenamil wereisolated from an alkalophilic Bacillus. Thesemutants suggested that amiloride and phenamilhave different interaction sites in the motor (Atsumi
et al., 1990), because the sensitivity to amiloride was
not affected. This difference between the two
species might reflect differences in the mutations orin the Na+ binding regions.
It has been observed that the motor rotation rate
fluctuates in the presence of phenamil but notamiloride or CCCP (Muramoto et al., 1996) and it
was suggested that this phenomenon is due to thelonger duration of phenamil binding to the motor.
If there are several force-generating units in one
motor and each unit functions independently, then
the number of functional units is proportional to
the rotation rate and changes in this number cause
the fluctuations of rotation rate. In the case ofphenamil, since the dissociation rate constantshould be small, phenamil binds stably to themotor. So unstable rotation at subnormal speed is
detected by LDM. On the other hand, theinteraction of amiloride with the force-generating
units should be more rapid, and an average speed
corresponding to the average number of active
force-generating units is detected. The fluctuationsof rotation rate, given by the tethering technique, inthe H+-motor of E. coli were explained by a model
incorporating dissociation and association of theforce-generating units (Kara-Ivanov et al., 1995).

314

Figure 4. Effect of various concentrations of phenamilon the estimated Na+ influx through the motors of Mpas
(upper) and Mpar (lower). Cells were suspended in TNM
medium containing 6.25 mM (W), 25 mM (w), or 100 mM(Q) NaCl and various concentrations of phenamil wereadded. The swimming speed was measured as describedin Materials and Methods and the Na+ influx was
estimated as described previously (Sugiyama et al., 1988).
The sodium motive force was calculated by using themembrane potential of −110 mV and the intracellar Na+
of 50 mM (Tokuda et al., 1988). A reciprocal plot (Dixon
plot) of those data was performed. The units in theordinate are given as mV s2 mm−2.

The model is essentially the same as our model
though the time resolution is extremely different.
The phenamil resistance of the mutant isolated inthis work can be explained by an increase in
the dissociation rate of phenamil, a decrease in theassociation rate, or both. If the dissociation rateincreases, the binding time of phenamil will be
shorter, reducing the amplitude of the speed
fluctuations. If the association rate decreases, thenthe binding time should not change, and fluctuation
would not be affected. The LDM analysis indicates

Phenamil-resistant V. alginolyticus Mutants

Figure 5. Mpas and Mpar flagellar rotation detected by
LDM. Peak intervals of the intensity changes of thescattered light from the rotating flagella correspond to
the rotation period. Cells harvested at late logarithmic
phase were stuck on the cover glass and the rotation of
single flagella were measured by LDM for about six
seconds in the presence of phenamil or amiloride which
decrease the rotation rate to about 200 r.p.s. (A) The Mpas
flagella with 1 mM phenamil; (B) the Mpar flagella with
5 mM phenamil; (C) the Mpas flagella with 0.5 mM
amiloride; (D) the Mpar flagella with 0.5 mM amiloride.

that the Mpar strain shows significantly smallerfluctuations than the Mpas strain. Accordingly, wesuggest that phenamil may dissociate from the
motor faster in the mutant and this causes theresistance to phenamil.
The interaction of phenamil and amiloride withthe motor is speculated as follows. Amiloride issuggested to associate with the same site in the
motor as do sodium ions, and quickly associate anddissociate with the motor, causing the competitive
inhibition with Na+. On the other hand, phenamilis assumed to interact at another high affinity siteand can bind more stably to the motor thanamiloride, causing the non-competitive inhibition
with Na+. Moreover, phenamil can also associatewith the amiloride binding site because ofstructural similarity. In the Mpar motor, we thinkthat the high affinity site for phenamil is mutated,

315

Phenamil-resistant V. alginolyticus Mutants

Figure 6. The rotation rate and its distribution of theMpas and Mpar flagella in the presence of phenamil andamiloride. The rotation rate calculated from the data of
Figure 5 for one second as described in Materials andMethods was shown in the left graphs. Histogram ofrotation rate for one second was shown in the rightgraphs. (A) The Mpas flagella with 1 mM phenamil; (B) the
Mpar flagella with 5 mM phenamil; (C) the Mpas flagellawith 0.5 mM amiloride; (D) the Mpar flagella with
0.5 mM amiloride. Average rotation rate (v) was 182
r.p.s. (A), 194 r.p.s. (B), 198 r.p.s. (C),188 r.p.s. (D). The
values of the fluctuation were 42% (A), 29% (B), 15% (C),
17% (D).

so phenamil additionally interacts with the amiloride binding site, causing phenamil to dissociate
more rapidly than in the Mpas motor and theintermediate inhibition profile between amiloride
and phenamil. The mutation might also cause
subtle structural changes around the common
interaction site, because inhibition by amiloride
was also slightly affected by the mutation.
Benzamil, which is larger than phenamil, canslightly interact with the high affinity site forphenamil and the mutation may not greatly affecttheir inhibition profile.
The interaction between amiloride analogs andtheir targets has been studied in various Na+-transporters and with anti-amiloride antibodies (Cragoe
et al., 1992; Lin et al., 1994). In the fibroblast Na+/H+
exchanger (NHE1), which has ten transmembranesegments, a mutant resistant to amiloride wasisolated (Counillon et al., 1993). This mutant haspoint mutations that convert Phe165 and Phe168
into Tyr. These residues are located in the middleof the fourth putative transmembrane segment.
However, in the case of the epithelial Na+ channel(ENAC), which is supposed to have six transmembrane segments, a recent report suggested that theamiloride-binding site resides on an extracellularloop of the a subunit and not the putative second
transmembrane domain which forms a channelpore (Li et al., 1995). The amiloride-binding site
may therefore be in very different locations indifferent proteins, although an additional possibility that the mutations in Na+/H+ exchanger
mapped in membrane segments might cause achange in conformation that affects an extracellularbinding site for amiloride cannot be eliminated. It
is also known that phenamil is a more potent
inhibitor of the Na+ channel than the Na+/H+
exchanger (Cragoe et al., 1992). Since the Na+driven motor is also inhibited potently byphenamil, it might have more features in common
with the Na+ channel. However, the additional highaffinity binding site of phenamil in the scheme
described above has not yet been identified in the
Na+ channel.
Recently, the motX and motY genes, which seemto encode components of the force-generating
units, were isolated from Vibrio parahaemolyticus(McCarter, 1994a,b). MotX is inferred to be acomponent of the Na+ channel of the motor because
overproduction of MotX was lethal in E. coli in
proportion to the external Na+ concentration, but
this effect was suppressed by the addition of
amiloride. The C-terminal domain of MotY is
homologous to many peptidoglycan-interacting
proteins. So, MotX and MotY, each of which has aputative single transmembrane region, form achannel component and a stator component,
respectively, as the H+ motor components of MotA
and MotB (Blair, 1995). The pom (polar flagellamotility) genes, whose mutants have paralysedpolar flagella, identical to motX and motY, weredetected in V. alginolyticus (Okunishi et al., 1996)
and novel pom genes have been isolated (Asai, Y.,
Okunishi, I., Kojima, S., Kawagishi, I. & Homma,M., unpublished). We expected that the Mpar
mutation would map to one of the genes coding forthe motor proteins, or to other components of theforce-generating units. Actually, the Mpar mutation
was mapped in a pom gene other than motX andmotY genes (Kojima, S., Asai, Y., Kawagishi, I. &Homma, M., unpublished). We hope to determine
the target of phenamil and thereby obtain
information about the mechanism by which Na+ isused to drive the motor.

Materials and Methods
Bacterial strains, growth conditions and media
V. alginolyticus VIO5 (Okunishi et al., 1996) was usedas the parent strain to isolate mutants. It is a Pof+ Laf− Rifr

316

Phenamil-resistant V. alginolyticus Mutants

Table 1. Summary of the results of effect of phenamil and amiloride on thestability of rotation rate of Mpar and Mpas strains obtained from four
independent cells
1

2

3

4

Average

Rotation rate (r.p.s.)Standard deviation (r.p.s.)
Fluctuation (%)

197
79
40

207
79
38

131
51
39

182
77
42

179
72
40

Rotation rate (r.p.s.)Standard deviation (r.p.s.)
Fluctuation (%)
Mpas (NMB136)
Rotation rate (r.p.s.)
0.5 mM Amiloride Standard deviation (r.p.s.)
Fluctuation (%)
Mpar (NMB157)
Rotation rate (r.p.s.)
0.5 mM Amiloride Standard deviation (r.p.s.)
Fluctuation (%)

170
47
28
198
30
15
188
32
17

221
42
19
138
23
17
270
30
11

160
34
21
162
20
12
258
29
11

194
57
29
158
19
12
258
33
13

186
45
24
164
23
14
243
31
13

s

Mpa (NMB136)
1 mM Phenamil
Mpar (NMB157)
5 mM Phenamilstrain derived from strain VIK4 (Okunishi et al., 1996),
which is a rifampicin-resistant derivative of the wild-typestrain 138-2 (Tokuda et al., 1988).
Cells were grown at 30°C with shaking in VG medium
containing 1% (w/v) polypeptone, 0.5% (w/v) glycerol,
0.4% (w/v) K2 HPO4 , and 0.5 M NaCl. To select motility
mutants, 0.25% (w/v) agar VD plates (0.25% agar in VD
medium containing 0.5% polypeptone, 0.2% glucose,
0.5% yeast extract, 0.4% K2 HPO4 , 50 mM NaCl, and
250 mM KCl) were used.
Vibrio buffer containing 10 mM Tris-HCl (pH 7.0),
10 mM MgCl2 , 350 mM NaCl was used to wash thecells and TNM medium containing 50 mM Tris-HCl(pH 7.5), 5 mM MgCl2 , 5 mM glucose, 50 mM NaCl,
250 mM KCl was used to measure the swimming speed.
When the concentration of Na+ in the motility medium
was varied, KCl was added to keep the salt concentration
constant.
Chemicals and anti-flagellar antibody
Amiloride and benzamil was purchased from Sigma
Chemical Co. and Research Biochemical Incorporated,
respectively. Phenamil, which was not commerciallyavailable, was synthesized by the method of Cragoe et al.
(1967).
Anti-polar flagellar antibody was prepared by injecting
a rabbit with purified polar flagella of V. alginolyticus
strain YM4 (Pof+ Laf− ) whose parent is the same as VIO5
(Kawagishi et al., 1995).
Isolation of motility mutants
Mutagenesis was carried out by using ethyl methanesulfonate (EMS). Cells of VIO5 were harvested bycentrifugation from 1.0 ml culture at late logarithmic
phase, washed once with Vibrio buffer, and resuspended
in 0.5 ml of Vibrio buffer. Then, 15 ml of EMS was added
to the suspension and incubated with mild shaking for
one hour at 30°C. After washing the cells once with Vibrio
buffer, they were resuspended in 0.5 ml of Vibrio buffer.
EMS-treated cells were streaked onto the surface of a
0.25% agar VD plate supplemented with 500 mM
phenamil, and the plate was incubated at 30°C for 12hours. Mutants that spread out from the point of
inoculation were isolated. Phenamil-resistant candidates
were tested for rifampicin resistance and also forreactivity to the anti-polar flagellar antibody.
Smooth swimming mutants were isolated from thephenamil-resistant mutants to easily measure theirswimming speeds. The che mutants were selected as
described previously (Homma et al., 1996).
Measurement of swimming speed
Cells were harvested at late logarithmic phase andresuspended in TNM medium at half the original culture
volume. Then a small amount of the cell suspension was
diluted about 100-fold into TNM medium, and motilityof the cells was observed under a dark-field microscopeand recorded on video tape. Swimming speed wasdetermined as described previously (Atsumi et al., 1996).
The average swimming speed was obtained by measuring more than 20 swimming tracks.
Measurement and analysis of single
flagellar rotation
High speed flagellar rotation was measured bylaser-dark field microscopy (LDM) as described previously (Kudo et al., 1990; Muramoto et al., 1995). Cells
were harvested at late logarithmic phase, washed oncewith TNM medium, and resuspended in the same
medium. The cell suspension was poured into the space
between slide and cover glass on which thin spacers wereplaced. After ten minutes, cells not stuck to the slide were
washed by the flow of TNM medium. Rotation of thepolar flagellum of a stuck cell was measured by using
LDM for about six seconds. When the effect of inhibitor
was measured, the stuck cells were washed with the flowof TNM medium supplemented with a suitable amountof inhibitor.
The photon counting gate time was set at 200 ms. The
rotation rate was calculated as a reciprocal of the periodof peak intervals, and average rotation rate (v̄) and its
standard deviation (sv ) for one second were calculatedby the following equations (Muramoto et al., 1996).
Ns v(k)t(k)

v̄ =

k=1

=

N

s t(k)

N
t0

(1)

k=1

N

s (v(k) − v̄)2t(k)

sv =

k=1
N

s t(k)
k=1.

(2)

Phenamil-resistant V. alginolyticus Mutants
Here, v(k) and t(k) are the rotation rate and period for kth
revolution, t0 is the duration of measurement, N is thetotal number of revolutions.

Acknowledgements
We thank Dr Robert M. Macnab for invaluable
discussions and Dr Kohtaro Tomizawa for assistancewith the phenamil synthesis. We especially thank
Dr David F. Blair for critical reading of the manuscript.
This work was supported in part by grants-in-aid forscientific researches (to T.A., I.K. and M.H.) from the
Ministry of Education, Science and Culture of Japanand by R & D program from NEDO of Japan (to I.K.
and M.H.).

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Edited by J. Karn
(Received 5 July 1996; received in revised form 21 October 1996; accepted 28 October 1996)