Molecular Microbiology (2009) 71(4), 825–835 䊏

doi:10.1111/j.1365-2958.2008.06569.x
First published online 13 January 2009

Sodium-dependent dynamic assembly of membranecomplexes in sodium-driven ﬂagellar motors
Hajime Fukuoka,1† Tomoyuki Wada,2† Seiji Kojima,2Akihiko Ishijima1 and Michio Homma2*
1
Institute of Multidisciplinary Research for Advanced
Materials, Tohoku University, Aoba-ku, Sendai,
980-8577, Japan.
2
Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya,464-8602, Japan.

Summary
The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H+ or Na+. The
motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na+ motor of Vibrio
alginolyticus, is thought to be a torque generator converting the energy of ion flux into mechanical power.
We found that specific mutations in PomB, including
D24N, F33C and S248F, which caused motility defects,
affected the assembly of stator complexes into thepolar flagellar motor using green fluorescent proteinfused stator proteins. D24 of PomB is the predicted
Na+-binding site. Furthermore, we demonstrated thatthe coupling ion, Na+, is required for stator assembly
and that phenamil (an inhibitor of the Na+-driven
motor) inhibited the assembly. Carbonyl cyanide
m-chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in thisorganism at neutral pH, also inhibited the assembly.
Thus we conclude that the process of Na+ influx
through the channel, including Na+ binding, is essential for the assembly of the stator complex to theflagellar motor as well as for torque generation.

Introduction
Many kinds of proteins with diverse and essential cellularfunctions such as ion channels, transporters and receptors exist in the bacterial cell membrane. Many membraneproteins function in complexes that are localized at
specific sites. Knowing how these membrane proteins
Accepted 4 December, 2008. *For correspondence. E-mailg44416a@cc.nagoya-u.ac.jp;
Tel.
(+81) 52 7892991;
Fax(+81) 52 7893001. †These authors contributed equally to this work.

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd

assemble and localize at specific locations is essential tounderstand their functions, but little is known about themechanisms responsible. The bacterial flagellum is a
locomotive organelle and a supramolecular complex penetrating the bacterial cell envelope, including the cytoplasmic and the outer membranes. The flagellum contains arotary motor driven by the electrochemical potential of H+
or Na+ (Yorimitsu and Homma, 2001; Blair, 2003). The
assembly mechanism of the flagellum has been intensively studied by using the H+-driven type present in Salmonella and Escherichia coli. The flagellum consists of abasal body (rotary motor), a helical filament (propeller)
and a hook (universal joint), and cells swim towards
favourable conditions by rotating the flagella (Macnab,
1996). The basal body consists of a rod structure andseveral rings, termed the L-, P-, MS- and C-ring, which areembedded in the outer membrane, peptidoglycan, cytoplasmic membrane, and attached on the cytoplasmic
surface of the MS-ring respectively. In the Na+-driven
motor of Vibrio alginolyticus, a novel ring structure, called
the T-ring, was recently found (Terashima et al., 2006).
The T-ring structure is located beneath the LP-ring and iscomposed of MotX and MotY. Loss of this ring by mutationof the motX and motY genes does not affect the assemblyof the flagellar structure but the flagellar motor does not
rotate.
The motor consists of a rotor and stator, and theirinteraction is thought to generate the motor rotation. The
stator is a complex of four molecules of an A subunit andtwo molecules of a B subunit (PomA and PomB in the
Na+-driven motor of V. alginolyticus, MotA and MotB in
the H+-driven motor of E. coli ) and functions as an ion
channel (Sato and Homma, 2000; Kojima and Blair,2004). The proteins PomB and MotB each contain a large
periplasmic domain that has a putative peptidoglycanbinding motif through which the stator unit is anchoredaround the rotor. PomB and MotB each also have an
essential aspartic acid residue in the putative membrane
spanning region that is likely to participate directly in ion
conduction (Zhou et al., 1998; Yakushi et al., 2004). In
PomB, the Asp-24 residue was shown to be the only
essential negatively charged residue for motor function
and this residue is thought to be a Na+-binding site. On theother hand, Asp-32 of MotB is the predicted H+-bindingsite in E. coli. It has been speculated that the substitution

826 H. Fukuoka et al. 䊏of Asp to Asn mimics the H+-binding state, and the ion
binding/dissociation at this residue triggers conformational changes in the stator that act on the rotor to drive
rotation (Zhou et al., 1998; Braun et al., 1999; Kojima andBlair, 2001). It has been recently suggested that at least
11 independent torque-generating units or stator units,
composed of the motor proteins, surround the basal bodyor the rotor (Reid et al., 2006).
In V. alginolyticus, the Na+-driven motor rotates a single
polar flagellum at the cell pole. Therefore, we can easily
monitor the stator assembly to determine its polar localization. In fact, we have shown that green fluorescentprotein (GFP)-fused stator proteins, GFP-PomA and GFPPomB, localize at the base of the polar flagellum with thepartner protein, PomB or PomA and found that their localization depends on the presence of the flagellar rotor(Fukuoka et al., 2005). Furthermore, MotX and MotY of the
T-ring are also required for localization of the stator proteins (Terashima et al., 2006), suggesting that the T-ring is
involved in the incorporation of the stator complex into theflagellar motor, or involved in the stabilization of the stator
complex in the flagellar motor. We have shown that MotX
probably interacts with PomB and that the N-terminal
region of MotY is essential for the association of the stator
complexes around the rotor (Okabe et al., 2005; Kojima
et al., 2008). These stator units are able to assemble to thebasal structure after the flagellar structure is built, and areable to exchange with the new unit in the functioning
motor, suggesting that the stator behaves more dynamically than previously expected (Leake et al., 2006). In thisstudy, we showed that mutation of the putative Na+-bindingsite in PomB affected the assembly of stator complexes
into the polar flagellar motor, and that the coupling ion,
Na+, was required for the stator assembly around theflagellar rotor. The evidence provides a new concept: thecoupling ion is not only used for generating rotational force
but also required for motor assembly.

Results
Fig. 1. GFP-fused stator proteins.

GFP-fused motor proteins
To express GFP-fused stator proteins, the gfp gene was
genetically fused to the 5′ end of the pomA or pomB geneto form gfp-pomA and gfp-pomB respectively (Fig. 1A).
The genes were placed under the control of an arabinoseinducible promoter in pBAD33 derivative plasmids. When
GFP-PomA was produced in DpomA cells, a single punctate fluorescent signal was observed at the base of thepolar flagellum (Fig. 1B). Similarly, GFP-PomB localized
at the base of the polar flagellum of DpomB cells. In
DpomAB cells producing both GFP-PomB and wild-type
PomA from a plasmid, cells swarmed slightly in semisolid
agar (Fig. 1C and D). The swimming speed and fraction of

A. Diagram of the flagellar motor structure of Vibrio alginolyticus
with GFP-fused stator proteins. Blue, red and green indicate PomA,
PomB and GFP respectively. The orange circle in PomB indicatesthe aspartic acid residue that serves as the Na+-binding site.
B. Colocalization of the polar flagellum and GFP-fused stator
proteins. Cells were treated with rabbit anti-polar flagellum antibody
and rhodamine-conjugated anti-rabbit secondary antibody (red).
The green spots localized at the base of polar flagellum represent
GFP-PomA in DpomA cells (left) and GFP-PomB in DpomB cells(right) respectively.
C. Swarming behaviour of cells producing GFP-fused stator
proteins. DpomAB cells producing PomA and PomB, GFP-PomA and
PomB, GFP-PomB and PomA, and GFP were grown on VPG5000.25% agar plates containing 0.006% arabinose at 30°C for 7 h.
D. Magnified image of C. The small faint ring around the tightly
packed colony is swarmed cells producing GFP-PomB and wild-type
PomA (left). On the right is the colony of cells producing GFP.

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Dynamic assembly of flagellar stator 827

DpomAB cells producing GFP-PomB and wild-type PomA
in 500 mM Na+ were 16 mm s-1 and 20% respectively. On
the other hand, the swimming speed and fraction of
DpomAB cells producing wild-type PomA and PomB
from a plasmid were 74 mm s-1 and 64%. Using oblique
epi-illumination fluorescence microscopy, GFP-PomB
appeared to localize at the base of the polar flagellum incells rotating about tethered flagella (see Movies S1 andS2). Thus GFP-PomB is incorporated into the flagellar
motor and retains weak function. However, cells producing GFP-PomA did not confer any motility. This suggeststhat the fused GFP does not inhibit the assembly of thestator but it abolishes the function of PomA.
Effects of various PomB mutations on the polar
localization of motor proteins
To investigate the effects of PomB mutations on theassembly of the stator, we mutated Asp-24, which is theputative Na+-binding site (D24C, D24N), Phe-33, which islocated in the putative transmembrane segment (F33C),
and Thr-231 and Ser-248, which are located in the putative peptidoglycan-binding motif (T231I, S248F) based on
previous information (Blair et al., 1991; Yakushi et al.,2004). First, we determined the effects of the mutations
on motility. Mutant PomB proteins and PomA were coexpressed from a plasmid in the DpomAB strain and motility
was investigated on soft agar plates (Fig. 2A). The D24C,
D24N and S248F mutations did not confer any motility,
whereas F33C showed very week motility as reported
previously (Yakushi et al., 2004). T231I did not affectmotility, which was unexpected because the corresponding mutation in MotB (T196I) caused a loss of motility(Blair et al., 1991). Each mutation was introduced into
GFP-PomB or PomB and coexpressed with PomA or
GFP-PomA from a plasmid in the DpomAB strain, andpolar localization of the GFP-fused stator complex was
observed by fluorescence microscopy. The fraction ofFig. 2. A. Swarming behaviour of cells producing mutant PomB proteins. All mutant PomB proteins were produced in DpomAB cells withwild-type PomA. Cells were grown on VPG500-0.25% agar plates at 30°C for 5 h. Mutations in PomB are shown on the side.
B. The effects of the pomB mutations on the polar localization of stators. DpomAB cells producing GFP-PomA/PomB or GFP-PomB/PomA withvarious PomB mutations (D24N, F33C, T231I and S248F) were observed in 500 mM Na+. Polar localized fractions of GFP-PomB are shown
under the pictures. n is the total number of cells that were analysed to obtain the fractions.
C. Immunoblot of GFP-PomB variants and wild-type PomA in whole-cell extracts. Proteins were detected with anti-PomA1312 andanti-PomB93 antibodies respectively. Molecular mass values (kDa) are shown on the left side of the panels. Arrowheads marked with 1, 2, 3
and 4 indicate bands of PomA, GFP-PomB variants, GFP-PomA and PomB respectively. Arrowheads marked with an asterisk indicate
non-specific bands detected by each antibody. Vibrio cells were grown in VPG500 medium containing 0.006% arabinose at 30°C for 4 h.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

828 H. Fukuoka et al. 䊏

Fig. 3. Na+-dependent polar localization of
stators. Localization of GFP-fused stator
proteins in 0 mM or 500 mM Na+. DpomAB
cells producing GFP-PomA and PomB
(upper), GFP-PomB and PomA (middle), andDfliG cells producing GFP-FliG (bottom) wereobserved. The cells were suspended in
500 mM Na+, resuspended in 0 mM Na+ andresuspended again in 500 mM Na+.

fluorescent dots that localized to the pole was drasticallyreduced by the D24 mutation; the fractions of GFP-PomBD24N and GFP-PomB-D24C in the stator were 5%
and 1%, respectively, in media containing 500 mM Na+(Fig. 2B, data not shown). In these strains, similar
amounts of GFP-PomB variants and wild-type PomA weredetected by immunoblotting (Fig. 2C). These results
suggest that Asp-24 is essential for polar localization of
stator proteins.
The polar localized fractions of GFP-PomB-F33C, GFPPomB-T231I and, GFP-PomB-S248F with PomA were32%, 75% and 2% respectively, and the polar localized
fractions of GFP-PomA with the mutant PomB weresimilar (Fig. 2B). Similar amounts of GFP-PomB variants
and wild-type PomA were detected in all strains by immunoblotting (Fig. 2C). There was good correlation between
cell motility and the polar localization of the mutant PomB
proteins investigated in this study. This correlation may
suggest that the assembly of mutant PomB proteins into
the flagellar motor was appropriately evaluated by determining the polar localization of GFP fusions.
Sodium-dependent polar localization of motor proteins

500 mM K+) following centrifugation, the polar localized
fraction decreased to 5%. However, the polar localization
was restored to 71% when cells were resuspended in
500 mM Na+. Similar results were observed for GFPPomA (Fig. 3, upper panels). In control cells producing
GFP-FliG (Yorimitsu et al., 2003), which is one of the rotor
components, fluorescent puncta were observed at the cellpole in about 80% of cells in either 500 mM or 0 mM Na+(Fig. 3, bottom panels). These results indicate that punctate fluorescent signals from GFP-stator proteins are
dynamically localized and dispersed at the poles and thepolar localization of PomA and PomB is dependent on
Na+. The relocalization was not affected by the addition of
kanamycin, an inhibitor of new protein synthesis, at
1 mg ml-1 which is a concentration 10 times higher thanthe minimum inhibitory concentration for growth of
V. alginolyticus cells (data not shown). Thus, new protein
synthesis is not necessary to restore polar localization.

Table 1. Polar localized fractions of GFP-PomB under conditions
that inhibit polar flagellar rotation.
Conditions+

To examine the dependence of the polar localization of
GFP stators on Na+, localization was observed in thepresence or absence of Na+ using normal epifluorescencemicroscopy. In a medium containing 500 mM Na+, polar
localization of GFP-PomB was observed in approximately
80% of cells (Fig. 3, middle panels; Table 1). When themedium was replaced with Na+-free medium (containing

Na 500 mM
Na+ 0 mM
0 mM CCCP
20 mM CCCP
0 mM phenamil
100 mM phenamil
300 mM phenamil

Polar localized fraction (%)
78 (n = 502)
5 (n = 215)
49 (n = 1257)
11 (n = 2176)
53 (n = 1142)
28 (n = 1054)
21 (n = 871)

n is the total number of cells that were analysed.

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

Dynamic assembly of flagellar stator 829

is due to the dissociation of the stator proteins from theflagellar motor.
Stator complexes in the absence of sodium ionsFig. 4. Relationship between the polar localization of stators andthe sodium ion concentration. The polar localized fraction of
GFP-PomB (squares) and swimming fraction of VIO5 cells (circles),versus [Na+], relative to values in 500 mM Na+, are shown.
GFP-PomB was produced in DpomAB cells with PomA.
Michaelis–Menten fits for polar localization and swimming fractions
are depicted by solid and dotted lines respectively.

To detect the motion of GFP-PomB at higher time resolution, we observed the localization by oblique illuminationepifluorescence microscopy using an EMCCD camera. In
this microscopic system, a fluorescent punctum wasobserved at the cell pole and we observed many fluorescent puncta diffusing along the cell body (see Movie S3).
Thus it appears that stator complexes that are not incorporated into the flagellar motor diffuse in the membranepool as proposed by Leake et al. (2006). Stator complexes diffusing in the cytoplasmic membrane in Na+-free
medium would relocalize to the cell pole upon additionof Na+.

Previously, we reported that individually, the stator proteins PomA and PomB do not localize at the cell pole but
the stator complex PomA/PomB does (Fukuoka et al.,2005). Therefore, we examined the size of the PomA/HisGFP-PomB complex purified from the cell membrane in
the presence and absence of Na+ by size exclusion
chromatography. In both ionic conditions, the elution profiles and the complex size of PomA and GFP-PomB
appeared the same; GFP-PomB and PomA were mainly
eluted in fractions 10–15, and in fraction 10 and 12
respectively (Fig. 5, bottom panels). These results
suggest that the PomA/PomB complex is retained in theabsence of Na+, and that stator proteins move to the cellpole in the form of stator complexes.
Requirement of sodium motive force for the polar
localization of motor proteins
In V. alginolyticus cells, at neutral pH, sodium motive
force (smf) is secondarily generated by the H+/Na+
antiporter using proton motive force (pmf) (Yorimitsu
and Homma, 2001). Under these conditions, additionof the proton-ionophore CCCP (carbonyl cyanide

Relationship between cell motility and polar localizationof motor proteins
If Na+ affects the incorporation of stator complexes into
the flagellar motor, there should be a correlation between
the polar localization of stators and cell motility. We investigated the polar localization of GFP-PomB and the motility of VIO5 cells in various concentrations of Na+. Polar
localization of GFP-PomB and the motile fraction of VIO5
cells were approximately constant in media containing
from 100 mM to 500 mM Na+. In media with lower than
100 mM Na+, the localization and fraction of swimming
cells were coordinately reduced (Fig. 4). There seems to
be a correlation between the polar localization of stator
proteins and cell motility. We assumed that the reductionof the swimming fraction at lower concentrations of Na+

Fig. 5. Size exclusion chromatography. Proteins eluted from theSuperdex 200 10/300 GL column in fractions were condensed by
precipitation with trichloroacetic acid and were analysed by
SDS-PAGE followed by immunoblotting. PomA was detected with
anti-PomA1312 antibody, and PomB and GFP-PomB were detectedwith anti-PomB93 antibody. Numbers shown on top of the columns
indicate fraction numbers from the size exclusion column elution.
Arrowheads indicate molecular markers used in the size exclusion
experiment.

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

830 H. Fukuoka et al. 䊏compared with the control experiments is probably due tothe solvent for phenamil, dimethyl sulphoxide (DMSO).
With 100 mM and 300 mM phenamil, the polar localized
fraction of GFP-PomB was reduced to 28% and 21%
respectively (Fig. 7, Table 1). In the presence of phenamil,
most VIO5 cells did not swim but we observed a few
moving cells. Phenamil affected the Na+-dependent
assembly of stator proteins into the motor. Polar localization of GFP-PomB was rarely observed in Na+-free
medium with or without 100 mM phenamil (data notshown). The results indicate that the sodium-channel
inhibitor prevents the polar localization of the stator
complex, although the effect is not perfect.
Fig. 6. Effect of CCCP on the Na+-dependent polar localization of
stators. DpomAB cells producing GFP-PomB and PomA in 50 mM
Na+ (pH 7.0) were observed in the presence of 0 mM, 20 mM CCCP.
White arrowheads indicate polar localization in the presence of
CCCP.

m-chlorophenylhydrazone) collapses pmf and hence
smf. In medium containing 50 mM Na+ (only this experiment was carried out at pH 7.0), the polar localized fraction of GFP-PomB was 49% in the absence of CCCP.
On the other hand, the polar localized fraction was drastically decreased to 11% in the presence of 20 mM
CCCP (Fig. 6, Table 1). In the presence of CCCP, most
VIO5 cells did not swim, although a small fraction ofcells was very slowly motile. smf seemed to be required
for stator assembly around the rotor in the sodiumdriven motor.
Effect of sodium-driven motor inhibitor on the polar
localization of motor proteins
The rotation of Na+-driven flagellar motors is specificallyinhibited by the non-competitive inhibitor phenamil(Atsumi et al., 1990; Kojima et al., 1999). We investigatedwhether phenamil affects the polar localization of thestator in medium containing 500 mM Na+. In the absenceof phenamil, the polar localized fraction of GFP-PomB
was 53% (Fig. 7, Table 1). This reduced polar localization

Discussion
The bacterial flagellar motor, which is driven by theelectrochemical potential of specific ions, H+ or Na+,is a supramolecular complex embedded in the cell
membrane. The stator, which is composed of PomA and
PomB, functions as an ion channel and is thought to be atorque generator converting the energy of ion flux intomechanical power via the interaction with the rotor. Multiple stator units assemble around the rotor and theseunits are able to exchange with new units even after theflagellar structure has been built. Leake et al. (2006)
reported that the stator complex can be exchanged in thefunctional motor, indicating that each stator behaves more
dynamically than previously expected. When a mutation is
introduced at the Asp-24 residue of PomB, which is theputative Na+-binding site, the polar localization of the fluorescent punctum was drastically reduced even in media
containing 500 mM Na+. It is likely that in this mutant, thestator complex might not be assembled into the motor
because Na+ ions do not bind to their binding site. In the
proton-driven motor of MotB, which is a PomB homologue, the substitution of Asp to Asn in the putative ionbinding site is assumed to mimic a H+-binding state (Zhouet al., 1998; Kojima and Blair, 2001). If this assumption isapplied to the Na+-driven motor, the polar localization ofFig. 7. Effect of phenamil on the
Na+-dependent polar localization of stators.
DpomAB cells producing GFP-PomB and
PomA in 500 mM Na+ were observed in thepresence of 0 mM, 100 mM and 300 mM
phenamil.

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

Dynamic assembly of flagellar stator 831

the stator complex may not be reduced by the D24N
substitution. Our data suggest that the substitution of
Asp-24 to Asn may not mimic the ion-binding state in thesodium motor because the size of a sodium ion is larger
than that of a proton. The polar localization was reducedby the mutation of PomB-F33 which is a bulky side-chainthat is located in the transmembrane segment of PomB.
However, the complex containing the PomB-F33C mutation still localized at the cell poles. This residual localization is consistent with the fact that PomB-F33C confers
very weak motility. The assembly of stator complexes into
the flagellar motor does not seem to be severely affectedby the mutation of PomB-F33C, but the motility was significantly reduced. So, we speculate that this mutation
more severely affects energy transduction than statorassembly around the rotor. The Ser-248 residue is locatedin the peptidoglycan-binding motif of PomB and both thefunction and polar localization of PomB were lost in the
S248F mutant. The S248F mutation may abolish bindingof PomB to peptidoglycan or the T-ring, and consequently
the stator complex could not assemble around the flagellar motor.
Next, we found that the polar localization of stator proteins depends on the extracellular Na+ concentration. We
could easily and reversibly regulate the polar localizationof the stator complex by changing the concentration of
Na+ in the buffer. In addition, the polar localization of stator
proteins was drastically reduced under conditions thatcollapsed the smf even when Na+ was present. We examined whether the stator complex, 4PomA:2PomB, wasdissociated in the absence of Na+. We isolated stator
complexes with similar molecular size both in the presence or absence of Na+, indicating that the complex isretained without Na+. In the presence of phenamil, which
is a specific non-competitive inhibitor of the Na+ channelor the Na+-driven flagellar motor, the polar localization of
GFP-PomB was reduced. These results indicate that theassembly of the stator complex into the flagellar motor
requires not only Na+ but also smf in the Na+-driven motorof V. alginolyticus. Now we realize that motor rotation isregulated by the ion flux as well as by the association/
dissociation of stator complexes via the coupling ion in the
motor complex. Similar regulation was found in a polar
flagellar motor of Shewanella oneidensis that has two
potential stator systems, PomAB for Na+-driven motor and
MotAB for H+-driven motor (Paulick et al., 2009). It has
been shown that the polar flagellum is powered by both of
the stator systems and their localizations are regulated in
response to sodium concentrations.
In reconstituted Na+-driven motors in E. coli, we
observed the stepwise increase of rotational speed withtime after the replacement of 0.1 mM Na+ with 85 mM Na+
in the medium (Sowa et al., 2005). This result is consistent with our interpretation that the assembly of stator

complexes into the flagellar motor depends on their coupling ion, because if the stator complex is kept in the
motor at low Na+ concentrations, the rotational speed
should be restored immediately to the original speed anda stepwise increase in the rotational speed should not be
detected. The following physiological evidence also supports our results. The motility of Rhodopseudomonas
sphaeroides is lost when the pmf is dissipated by the darkwith addition of a H+ uncoupler. Cell motility can be recovered upon regeneration of the pmf following illumination.
However, cell motility did not immediately restart upon
redevelopment of pmf (Armitage and Evans, 1985; Evansand Armitage, 1985). In these reports, authors discussed
the possibility that the delay in re-initiation of motility iscaused by the dissociation of stator units from the motor
under the 0 pmf conditions, or by the inhibition of H+ flowby direct interaction between the H+ uncoupler and the
motor. In the H+-driven motor of E. coli, the recover ofmotor rotation also appears to be delayed after the pmf isrecharged (Fung and Berg, 1995). These observations
may suggest that the membrane potential is required forthe assembly of stator proteins into the flagellar motor in
the H+-driven motor as we have shown in the Na+-driven
motor.
Based on the present results and the evidence as
described above, we propose a hypothetical model ofstator assembly onto the flagellar motor (Fig. 8). At first,
PomA and PomB form the stator complex in the cytoplasmic membrane, and Na+ does not flux into the cytoplasm.
Hosking et al. (2006) proposed a mechanism that prevents H+ flow into the cytoplasm via unincorporated stator
complexes. When the Na+ concentration outside the cytoplasmic membrane is sufficient (in the presence of smf),
the stator complex interacts with the rotor (flagellar basalbody), for example, via interactions between PomB and
the T-ring, or between PomA and FliG. As a result of thisinteraction, the sodium ion channel is opened and Na+
binds to its binding site. The bound Na+ is released fromthe complex and fluxed into the cytoplasm, and consequently, rotational force is generated. Subsequently, Na+
immediately binds to its binding site (Asp-24), and thenthe Na+-bound form of the stator complex again interacts
with the rotor. So, the stator complex is retained in the
motor by continuous Na+ influx. In the absence of smf, thestator complex may not interact strongly with the rotor.
In the present study, we demonstrated that regulation of
motor function occurs by the association/dissociationof stator complexes via their coupling ion. This type of
regulation has been proposed for V-ATPase, which is arotary motor that pumps out protons using the energyof ATP hydrolysis; the function of V-ATPase is regulated
via reversible association/dissociation of the V1 sector(soluble) and the Vo sector (membrane bound) (Kaneand Smardon, 2003). V-ATPase is only active when

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

832 H. Fukuoka et al. 䊏dence that the function of the flagellar motor is regulated
by dynamic coupling ion-dependent assembly of thestructure. We predict that other supramolecules in themembrane, such as transporters or respiratory complexes, may be regulated in a similar way.

Experimental procedures
Vibrio strains, plasmids and cell preparation
Bacterial strains and plasmids used in this study are shown in
Table 2. Vibrio strains were derived from strain VIO5, which
has wild-type polar flagella (Okunishi et al., 1996). NMB190
was transformed with pHFGA, NMB191 was transformed
with pHFGAB or pHFGBA2, NMB192 was transformed with
pJN227, and NMB198 was transformed with pTY201 respectively (Yorimitsu et al., 2003; Fukuoka et al., 2005). To
investigate the function of PomB mutants, NMB191 wastransformed with derivatives of plasmid pJN152 (Fukuoka
et al., 2005). All point mutations were introduced by PCR
using pfu DNA polymerase (Stratagene). Cells were precultured in VC medium and then grown in VPG500 medium.
Chloramphenicol and kanamycin were added to final concentrations of 2.5 mg ml-1 and 100 mg ml-1 respectively. For
swarming assays, overnight cultures were spotted onto
VPG500 plates containing 0.25% agar.

Fig. 8. Model of the stator assembly in the sodium-driven polar
flagellar motor of Vibrio. PomA and PomB are coloured light grey
and dark grey respectively. White circles with D in the PomBtransmembrane region indicate the aspartic acid residue that binds
Na+ ions. Black circles indicate Na+ ions.
A. PomA and PomB form a 4A:2B complex independent of Na+.
B. The PomA/PomB complex diffuses in the inner membrane, and
the PomA/PomB complex can locate around the rotor through theassociation with MotX in the T-ring or FliG. Then, Na+ binds to its
binding site (Asp-24).
C. The bound Na+ is released from the complex and generates arotational force. The stator complex is released from the rotor.
Subsequently Na+ immediately binds to its binding site (Asp-24),
and the Na+-bound form of the stator complex interacts with therotor again. So, the stator complex is retained in the motor by thecontinuous Na+ influx.

both sectors are assembled in the plasma membrane.
Although the mechanism of association/dissociation is notwell understood, their dissociation is known to be caused
by glucose deprivation or a change in pH. Thus the activityof V-ATPase is regulated in response to environmental
signals. Regulation of molecular machines by association/
dissociation of relevant components may make it possible
to rapidly respond to environmental changes and allow
the recycling of the components without the requirementfor de novo synthesis. The control of a molecular machineby association/dissociation of its components might be a
strategy that evolved to allow organisms to live in varying
environments. Our study provides clear and direct evi-

Observation of localization with fluorescencemicroscopy
Overnight cultures were inoculated into VPG500 medium(1/100 volume) containing 0.006% arabinose for productionof GFP-fused stator proteins and 0.01% arabinose for production of GFP-FliG, and cells were grown for 4 h at 30°C. To
analyse the recovery of polar localization, 6 ml of cultures
was harvested by centrifugation and suspended in 1.2 ml of
TMN500 medium [50 mM Tris-HCl (pH 7.5), 5 mM MgCl2,
5 mM glucose, 500 mM NaCl], and incubated for 15 min. One
hundred microlitres of cell suspension was retained forobservation. The remaining 1.1 ml was centrifuged and cells
were suspended in 1 ml of TMN0 medium [50 mM Tris-HCl(pH 7.5), 5 mM MgCl2, 5 mM glucose, 500 mM KCl]. Cells
were harvested by centrifugation and suspended in 360 ml
of TMN0 and incubated for 15 min. One hundred microlitresof this cell suspension was retained for observation. The
remaining 260 ml was centrifuged and cells were suspended
in TMN500 medium. One milligram per millilitre of kanamycin
was added to motility media to prevent synthesis of GFPfusion proteins.
To investigate the effect of various concentrations of Na+,
1 ml aliquots of cell cultures were centrifuged and cell pellets
were suspended in 1 ml of TMN0 (500 mM KCl), TMN10(10 mM NaCl, 490 mM KCl), TMN50 (50 mM NaCl, 450 mM
KCl), TMN100 (100 mM NaCl, 400 mM KCl), TMN300(300 mM NaCl, 200 mM KCl) and TMN500 (500 mM NaCl)
respectively. Cells were harvested by centrifugation and suspended in 200 ml of the same medium.
To investigate the effect of phenamil, 1 ml aliquots of cell
culture were centrifuged and cell pellets were suspended in© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

Dynamic assembly of flagellar stator 833

Table 2. Bacterial strains and plasmids.
Strain or plasmid
V. alginolyticus
VIO5
NMB190NMB191NMB192
NMB198Plasmid
pSU41
pBAD33
pHFGA
pJN227
pHFGBA2
pHFAB
pJN152

Description

Source or reference

Rifr, Pof+, LafRifr, Pof+, Laf- DpomA
Rifr, Pof+, Laf- DpomAB
Rifr, Pof+, Laf- DpomB
Rifr, Pof+, Laf- DfliG

Okunishi et al. (1996)
Asai et al. (1999)
Yorimitsu et al. (1999)
Asai et al. (2003)
Yorimitsu et al. (2003)

Kmr, Plac, lacZa
Cmr, PBAD
his6-gfp-pomA in pBAD33
his6-gfp-pomB in pBAD33
his6-gfp-pomB and pomA in pBAD33
pomA and pomB in pBAD33
his6 -pomA and pomB in pSU41

Bartolome et al. (1991)
Guzman et al. (1995)
Fukuoka et al. (2005)
Fukuoka et al. (2005)
Fukuoka et al. (2005)
Fukuoka et al. (2005)
Fukuoka et al. (2005)

Rifr, rifampicin-resistant; Kmr, kanamycin-resistant; Cmr, chloramphenicol-resistant; Plac, lac promoter; PBAD, araBAD promoter; Pof+, normal
polar flagellar formation; Laf-, defective in lateral flagellar formation.

1 ml of TMN500 respectively. Cells were harvested by centrifugation and resuspended in 100 ml of TMN500. One
microlitre of DMSO solution containing 30 mM, 10 mM, or no
phenamil was added to each cell suspension.
To investigate the effect of CCCP, 1 ml aliquots of cell
culture were centrifuged and cell pellets were suspended in
1 ml of TMN50 (pH 7.0) respectively. Cells were harvested bycentrifugation and resuspended in 100 ml of TMN50 (pH 7.0).
One microlitre of DMSO containing 2 mM or no CCCP wasadded to each cell suspension.
For observation of polar localization, cell suspensions(20 ml each) were loaded into the space between apolylysine-coated coverslip and a microscope slide with aspacer. In this space, about 20 ml of solution can be retained.
Additional motility medium was loaded into the space
between the coverslip and the slide to remove the remaining
unattached cells, and the samples were incubated for 15 minat room temperature. Cells were observed by normal
epi-illumination fluorescence microscopy (B¥50, Olympus).
Images were recorded every 1 s (exposure time) and processed using a CCD camera (Orca-ER, Hamamatsu Photonics) and imaging software (IPLab, ver. 3.9.5r2, Scanalytics).
For observation of the diffusion of GFP-PomB, we used
Olympus IX71 based oblique-illumination fluorescencemicroscopy (Fukuoka et al., 2007) and images were recordedby using EMCCD camera by video rate (DV860-BV, Andor
Technology).

Size exclusion chromatography
Membrane fractions were prepared as described (Fukuoka
et al., 2005). Five milligram per millilitre of membrane proteins was solubilized in each condition. In the presenceof Na+, solubilization was carried out in 20TNPDG [20 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 mM PMSF, 1 mM DTT,
10% (w/v) glycerol] containing 5 mM imidazole and 2.5%(w/v) Chaps {3-[(3-cholamidopropyl)-dimethylammonio]-1propanesulphonate}. Ni-NTA agarose with his-tagged proteins bound was washed with the same buffer containing 1%
Chaps, and the associated proteins were eluted with 750 mlof 20TNPDG containing 200 mM imidazole and 1% Chaps.
For the Na+-free condition, stator proteins were isolated in thesame way, but we used 20TKPDG [20 mM Tris-HCl (pH 8.0),
150 mM KCl, 0.5 mM PMSF, 1 mM DTT, 10% (w/v) glycerol]
instead. Five hundred millilitres of eluted proteins was loaded
on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with 20TNPDG or 20TKPDG containing 1% Chaps,and run at a flow rate of 0.5 ml min-1 with the appropriate
buffer respectively. Eluted proteins in fractions (500 ml each)were precipitated with trichloroacetic acid, washed withacetone, and then analysed by SDS-PAGE followed byimmunoblotting. Thyroglobulin (669 kDa), Ferritin (440 kDa),
Aldolase (158 kDa) and Conalbumin (75 kDa) were used forsize markers.

Immunoblotting whole cell extracts
Measurement of motility
Cells were harvested by centrifugation and were suspended
TMN0, TMN10, TMN50, TMN100, TMN300 and TMN500.
Cells were harvested by centrifugation and resuspended in
the same medium. Cell suspensions were diluted 100-fold inthe same motility medium and incubated for 15 min. Finally, a
1 M serine solution was added to a final concentration of
20 mM to suppress changes in swimming direction. Cell
motility was observed by dark-field microscopy and recorded
on a videotape. Swimming speed was determined as
described (Atsumi et al., 1996).

DpomAB cells producing GFP-PomB variants and PomA
were grown in VPG500 containing 0.006% arabinose at
30°C for 4 h. Cells were harvested by centrifugation. Cell
pellets were suspended in SDS-loading buffer containing
b-mercaptoethanol. Samples were separated by SDS-PAGE
and proteins were detected with anti-PomB93 and antiPomA1312 antibodies.

Acknowledgements
We thank Y. Sowa for useful comments and K.M. Thormann
for exchanging unpublished results of a submitted paper. This

© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835

834 H. Fukuoka et al. 䊏work was supported in part by grants-in-aid for scientificresearch from the Japan Society for the Promotion of Science(to M.H. and S.K.); from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (to A.I.); and fromthe Japan Science and Technology Agency (to M.H., S.K. andA.I.).

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© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 71, 825–835