JOURNAL OF BACTERIOLOGY, July 2006, p. 5308–5314
0021-9193/06/$08.00!0 doi:10.1128/JB.00187-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 188, No. 14

Roles of the Intramolecular Disulfide Bridge in MotX and MotY,
the Specific Proteins for Sodium-Driven Motors in Vibrio spp.
Jin Yagasaki, Mayuko Okabe,† Rie Kurebayashi, Toshiharu Yakushi,‡ and Michio Homma*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan
Received 4 February 2006/Accepted 24 April 2006

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na!-driven flagella inVibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide[CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented
the formation of an intramolecular disulfide bond, which is presumably important for protein stability.
Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which
did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide
bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge forproper function.

somes (29, 30, 36). In a proton-driven motor of Escherichia
coli, the torque-generating unit is composed of four MotA
subunits and two MotB subunits, which are orthologues of
PomA and PomB, respectively (9, 16). Unlike the sodiumdriven polar-flagellar motor, however, the proton-driven motor of Escherichia coli does not require MotX and MotY for
torque generation.
MotX and MotY were identified in Vibrio spp. as proteinsspecific for the sodium-driven motor (20, 21, 26, 28). Additionally, MotY homologues were recently reported to be part ofthe lateral proton-driven flagellar system of Vibrio spp. and theflagellum of Pseudomonas aeruginosa (12, 32). As with MotB
and PomB, MotY contains a C-terminal peptidoglycan-binding
motif (28). We have shown that the N-terminal segments of
MotX and MotY contain secretion signals and that cleavage ofthese signals leaves the mature proteins outside the inner
membrane (27). We also recently demonstrated direct interaction between MotX and MotY and that in the absence ofMotY, the overproduction of MotX affected the membrane
localization of PomB and the PomA-PomB complex, suggesting interaction between MotX and PomB (25).
Although there are several lines of evidence for the membrane localization and direct interaction of MotX and MotY,
the precise roles of these proteins are still unclear. Since overproduction of MotX partially restored the motility of motY
strains (26), we hypothesized that MotX is more directly involved than MotY in torque generation. In this report, toelucidate the functions of MotY, we randomly mutagenized amotY gene cloned on a plasmid and isolated three missense
mutations that caused various swimming defects. Two of themutations were found to change the two cysteine residues in
MotY. Coincidently, MotX also has two cysteine residues, andMotX and MotY each contain a tetrapeptide sequence thatbegins with a cysteine (CQLV) (20). Both of the cysteine
residues are highly conserved in either MotX or MotY ofvarious species. To investigate the roles of the cysteine residues, we constructed mutants of MotX and MotY in which thecysteine residues were replaced by serine residues. We thencharacterized these mutants in terms of the swimming andRotation of a helical flagellar filament is the most prevalentmeans of propulsion in bacteria (5, 6, 18). The driving force forthis flagellar rotation is coupled to the flux of a specific ion
across the cytoplasmic membrane (19). To date, proton andsodium ions have been shown to be used in this energy-coupling step (34). The motor, which is embedded in the cytoplasmic membrane at the base of a flagellum, is composed of statorand rotor components. In gram-negative bacteria, the basalbody structure consists of a rod and four surrounding ring
structures called the L ring, the P ring, the MS ring, and the C
ring (11). It is thought that multiple stator units surround theMS ring (7, 14, 24) and that the rotational force is generated bya stepping process (31).
Vibrio alginolyticus has two types of flagella in each bacterial
cell: a lateral flagellum with a proton-driven motor and a polar
flagellum with a sodium-driven motor (2, 13). The most extensively studied sodium-driven motors are the polar flagella ofVibrio spp. (22, 34). In these sodium-driven motors, PomA,
PomB, MotX, and MotY have been identified as essential
proteins for torque generation. PomA and PomB are membrane proteins that have four and one transmembrane segments, respectively. Furthermore, the C-terminal periplasmic
domain of PomB contains a segment with high sequence similarity to the peptidoglycan-binding motif (1). Thus, PomB is
believed to anchor and immobilize the PomA-PomB complex
to the peptidoglycan layer in the inner membrane. A PomAPomB complex contains four PomA subunits and two PomB
subunits, and purified complexes have been shown to catalyze
sodium influx when they were reconstituted into proteolipo-* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan. Phone: 81-52-789-2991. Fax: 81-52-789-3001.
E-mail: g44416a@cc.nagoya-u.ac.jp.
† Present address: Molecular Biophysics and Biochemistry, Yale
University, 266 Whitney Avenue, Bass 225, New Haven, CT 065208114.
‡ Present address: Department of Bioscience and Biotechnology,
Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano399-4598, Japan.
5308

VOL. 188, 2006

NOTES

5309

TABLE 1. Bacterial strains and plasmids
Strain or plasmid

V. alginolyticus strains
VIO5
VIO542
NMB94
GRF2
E. coli strains
JM109
BL21(DE3)
Plasmids
pSU38
pSU41
pIO2
pIO6
pRK101
pRK102
pRK103
pRK201
pRK202
pRK203
pKS202
pMO401
pMO404pMO405
pMO413

Genotype or descriptiona

Source or reference

VIK4 laf (Rif r Pof! Laf")
VIO5 motY (Rif r Laf" Mot")
VIO5 motX (Rif r Laf" Mot")
VIO5 #motY (Rif r Laf" Mot")

28
28
26
This study

recA1 endA1, gyrA96 thi hsdR17 relA1 supE44 $" #(lac-proAB)
F% traD36 proAB lacIq #M15

33

kan (Kmr) Plac lacZakan (Kmr) Plac lacZa
pSU21; 20-kb SalI fragment (motY !)pSU38; 1.0-kb HindIII-XbaI fragment (motY !)pIO6; motY (C147Y)
pIO6; motY (R262Q)
pIO6; motY (C25Y; "3S of signal sequence to N)
pSU38; 1.0-kb HindIII-XbaI fragment (motY C25S)
pSU38; 1.0-kb HindIII-XbaI fragment (motY C147S)
pSU38; 1.0-kb HindIII-XbaI fragment (motY C25S/C147S)
pSU41; 1.0-kb EcoRI-XbaI fragment (6His-motY)
pSU41; 700-bp ClaI-BamHI fragment (motX!)
pSU41; 700-bp ClaI-BamHI fragment (motX C35S)
pSU41; 700-bp ClaI-BamHI fragment (motX C66S)
pSU41; 700-bp ClaI-BamHI fragment (motX C35S/C66S)

4
4
28
28
This study
This study
This study
This study
This study
This study
This study
26
This study
This study
This study

Stratagene, San Diego, CAa
Rif r, rifampin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Plac, lac promoter; Pof!, normal polar-flagellar formation; Laf", defective inlateral-flagellar formation.

swarming abilities of the bacteria, protein stability, and the
MotX-MotY interaction.
Isolation of motY mutants and characterization of their motility. Random mutagenesis of the motY gene was carried out
by treating the plasmid pIO6 with hydroxylamine as described
previously (17). The mutagenized plasmids were introduced
into the motY mutant VIO542 (28), which possesses a nonmotile polar flagellum, though the mutation site has not been
determined yet (15). We then isolated mutants that were completely or partially impaired in their polar-flagellar motility ina semisolid agar. Each of the nucleotide changes in the motY
alleles was determined by DNA sequencing. In 10 isolates, nonucleotide change was found in the motY coding region of theplasmid, suggesting that the causative mutations likely affected
the expression of motY. Introduced termination codons were
found in an additional 16 isolates. We identified two isolates
carrying single amino acid substitutions and one isolate with
two amino acid substitutions: MotY-C147Y, MotY-R262Q,
and MotY-C25Y(S"3N). It is noteworthy that two of the missense mutations affected one of the two cysteine residues
found in MotY. The arginine residue at amino acid position
262 is located in a peptidoglycan-binding motif (28). The peptidoglycan-binding motif is conserved among the motor proteins MotB, PomB, and MotY, as well as several outer membrane proteins, such as PAL and OmpA (8, 10). Peptidoglycan
binding by any of the flagellar-motor proteins, however, has
not been demonstrated in vivo or in vitro; thus, the significanceof these peptidoglycan-binding motifs for motor function remains unknown.
MotY-C147Y, MotY-R262Q, and MotY-C25Y(S"3N) were

produced in VIO5 (a strain with wild-type polar flagella) (28)and GRF2 (a #motY strain constructed from VIO5 in thisstudy) (Table 1) cells, and the motility of the cells in 0.25%
agar was analyzed (Fig. 1 and Table 2). Whereas wild-type
MotY complemented the motility defect of the GRF2 cells,
none of the three mutants was able to produce a wild-type levelof motility in GRF2 cells. Among these MotY mutants, MotYR262Q expression resulted in the most motile bacteria andalso produced a negative dominance by multicopy effects(slower swarming speed) when it was coexpressed in VIO5
cells with the genomic copy of wild-type MotY.
Conservation of cysteine residues in MotX and MotY. The
above discussion mentioned the importance of two cysteine
residues of MotY. The fact that MotX and MotY of V. alginolyticus share the same CQLV sequence and have two cysteine
residues caused us to hypothesize the evolutionary importanceof these cysteine residues for MotX and MotY. So far, MotX
and/or MotY have been found or assigned only in some speciesof gammaproteobacteria, i.e., Vibrio species and Pseudomonas
species. The CQLV sequence in MotY is conserved only in V.
alginolyticus and Vibrio parahaemolyticus. According to theamino acid sequence alignment of these MotX or MotY proteins, the cysteine residues are conserved in all MotX andMotY proteins found (data not shown). These cysteine residues seem to be important for the proper function of MotX
and MotY.
Intramolecular disulfide bond in MotY. Since native MotY
contains two cysteine residues, it is possible that these twocysteine residues are involved in intramolecular or intermolecular disulfide bond formation, which might be important for

5310

NOTES

J. BACTERIOL.

FIG. 1. Characterization of MotY mutants. (A) VPG (1% polypeptone, 0.4% K2HPO4, 3% NaCl, and 0.5% [wt/vol] glycerol) plates containing
0.25% agar and 100 &g/ml kanamycin were inoculated with fresh colonies of the wild-type strain (VIO5) or the #motY mutant strain (GRF2)
expressing the indicated proteins from plasmids. They were incubated at 30°C for 4 h or 15 h. The plasmids and strains used are listed in Table
1. (B) Whole-cell lysates and the membrane fractions of GRF2 cells (#motY) expressing the indicated proteins from plasmids and the wild-typestrain [VIO5; wt (genomic)] were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as
described previously (35) using anti-MotY antibodies (MotYB0079) in the absence (left) or the presence (right) of 2-mercaptoethanol (2ME). The
asterisks (upper left and upper right) indicate that those samples were 1/10 the volume of the other samples. The anti-MotY antibody was raised
against purified MotY produced from pKJ503 (25), which encoded C-terminally histidine-tagged MotY.

the function of the protein. To investigate this possibility, weconstructed three motY mutants in which either or both cysteine residues were replaced by serine residues (pRK201[C25S], pRK202 [C147S], and pRK203 [C25S/C147S]). The

MotY-C25S mutant was not motile either on 0.25% agar plates
or in broth medium, whereas the MotY-C147S and MotYC25S/C147S mutants showed significantly reduced but observable spreads in the swarm assay after 15-h incubations (Fig.

VOL. 188, 2006

NOTES
TABLE 2. Swimming fractions

Host/plasmid

Description

Swimming
fraction (%)aVIO5/pRK102
GRF2/pIO6
GRF2/pRK102
GRF2/pRK201
GRF2/pRK202
GRF2/pRK203

Wild type/MotY-R262Q#motY/wild-type MotY
#motY/MotY-R262Q#motY/MotY-C25S#motY/MotY-C147S#motY/MotY-C25S-C147S

40 (n ' 193)
61 (n ' 208)
29 (n ' 192)
0
(1
(1

a
Cells were grown at 30°C for 4 h in VPG broth medium. The swimming cells
were counted under a dark-field microscope. The numbers in parentheses indicate the total cells counted. GRF2/pRK201 did not produce any motile cells,
whereas GRF2/pRK202 and GRF2/pRK203 produced low but observable numbers of motile cells.

1A). Since we did not find any revertant or pseudorevertantthat would swarm at the rate of the wild type, it is not likelythat those swimmers were the reverted wild-type cells. The
swimming fraction of MotY-C147S and the double cysteine
mutant in broth medium were below 1% (Table 2).
Next, we performed immunoblot assays to detect the mutant
proteins. In the presence of the reducing agent 2-mercaptoethanol, all the MotY derivatives overexpressed with the lacpromoter for enhanced visualization appeared as an approximately 30-kDa band, which was consistent with the expected
size of the mature MotY protein. In the absence of 2-mercaptoethanol, however, there were slight differences in the mobilities of the wild-type and the MotY Cys mutant proteins (Fig.
1B). In the presence of a reducing agent, the native MotY band
shifted upward to align with the other proteins. The faster
mobility of the wild-type band in the absence of a reducing
agent can be explained by a more compact conformation of thewild-type protein due to intramolecular disulfide bond formation. There were also significant differences in the band intensities. For the whole-cell preparations, the intensity of theoverproduced wild-type MotY band was much stronger than
the intensity of any of the Cys mutant protein bands. The
differences in the band intensities from membrane fraction
samples, however, were moderate. These results strongly suggest that MotY contains an intramolecular disulfide bridge.
The reduced levels of the MotY Cys mutants suggest that the
intramolecular disulfide bridge in MotY is required to maintain a stable conformation; without the disulfide bond, MotY is
more susceptible to degradation and the amount of the protein
is greatly reduced. However, the band intensities of the overproduced proteins with Cys replaced were still far greater thanthat of chromosomally expressed wild-type MotY. This might
imply that the conformation achieved by the intramolecularcross-linking is necessary for the proper function of MotY, in
addition to resistance to degradation. Whereas wild-type
MotY did not show a sign of intermolecular cross-linking,MotY-C25S and MotY-C147S produced intermolecular-crosslink products in immunoblot assays; in particular, MotY-C25S
showed numerous extra bands (data not shown). We speculatethat the cysteine residue at 147 is more prone to a cross-linkformation with other periplasmic proteins than C25 when an
intramolecular cross-link is not possible and that the homo- or
heterodimer formation interferes with its function.
Prevention of MotX degradation by MotY. It has beenshown that MotY directly interacts with MotX and that in the5311

absence of MotY expression, MotX is not detectable by immunoblotting (25, 26). These lines of evidence led us to hypothesize that MotY acts to protect MotX against degradation.
To evaluate our MotY mutants, we examined whether our
MotY derivatives could prevent the degradation of MotX. As
expected, increasing the amount of wild-type MotY elevatedthe level of detectable MotX without changing the motX expression on the chromosome (Fig. 2). Overexpression of
MotY-R262Q also increased the detectable MotX level; it islikely that the R262 residue does not affect the interaction with
MotX. On the other hand, although the levels of the Cyssubstituted MotY mutants were greater than that of genomically expressed wild-type MotY, no MotX band was observed
from cells expressing these mutant MotY proteins (Fig. 1B).
This result implies that these Cys mutants are impaired in theability to interact with MotX, and we postulate two distinct
consequences: elevated susceptibility to periplasmic digestion
and loss of a docking site for MotX. Since MotX appears to be
more directly involved in flagellar-motor function than MotY(26), the direct reason for swimming deficiencies of these
MotY Cys mutants seems to be the loss of MotX due todegradation.
Characterization of the MotX Cys mutants. We constructed
three motX mutants in which either or both of the cysteine
residues were changed to serine residues by site-directed mutagenesis (pMO404 [C35S], pMO405 [C66S] and pMO413[C35S/C66S]). All three MotX Cys mutant proteins conferredswarming ability on motX mutant cells (NMB94) (Fig. 3A). In
the absence of the reducing agent dithiothreitol (DTT), thedouble mutant MotX-C35S/C66S produced a swarm ring thatwas unexpectedly larger than the one that resulted from overproduction of wild-type MotX. In the presence of DTT, MotXC35S-expressing NMB94 cells formed a larger swarm ring thanin the absence of DTT. For the other cells, the levels of thewild-type and C66S mutant proteins did not seem to be affected by the addition of DTT.
We performed immunoblot assays to detect the MotX proteins in the presence or absence of the reducing agent 2-mercaptoethanol. In all the mutant and wild-type MotX samples,
we detected 24-kDa bands, which corresponded to the mature
MotX protein (Fig. 3B). In the absence of the reducing agent,

FIG. 2. Detection of MotX in strains coexpressing various MotY
mutant proteins. Whole-cell lysates of the GRF2 strain (#motY) expressing the indicated proteins from plasmids and the wild-type strain[VIO5; wt (genomic)] were prepared and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunoblotting as described previously (35) using anti-MotX antibodies (MotXB0080).

5312

NOTES

J. BACTERIOL.

FIG. 3. Characterization of the MotX mutants. (A) VPG plates containing 0.25% agar, kanamycin, and various concentrations of DTT were
inoculated with fresh colonies of motX mutant cells (NMB94) expressing the indicated proteins from plasmids and incubated at 30°C for 4 h.
(B) Whole-cell lysates of the NMB94 strain expressing the indicated proteins from plasmids were prepared and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunoblotting as described previously (35) using anti-MotX antibodies (MotXB0080) in theabsence (") and the presence (!) of 2-mercaptoethanol (2ME). The anti-MotX antibody was raised against purified MotX produced from pKJ402,
which encoded C-terminally histidine-tagged MotX.

MotX-C35S and MotX-C66S produced extra bands of various
sizes, probably due to the intermolecular-cross-link formation.
As in the case of MotY Cys mutants, we can see that C66 ismore likely than C35 to form intermolecular cross-links, whichmight explain the slower swarming of MotX-C35S than theother two mutants and its greater recovery by the addition of
the reductant. Although the double Cys mutant (MotX-C35S/
C66S) conferred better swarming ability than the wild-type
protein when overproduced, the level of the double mutant
was significantly reduced. It has been reported that an excessive amount of MotX forms aggregates in V. alginolyticus (25).

We speculate that aggregation and/or intermolecular crosslinking by single Cys mutants of MotX can raise its resistanceto degradation. Therefore, among these MotX derivatives,
only the double Cys mutant showed significant reduction in theamount detected. It has also been reported that the overexpression of MotX in E. coli kills the cells (20). In addition,
Vibrio cells overexpressing MotX exhibit aberrant morphologies (data not shown). These types of evidence suggest that theaggregation of MotX affects the shape of the cells, possibly by
disrupting the peptidoglycan layer or outer membrane integrity. We have also observed that MotX overproduction affectedVOL. 188, 2006

NOTES

5313

FIG. 5. Schematic diagram of MotX and MotY. MotX and MotY
contain two cysteine residues and the conserved tetrapeptide CQLV.
The arrowheads indicate the cleavage sites of the signal sequences(gray sections). The double-headed arrow demarcates the region containing the putative motif for peptidoglycan binding (PG motif). a.a.,amino acids.

FIG. 4. Biotin maleimide labeling of MotX cysteine residues.
NMB94 harboring pMO401 (MotX wild type) was treated with biotin
maleimide (BM) and DTT or CuCl2. The immunoprecipitates wereseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Biotinylated MotX was detected with streptavidin-conjugated horseradish peroxidase and chemiluminescence (A) or immunoblotting using anti-MotX
antibodies (B).

the motility—both the swimming and swarming abilities—of
the cells (data not shown). The better swarming ability of the
MotX-C35S/C66S-expressing cells than the cells overproducing wild-type MotX, therefore, might be explained by the increased susceptibility of this mutant protein to degradation,
which reduced the damaging effects of the aggregation.
Unlike MotY, wild-type MotX exhibited the same gel mobilities in the presence and absence of the reductant. We thentreated MotX with biotin maleimide, which binds to free thiolgroups, and the labeled biotin was detected using horseradish
peroxidase-conjugated streptavidin. The labeled biotin was detected only when DTT was added to the sample (Fig. 4). Since
the wild-type protein seemed to be a monomer in the absenceof the reductant (Fig. 3B), biotin labeling only in the presenceof DTT suggests that MotX contains an intramolecular disulfide bond. In conclusion, the presence of the intramoleculardisulfide bond is not essential for the function of MotX but
instead affects the susceptibility of the protein to degradation.
Concluding remarks. MotX and MotY each contain two
cysteine residues (Fig. 5), which are highly conserved among
several species of gammaproteobacteria. We speculated thatthese cysteine residues are critical for the functions of these
proteins and determined that both MotX and MotY form
intramolecular disulfide bonds. In vivo, disulfide bond formation in the prokaryotic periplasm is a catalyzed process, whichis crucial for the folding and stability of many proteins andprotein complexes (23). Failure to form proper disulfide bonds
is likely to lead to protein aggregation and degradation byproteases. Disulfide bonds are donated directly to unfolded
polypeptides by the DsbA protein, whereas DsbA is reoxidized
by DsbB. For example, dsbA mutant strains exhibit reduced

levels of alkaline phosphatase, )-lactamase, or the outer membrane protein OmpA (3). It seems that both MotX and MotY
acquired the cysteine residues for the same reason the othersecreted proteins did. However, we have also shown that disulfide bond formation is not sufficient for MotX to be fullystabilized. The interaction with MotY is the key for MotX to
fold correctly and to function. For example, when MotX is far
in excess of MotY, MotX aggregates, which in turn affects
swimming ability, cell shape, and cell growth, as stated above.
Therefore, we speculate that the MotX-MotY interaction isanother level of control on the amount of MotX in the
periplasm. As MotX is essential for motor rotation in Vibrio
and is likely to interact with PomB (25), we believe that MotX
modulates the activity of the sodium channel, a stator complex
composed of PomA and PomB. On the other hand, the role of
MotY may be limited to the anchoring and/or stabilizationof MotX.
We are grateful to Seiji Kojima for critically reading the manuscript.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan;
from the Japan Science and Technology Corporation (to M.H. andT.Y.); and from the Soft Nano-Machine Project of the Japan Scienceand Technology Agency (to T.Y. and M.H.).
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