JOURNAL OF BACTERIOLOGY, Aug. 1997, p. 5104–5110
0021-9193/97/$04.0010
Copyright © 1997, American Society for Microbiology

Vol. 179, No. 16

Putative Channel Components for the Fast-Rotating SodiumDriven Flagellar Motor of a Marine Bacterium
YUKAKO ASAI, SEIJI KOJIMA, HARUKI KATO, NORIKO NISHIOKA, IKURO KAWAGISHI,
AND MICHIO HOMMA*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-01, Japan
Received 8 April 1997/Accepted 13 June 1997

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) byusing a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor havebeen identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to thegenes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX wasproposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA andpomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively,
especially in the predicted transmembrane regions. These results suggest that PomA and PomB may besodium-conducting channel components of the sodium-driven motor and that the motor part consists of the
products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by
the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components
of a force-generating unit, were successively integrated into the defective motor complexes. These findings
imply that Na1-driven flagellar motors may have similar structure and function as proton-driven motors, butwith some interesting differences as well, and it is possible to compare and study the coupling mechanisms of
the sodium and proton ion flux for the force generation.
it has been shown that the rotation of the polar flagella is very
fast in Vibrio alginolyticus; speeds of ca. 600 revolutions per s(rps) are observed in 50 mM NaCl at room temperature, andsurprisingly, the speeds increase up to ca. 1,700 rps in 300 mM
NaCl at 35°C (28, 29, 37).
The rotation of the sodium-driven motor seems to be very
stable at high speed (37). When the rotation rate is reduced byamiloride, the motor still rotates smoothly though the rotation
rate tends to fluctuate. On the other hand, polar flagellashowed remarkably larger speed fluctuations upon addition ofphenamil, an amiloride analog (38). The speed fluctuations
induced by phenamil were explained by a low rate of dissociation of the inhibitor from the force-generating unit. Mutants
resistant to phenamil were isolated, and the Ki value for phenamil in the resistant strain was estimated to be five times larger
than that in the wild-type strain. By analysis of motor speed
fluctuations, it was suggested that a phenamil-specific bindingsite of the motor was mutated in the resistant strain (24). The
resistance mutation may map to one of the genes coding forthe motor proteins.
Recently, the motX and motY genes, which seem to encode
components of the force-generating units of the Na1 motor,
were isolated from Vibrio parahaemolyticus (30, 31). However,the predicted products have no homology to MotA and MotB,
which are components of the force-generating units of H1
motors and are thought to form a H1 channel (6, 8, 44). On theother hand, the C-terminal domains of MotY and MotB do
have sequence similarities to peptidoglycan-interacting proteins. MotX is inferred to be a component of the Na1 channelof the motor because overproduction of MotX was lethal toEscherichia coli in proportion to the external Na1 concentration, and this effect was suppressed by the addition of amiloride (31). Genes identical to the motX and motY Na1 motor
genes were also detected in V. alginolyticus (reference 40 and
unpublished data). So, it had been thought that the forcegenerating units of H1 and Na1 motors were very different.
MotX and MotY of the Na1 motor, each of which has a

The bacterial flagellar motor rotates the helical flagellar
filament that extends from the cell body to propel the cell. Theenergy source for motor rotation is the electrochemical gradient of a specific ion (either a proton or a sodium ion) across thecytoplasmic membrane (6). Thus, the flagellar motor is a molecular machine that couples ion flux to force generation. The
proton-driven motors of bacterial flagella have been extensively studied in terms of structure, genetics, biochemistry, andbiophysics. Five proteins, MotA, MotB, FliG, FliM, and FliN,
appear to be involved in force generation of proton-driven
motors. It has been proposed that FliG, FliM, and FliN makea switch complex and function as the rotor of the flagellar
motor (27, 51). They form the C ring, which was newly foundon the cytoplasmic face of the MS ring (13, 53). MotA and
MotB, which are the membrane proteins and have four transmembrane segments and one transmembrane segment, respectively, are associated in a complex and function as a protonconducting channel (6, 8, 47). MotB is believed to act as ananchor to the cell wall (11, 12) and to work with MotA as thestator of the flagellar motor. It has been shown that MotA
interacts, but only weakly, with FliG and FliM of the switch
complex (49).
Sodium-driven motors have advantages for experimental
analysis, since sodium motive force can be manipulated more
readily than proton motive force (18, 19, 52). Moreover, amiloride and its analogs, which specifically and strongly inhibit thepolar-flagellum motors, are available and are thought to interact with a sodium channel of the flagellar motor (1, 3, 48).
Some marine Vibrio species have two types of flagella, polar(Pof) and lateral (Laf), which are rotated by sodium- andproton-driven motors, respectively, in the same cell (2, 21).
The polar flagella work better for swimming in a liquid or a
low-viscosity environment (4). By laser dark-field microscopy,* Corresponding author. Phone: 52-789-2992. Fax: 52-789-3001. Email: g44416a@nucc.cc.nagoya-u.ac.jp.
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TABLE 1. Bacterial strains and plasmids

V. alginolyticus strains
138-2
VIK4
VIO5
VIO586
V. parahaemolyticus
LM4170LM4171
E. coli strains
DH5a
XL1-BlueS17-1
Plasmids
pSU21
pSU41
pIO3
pYA101
pYA201
pYA202
pYA2021
pYA2022
pYA2023
pYA203
pYA2031
pYA2032
pHK2
pHK3
pSK1
pSK1-D28
pMMB206

Referenceor source

Genotype or descriptiona

Strain or plasmid

Wild type138-2 rif (Rifr Pof1 Laf1)
VIK4 laf (Pof1 Laf2)
VIO5 pomA (Laf2 Pom2)

50
40
40
This work

lafX313 motX118 (Laf2 Pom2)
lafX313 motY141 (Laf2 Pom2)

3130

F2 l2 recA1 hsdR17 endA1 supE44 thi-1 relA1 gyrA96 D(argF-lacZYA)U169 f80dlacZDM15
recA1 hsdR17 endA1 supE44 D(lac-proAB) {F9::Tn10 proAB lacIqZDM15}
recA hsdR thi pro ara RP-4 2-Tc::Mu-Km::Tn7 (Tpr Smr)

16
Stratagene46

cat (Cmr) Plac lacZakan (Kmr) Plac lacZa (MCS same as that in pSU21)
pSU21 0.4-kb and 6.0-kb HindIII fragment (motY1)
pSU21 5.0-kb and 3.6-kb HindIII fragment (pomA)
pSU21 5.2-kb and 3.3-kb EcoRI fragment (pomA1)
pSU21 3.3-kb EcoRI fragment (pomA1)
pSU21 2.7-kb EcoRI-HindIII fragment (pomA)
pSU21 1.9-kb EcoRI-XbaI fragment (pomA)
pSU21 1.6-kb EcoRI-SacI fragment (pomA)
pSU21 3.3-kb EcoRI fragment (pomA1) (containing the same fragment as pYA202, but inthe opposite orientation)
pSU21 2-kb SacI-EcoRI fragment (pomA1)
pSU21 1.45-kb XbaI-EcoRI fragment (pomA1)
pSU21 3.3-kb SacI fragment (pomAB1)
pSU21 1.2-kb HindIII-SacI fragment pomA pomB1)
pSU41 1.45-kb XbaI-EcoRI fragment (pomA1)
pSU41 with 329 bp deleted from XbaI site of pSK1; lack of the native promotercat (Cmr) IncQ lacIqDbla Ptac-lac lacZa rrnB

5540This work
This work
This work
This work
This work
This work
This work
This work
This study
This work
This work
This work
This work
35aCmr, chloramphenicol resistant; Kmr, kanamycin resistant; MCS, multicloning site; Plac, lac promoter; Ptac-lac, tandemly located tac and lac promoters; Rifr, rifampin
resistant; Smr, streptomycin resistant; Tpr, trimethoprim resistant.

putative single transmembrane region, were thought to form achannel component and/or a stator component, functionallyhomologous to the H1 motor components MotA and MotB.
However, in this study, we isolated novel pom (polar flagellum
motility) genes whose predicted products are structurally homologous to the H1 motor components, MotA and MotB.
This shows that the channel components of the Na1 motor are
similar to those of the H1 motor.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The strains and plasmids
used are shown in Table 1. V. alginolyticus and V. parahaemolyticus cells were
cultured at 30°C in VC medium (0.5% polypeptone, 0.5% yeast extract, 0.4%
K2HPO4, 3% NaCl, 0.2% glucose) or VPG medium (1% polypeptone, 0.4%
K2HPO4, 3% NaCl, 0.5% glycerol). When necessary, chloramphenicol and kanamycin were added at the final concentrations of 2.5 and 100 mg/ml, respectively.
DNA manipulations and sequencing. Routine DNA manipulations were carried out according to the standard procedures (41). Restriction endonucleases
and other enzymes for DNA manipulations were purchased from Takara Shuzo(Kyoto, Japan) and New England Biolabs (Beverly, Mass.). The nucleotide
sequence was determined by the dideoxy chain termination method using theSequenase version 2.0 DNA sequencing kit (United States Biochemical Corp.,
Cleveland, Ohio). [a-32P]dCTP (3,000 mCi/mmol; Amersham Japan, Tokyo,
Japan) was used for radioactive labeling.
Electroporation. Transformation of Vibrio cells by electroporation was carriedout as described previously (22). The cells were subjected to osmotic shock andwere washed thoroughly with 20 mM MgSO4. Electroporation was carried out
with the Gene Pulser electroporation apparatus (Japan Bio-Rad Laboratories,
Tokyo, Japan) at an electric field strength between 5.0 and 7.5 kV/cm.
Isolation of pom mutants. From the lateral flagellum-defective (Pof1 Laf2)mutants VIO5 (40), YM4 (17), and NMB201 or NMB205 (24), swarm-deficientmutants were isolated and pom mutants were selected from the swarm-deficientmutants as described previously (40).
pom gene cloning. Plasmid pSU21 (5) and chromosomal DNA from V. alginolyticus VIO5 were digested with EcoRI or HindIII and ligated. These DNA
libraries were transferred into pom mutant (VIO586) cells by electroporationand selected as described previously (40). Cells were incubated at 30°C on 0.3%
agar–VPG plates containing chloramphenicol.
Conjugation. Plasmid transfer by conjugation was carried out according to themethod of Okunishi et al. (40). Aliquots (20 ml each) of fresh overnight culturesof E. coli S17-1 cells carrying plasmid pMMB206 (donor) and Vibrio cells weremixed on a 1.25% agar–VC plate and incubated at 30°C overnight. Cells were
scraped from the plate and suspended in 100 ml of VC medium. To selecttransconjugants, the suspension was plated on a 1.25% agar–VC plate supplemented with 2.5 mg of chloramphenicol per ml.
Measurement of swimming speed. Cell culture was diluted about 100-fold into
Tris motility buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM glucose, 50
mM NaCl, 250 mM KCl), and motility of the cells was observed under a darkfield microscope and recorded on videotape. Swimming speed was determined as
described previously (17).
Nucleotide sequence accession number. The nucleotide sequences for the
pomA and pomB genes from V. alginolyticus have been deposited in DDBJ underaccession no. AB004068.

RESULTS
Cloning of a pom gene required for polar flagellar rotation.
We isolated pom (polar flagellar motility) mutants which haveparalyzed polar flagella (40). Using a pom mutant (VIO586) asa recipient for shotgun cloning, we cloned a pom gene in
pYA101 and pYA201 by using HindIII and EcoRI, respectively. When these plasmids were introduced into VIO586
cells, the cells swarmed in the 0.3% agar–VPG plate (Fig. 1A

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FIG. 2. The promoter region of pomA (A), the noncoding region between
pomA and pomB (B), and the terminator region of pomB (C) in the 2,196-bp
XbaI-SacI fragment. The sequences enclosed by boxes indicate the predictedpromoter region, Shine-Dalgarno (SD) sequences, and start codons. The complementary sequences are shown by arrows.

FIG. 1. Isolation of novel pom genes. (A and B) Swarming abilities of
VIO586 cells carrying various plasmids. Fresh colonies were inoculated in 0.3%
agar–VPG plates containing chloramphenicol and incubated at 30°C for 5 (A)and 9 (B) h. (C) Restriction map of plasmids. The swarm recovery of VIO586 bytransformation of the plasmids in 0.3% agar–VPG plates is shown on the rightside of the map. The chromosomal DNA fragments are indicated by solid lines.
CAT indicates the chloramphenicol acetyltransferase gene from the vector
pSU21 (5). The open arrows show the coding regions of PomA and PomB. A
plasmid (pYA2021) containing a 2.7-kb HindIII-EcoRI fragment made small
swarms after a long incubation (2p). Probably the functional pom gene wasformed by homologous recombination between the mutant pom gene on thechromosome and a part of the pom gene on the plasmid. Restriction sites areshown as follows: E, EcoRI; H, HindIII; Sc, SacI; Xb, XbaI.

and 1B). Cells with pYA201 could swarm in 5 h; however, cellswith pYA101 could make only small swarms in 9 h. pYA101
seemed not to contain the complete gene. Therefore, we used
pYA201 for further analysis. The fragment in pYA201 did notcomplement motX and motY mutants of V. parahaemolyticus ora motY mutant of V. alginolyticus (data not shown), and theplasmid pIO3 carrying the motY gene of V. alginolyticus (40)
did not complement the pom mutant. So, this gene must be anovel one related to the sodium-driven motor. A 3.2-kb EcoRI
fragment, which still complemented the pom mutant, was subcloned, and the resulting plasmid was named pYA202. Next,
we made restriction maps of pYA202 and pYA203 (which has
the same fragment as pYA202 but in the opposite orientation)and constructed various deletion derivatives. Complementation assays using deletion derivative plasmids revealed that thepom gene was located in the 1.45-kb XbaI-EcoRI fragment
containing the HindIII site (Fig. 1C). The plasmid containing
the fragment is pYA2032. After sequencing the fragment (seebelow), we found that it also contained part of a second pom
gene. To clone the whole gene, shotgun cloning and screening
were done with SacI, which cut out a ca. 3.0-kb chromosomal

fragment which hybridized to the 1.45-kb XbaI-EcoRI fragment (data not shown). Plasmid pHK2, containing the ca.
3.0-kb SacI fragment, complemented the mutation of VIO586.
Nucleotide sequence of the pom genes. We determined thenucleotide sequence of the 2,196-bp XbaI-SacI fragment andidentified two open reading frames, which we named pomAand pomB; they encode proteins containing 253 and 315 aminoacids, respectively. A consensus of ribosome binding sites wasfound at 12 and 11 bp upstream from the start codons of pomAand pomB, respectively (Fig. 2A and B). There are only 15 bp
between the two genes (Fig. 2B). Thirty-five base pairs downstream of the stop codon of pomB, there is a consensus rindependent transcription termination site (Fig. 2C). A potential promoter sequence of TAAA(N15)GTCGATAC, which isvery similar to the s28 promoter consensus of TAAA(N15)GC
CGATAA of Salmonella typhimurium and E. coli (32, 40), wasfound 67 bp upstream of the start codon (Fig. 2A). Thus, thetwo genes seem to constitute a single transcription unit. A
plasmid, pHK3 (Fig. 1C), containing only the pomB gene complemented the pom strains NMB104, NMB152, and NMB161.
This indicates that the three strains are pomB mutants and that
PomB is actually required for polar flagellar motility.
Comparison of PomA and PomB to MotA and MotB of H1
motor. The PomA and PomB proteins were found to be homologous to the proton-driven motor proteins, MotA and
MotB, respectively, of Rhodobacter sphaeroides, Bacillus subtilis, and E. coli (Fig. 3). The hydropathy profiles of PomA and
PomB are very similar to those of MotA and MotB of E. coli
(data not shown), which have been shown to have four and one
membrane-spanning helices, respectively. As with motA andmotB in E. coli or B. subtilis, pomA and pomB are in the same
operon and have a s28-type promoter sequence upstream. In
E. coli, cheA and cheW reside downstream of the mot genes inthe same operon. In V. alginolyticus, as in R. sphaeroides, theoperon contains only the two motor genes. Upstream of pomA,
we found genes homologous to E. coli xseB and ispA, which

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FIG. 3. Amino acid alignments of PomA (A) and PomB (B) with homologous proteins of various species. Dotted bars indicate membrane-spanning regions of E.
coli MotA and MotB. Abbreviations: VaPomA, V. alginolyticus PomA; RsMotA, R. sphaeroides MotA; BsProA, B. subtilis hypothetical protein A; BsMotA, B. subtilis
MotA; VpLafT, V. parahaemolyticus LafT; EcMotA, E. coli MotA; VaPomB, V. alginolyticus PomB; RsMotB, R. sphaeroides MotB; BsProB, B. subtilis hypotheticalprotein B; BsMotB, B. subtilis MotB; VpLafU, V. parahaemolyticus LafU; EcMotB, E. coli MotB. White letters in black boxes show identical residues.

encode the exoDNase small subunit and the farnesyl diphosphate synthase (14), respectively. The other sodium motor
genes, motX and motY, are localized elsewhere on the chromosome (30, 31, 40).
Figure 3A shows the alignments of PomA with various
MotA sequences. The four membrane-spanning regions, especially the third and fourth, are more similar than the otherregions and, in MotA, are thought to form the proton channel.
It has been shown that nonmotile or severe mot mutations are
localized in the four membrane-spanning regions of MotA of
E. coli (9). PomA is more similar to MotA of B. subtilis than to
MotA of E. coli, even though the latter is phylogeneticallycloser to V. alginolyticus. PomA and R. sphaeroides MotA (43)are extremely similar and contain the same number of residues. It would be interesting to see whether these motor components are functionally interchangeable. For example, thechannel components of proton-type and sodium-type F0F1
ATPases are similar, and it has been shown that the substitution of only two amino acids changes the ion specificity of
F0F1 ATPase (20).
PomB consists of 315 amino acids, and the N-terminal andthe C-terminal regions are similar to those of MotB in otherspecies (Fig. 3B). In those C-terminal regions of V. alginolyticus
and E. coli, the peptidoglycan-binding motif is highly conserved but R. sphaeroides does not have the motif (42). The
membrane-spanning region at the N terminus of MotB isthought to complex with the transmembrane regions of MotA
and form a part of the proton channel (45). The N-terminal
regions of PomB and R. sphaeroides MotB (42) are very similar. These results suggest that PomB also has only one membrane-spanning region and interacts with the cell wall peptidoglycan via its C-terminal domain.

Controlled expression of pomA in V. alginolyticus. The XbaIEcoRI fragment of pYA2032 was subcloned into the pSU41
vector (5). From the resultant plasmid, pSK1, various lengthsof deletion were carried out with exonuclease III and mung

FIG. 4. Swarming profile by controlled expression of pomA. After pSK1-D28,
pSK1, and pSU41 were introduced into VIO586/pMMB206, fresh overnightcolonies were spotted on 0.25% agar–VC plates containing 2.5 mg of chloramphenicol per ml, 100 mg of kanamycin per ml, and various concentrations of
IPTG. The plates were incubated for 5 h at 30°C.

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FIG. 6. Motor genes of membrane components for the proton-driven- andsodium-driven-type flagella and the predicted membrane topology of the four
pom gene products.

FIG. 5. Motility recovery by controlled expression of pomA. (A) For motility
assay, VIO586/pMMB206/pSK1-D28 was grown in VPG medium containing 50
mM NaCl and 250 mM KCl at 30°C. At late logarithmic phase, 100 mM IPTG
was added to the medium (indicated by arrows). At various times, swimming
speeds of motile cells were measured. The average swimming speed was obtainedby measuring at least 20 cells. The motile fraction was determined by counting
the number of motile cells among the total cells in one video image and averaging at least three images in one condition. (B) The distributions of swimming
speeds in panel A at 4, 6, 10, and 20 min are shown as the fractions of the total
counted cells.

bean nuclease, and pomA was placed under the control of a lac
promoter-repressor system that had been established previously in V. alginolyticus (40) (Fig. 4). The putative s28 promoter sequence was deleted in plasmids pSK1-D28 and pSK1D32 (with deletion endpoints at 329 and 346 bp, respectively, inFig. 2A). The motility of VIO586/pMMB206 cells carrying
either of the plasmids was clearly dependent on the concentration of IPTG (isopropyl-b-D-thiogalactopyranoside). Figure
5A shows the restoration of the swimming speed and motile
fraction of VIO586/pMMB206/pSK1-D28 cells after the induction of pomA expression by IPTG. At 4 min, cells began toswim slowly, and the swimming speed gradually increased,reaching wild-type level at 20 min. The motile fraction showedalmost the same profile as the swimming speed. After induction, the peak of the distribution progressively shifted from low
swimming speed to high (Fig. 5B). The number of polar flagella is usually only one in a Vibrio cell, and the swimming
speed of the cell reflects the motor property. These results
suggest that newly synthesized PomA proteins were successively integrated into the defective motor complexes as shownin the H1-driven motor of E. coli (7).
DISCUSSION
Until now, two sodium-driven motor genes, motX and motY,
have been identified in V. parahaemolyticus and V. alginolyticus.
Their products are both predicted to have one transmembraneregion. It is believed that MotX is a sodium channel component and that MotY connects the motor with peptidoglycan,
because its C-terminal region is significantly similar to that of
OmpA in E. coli. In this study, we cloned two novel sodiumdriven motor genes, pomA and pomB, which are homologous
to the motA and motB genes of proton-driven motors, respectively. So, the sodium-driven motor consists of the products offour genes, pomA, pomB, motX, and motY (Fig. 6). The genes,
lafT and lafU, of the lateral flagellar motor which is proton
driven have been isolated, and the predicted products areshown to be homologous to the motA and motB products of E.
coli, respectively (34).
By inducing the E. coli motA and motB genes in tethered
motA and motB mutant cells, respectively, restoration of
torque took place in a series of equally spaced steps (7, 10). On
the other hand, the rotation rate decreased stepwise in thetethered alkalophilic Bacillus cells by a photoactivated amiloride analog which covalently binds to the Na1-interaction siteof a channel (36). These results suggest that the motor has
multiple force-generating units and that each force-generating
unit functions independently. Moreover, the torque generated
by a motor is the sum of the torque generated by each unit. It
has been estimated that the H1-driven motor of E. coli and the
Na1-driven motor of alkalophilic Bacillus have 8 and 5 to 9
force-generating units, respectively. In our induction systemfor pomA, the swimming speed and motile fraction of thepomA mutant cells were restored to wild-type levels in 20 min,suggesting that newly synthesized PomA proteins were successively integrated into the defective motor complexes. We haveshown that the swimming ability of the motY mutant increased
with the motY induction (40). The independent force-generating unit probably functions as described above in the Na1driven motor of V. alginolyticus. We plan to analyze the rotation speed at low numbers of the force-generating units bylaser dark-field microscopy (38) and to investigate the stabilityor the stepping of the speed.
From the homology to the proton motor components andtheir properties (15, 39, 44, 45, 54), we expect that the fourtransmembrane regions of PomA and the one transmembraneregion of PomB also form a complex and function as a sodium
channel. An Asp residue in the transmembrane region of E.
coli MotB has been proposed to act as the donor in H1 transfer

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GENES OF SODIUM-DRIVEN FLAGELLAR MOTOR

to a recipient near the cytoplasmic side of the protein (45). The
Asp is conserved in the PomB transmembrane region and may
be the donor in Na1 transfer to a neighboring protein. It is also
possible that a sodium channel is formed from the four pom
products, PomA, PomB, MotX, and MotY, because it has beensuggested that MotX is the channel component of the motorand interacts with MotY (30). It is not ruled out that MotX andMotY might form a different sodium channel.
In the proton-driven motor, MotB is thought to function asa stator by interaction with peptidoglycan. PomB presumably
functions in a similar way. In addition to PomB, the sodiumdriven-type motor apparently needs another component,
MotY, which has a peptidoglycan binding motif but no further
similarity to MotB. These two possible stators, PomB and
MotY, are required for polar motility of V. alginolyticus.
Why does the sodium-driven motor need two stators? And
why does it need four motor components, whereas the protondriven motor needs only two? Vibrio has two types of flagella,polar and lateral. The polar flagellum is thought to function as
a viscosity sensor (33) that regulates expression of lateral
flagellar genes. Recently, we have shown that Vibrio cells sensea decrease in the rotation rate of (or the sodium influx
through) the polar flagellar motor (23). Therefore, we speculate that MotX and MotY might sense the rotation rate or thesodium flux. Alternatively, they may be needed for the very fast
rotation of the sodium-driven motor. It has been reported thatits speed is ca. 600 rps in 50 mM NaCl at room temperatureand increases up to ca. 1,700 rps in 300 mM NaCl at 35°C (28,29, 37), whereas the speed of the proton-driven motor is 200 to
300 rps at most in S. typhimurium or E. coli (25, 26).

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ACKNOWLEDGMENTS
We thank Linda McCarter for providing us with the strains and I.
Okunishi for assistance with the pom mutant isolation. We especiallythank Robert M. Macnab for critical reading of the manuscript.
This work was supported in part by grants-in-aid to I.K. and M.H.
from the Ministry of Education Science and Culture of Japan and by
the research and development program of NEDO of Japan.
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