JOURNAL OF BACTERIOLOGY, Apr. 1996, p. 2409–2415
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology

Vol. 178, No. 8

Cloning and Characterization of motY, a Gene Coding for a
Component of the Sodium-Driven Flagellar
Motor in Vibrio alginolyticus
ISAO OKUNISHI, IKURO KAWAGISHI,* AND MICHIO HOMMA
Department of Molecular Biology, Faculty of Science, Nagoya University,
Chikusa-ku, Nagoya 464-01, Japan
Received 31 July 1995/Accepted 8 February 1996

The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation
for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motY) encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus. Nucleotide sequence analysis revealed
that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is very
similar (94.5% identity) to the recently described MotY of V. parahaemolyticus. Their C-terminal domains weresimilar to the C-terminal domains of many peptidoglycan-interacting proteins, e.g., Escherichia coli MotB and
OmpA, suggesting that MotY may interact with peptidoglycan for anchoring the motor. By using the lacpromoter-repressor system, motY expression was controlled in V. alginolyticus cells. Swimming ability increased
with increasing concentrations of the inducer isopropyl-b-D-thiogalactopyranoside, and the swimming fraction
increased after induction. These results are consistent with the notion that MotY is a component of theforce-generating unit. V. alginolyticus motY complemented the motY mutation of V. parahaemolyticus. However,motY appeared to lack a region corresponding to the proposed motY promoter of V. parahaemolyticus. Instead,sequences similar to the s54 consensus were found in the upstream regions of both species. We propose thatthey are transcribed from the s54-specific promoters.
motive force, and the lateral flagellar motor is driven by proton
motive force (4, 23). A single cell possessing two types ofmotors with two different coupling ions provides an excellent
experimental system. Moreover, the polar flagellar motor has
the following interesting properties. First, it is one of the fastest biological motors (up to 1,700 rps at 378C) known to date(28). Second, it functions not only as a locomotive organelle
but also as a viscosity sensor that regulates expression of lateral
flagellar genes which are induced only under viscous conditions(7, 30).
Using transposon mutagenesis and a cosmid library, McCarter (32, 33) has recently identified two V. parahaemolyticus
genes (motX and motY) encoding components of the polar
flagellar motor. She has proposed that their gene products,
MotX and MotY, each of which has a single membrane-spanning segment, are a channel component and a stator component, respectively. Surprisingly, they are not related to MotA
and MotB.
We have already established a reliable procedure for electroporation of Vibrio alginolyticus (24), which is closely related
to but is less pathogenic than V. parahaemolyticus and is wellcharacterized in terms of respiration-coupled primary sodium
pumps (47, 51). In this study, we cloned and characterized a V.
alginolyticus gene coding for a component of the sodium-driven
motor, using a mutant defective in polar flagellar motor rotation as a recipient for electroporation.

The flagellar motor rotates the flagellar filament that extends from the cell body to propel the cell (for reviews, seereferences 8, 21, 29, and 42). The energy source for motor
rotation is the electrochemical gradient of a specific ion (proton or sodium ion) across the cytoplasmic membrane. Thus,
the flagellar motor is a molecular machine that couples ion flux
to force generation. The genes (motA and motB) that code forthe transmembrane components of the proton-driven motorsof several species, including Escherichia coli, Bacillus subtilis,
and Vibrio parahaemolyticus, have been cloned and sequenced(16, 34, 36, 48). It has been suggested that MotA constitutes a
proton-conducting channel (10) and MotB interacts with peptidoglycan (also known as the cell wall or the murine layer) toact as a stator (15). Three other proteins (FliG, FliM, andFliN), which are located peripherally on the cytoplasmic sideof the basal body, are involved in motor rotation and switchingof the rotational sense (for details, see the reviews cited
above). However, the molecular mechanism of energy transduction has not been elucidated.
For investigation of the energy-coupling mechanism, thesodium-driven motor is promising (19), since sodium concentration can be manipulated more readily than proton concentration, and amiloride and its analogs inhibit motor rotation ina fairly specific manner (3, 5, 41, 50). However, this type of
motor has been poorly characterized in terms of genetics andbiochemistry. We therefore wanted to clone and characterize
genes coding for components of the sodium-driven motor.
Certain marine Vibrio species are suitable organisms for thispurpose. Vibrio species have two types of flagella: a single polar
flagellum suited for swimming in liquids and numerous lateral
flagella suited for swarming over animate or inanimate surfaces (1, 32, 44). The polar flagellar motor is driven by sodium

MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The strains and plasmids
used in this study are listed in Table 1. It should be noted that pSU38 and pSU41
have different multicloning sites. V. alginolyticus and V. parahaemolyticus cells
were cultured at 308C 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). For compact colony formation andswarm assays, a VC–1.5% agar plate and a VP (0.5% polypeptone, 0.4%
K2HPO4, 3% NaCl)–0.3% agar plate, respectively, were used. When necessary,chloramphenicol and kanamycin were added at final concentrations of 1.25 and* Corresponding author. Phone: 81-52-789-2993. Fax: 81-52-7893001. Electronic mail address: i45406a@nucc.cc.nagoya-u.ac.jp.
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TABLE 1. Bacterial strains and plasmids used
Strain orplasmidV. alginolyticus
strains
138-2
VIK4
VIO5
VIO542
V. parahaemolyticus
strains
BB22
LM4170LM4171
E. coli strains
DH5 a

XL1-BlueS17-1
Plasmids
pSU21
pSU38
pSU41
pIO2
pIO3
pIO5
pIO6
pIO7
pMMB206

Genotype or descriptiona

Referenceor source

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

52
This work
This work
This work

Wild typelafX313::lux motX118::mini-Mu lac(Tets)
lafX313::lux motY141::mini-Mu lac(Tets)

7
33

recA1 hsdR17 endA1 supE44 thi-1
relA1 gyrA96 D(argF-lacZYA)U169
f80dlacZDM15
recA1 hsdR17 endA1 supE44 D(lacproAB) (F9::Tn10 proAB
lacIqZDM15)
recA hsdR thi pro ara
RP-4-2-Tc::Mu-Km::Tn7 (Tpr Smr)18

cat (Cmr) Plac lacZakan (Kmr) Plac lacZa (MCS different
from that in pSU21)
kan (Kmr) Plac lacZa (MCS same asthat in pSU21)
pSU21 20-kb SalI fragment (motY1)
pSU21 0.4- and 6.0-kb HindIII fragments (motY1)
pSU21 977-bp HindIII-SphI fragment(motY1)
pSU38 1.0-kb HindIII-XbaI fragment(motY1)
pSU41 1.0-kb HindIII-XbaI fragment(motY1)
IncQ lacIq Dbla cat (Cmr) Ptac-lac
lacZa rrnB

32

Stratagene

46

6
6
6
This work
This work
This work
This work
This work
37aCmr, 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; Tets, tetracycline sensitive; Tpr,
trimethoprim resistant.

100 mg/ml, respectively. E. coli cells were cultured at 308C in LB medium (1%tryptone, 0.5% yeast extract, 0.5% NaCl) or 23 YT medium (1.6% tryptone, 1%
yeast extract, 0.5% NaCl).
Isolation of Laf2 and Pom2 mutants. An overnight culture of V. alginolyticus
cells was inoculated into 1.5 ml of VC medium (1:50 dilution) and cultured for
2 h at 308C with vigorous shaking. Cells were harvested by centrifugation (5,000
3 g for 5 min) at room temperature and washed with 1 ml of ice-cold minimal
AV medium [3% NaCl, 1.05% K2HPO4, 0.45% KH2PO4, 0.1% (NH4)2SO4,
0.05% sodium citrate]. Cells were resuspended in 1 ml of minimal AV, and 15 mlof ethyl methanesulfonate was added. The suspension was incubated at 308C for
1 h with gentle shaking. Cells were washed twice with 1 ml of minimal AV
medium and resuspended in 10 ml of VC medium. This suspension was divided
into 10 test tubes and cultured overnight. About 5 ml of each culture was spottedat the center of a VC–1.5% agar or VM–0.3% agar plate, and the center of theswarm was picked for single-colony isolation. Single colonies were inoculated onplates to test for swimming or swarming abilities.
Measurement of motility. A fresh overnight culture was inoculated into VPG
medium (1:30 dilution) supplemented, if necessary, with 1.25 mg of chloramphenicol per ml and cultured for 3 h. Cells were harvested and suspended in
TNM buffer (50 mM Tris-HCl [pH 7.5], 5 mM glucose, 5 mM MgCl2, 50 mM
NaCl, 250 mM KCl) supplemented with 10 mM L-serine, a chemoattractant.
Addition of 10 mM serine suppressed the directional change of swimming for atleast 1 min. When the Na1 concentration in the medium was varied, KCl wasadded to keep the salt concentration constant. Swimming speed and motile
fraction of the cells were measured by the photographic method described
previously (20).

Electroporation. Transformation of Vibrio cells by electroporation was carriedout as described previously (24). In brief, Vibrio cells harvested at late exponential to early stationary phase were subjected to osmotic shock to remove theperiplasmic DNase and were washed thoroughly with 10 mM MgSO4. Plasmids
having the P15A replicon and the chloramphenicol or kanamycin resistance gene
were used for vectors. Electroporation was performed with a Gene Pulser electroporation apparatus (Japan Bio-Rad Laboratories, Tokyo, Japan) at an electricfield strength of between 5.0 and 7.5 kV/cm.
DNA sequencing and PCR. The nucleotide sequence was determined by the
dideoxy-chain termination method, using a Sequenase version 2.0 DNA sequencing kit (United States Biochemical Corp., Cleveland, Ohio). [a-32P]dCTP (3mCi/mmol; Amersham Japan, Tokyo, Japan) was used for radioactive labeling.
Oligonucleotide primers for PCR were synthesized at the Center for Gene
Research at Nagoya University. PCR amplification was carried out with the
TaKaRa Taq kit (Takara Shuzo, Kyoto, Japan) and Thermal Cycler (PerkinElmer Japan, Chiba, Japan) as recommended by the distributors. Reactions wererepeated for 25 cycles of 30 s at 948C, 60 s at 608C, and 60 s at 728C. The PCR
products were analyzed by 1.7% agarose gel electrophoresis.
Conjugation. Aliquots (20 ml each) of fresh overnight cultures of E. coli S17-1
cells carrying plasmid pMMB206 (donor) and VIO54 cells carrying plasmid pIO6
or pIO7 (recipient) were mixed on a VC–1.5% agar plate and incubated at 308C
overnight. Cells were scraped from the plate and suspended in 100 ml of VC
medium. To select transconjugants, the suspension was plated on a VC–1.5%
agar plate supplemented with 1.25 mg of chloramphenicol per ml and 100 mg ofkanamycin per ml. As a control, the cultures of the donor and recipient wereplated separately on VC–1.5% agar plates containing the two antibiotics. Neither
VIO542/pIO6, VIO542/pIO7, nor S17-1/pMMB206 cells grew on the selection
plate.
Nucleotide sequence accession number. The sequence data reported in thispaper have been deposited in the GenBank database.

RESULTS
Cloning of a pom gene required for polar flagellar rotation.
We isolated pom (polar flagellar motility) mutants, which haveparalyzed polar flagella, from strain VIO5, which is defective inlateral flagellar formation (Pof1 Laf2; Pof for polar flagellation and Laf for lateral flagellation). VIO5 cells were mutagenized with ethyl methanesulfonate, and 65 mutants that
could not spread in a VP–0.3% agar plate were isolated. We
observed these mutant cells by high-intensity dark-field microscopy and identified four pom mutants. One of them, strain
VIO542, was used as a recipient for shotgun cloning.
Plasmid pSU21 and chromosomal DNA from V. alginolyticus
138-2 were digested with EcoRI, SalI, or HindIII and ligated.
These DNA libraries were transferred into VIO542 cells byelectroporation, and the cells were plated onto a VC–1.5%
agar plate supplemented with chloramphenicol. Colonies werethen inoculated on a VP–0.3% agar plate containing chloramphenicol. Two colonies that spread in the plates were isolated,
and the plasmid DNA from these clones was recovered. They
contained the same 20-kb SalI fragment. One of them wasnamed pIO2 and used for further analysis. When plasmid pIO2
was introduced into VIO542 cells, the resulting cells spread ina VP–0.3% agar plate as fast as wild-type cells. Therefore, we
concluded that the SalI fragment contains the pom gene that is
defective in VIO542.
We then subcloned the pom1 DNA fragment. Plasmid pIO2
DNA was digested with HindIII and ligated. The ligation mixture was introduced into VIO542 cells, and 22 clones thatspread in a VP–0.3% agar plate containing chloramphenicol
were isolated. Three of the pom1 clones were found to carryplasmids containing two HindIII (5.6- and 0.4-kb) fragments.
One of the plasmids was named pIO3 (Fig. 1A). It should be
noted that the two HindIII fragments and the HindIII-SalI
fragment may not be contiguous on the Vibrio chromosome.
Various deletion derivatives were constructed from plasmid
pIO3 (Fig. 1A). The complementation assays using the resulting plasmids located the pom gene in the 977-bp HindIII-SphI
fragment (Fig. 1A and B). The plasmid containing the fragment was named pIO5.
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SODIUM-DRIVEN FLAGELLAR MOTOR GENE

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Only one open reading frame of 293 amino acids was found in
the region. This gene was named motY because the nucleotide
sequence was found to be very similar to that of V. parahaemolyticus motY (see below). The hydropathy analysis (26) of
MotY revealed that there is only one weakly hydrophobic
stretch of 21 amino acids very close to the N terminus. Thismay constitute a membrane-spanning region. The C-terminal
region of MotY shows similarities to the C-terminal regions ofa number of membrane proteins, such as E. coli and B. subtilis
MotB, E. coli OmpA, and E. coli Pal (Fig. 3). The C-terminal
domains of these proteins are known or presumed to interact
with the cell wall peptidoglycan (14, 27). Therefore, the Cterminal domain of MotY may also interact with peptidoglycan
to anchor the force-generating components.
While this work was in progress, the nucleotide sequence of
V. parahaemolyticus motY was published (32). The amino acidsequence of MotY was found to be strikingly similar (94.5%
identity) to that of MotY (data not shown). Interestingly, thesequence similarity of the putative transmembrane region between the two proteins is relatively low (71.4% identity). To
test whether V. alginolyticus motY complements the motY mutation of V. parahaemolyticus, plasmid pIO5 or the vector pSU21
was introduced into V. parahaemolyticus LM4171 (motY) andLM4170 (motX). LM4171 cells carrying pIO5 formed small
swarm colonies in VC 0.3% agar, whereas LM4170/pIO5 cells
FIG. 1. Deletion mapping of the pom gene. (A) Restriction map of plasmid
pIO3 and its deletion derivatives. The plasmids carry the fragments indicated bysolid lines. Their abilities to complement the defect of strain VIO542 are indicated on the right. Note that the two HindIII fragments and the HindIII-SalI
fragment may not be contiguous on the Vibrio chromosome. Symbols: A, AccI; E,
EcoRV; Ec, EcoRI; H, HindIII; M, MluI; Nd, NdeI; Nh, NheI; S, SphI; Sa, SalI;
Se, SpeI; X, XhoI; MCS, multicloning site in vector pSU21; 1, swarmed in
VP–0.3% agar plates; 2, did not swarm; 6, did not swarm initially but swarms
appeared after prolonged incubation. (B and C) Swimming abilities of VIO542
cells carrying various plasmids. Overnight cultures were spotted on VP–0.3%
agar plates containing chloramphenicol and incubated at 308C.

swarm in a VP–0.3% agar plate, but tiny swarm colonies appeared after a few hours of incubation (Fig. 1C). The cells
purified from these tiny swarm colonies spread in a VP–0.3%
agar plate as fast as wild-type cells (data not shown). These
results suggest that plasmids pIO303 and pIO304 do not haveintact pom genes but have parts of it and that the functional
pom gene was formed by homologous recombination between
the mutant pom gene on chromosome and the parts of pom
gene on the plasmids.
Effects of Na1 concentration and inhibitors on the swimming speed of VIO542 cells carrying the pom1 plasmid pIO5.
To characterize the polar flagellar motor of strain VIO542
transformed with the pom1 plasmid, we examined the effects
of sodium ion concentrations and specific inhibitors on theswimming speed of VIO542 cells carrying pIO5. VIO542/pIO5
cells did not swim in the absence of external Na1, and theiraverage swimming speed increased as the Na1 concentration
increased (Fig. 2A). There was no significant difference in thesodium dependency of swimming speed between VIO542/
pIO5 and VIO5 (Pom1 Laf2) cells. Effects of benzamil (Fig.
2B) and of amiloride and phenamil (data not shown) were also
examined. There was little difference, if any, in the effects of
the inhibitors between the two strains. These results suggestthat strain VIO542/pIO5 has a polar flagellar motor indistinguishable from that of strain VIO5 and that the pom gene on
the multicopy plasmid pIO5 can function normally in Vibrio
cells.
Nucleotide sequence of the pom gene. We determined thenucleotide sequence of the 977-bp HindIII-SphI fragment.

FIG. 2. Effects of sodium concentration and the inhibitor benzamil on swimming speed of VIO542 cells carrying plasmid pIO5. (A) Effect of sodium concentration. VIO542/pIO5 or VIO5 cells were grown at 308C for 3 h in VPG
medium supplemented with chloramphenicol when necessary. Cells were washedand resuspended in TNM buffer containing various concentrations of NaCl. The
total salt concentration was kept constant by adjusting the KCl concentration.
(B) Effect of benzamil. Cells were suspended in TNM medium containing 50 mM
NaCl and 250 mM KCl. Swimming speeds were measured immediately after the
addition of benzamil.

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FIG. 3. Alignment of the C-terminal regions of MotY, MotY, MotB, OmpA, Pal, and OrfB. Residues identical or similar to those in V. alginolyticus MotY are
indicated by boldface letters and enclosed by boxes. Abbreviations: VAMOTY, V. alginolyticus MotY; VPMOTY, V. parahaemolyticus MotY; EMOTB, E. coli MotB;BMOTB, B. subtilis MotB; BORFB, B. subtilis OrfB (a MotB-related protein but not a motor component); EPAL, E. coli peptidoglycan-associated protein (Pal);
EOMPA, E. coli OmpA.

did not spread at all (data not shown). Microscopic observation
revealed that LM4171/pIO5 cells swam by polar flagella withnotably low tumbling frequency. Although the reason for thissmooth-swimming bias is not clear, these results indicate thatthe motY genes of the two species are functionally exchangeable.
Comparison of the 5* upstream sequences of the motY coding regions of the two Vibrio species. The V. alginolyticus motY
coding region starts with the ATG codon that is preceded by asequence resembling the E. coli ribosome binding site (43)(Fig. 4). The HindIII-SphI fragment contains only 70 bp of 59
upstream sequence of the motY coding region, within which we
could not identify a possible promoter sequence. We therefore
determined 648 bp of additional upstream sequence. The V.
alginolyticus sequence (about 1.6 kb) matched well that of V.
parahaemolyticus. However, the sequence that was proposed tobe the s28-dependent promoter (TAATN15GCGCTTGA) for
V. parahaemolyticus motY (32) was not identified in the motY
upstream region of V. alginolyticus (Fig. 4). The corresponding
region (about 80 bp) seemed to be deleted. Instead, we founda sequence (TGGCGAN5TTGCA) similar to the s54-dependent promoter consensus sequence (TGGCACN5TTGCA)(38) in the 285 to 271 region (when the first residue of theputative initiation codon is numbered 11; Fig. 4). Also in V.
parahaemolyticus, a similar sequence (TGGCGGN5TTGCA)
was found in the bp 284 to 270 region (Fig. 4).
We examined the motY upstream region on the chromosomes of the two Vibrio species by using PCR. The two PCRprimers were designed as shown in Fig. 5. PCR was performed
with pIO3 DNA and genomic DNAs of V. alginolyticus wildtype strain 138-2 and V. parahaemolyticus wild-type strain
BB22. The products from pIO3 and genomic DNA of strain138-2 were the same size (about 820 bp; Fig. 5). This size
agreed well with the size (823 bp) predicted from the nucleotide sequence. On the other hand, the product from BB22
DNA was about 920 bp (Fig. 5). Again, the size agreed with thepredicted size (908 bp). These results indicate that the 80-bp
deletion described above was not a cloning artifact and that V.
alginolyticus motY does not have a sequence similar to that of
the s28-dependent promoter.
PCR products from genomic DNAs of the motY mutant andthe wild-type strains were the same size (data not shown),
indicating that the motY mutation is a point mutation or smalldeletion. The complementation assays described above indi-cated that a part of motY gene on pIO303 but not on pIO302
can recombine with the mutant motY on VIO542 chromosome
to yield functional motY (Fig. 1A and C). Therefore, this mutation site is presumed to be in the 462-bp SpeI-MluI region.
We also attempted to identify MotY protein expressed fromplasmids in V. alginolyticus and in E. coli. However, MotY
could not be detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie brilliant blue staining(data not shown). The amount of MotY may be too small for
detection in this system.
Controlled expression of motY in V. alginolyticus. To control
motY expression in V. alginolyticus, we constructed plasmid
pIO6, in which motY is placed downstream of the lac promoter,by subcloning the 982-bp HindIII-XbaI fragment (containing
motY1 HindIII-SphI region) of plasmid pIO5 into vector
pSU38 carrying the kanamycin resistance gene. As a control,
plasmid pIO7, in which motY is in the opposite orientation, wasconstructed by subcloning the same fragment into vector
pSU41. The relative positions of the unique HindIII and XbaI
cloning sites of pSU41 are opposite those of pSU38. Strain
VIO542 carrying plasmid pIO6 or pIO7 was conjugated with E.
coli S17-1 carrying plasmid pMMB206 (resistant to chloramphenicol) which contained the lacIq gene. Swimming abilitiesof the resulting strains, VIO542/pIO6/pMMB206 and VIO542/
pIO7/pMMB206, were examined in VP–0.3% agar plates supplemented with chloramphenicol, kanamycin, and various concentrations of isopropyl-b-D-thiogalactopyranoside (IPTG)(Fig. 6). In the absence of IPTG, VIO542/pIO6/pMMB206
cells did not spread well from the position where they were
inoculated. As the IPTG concentration increased, the cells
spread better. The swimming ability was maximal in the presence of 10 mM or higher IPTG. However, IPTG did not affectthe swimming ability of VIO542/pIO7/pMMB206 cells. Swimming of this strain was rapid at all IPTG concentrations, owing
to constitutive expression of motY from some promoter onvector pSU41. These results indicate that expression of the
motY gene in Vibrio cells can be artificially controlled by using
a lac promoter-repressor system. The presence or absence of 1
mM IPTG did not affect growth of VIO542/pIO6/pMMB206
cells significantly (data not shown), indicating that expressionof the motY gene does not affect cell growth and that thedifference in swimming ability described above did not result
from the difference in growth rates.

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FIG. 4. Alignment of 59 upstream regions of V. alginolyticus (VA) and V. parahaemolyticus (VP) motY. The first nucleotide of the initiation codon of each motY is
numbered 11. ATG and CAT in boldface indicate the initiation codons. Underlines indicate Shine-Dalgarno sequences and the motY promoter sequence proposedby McCarter (32). In both species, a gene oriented divergently to motY and encoding RNase T is located 59 upstream of motY (32). Sequences similar to the consensus
sequence for s54-dependent promoters are indicated by boxes. Below the alignment, possible promoter sequences are listed together with the consensus sequences.

We next examined the effect of motY expression on freeswimming cells (data not shown). VIO542/pIO6/pMMB206
cells were cultured in the absence of IPTG, and 1 mM IPTG
was added at late exponential phase. The motile fraction wasmonitored before and after IPTG induction. Cells did not swim
at all in the absence of IPTG, suggesting that the recombination described above was negligible in this type of experiment.
After induction, the motile fraction reached a maximum within
20 min, although many cells remain nonmotile for unknown
reasons. At this time, essentially all motile cells swam at maximal speed. In contrast, when IPTG concentration was decreased, at least some fraction of cells was observed to swim at
significantly slower speeds. However, we have not done a quantitative analysis in this regard because the relatively low motile
fraction of the cells even after full induction of motY made itdifficult.
DISCUSSION
Using a previously established electroporation protocol (24),
we carried out shotgun cloning of a gene complementing aparalyzed polar flagellum mutation and identified and characterized the gene motY, which codes for a putative stator component of the sodium-driven flagellar motor.
The nucleotide sequence of V. alginolyticus motY is very
similar to that of V. parahaemolyticus motY. The 59 upstream
regions are also similar, but there is a significant difference. A
s28-dependent promoter sequence is suggested for the latter
species (32), but the sequence is not conserved in the 59 upstream region of motY in V. alginolyticus: the corresponding
region is deleted. We demonstrated by PCR that this deletion
is not a cloning artifact (Fig. 5). Instead, we found a sequence(TGGCGAN5TTGCA) that is very similar to the proposed
consensus sequence (TGGCACN5TTGCA) for s54-dependent
promoter (38) (Fig. 4). A very similar sequence is found in V.
parahaemolyticus motY (Fig. 5). Within the consensus, a GG
motif is invariant and a GC motif is almost completely conserved in the s54-dependent promoter family (35). Both motifs
are perfectly conserved in sequences upstream of motY in both
species. Furthermore, the V. alginolyticus gene (rpoN) encoding s54 has recently been cloned as a gene essential for polar

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FIG. 5. PCR amplification of the 59 upstream regions of V. alginolyticus andV. parahaemolyticus motY from chromosomal DNAs. PCR primers MotY2 (dA
CCTTGTCTTTCGTAGTGCAAAATCG) and MotY3 (dTGTGTGACGAAGGCTGC) were designed as indicated. This primer pair is presumed to amplify
the region containing the proposed promoter sequences and SpeI and MluI
restriction sites. The gene rnt encodes a polypeptide similar to E. coli RNase T(32; see also Fig. 4). Lanes: Marker, fX174 DNA digested with HaeIII; pIO3,
PCR products from plasmid pIO3; 138-2, PCR products from chromosomal
DNA of V. alginolyticus wild-type strain 138-2; BB22, PCR products from chromosomal DNA of V. parahaemolyticus wild-type strain BB22.

flagellar synthesis (23a). From the available data, we suggestthat motY is transcribed from s54-dependent promoters inboth species. In several other polarly flagellated bacteria, suchas Caulobacter crescentus and Pseudomonas aeruginosa, some
flagellar genes are transcribed from s54-dependent promoters(13, 39, 49, 53).
As demonstrated for V. parahaemolyticus MotB by McCarter(32), the amino acid sequence of the carboxyl-terminal regionof V. alginolyticus MotY (about 60 amino acids in length)shows remarkable similarity to sequences of a number of outermembrane proteins known to associate with peptidoglycan,
including a major outer membrane protein OmpA and peptidoglycan-associated lipoprotein Pal. The C-terminal portionsof OmpA and Pal are believed to be located in the periplasm
to interact with peptidoglycan (14, 27). As discussed by De
Mont and Vanderleyden (17), the motB products of E. coli (aJ. BACTERIOL.

gram-negative bacterium) and B. subtilis (a gram-positive bacterium), the former of which has been shown to span thecytoplasmic membrane once in the region close to its N terminus and the C-terminal domain is located in the periplasmic
space (15), have features in common with this C-terminal domain. The positively charged arginine residues in this domain
are postulated to be involved in noncovalent linkage of Pal
with peptidoglycan (27) and are essential for MotB function in
E. coli (11). As in MotB (17), almost all of these residues areconserved in MotY. Therefore, MotY is likely to interact withpeptidoglycan to anchor the sodium-driven flagellar motor.
McCarter (33) has identified motX encoding another component of the V. parahaemolyticus polar flagellar motor thathas a single putative transmembrane segment and suggested
that MotX forms a sodium channel because expression of
motX in E. coli resulted in growth retardation and this inhibitory effect was reversed by amiloride. We identified by PCR a
motX-related gene in V. alginolyticus (unpublished results).
Why are MotX and MotY so different from MotA and
MotB? Difference of the coupling ions may be one reason.
However, for example, sodium-translocating F0F1-ATP synthetase of Propionigenium modestum is related to the protontranslocating enzyme of E. coli (2, 22). Therefore, we may also
have to account for the facts that the polar flagellum of Vibrio
cells is covered by a sheath (1) and that its rotation is very fast
(28, 40). These motor components might be evolved to meetwith a large mechanical force required for rotation. In thisregard, it is interesting that MotX and MotY are similar inmembrane topology and have identical four-residue sequencesat the corresponding positions (33). These residues are perfectly conserved in MotY.
As an initial step for further investigations of these motor
components, we succeeded in regulating motY gene expression
in V. alginolyticus by using a lac promoter-repressor system (toour knowledge, this is the first case in which expression of any
gene is controlled artificially in marine Vibrio cells). Swimming
ability increased with increasing concentrations of the inducer
IPTG, and swimming fraction increased after addition of
IPTG. Using this system, we observed that some cells swamwith suboptimal speeds at lower concentrations of IPTG. Thisfinding is consistent with the prediction that MotY may be aconstituent of the force-generating unit. To clarify this, it will
be necessary to examine in detail the relation between motY
expression and torque generation. It will be interesting to see
whether the rotation rate of a single motor, which can bemonitored by a tethered cell method (45) or by laser dark-fieldmicroscopy (25, 40), increases stepwise when increasing numbers of MotY subunits are incorporated into a motor, as observed for E. coli MotA and MotB (9, 12).
ACKNOWLEDGMENTS
We thank L. McCarter and V. M. Morales for kindly providing
strains and plasmids and S. Kudo for helpful discussion. We especiallythank R. M. Macnab for critically reading the manuscript.
This work was supported in part by grants-in-aid for scientific research to I.K. from the Ishida Foundation and to I.K. and M.H. from
the Ministry of Education, Science and Culture of Japan.

FIG. 6. Controlled expression of motY in V. alginolyticus. VIO542/pIO6/
pMMB206 and VIO542/pIO7/pMMB206 colonies were picked and inoculated
onto VP–0.3% agar plates containing chloramphenicol, kanamycin, and various
concentrations of IPTG. The plates were incubated at 308C for 5 h. Orientationof motY relative to the lacUV5 promoter is indicated below the photographs.

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