JOURNAL OF BACTERIOLOGY, Mar. 1999, p. 1927–1930
0021-9193/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 6

The Polar Flagellar Motor of Vibrio cholerae Is Driven
by an Na1 Motive ForceSEIJI KOJIMA,1 KOICHIRO YAMAMOTO,2 IKURO KAWAGISHI,1 AND MICHIO HOMMA1*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602,1 and Department of Nutritional Science, Faculty of Healthand Welfare Science, Okayama Prefectural University,
Soja, Okayama 719-1197,2 Japan
Received 27 October 1998/Accepted 12 January 1999

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing
project of this organism is in progress, and the genes that are highly homologous to the essential genes of theNa1-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. Theenergy source of its flagellar motor was investigated. We examined the Na1 dependence and the sensitivity to
the Na1 motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar
flagellar motor of V. cholerae is driven by an Na1 motive force.
human small intestine. During the free-swimming phase, theorganism is highly motile by means of a single polar flagellum(14). Motility is thought to contribute to the virulence ofV. cholerae, but the relationship between motility and virulence
is not yet understood (14, 27) and basic studies of its flagellar
motor have not been done. As described above, the flagellar
motor has been intensively investigated in other Vibrio species,
namely V. alginolyticus and V. parahaemolyticus. They have two
distinct types of flagella, a polar flagellum used for swimmingin a liquid medium and numerous lateral flagella used forswarming on the surface of solid substrate (5, 20, 22, 32, 33).
Polar and lateral flagella rotate by Na1- and H1-driven flagellar motors, respectively (2, 20). When grown in a liquid medium, the organism mainly expresses a single polar flagellum
and swims rapidly. Therefore, the cell shape and motility ofthese Vibrio are very similar to those of V. cholerae in liquid. It
has also been shown that the flagellar basal body of V. alginolyticus resembles that of V. cholerae (4, 13). In addition, fromthe genome sequencing project of V. cholerae, the partial sequences of genes homologous to pomA, pomB, motX, andmotY, essential for the rotation of the Na1 motor, have beenfound in the database. Gardel and Mekalanos (15) reported
that the disruption of the pomB homolog of V. cholerae results
in a flagellated but nonmotile strain (the authors described
pomB as motB, and the partially deduced amino acid sequenceof MotB showed 84% identity to PomB of V. alginolyticus),suggesting that the pomB homolog of V. cholerae is also essential for force generation in the motor. Therefore, we think thatthe polar flagellar motor of V. cholerae might be driven by an
Na1 motive force. In this study, we examined Na1 dependence
and the effects of specific inhibitors on motility in V. choleraeand present evidence that the polar flagellar motor is driven by
an Na1 motive force.
V. cholerae O139 strain 1854 is highly motile. We examined
the swarming abilities of the following V. cholerae strains: 1854
(O139; Bengal serovar [18]), E8499 (non-O1 [11]), S7 (O37[35]), N86 (O1; El Tor biotype [36]), and 569B (O1; Classicalbiotype [11, 21]). Fresh colonies on LB solid agar plates (1%tryptone, 0.5% yeast extract, 0.5% NaCl, 1.25% agar) were
inoculated onto LB semisolid plates (0.3% agar) and incubatedfor 4 h at 37°C. As shown in Fig. 1, strains E8499, 569B, and1854 made swarm rings (the ring of strain 1854 was the larg-

Many motile bacteria move by rotating their flagella, thefilamentous organelles that extend from the cell body. Flagellar rotation is carried out by a reversible rotary motor (about
20 nm in diameter) embedded in the cytoplasmic membrane at
the base of each flagellar filament. These motors are poweredby an electrochemical gradient of specific ions across the cytoplasmic membrane and are classified into two types of couplingions: an H1-driven motor (in Escherichia coli, Salmonella typhimurium, and Bacillus spp.) and an Na1-driven motor (inalkalophilic Bacillus and marine Vibrio spp.) (6, 19). Thus, theflagellar motor is a tiny and elaborate molecular machine
which converts electrochemical energy into mechanical work.
However, the mechanism of this energy conversion is not clarified at the molecular level.
The study of the flagellar motor has been extensively done
for the H1-driven motor of E. coli and S. typhimurium, and it
has been shown that two integral membrane proteins, MotA
and MotB, are essential for force generation in the motor (12,29). They have four and a single transmembrane segment(s),respectively (10, 38), and form a proton-conducting channel
complex responsible for coupling ion translocation to force
generation in the motor (7, 16, 28, 30). The MotA-MotB channel complex is anchored to the cell wall by a peptidoglycan
binding domain of MotB. Some critical residues involved intorque generation have recently been identified by intensive
mutational studies (8, 9, 37, 39). In the Na1-driven motor,
it was shown that four integral membrane proteins, PomA,
PomB, MotX, and MotY, are essential for force generation in
the polar flagellar motor of the marine bacterium Vibrio alginolyticus (1, 26). MotX and MotY have also been identified in
Vibrio parahaemolyticus (23, 24). PomA and PomB have similarities to MotA and MotB, respectively, and so they might also
form an Na1 channel complex and play an important role inforce generation in the motor.
Vibrio cholerae is the bacterium that causes cholera. It has apathogenic cycle consisting of a free-swimming phase outside
its host and a sessile virulent phase when it is colonizing the* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan. Phone: 052-789-2991. Fax: 052-789-3001. Email: g44416a@nucc.cc.nagoya-u.ac.jp.
1927

1928

NOTES

FIG. 1. Swarming abilities of V. cholerae strains. Fresh single colonies of theindicated V. cholerae strains were inoculated onto an LB semisolid plate (0.3%
agar) and incubated for 4 h at 37°C.

est), but S7 and N86 showed very little swarming ability. Whencells were cultured in LB broth to directly observe motility,strain 1854 demonstrated the best motility of the strains used,consistent with the results of swarming ability; most of the cells
swam very rapidly and the fraction of cells that were motile wasvery large compared with that of the other strains (data notshown). Some of the other strains were also motile, but thefraction of motile cells was smaller than that of strain 1854,
because of the aggregation or elongation of the cells (data notshown). Therefore, we mainly used strain 1854 for motility
analysis in this study.
Effect of ionic strength on the swimming speed of V. cholerae
1854. A previous study of the 5S rRNA sequence suggested
that V. alginolyticus, which has an Na1-dependent polar flagellum, is closely related to V. cholerae (4). Therefore, we compared the motilities by polar flagella of V. cholerae and V. alginolyticus. These two Vibrio spp. are usually grown in different
salt concentrations. In LB medium, which contains 0.5% NaCl,commonly used for growing V. cholerae, the V. alginolyticus
strain VIO5 (wild type in polar flagellar motility [26]) could not
grow (data not shown). However, in VPG medium (1% polypeptone, 0.5% glycerol, 0.4% K2HPO4, 3% NaCl), commonly
using for growing V. alginolyticus, V. cholerae 1854 grew comparably to V. alginolyticus VIO5. We compared the effects ofionic strength on the motility of V. cholerae 1854 cells cultured
in these two media. Strain 1854 cells were grown in both LB
and VPG media, and as a control V. alginolyticus VIO5 cells
were cultured in VPG medium. At late log phase, cells were
harvested and resuspended in TMN medium containing 50
mM Tris-HCl, 5 mM MgCl2, 5 mM glucose, and various concentrations of salts, and the motility of the cells was observed.
As shown in Table 1, when the ionic strength of TMN medium
was fixed at 300 mM, the motility of LB medium-cultured 1854
cells deteriorated and this deterioration became more severeas the concentration of NaCl increased. At 300 mM NaCl, boththe motile fraction and the swimming speed were greatly reduced. LB medium-cultured 1854 cells resuspended in mediaof lower ionic strength (100 or 200 mM NaCl) showed a larger
motile fraction and a greater swimming speed. However, theVPG-cultured V. cholerae 1854 and V. alginolyticus VIO5 cells
showed nearly the same motile fractions regardless of ionic

J. BACTERIOL.

strength, and their swimming speeds increased as the NaCl
concentration of the medium was increased. However, themotile fractions were significantly smaller than that of LBcultured 1854 cells at 100 mM NaCl. We note that VPGcultured 1854 cells showed very tumbly-biased swimming, andattractants, such as serine or Casamino Acids, were not effective in suppressing the directional change of swimming. These
results indicate that V. cholerae 1854 can grow in media of a
relatively wide range of ionic strengths, but the greatest motility of V. cholerae 1854 cells is achieved with an ionic strengthof 100 mM when the cells are cultured in LB medium containing 0.5% NaCl. Therefore, in the following experiments, thetotal ionic concentration of Na1 and K1 was fixed to 100 mM
in TMN medium.
Polar flagellar motor of V. cholerae 1854 is driven by the sodium motive force. We next examined the potential for Na1 as
an energy source of the polar flagellar motor of strain 1854.
V. cholerae 1854 and V. alginolyticus VIO5 cells were cultured
in LB and VPG media, respectively, and motility was measured
in TMN medium containing various concentrations of NaCl,with a salt concentration of 100 mM attained by adding KCl.
As shown in Fig. 2A, both 1854 and VIO5 cells clearly showedNa1-dependent motility and 1854 cells swam significantly faster than VIO5 cells did under all conditions. LB medium-cultured 1854 cells also showed a similar Na1-dependent motility
in the medium whose ionic strength was fixed at 100 mM by theaddition of choline chloride instead of KCl (data not shown).
We also examined the effect of the Na1 motor-specific inhibitor, phenamil, on the motilities of both strains. Phenamil is
known to inhibit the Na1 motor in a noncompetitive mannerwith Na1 in the medium (3). As shown in Fig. 2B, the motilities
of both strains were also clearly inhibited by phenamil in thepresence of 50 mM NaCl, although the concentration of phenamil required to completely inhibit the motility of V. cholerae
was slightly higher than that needed for V. alginolyticus. AmiloTABLE 1. Effect of ionic strength on the swimming speedof V. cholerae 1854 and V. alginolyticus VIO5
Strain–mediuma

NaCl(mM)

1854-LB

300
200
100
200
100

1854-VPG

300
200
100
200
100

VIO5-VPG

300
200
100
200
100

KCl
(mM)

100
200

100
200

100
200

Motile fractionb(motile cells/total cells)

Swimming
speedc (mm/s)

0.19
0.33
0.43
0.65
0.68

44.7
56.8
61.4
83.0
99.1

0.44
0.38
0.40
0.37
0.40

84.0
79.1
71.7
75.2
74.2

0.46
0.43
0.54
0.47
0.50

61.2
61.4
58.5
61.8
60.1

a
V. cholerae 1854 and V. alginolyticus VIO5 were cultured until late log phasein the indicated medium.
b
Cells harvested at late log phase were resuspended in TMN medium (pH 7.5)
containing various concentrations of salts, and motility was measured and recorded on videotape. To suppress the directional change of swimming, 10 mM
serine was added to the medium as an attractant. The motile fraction wasdetermined by counting the number of motile cells among the total cells in one
video image and averaging the findings in at least three images in one condition.
c
The average swimming speeds were obtained by measuring at least 20 swimming tracks of cells generated from the integrated video images.

VOL. 181, 1999

NOTES

1929

FIG. 2. Na1-dependent and phenamil-sensitive motility of V. cholerae 1854. V. cholerae 1854 and V. alginolyticus VIO5 cells were cultured in LB or VPG medium,
respectively. At late log phase, cells were harvested and resuspended in TMN medium containing various concentrations of NaCl (A) or in TMN medium (pH 7.5)
containing 50 mM NaCl, 50 mM KCl, and various concentrations of phenamil (B). In the experiment shown in panel A, the total concentration of salts in TMN medium
was adjusted to 100 mM by the addition of KCl. The swimming speeds of the cells were obtained as described in Table 1, footnote c.

ride, which is the analog of phenamil and which specificallyinhibits the Na1 motor in a competitive manner with Na1 in
the medium (31), also inhibited the motilities of both strains(data not shown). These results suggest that the polar flagellar
motor of V. cholerae 1854 is driven by the Na1 motive force.
It is known that V. alginolyticus has an Na1-dependent
NADH-quinone reductase (NQR) that functions as a respiration-coupled Na1 pump only in an alkaline pH range (34). Ata neutral pH, the Na1 motive force is secondarily generated bya Na1/H1 antiporter from the H1 motive force. In this case,the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) collapses the H1 motive force andhence the Na1 motive force. At an alkaline pH, CCCP collapses the H1 motive force but not the Na1 motive force,
which is generated and maintained by the Na1 primary pump.
The Na1 pump can be specifically inhibited by 2-heptyl-4hydroxyquinoline-N-oxide (HQNO). We examined the effectsof CCCP and HQNO on motility in neutral or alkaline conditions in order to confirm the Na1-dependent motility of
V. cholerae. As shown in Table 2, the swimming speeds of both1854 and VIO5 cells in the medium containing 20 mM CCCP
at pH 7.5 were severely reduced. At pH 8.5, both 1854 andVIO5 cells were still motile in the presence of 20 mM CCCP.
However, the motility of both strains was completely inhibitedby the addition of 20 mM HQNO to this medium. These results
TABLE 2. Effect of CCCP and HQNO on the swimming speedof V. cholerae 1854 and V. alginolyticus VIO5
Swimming speed (mm/s)apH of
medium

CCCP
(mM)

HQNO
(mM)

Strain 1854

Strain VIO5

7.5

0
20

0
0

90
6

53
0

8.5

0
20
0
20

0
0
20
20

101
35
89
0

55
21
55
0

a
Cells were grown to late log phase in LB (strain 1854) or VPG (strain VIO5)
medium. Motility was measured in TMN medium (pH 7.5) containing 50 mM
NaCl and 50 mM KCl. The average swimming speeds were obtained by measuring at least 20 cells.

showed that V. cholerae as well as V. alginolyticus cells are
motile only in the presence of the Na1 motive force, and wehave demonstrated that the polar flagellar motor of V. cholerae
is powered by an Na1 motive force. These results also suggestthat the respiratory chain of V. cholerae contains NQR, similar
to that of V. alginolyticus. This enzyme in V. alginolyticus wasrecently reported to be composed of six subunits, NQR1 to
NQR6 (17, 25). Partial sequences of the genes homologous tonqr1 and nqr2 were also found in the genome database of
V. cholerae, consistent with our physiological results. Actually,
nqrB (5nqr2) was recently identified in V. cholerae (16a). Considering the evolutionarily close relation between these two
Vibrio spp. revealed by the analysis of 5S rRNA sequences (4),they should have many other similar physiological characters.
Some distinct aspects between them were found in the present
study. For example, V. cholerae cells swim faster than V. alginolyticus cells under certain conditions. The genome sequenceof V. cholerae revealed that it has homologous genes coding forthe essential components of the Na1 motor, i.e., pomA, pomB,
motX, and motY, which are all of the Na1 motor genes identified until now. These two Vibrio spp. have very similar polar
flagellar basal body structures (4). Thus, subtle differences inthese motor components between these species might cause
differences in the efficiency of energy conversion in the Na1
motor. We hope that the comparative studies of Na1 motors inthese two species will reveal some cues for clarifying themechanism of mechanochemical coupling in Na1-driven
flagellar motors.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan(to I.K. and M.H.) and from the Japan Society for the Promotion of
Science (to S.K.).
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