doi:10.1016/j.jmb.2005.06.037

J. Mol. Biol. (2005) 351, 707–717

Assembly of Motor Proteins, PomA and PomB, in the
NaC-driven Stator of the Flagellar Motor
Hajime Fukuoka, Toshiharu Yakushi, Akiko Kusumoto andMichio Homma*
Division of Biological Science
Graduate School of Science
Nagoya University, Chikusa-Ku
Nagoya 464-8602, Japan

PomA and PomB are transmembrane proteins that form the stator complex
in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed
to surround the rotor part of the flagellar motor. We constructed andobserved green fluorescent protein (GFP) fusions of the stator proteins
PomA and PomB in living cells to clarify how stator proteins are assembled
and installed into the flagellar motor. We were able to demonstrate that
GFP-PomA and GFP-PomB localized to a cell pole dependent on thepresence of the polar flagellum. Localization of the GFP-fused stator
proteins required their partner subunit, PomA or PomB, and the
C-terminal domain of PomB, which has a peptidoglycan-binding motif.
Each of the GFP-fused stator proteins was co-isolated with its partner
subunit from detergent-solubilized membrane. From these lines ofevidence, we have demonstrated that the stator proteins are incorporated
into the flagellar motor as a PomA/PomB complex and are fixed to the cellwall via the C-terminal domain of PomB.
q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: GFP; membrane protein; energy transduction; sodium motive
force; Vibrio

Introduction
Many bacteria can swim by rotating their helicalflagellar filament in a liquid environment. The
bacterial flagellum consists of a flagellar filament,a hook, and a basal body. The helical filament acts as
a propeller and is connected to the hook that serves
as the universal joint connecting the filament andbasal body. The basal body is a part of the flagellar
motor embedded in the cytoplasmic membrane.1
The torque of flagellar rotation is generated by theinteraction of the rotor and stator parts of thismotor. The energy source for flagellar motor
rotation is the electrochemical potential of either
protons or sodium ions across the cell membrane;these driving ions define the two major subtypes offlagellar motor.2,3 The flagellar motor of Escherichia
coli is proton-driven,4 whereas the flagellar motor ofalkalophillic Bacillus and the polar flagellar motor of
Vibrio species are sodium-driven.5–7 It was reported
recently in Bacillus subtilis that the distinctive stators

Abbreviation used: GFP, green fluorescent protein.
E-mail address of the corresponding author:
g44416a@cc.nagoya-u.ac.jp

for the different ion-coupling, sodium ion or proton,
are functional in a bacterial cell.8
In Gram-negative bacteria, the basal body of theflagellum consists of several rings, termed L-ring,
P-ring, MS-ring, and C-ring, which are embeddedin the outer membrane, the peptidoglycan layer, thecytoplasmic membrane, and attached on thecytoplasmic surface of the MS-ring, respectively.
The rod structure pierces these rings.1 During
flagellar construction, FliF proteins first assemble
to form the MS-ring in the cytoplasmic membrane.9
The C-ring, which consists of FliG, FliM, and FliN,
is constructed together with an export apparatus on
the cytoplasmic surface of the MS-ring.10–14 Components of the rod and hook are transported by thisexport apparatus and assembled onto the periplasmic side of the MS-ring. Finally, flagellinmolecules are exported and self-assemble to makeup the flagellar filament. These axial flagellar
proteins are transported through the central
channel of the flagellum.15,16 FlgH and FlgI, whichare L-ring and P-ring proteins, are transported into
the periplasm by the Sec machinery.17
MotA and MotB, components of proton-driven
flagellar motor of E. coli, or PomA and PomB,
components of the sodium-driven flagellar motorof Vibrio alginolyticus, are referred to as stator0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

708

Assembly of Stator Components in Flagellar Motorproteins, because MotB and PomB have peptidoglycan-binding motifs in their C-terminal regions
through which the motor is anchored to the cellwall.18,19 MotA and MotB, or PomA and PomB,
which are the four transmembrane and one
transmembrane proteins, are predicted to form acomplex with 4A:2B stoichiometry and to function
as a specific ion channel.18,20–23 The putative ionbinding site is located in the transmembranesegment of the B subunit.18,24 The stator complex
is thought to interact electrostatically with FliG, oneof the rotor components, to generate torque for theflagellar motor.25 Stator and rotor components ofthe sodium-driven and proton-driven types arethought to work in a similar way,26 althoughcharged residues critical for flagellar rotation
appear to be different between the Vibrio motor
and the E. coli motor, or additional charged residues
may be required in the Vibrio motor.27,28
A circular array of particles surrounding a larger
particle was observed in freeze-fracture images ofcytoplasmic membrane in E. coli; these particles arethought to represent stator complexes incorporated
into flagellar motor.29 The stator proteins areincorporated into the motor in the last step offlagellar assembly. The system of flagellar gene
expression is hierarchical and tightly regulatedon the transcriptional, translational, and posttranslational levels. This hierarchy was classified
into three classes by the expression order, and thestator genes are encoded as the last class, and theirprotein products are produced after the completionof hook–basal body assembly or rotor construction.30–34 The stator proteins can be installed and arefunctional even after the completion of the entire
flagellar structure.35,36 However, the knowledge on
the process of incorporation of the stator proteins
into the flagellar motor is very limited. In this study,
we developed green fluorescent protein (GFP)fusion constructs of the stator proteins PomA and
PomB, and observed them to clarify the localizationof stator proteins in the living cells and how stator
proteins are assembled and incorporated into theflagellar motor. We demonstrated that PomA and
PomB were localized to the cell pole that is on thesame side as the polar flagella generation, and theymust interact with each other to be localized
properly. We showed that the flagellar structure
and C-terminal domain of PomB are required forthe localization.

GFP-PomB, respectively. The genes encoding the
GFP-fused motor proteins were placed under thecontrol of an arabinose-inducible promoter. They
were expressed in NMB190 and NMB192 cells,
which were isolated previously as pomA and pomB
deletion strains, respectively. To examine thefunction of GFP-PomA and GFP-PomB proteins,
we performed a swarm assay (Figure 1(b)).
NMB190 cells producing GFP-PomA did notswarm at all. On the other hand, NMB192 cells
producing GFP-PomB swarmed slightly. Under
light microscopy, we could detect the swimming
NMB192 cells producing GFP-PomB; however, theirswimming ability was much lower than that ofwild-type cells. The swimming speed and thefraction of the cells producing GFP-PomB were
17 mm/s and 26%, compared to 75 mm/s and 64%
for cells producing wild-type PomB.
In immunoblot analysis of whole cell extracts,
GFP-PomA and GFP-PomB proteins were detected
at the expected molecular mass values of about
45 kDa and 66 kDa (estimated values from the gel,
50 kDa and 69 kDa, respectively) (Figure 1(c)).
Some GFP-PomB degradation products with apparent sizes of 62 kDa and 32 kDa were detected byanti-GFP antibody (Figure 1(c), arrowheads marked
as *1 and *2). On the other hand, the degradedproducts were not detected by anti-PomA or antiPomB antibodies. GFP-PomB might be marginallyfunctional in the flagellar motor of V. alginolyticus;
however, we could not rule out the possibility that
GFP-PomB proteins were cleaved into functional
PomB proteins.
Cells harvested at mid-log phase were observedby fluorescence microscopy as described inMaterials and Methods. In NMB190 cells producing
GFP-PomA, a fluorescent dot was observed at thecell pole (Figure 2, GA) in medium containing
0.006% (w/v) arabinose. Similarly, a fluorescent dot
at the cell pole was observed in NMB192 cells
producing GFP-PomB (Figure 2, GB). Such fluorescent dots were observed in about 50% of cells
expressing GFP-PomA or GFP-PomB at either of thepoles. In a small number of cells, fluoresent dots
were observed at both poles. When both strains
were made to express GFP alone, which is a soluble
protein, the fluorescence was diffuse throughout thecell (Figure 2, GFP).

Results

It has been shown that PomA interacts with
PomB to form a complex, and that the PomA/PomB
complex functions as a sodium ion channel in thebacterial cytoplasmic membrane.21,22 To examine
whether the localization of GFP-fused stator proteins to a cell pole requires their partner subunit,
this fusion protein was produced in NMB191,
which is a pomA/pomB deletion strain. When GFPPomA or GFP-PomB was produced in a pomA or apomB deletion strain, fluorescent dots were detected
at cell poles as shown above. On the other hand,

Subcellular localization of GFP-PomA and
GFP-PomB
To investigate the localization of PomA and
PomB proteins, which are the polar flagellar motor
stator components of V. alginolyticus, we constructed expression systems for GFP-fused PomA
and PomB proteins (Figure 1(a)). These N-terminalfused constructs were designated GFP-PomA andPolar localization of the stator requires both
PomA and PomB

Assembly of Stator Components in Flagellar Motor

709

Figure 2. Subcellular localization of GFP-fused stator
proteins. Cells producing GFP-fused stator proteins(abbreviated as in Figure 1) or GFP were grown at 30 8C
for four hours in medium containing 0.006% (w/v)
arabinose and were observed under a fluorescencemicroscope as described in Materials and Methods.

Figure 1. (a) Plasmid constructs for GFP-fused stator
genes. All gfp-stator genes are under the control of thearaBAD promoter in pBAD33. Dark gray and light grayboxes indicate the regions of gfp and pomA or pomB,

respectively. BDC indicates the pomB gene in which the
C-terminal domain is deleted (PomBDC). GFP and PomA
are linked by five glycine residues (denoted as g5), and
GFP and PomB are fused without a linker. A hexahistidine tag (his6) was fused to the N terminus of each
GFP-fused protein. The checked box following the gfppomB gene indicates the transcriptional terminator. The
plasmids were designed to encode GFP-PomA (GA),GFP-PomA and PomB (GA/B), GFP-PomA and PomBDC(GA/BDC), GFP-PomB (GB), GFP-PomB and PomA
(GB/A), and GFP-PomBDC and PomA (GBDC/A).
(b) Swarming behavior in cells expressing GFP-fusedstator genes. NMB190 (DpomA) cells producing GFPPomA (GA), NMB192 (DpomB) cells producing GFPPomB (GB), and NMB191 (DpomAB) cells producing GFPor PomA and PomB (PomA/PomB) were grown in 0.25%(w/v) soft agar medium containing 0.01% (w/v) arabinose at 30 8C for seven hours. (c) Immunoblot of GFPfused stator proteins in whole cell extracts. Proteins weredetected with an anti-PomA1312 antibody, anti-PomB93
antibody, or anti-GFP antibody. GA* indicates GFP-PomA
produced in NMB190 cells and GB* indicates GFP-PomB
produced in NMB192 cells, and all other proteins (GFP,
GFP-fused stator proteins, PomA, and PomB) were
produced in NMB191 cells. Cells were grown at 30 8C
for four hours in medium containing 0.01% arabinose.
Arrowheads marked as *1 and *2 indicate degradedproducts detected by anti-GFP antibody. Molecular mass
values (kDa) are shown on the left or right side of thesepanels.

710

Figure 3. Subcellular localization of GFP-fused stator
proteins (abbreviated as in Figure 1) in a pomA/pomB
deletion strain. Cells were grown at 30 8C for four hours in
medium containing 0.006% (w/v) arabinose.

when GFP-PomA was produced alone in the pomA/
pomB double mutant, fluorescent dots were notdetected at the cell poles, but some fluorescentclusters were observed throughout the cell (Figure 3,
GA). Similarly, GFP-PomB was produced alone in
the pomA/pomB double mutant, fluorescent dots
were not detected at the cell poles and fluorescence
was diffused all over the cell (Figure 3, GB). When
GFP-PomA or GFP-PomB was produced together
with their wild-type partner subunit from the same
plasmid in the pomA/pomB deletion strain, thefluorescent dots were localized at the cell poles(Figure 3, GA/B and GB/A).
By immunoblot analysis, we detected a similar
amount of GFP-PomA protein in the pomA andpomA/pomB deletion strains (Figure 1(c), lanes GA*
and GA). A similar amount of GFP-PomB protein
was detected in the pomB and pomA/pomB deletionstrains (Figure 1(c), lanes GB* and GB). These
results suggest that the polar localization of PomA
and PomB requires their partner subunit.
Polar localization of stator depends on flagellar
assembly
To investigate whether the fluorescent dot
colocalizes with flagellar structures, the polar
flagellum was observed using a primary antibody
against the polar flagellum and a secondary antirabbit IgG conjugated with rhodamine. As shown in
Figure 4(a), the fluorescent dots from GFP-fusedstator proteins and the polar flagellum wereobserved at the same pole. This was true for 84%(GFP-PomA) and 77% (GFP-PomB), respectively, of
all cells that exhibited a fluorescent spot. Only asmall percentage possessed either a fluorescent dot

Assembly of Stator Components in Flagellar Motor

Figure 4. Polar localization of stator proteins dependenton flagellar assembly. (a) Co-localization of the polar
flagellum and GFP-fused stator proteins. GFP-PomA wasproduced in NMB190 (DpomA) cells (upper panels), and
GFP-PomB was produced in NMB192 (DpomB) cells(lower panels). Cells were treated with anti-polar flagellar
antibody and rhodamine-conjugated anti-rabbit IgG
antibody and the fluorescence of rhodamine and GFP
was observed as described in Materials and Methods.
(b) Subcellular localization of GFP-fused stator proteins ina non-flagellated strain. NMB196 (DfliF) cells producing
GFP-PomA and wild-type PomB (left), and those
producing GFP-PomB and wild-type PomA (right).
Cells were grown at 30 8C for four hours in medium
containing 0.006% (w/v) arabinose.

at a pole without a polar flagellum, or a fluorescent
dot localized opposite to the flagellar pole (Table 1).
To investigate whether localization of GFP-PomA
or GFP-PomB depends on the presence of the polar
flagellum, each of the GFP-fused stator proteins wasproduced in a non-flagellated strain, NMB196,
which is the fliF deletion mutant.37 FliF is acomponent of the MS-ring, which is thought to be
the first assembled component in the flagellar
construction.9 It has been shown that flagellar
genes are regulated hierarchically, and that thelate products of flagellar synthesis are not produced
in basal body mutants. 30–34 To rule out theTable 1. Cells containing fluorescent dots at the flagellated poles (%)

GA(DpomA)
GB(DpomB)

Flagellatedpole

Non-flagellated pole

Non-flagellated cells

84
77

0
5

16
18

The percentage of cells containing a fluorescent dot co-localizing
with the polar flagellum, localizing to the opposite pole, or either
pole of non-flagellated cells is given as a fraction of all cells with afluorescent dot.

Assembly of Stator Components in Flagellar Motor

possibility that PomA and PomB proteins (encodedas part of the late class of genes) were not producedin the fliF mutant, either GFP-PomA and wild-type
PomB, or GFP-PomB and wild-type PomA were
produced from a single plasmid under control of
the arabinose promoter. In this non-flagellated
strain (the fliF mutant), GFP-PomA was localized
diffusely and appeared to form some clusters
throughout the cell, and GFP-PomB was diffuse allover the cell. Neither GFP-PomA nor GFP-PomB
localized at cell poles (Figure 4(b)). Immunoblot
analysis revealed similar amounts of GFP-PomA or
GFP-PomB protein as well as their wild-type
partners in the pomA/pomB deletion and nonflagellated strains (data not shown). These results
indicate that PomA and PomB are being produced
in a fliF mutant, and their polar localization of thestator proteins requires the presence of the polar
flagellum or at least some of its components.
Inhibition of the motor rotation by the GFP-fusedstator proteins
GFP-fused stator proteins were produced with
their partner subunit in VIO5 cells, which have awild-type polar flagellum. In a soft agar platecontaining no arabinose, strains harboring a711
plasmid encoding either GFP-PomA and PomB, or
GFP-PomB and PomA swarmed as well as the cells
harboring GFP alone (Figure 5(a), left). On the other
hand, in a soft agar plate containing 0.02%
arabinose, the swarming ability of the cells producing GFP-fused stator proteins was reduced incomparison with that of the cells producing GFP
alone (Figure 5(a), right). The motile fraction of
VIO5 cells producing GFP-PomA and PomB grownin liquid medium containing 0.02% arabinose was
39% versus 66% without arabinose. The reduced
swarming ability, which probably derived from thedefect of motor rotation, suggests that a fraction of
GFP-fused proteins had been incorporated into thestator complex.
In most VIO5 cells producing GFP-PomA and
PomB with 0.006% arabinose induction, the fluorescent signal was diffuse throughout the cell,although a small number of cells had fluorescentdots at the cell poles (Figure 5(b), GA/B). Similarly,
in VIO5 cells producing GFP-PomB and PomA, thefluorescence was diffuse in the entire cell and polar
dots were observed rarely (Figure 5(b), GB/A). In
medium containing 0.02% arabinose, fluorescentdots were not observed in VIO5 cells producing
GFP-fused stator proteins; rather, strong fluorescence was observed all over the cell (data notshown). This high background expression may
have obscured the fluorescent dots at the cell pole.
Polar localization of stator proteins requires the
C-terminal domain of PomB
MotB of E. coli and PomB of V. alginolyticus have
peptidoglycan-binding motifs in their C-terminal
domains, and it has been proposed that the stator isfixed to the cell wall via this motif.18,19 It has beenshown that a PomB protein missing this C-terminal
domain, designated PomBDC, can form a complex
with PomA, but it is not functional and does nothave a dominant effect on the wild-type strain.38 In
order to investigate the effect of the C-terminal
domain of PomB on the polar localization of stator
proteins, either GFP-PomA and PomBDC, or

Figure 5. Effects of GFP-PomA and GFP-PomB on
swarming and subcellular localization in wild-type cells.
(a) VIO5 cells (pomC) harboring the plasmid encoding
GFP-fused stator proteins (abbreviated as in Figure 1)
were swarmed at 30 8C for six hours on soft agar plates
containing 0% or 0.02% (w/v) arabinose. Nonmotile
NMB191 cells producing GFP were inoculated in thecenter of the plates. (b) Subcellular localization of GFPfused stator proteins (abbreviated as in Figure 1) in wildtype cells. Cells were grown at 30 8C for four hours in
medium containing 0.006% (w/v) arabinose.

Figure 6. Effect of the C-terminal deletion of PomB on
the polar localization of GFP-fused stator proteins. Cells
producing GFP-PomA together with PomBDC (left), andwild-type PomA together with GFP-PomBDC (right) weregrown at 30 8C for four hours in medium containing (GA/
BDC) 0.01% (w/v) arabinose or(GBDC/A) 0.006% (w/v)
arabinose.

712
GFP-PomBDC and PomA were produced in the
pomA/pomB deletion strain. In both cases, the GFP
signals were diffuse and appeared to form someclusters in the entire cell region, without localizing
to the cell poles (Figure 6). This suggests that the
C-terminal region of PomB is necessary for the polar
localization of the stator complex.
Next, we confirmed the interaction between
GFP-PomA and wild-type PomB or PomBDC, andbetween wild-type PomA and GFP-PomB or
GFP-PomBDC. Six histidine residues were added
to the N termini of each GFP-fused stator protein.
After isolation of the membrane fraction andsolubilization of the GFP-fused stator protein with
Chaps {3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate}, the His-tagged GFP-fusedstator proteins were bound to Ni-NTA agaroseand eluted. Both wild-type PomB and PomBDC
co-eluted with GFP-PomA (Figure 7, left panels).
The signal intensities of wild-type and deletion
PomB were similar. Wild-type PomA co-eluted with
GFP-PomBDC as well as GFP-PomB (Figure 7, rightpanels). These results indicate that GFP-PomA and
PomB or PomBDC, and PomA and GFP-PomB or
GFP-PomBDC interact with each other andprobably can form complexes in the cytoplasmic
membrane. Some degraded products were detectedby anti-PomA and anti-PomB antibodies (arrowheads marked as *1 and *2), and degradation
products corresponding to monomeric PomA and
PomBDC were detected (arrowheads marked as *3
and *4).

Discussion
The bacterial flagella contain a rotary motor

Assembly of Stator Components in Flagellar Motordriven by the electrochemical potential of ions.
Motor rotation is generated by the interaction of thestator part with the rotor part, thus converting theenergy of ion flux into mechanical power. A circulararray of particles surrounding a larger particle wasobserved in freeze-fracture images of cytoplasmic
membrane in E. coli; these particles are thought torepresent stator complexes composed of MotA and
MotB.29 However, the stator parts or Mot proteins
were not associated with the rotor structure evenunder the mild condition used to isolate the C-ringwith the rotor structure.39 PomA and PomB, which
are the stator components of the sodium-driven
flagellar motor of V. alginolyticus, were likewisefound not to be associated with the purified rotor
structure (H. Terashima, T.Y. & M.H., unpublishedresults).
In the present study, to investigate the assemblyof the stator proteins PomA and PomB, weconstructed GFP-fused PomA and PomB proteins
to observe the proteins in living cells. GFP-PomB
rescued the motility of the pomB deletion mutant,albeit weakly, whereas GFP-PomA did not rescue
the motility of the pomA deletion mutant at all(Figure 1(b)). Stator proteins (PomA and PomB in
V. alginolyticus or MotA and MotB in E. coli) arethought to form a complex containing fourmolecules of subunit A and two molecules ofsubunit B,21–23 which then interacts electrostaticallywith FliG, one of the components of the rotor
structure, via the cytoplasmic surface of subunit
A.25 Given this stoichiometry of the motor complex,
the GFP-PomA/PomB complex and the PomA/
GFP-PomB complex should contain four and two
GFP molecules, respectively, in their cytoplasmic
surfaces. These GFP-fused stator complexes probably have little or no ability to interact with the rotor

Figure 7. Western blots showing
the interaction between PomA and
PomB. The GFP-fused stator
complex was prepared from
NMB191 cells as described inMaterials and Methods. PomA/B
indicates his-tagged PomA andwild-type PomB, and control indicates wild-type PomA and PomB
produced in NMB191 cells. PomA
and PomB variants were detectedwith anti-PomA1312 and antiPomB93 antibodies. The 45 kDaband marked by an arrowheadwith his-PomA* in the lane of
PomA/B is believed to be thedimer of his-PomA as observed
previously.50 The 45 kDa bands
detected in the lanes of GA/B and
GA/BDC are GFP-PomA marked
as GA with an arrowhead. The
arrowheads marked as GB and GBDC indicate GFP-PomB and GFP-PomBDC, respectively. T, total membrane; S,solubilized fraction; W, washed fraction; E, eluted fraction. Arrowheads marked with *1 and *2 indicate degradation
products that are 7 kDa shorter than the full-length GFP-fused stator proteins, and arrowheads marked with *3 and *4
indicate monomer-sized degradation products from GFP-PomA and GFP-PomBDC.

713

Assembly of Stator Components in Flagellar Motorbecause of steric interference by the GFP moleculeson the cytoplasmic surface of the stator.
In the present study, we found that GFP-PomA
and GFP-PomB proteins were localized at the cell
poles (Figure 2). The fluorescent dot co-localizedwith the base of polar flagellum, and this localization was dependent on the presence of the polar
flagellum. From these lines of evidence, we
speculate that the fluorescent dots probably represent the stator complex surrounding the rotor
complex. It has been postulated that eight to 12
stator units consisting of Pom or Mot complexes
surround each rotor structure.29,35,36 If this is true,
the polar-localized fluorescent dot may correspondto the fluorescence from about 40 (GFP-PomA) or 20(GFP-PomB) GFP molecules. These GFP-fusedstator proteins conferred an inhibition for the
motor function of wild-type cells when theexpression levels were increased by an inducer. By
inducing the expression of GFP-fused stator
proteins with a low concentration of arabinose(0.006%), we could observe fluorescent dots
localized at the poles of wild-type cells. However,the number of the wild-type cells that had polarlocalized fluorescent dots was much smaller than
the total number of pomA/pomB deletion cells that
had polar-localized fluorescent dots, and thefluorescence was diffuse throughout the cell in thewild-type. The wild-type PomA/PomB complexes
seemed to occupy most of the flagellar motors inwild-type cells. The GFP-fused stator complexes areprobably much less able than the wild-type stator
complexes to incorporate themselves into themotor. Polar localization of the GFP-fused stator
proteins required the presence of their partner
subunit. GFP-PomA or GFP-PomB without PomB
or PomA, respectively, did not localize to the cell
poles, and the resulting fluorescence was diffuse
throughout the cell; GFP-PomA and GFP-PomB
were likely spread out across the entire cell
membrane. We speculate that stator proteins areincorporated into the flagellar motor as a unit of thestator complex.
Prior reports have suggested that MotA and
MotB form a stable pre-assembly complex andincorporate themselves into the flagellar motor
through electrostatic interactions between a rotor
protein and MotA, fixing the stator complex to thecell wall through the C-terminal domain of MotB.40
We demonstrated that the polar localization of GFPfused stator proteins requires the C-terminal
domain of PomB, which contains a putative
peptidoglycan-binding motif. This domain is notnecessary for association of PomA and PomB, but isessential for flagellar motor function, and the
C-terminal-deleted PomB protein does not have adominant effect on the wild-type strain.38 Thus, it islikely that the stator complex lacking the C-terminal
domain of PomB is not incorporated into theflagellar motor. The PomA/PomB complexes areprobably fixed to the cell wall or the othercomponents via the C-terminal domain of PomBand stably remain near the rotor structures. In Vibrio

species, MotX and MotY are the essential components for the flagellar rotation. MotY has apeptidoglycan-binding motif similar to PomB,41
and MotX is thought to form a complex in theouter membrane with MotY. 42,43 We showedrecently that MotX interacts with the PomA/
PomB complex.44 In a motX or motY mutant strainof V. alginolyticus, the number of the cells that hadpolar localized GFP-fused stator proteins was
reduced; furthermore, the GFP-fused stator
proteins were little localized in the pomA/pomB/
motX/motY deletion strain of V. cholerae, themotility of which is recovered by the genes of
V. alginolyticus (unpublished results). MotX andMotY might be involved in the stabilization,
localization, or incorporation of the PomA/PomB
complex.
Several models for the polar localization of
membrane proteins have been proposed.45,46 We
have shown that the chemoreceptors of E. coli forma cluster at the cell pole with some Che proteins.47,48
The chemoreceptors are localized on the lateralcellular surface in a helical pattern similar to the
Sec machinery (D. Shiomi, M. Yoshimoto &I. Kawagishi, personal communication). The
chemoreceptors might be inserted into the cytoplasmic membrane by the Sec complexes and bedirected to the cell pole. Alternatively, IcsA, an
actin-nucleating protein of Shigella, is targeted
directly to the membrane of the old pole, andlaterally diffused IcsA molecules are digested by theaction of a specific protease.49 In this study, we
found that localization of stator proteins to a cellpole in V. alginolyticus was dependent on thepresence of the polar flagellum, but also that the
GFP-fused stator proteins appeared to be inserted
into the cytoplasmic membrane even in the flagelladefective strain. The stator proteins are produced
after the completion of hook–basal body assemblyor rotor construction,31–34 and can be incorporated
into the flagellar motor even after the whole
flagellum is assembled.35,36 This suggests that thestator proteins should be able to recognize theflagellar structure. The stator complex might beinserted into the cytoplasmic membrane at first andmove through the membrane randomly. Finally, thestator complex might be incorporated into theflagellar motor through its interaction with therotor.

Materials and Methods
Bacterial strains, growth conditions, and media
Bacterial strains are listed in Table 2. V. alginolyticus wascultured at 30 8C in VC medium (0.5% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.4% (w/v) K2HPO4,
3% (w/v) NaCl, 0.2% (w/v) glucose) or in VPG500
medium (1% (w/v) polypeptone, 0.4% (w/v) K2HPO4,
500 mM NaCl, 0.5% (w/v) glycerol). E. coli was cultured
in LB broth (1% (w/v) tryptone peptone, 0.5% (w/v)
yeast extract, 0.5% (w/v) NaCl). Chloramphenicol

714

Assembly of Stator Components in Flagellar Motor

Table 2. Bacterial strains and plasmids
Strain or plasmid
V. alginolyticus
VIO5
NMB190NMB191NMB192
NMB196
Plasmid
pSU21
pSU41
pBAD24
pBAD33
pTrcHisB
pYA301
pMK101
pYA303
pSK603
pTY200
pHFGA
pJN227
pJN152
pHFGAB
pHFGAB2
pHFGBA2
pHFGBA3
pHFA1
pHFA2
pHFAB

Description
Rifr, PofC, LafK
Rifr, PofC, LafK DpomA
Rifr, PofC, LafK DpomAB
Rifr, PofC, LafK DpomB
Rifr, PofK, LafK DfliF
Cmr, Plac, lacZa
Kmr, Plac, lacZa
Apr, PBAD
Cmr, PBAD
Apr, Ptrc
pomA in pSU41
pomA in pSU21
pomA and pomB in pSU41
pomB in pSU41
his6-gfp in pBAD33
his6-gfp-pomA in pBAD33
his6-gfp-pomB in pBAD33
his6-pomA and pomB in pSU41
his6-gfp-pomA and pomB in pBAD33
his6-gfp-pomA and pomBDC in pBAD33
his6-gfp-pomB and pomA in pBAD33
his6-gfp-pomBDC and pomA in pBAD33
pomA in pBAD24
pomA in pBAD33
pomA and pomB in pBAD33

Source or reference
41
52
50
26
37
53
53
54
54
Invitrogen
51
55
51
51
37
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work

Rifr, rifampicin-resistant; Kmr, kanamycin-resistant; Cmr, chloramphenicol-resistant; Apr, ampicillin-resistant; Plac, lac promoter; PBAD,
araBAD promoter; Ptrc, trc promoter; PofC, normal polar flagellar formation; LafK, defective in lateral flagellar formation.

was added to a final concentration of 2.5 mg/ml for
V. alginolyticus and 25 mg/ml for E. coli. For swarming
assay, overnight cultures were spotted onto 0.25% agarVPG500 plates containing 2.5 mg/ml of chloramphenicol
and appropriate concentrations of arabinose.
Plasmids
Plasmids and primers are listed in Tables 2 and 3,
respectively, and the plasmid constructs are shown in
Figure 1(a). Plasmids carrying motor genes under thecontrol of the araBAD promoter are pBAD33-based
plasmids. Fragments encoding pomA were amplified by
PCR using the sense primer BsrGI-5G-PomA and theantisense primer PomA-XbaI, and this fragment wasinserted into the BsrGI and XbaI sites of pTY200DH (theHindIII site in the multi-cloning site was deleted); thisplasmid was designated pHFGA. pHFA1 was constructed by inserting the EcoRI/XbaI fragment from
pMK101 (containing pomA gene) into the EcoRI/XbaIsite of pBAD24. The EcoRV/SalI fragment from pHFA1was inserted into the EcoRV/SalI sites of pBAD33DH (theHindIII site in the multi-cloning site was deleted); thisplasmid was designated pHFA2. A PCR fragment of
pomB, which was amplified using pYA303 as a template, auniversal primer, and the antisense-primer PomB-XbaI,
was inserted into the HindIII (located in the open reading
frame of pomA) and XbaI sites of pHFGA and pHFA1, andthe resulting plasmids were designated pHFGAB and
pHFAB, respectively. The XbaI/SacI fragment frompSK603 was inserted into the NheI/SacI sites of pTrcHisB
to create the plasmid pTricHisB-PomB. A DNA fragment
containing the pomB open reading frame was prepared by
PCR using pTricHisB-PomB as a template together withthe sense primer BsrGI-PomB and the pTrc-reverse
primer as the antisense primer. The resulting fragment
was cloned into pTY200 digested with BsrGI and HindIII,
and the resulting plasmid was designated pJN227 andcarried gfp-pomB. The plasmid pHFGBA2 carrying gfppomB and pomA were constructed as follows. To remove
the transcriptional terminator following the pomB coding
region, a DNA fragment including the pomB was
prepared by PCR using pJN227 as a template together
with the sense primer BsrGI-PomB and the antisense
primer PomB-SacI; the resulting fragment was re-cloned
into the BsrGI and SacI sites of pJN227. The BamHIdigested fragment containing of pomA gene of pMK101

Table 3. Primers
PrimerBsrGI-5G-PomA
PomA-XbaIPomB-XbaI
BsrGI-PomB
PomB-SacIPomBDC-XbaI
PomBDC-SacI

Sequence
5 0 -GCTGTACAAGGGAGG CGGAGGCGGAATGGATTTAGCAACC CTATTAG-3 0
5 0 -GCTCTAGATTACTCG TCAATCTCAAGGGC-3 0
5 0 -GCTCTAGAGCCTAGT TATTGAATTACCGGC-3 0
5 0 -GTCTGTACAAGGATG ATGAAGATAACAAAT GCG-3 0
5 0 -AGCGAGCTCGCCTAG TTATTGAATTACCGG C-3 0
5 0 -GCTCTAGATTATGCA CCTTTTTCACGCATA C-3 0
5 0 -AGCGAGCTCTTATGC ACCTTTTTCACGCAT AC-3 0

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Assembly of Stator Components in Flagellar Motor

was inserted into the BglII site of the resultant plasmid;
this new plasmid was designated pHFGBA2. A pomBfragment missing the C-terminal domain was amplified
using pYA303 as a template, the universal reverse primer,
and the PomB-DC-XbaI primer, and was cloned into the
HindIII and XbaI sites of pHFGAB; the resulting plasmid
was designated pHFGAB2. A pomB fragment missing the
C-terminal domain was amplified using pJN227 as the
template together with the primers BsrGI-PomB and
PomB-DC-SacI, and the resulting fragment was cloned
into the BsrGI and SacI sites of pHFGBA2; this plasmid
was designated pHFGBA3.
Detection of PomA and PomB proteins in Vibrio cells
Vibrio cells harboring plasmids encoding the stator
proteins were cultured at 30 8C for four hours in VPG500
medium in the presence of arabinose. The cells were
harvested by centrifugation and suspended in distilledwater to an A660 of 10. The same volume of SDS loading
buffer was added to the cell suspensions and boiled at
100 8C for five minutes. The proteins were separated by
SDS-PAGE and immunoblotting was performed using ananti-PomA antibody (PomA1312) and anti-PomB antibody (PomB93) as described.50
Detection of interaction between PomA and PomB
Vibrio cells harboring the plasmids coding the stator
proteins were cultured at 30 8C for four hours in VC
medium containing 0.02% arabinose. Six histidine
residues were added to the N terminus of each GFPfused stator protein. Cells were harvested by centrifugation, washed with V buffer (25 mM Tris–HCl (pH 7.5),
300 mM NaCl, 10 mM MgCl2), and resuspended in
20TMPD (20 mM Tris–HCl (pH 8.0), 5 mM MgSO4,
0.5 mM PMSF, 1 mM DTT) containing 5 mg/ml of DNase
I. The cells were disrupted by sonication. After unbroken
cells were removed by low-speed centrifugation (10,000g
for five minutes at 4 8C), the membrane was recovered byultracentrifugation (150,000g for one hour at 4 8C). The
membrane pellet was homogenized with 20TPD (20 mM
Tris–HCl (pH 8.0), 0.5 mM PMSF, 1 mM DTT) containing
20% (w/v) glycerol, and stored at K80 8C. The membranehomogenate was mixed with 20TNPD (20 mM Tris–HCl(pH 8.0), 150 mM NaCl, 0.5 mM PMSF, 1 mM DTT)
containing 10% (w/v) glycerol, 5 mM imidazole, and 2.5%(w/v) Chaps to a final total protein concentration of
5 mg/ml and incubated for ten minutes on ice. The
solubilized samples were centrifuged at 150,000g for
30 minutes at 4 8C. Ni-NTA agarose was added to thesupernatant, and the mixture was incubated for one hour
at 4 8C. The Ni-NTA agarose was washed by centrifugation three times with 20 TNPD containing 10% (w/v)
glycerol, 5 mM imidazole, and 1% (w/v) Chaps at 4 8C.
The associated proteins were eluted by 20TNPD containing 10% (w/v) glycerol, 200 mM imidazole, and 1% (w/v)
Chaps at 4 8C. The same volume of SDS loading buffer wasadded to the eluted solutions and boiled at 100 8C for fiveminutes. The proteins were separated by SDS-PAGE andimmunoblotting was performed using anti-PomA antibody(PomA1312) and anti-PomB antibody (PomB93) as above.
Observation of the subcellular localization of
GFP-PomA and GFP-PomB
Vibrio cells harboring plasmids encoding GFP-fusedproteins were grown at 30 8C for four hours in VPG500

medium containing arabinose. A sample (1 ml) of
cultured cells were harvested by centrifugation andsuspended in 200 ml of TMN500 (50 mM Tris–HCl(pH 7.5), 5 mM MgCl, 5 mM glucose, 500 mM NaCl).
A small aliquot of the cell suspension was spotted onto a
glass slide, and cells were fixed on the slide with an equalvolume of 0.5% agarose dissolved in TMN500. Cells wereobserved by fluorescence microscopy (Olympus. BX50).
The images were recorded and processed using a digital
camera (Hamamatsu Photonics, C4742-95) and imaging
software (Hamamatsu Photonics, AQUA-Lite, Ver. 1.0).
For observation of the polar flagellum, the cell suspension
was placed on the slide covered with a glass cover-slip
with spacers, washed with TMN500 twice, and the antipolar flagellum antibody and anti-rabbit IgG conjugatedwith rhodamine were applied. After washing with
TMN500, cells attached to the cover-slip were observed.
Measurement of swimming speed
Cells grown as described above were harvested andsuspended in TMN500. The cell suspensions were diluted
100-fold into TMN500 containing 20 mM serine, which
was used to suppress changes in swimming direction.
Cell motility was observed under a dark-field microscopeand recorded on videotape. Swimming speed wasdetermined as described.51 The average swimming
speed was obtained by measuring at least 20 swimming
tracks. The swimming fraction was measured from thesame videotape.

Acknowledgements
This work was supported, in part, by grants-inaid for scientific research from the Ministry of
Education, Science, and Culture of Japan; the Japan
Science and Technology Corporation (to M.H. andT.Y.); the Soft Nano-Machine Project of Japan
Science and Technology Agency (to M.H. andT.Y.); and the Strategic International Cooperative
Program of Japan Science and Technology Agency(to H.F.).

References
1. Macnab, R. M. (2003). How bacteria assemble flagella.
Annu. Rev. Microbiol. 57, 77–100.
2. McCarter, L. L. (2001). Polar flagellar motility of theVibrionaceae. Microbiol. Mol. Biol. Rev. 65, 445–462.
3. Yorimitsu, T. & Homma, M. (2001). NaC-driven
flagellar motor of Vibrio. Biochim. Biophys. Acta, 1505,
82–93.
4. Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M.
& van der Drift, C. (1977). A proton motive force
drives bacterial flagella. Proc. Natl Acad. Sci. USA, 74,
3060–3064.
5. Hirota, N., Kitada, M. & Imae, Y. (1981). Flagellar
motors of alkalophilic Bacillus are powered by anelectrochemical potential gradient of NaC. FEBS
Letters, 132, 278–280.
6. Chernyak, B. V., Dibrov, P. A., Glagolev, A. N.,
Sherman, M. Y. & Skulachev, V. P. (1984). A novel

716
type of energetics in a marine alkali-tolerant bacterium:
NaC-driven motility and sodium cycle. FEBS Letters,
164, 38–42.
7. Atsumi, T., McCarter, L. & Imae, Y. (1992). Polar andlateral flagellar motors of marine Vibrio are driven bydifferent ion-motive forces. Nature, 355, 182–184.
8. Ito, M., Hicks, D. B., Henkin, T. M., Guffanti, A. A.,
Powers, B. D., Zvi, L. et al. (2004). MotPS is the statorforce generator for motility of alkaliphilic Bacillus, andits homologue is a second functional Mot in Bacillus
subtilis. Mol. Microbiol. 53, 1035–1049.
9. Kubori, T., Shimamoto, N., Yamaguchi, S., Namba, K.
& Aizawa, S. (1992). Morphological pathway offlagellar assembly in Salmonella typhimurium. J. Mol.
Biol. 226, 433–446.
10. Fan, F., Ohnishi, K., Francis, N. R. & Macnab, R. M.
(1997). The FliP and FliR proteins of Salmonella
typhimurium, putative components of the type III
flagellar export apparatus, are located in the flagellar
basal body. Mol. Microbiol. 26, 1035–1046.
11. Francis, N. R., Sosinsky, G. E., Thomas, D. & Derosier,
D. J. (1994). Isolation, characterization and structure ofbacterial flagellar motors containing the switch
complex. J. Mol. Biol. 235, 1261–1270.
12. Katayama, E., Shiraishi, T., Oosawa, K., Baba, N. &Aizawa, S. (1996). Geometry of the flagellar motor in
the cytoplasmic membrane of Salmonella typhimurium
as determined by stereo-photogrammetry of quickfreeze deep-etch replica images. J. Mol. Biol. 255,
458–475.
13. Kubori, T., Okumura, M., Kobayashi, N., Nakamura,D., Iwakura, M. & Aizawa, S. I. (1997). Purificationand characterization of the flagellar hook-basal bodycomplex of Bacillus subtilis. Mol. Microbiol. 24, 399–410.
14. Minamino, T. & Macnab, R. M. (1999). Components of
the Salmonella flagellar export apparatus and classification of export substrates. J. Bacteriol. 181, 1388–1394.
15. Macnab, R. (1996). Flagella and motility. In Escherichia
coli and Salmonella (Neidhardt, F. C., ed.), pp. 123–
145, American Society for Microbiology, Washington,
DC.
16. Namba, K., Yamashita, I. & Vonderviszt, F. (1989).
Structure of the core and central channel of bacterial
flagella. Nature, 342, 648–654.
17. Homma, M., Komeda, Y., Iino, T. & Macnab, R. M.
(1987). The flaFIX gene product of Salmonella
typhimurium is a flagellar basal body component
with a signal peptide for export. J. Bacteriol. 169,
1493–1498.
18. Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi,
I. & Homma, M. (1997). Putative channel components
for the fast-rotating sodium-driven flagellar motor ofa marine bacterium. J. Bacteriol. 179, 5104–5110.
19. De Mot, R. & Vanderleyden, J. (1994). The C-terminal
sequence conservation between OmpA-related outermembrane proteins and MotB suggests a common
function in both gram-positive and gram-negative
bacteria, possibly in the interaction of these domains
with peptidoglycan. Mol. Microbiol. 12, 333–334.
20. Blair, D. F. & Berg, H. C. (1990). The MotA protein of
E. coli is a proton-conducting component of theflagellar motor. Cell, 60, 439–449.
21. Sato, K. & Homma, M. (2000). Functional reconstitution of the NaC-driven polar flagellar motor
component of Vibrio alginolyticus. J. Biol. Chem. 275,
5718–5722.
22. Yorimitsu, T., Kojima, M., Yakushi, T. & Homma, M.

Assembly of Stator Components in Flagellar Motor(2004). Multimeric structure of the PomA/PomB
channel complex in the NaC-driven flagellar motorof Vibrio alginolyticus. J. Biochem. 135, 43–51.
23. Kojima, S. & Blair, D. F. (2004). Solubilization andpurification of the MotA/MotB complex of Escherichia
coli. Biochemistry, 43, 26–34.
24. Kojima, S. & Blair, D. F. (2001). Conformationalchange in the stator of the bacterial flagellar motor.
Biochemistry, 40, 13041–13050.
25. Zhou, J. D., Lloyd, S. A. & Blair, D. F. (1998).
Electrostatic interactions between rotor and stator in
the bacterial flagellar motor. Proc. Natl Acad. Sci. USA,95, 6436–6441.
26. Asai, Y., Yakushi, T., Kawagishi, I. & Homma, M.
(2003). Ion-coupling determinants of NaC-driven and
HC-driven flagellar motors. J. Mol. Biol. 327, 453–463.
27. Yorimitsu, T., Sowa, Y., Ishijima, A., Yakushi, T. &Homma, M. (2002). The systematic substitutions
around the conserved charged residues of thecytoplasmic loop of NaC-driven flagellar motor
component PomA. J. Mol. Biol. 320, 403–413.
28. Fukuoka, H., Yakushi, T. & Homma, M. (2004).
Concerted effects of amino acid substitutions inconserved charged residues and other residues in
the cytoplasmic domain of PomA, a stator componentof NaC-driven flagella. J. Bacteriol. 186, 6749–6758.
29. Khan, S., Dapice, M. & Reese, T. S. (1988). Effects ofmot gene expression on the structure of the flagellar
motor. J. Mol. Biol. 202, 575–584.
30. Prouty, M. G., Correa, N. E. & Klose, K. E. (2001). The
novel s54- and s28-dependent flagellar gene transcription hierarchy of Vibrio cholerae. Mol. Microbiol. 39,
1595–1609.
31. Kim, Y. K. & McCarter, L. L. (2000). Analysis of thepolar flagellar gene system of Vibrio parahaemolyticus.
J. Bacteriol. 182, 3693–3704.
32. Komeda, Y., Kutsukake, K. & Iino, T. (1980). Definitionof additional flagellar genes in Escherichia coli K12.
Genetics, 94, 277–290.
33. Komeda, Y. (1986). Transcriptional control of flagellar
genes in Escherichia coli K-12. J. Bacteriol. 168,
1315–1318.
34. Kutsukake, K., Ohya, Y. & Iino, T. (1990). Transcriptional analysis of the flagellar regulon of Salmonella
typhimurium. J. Bacteriol. 172, 741–747.
35. Blair, D. F. & Berg, H. C. (1988). Restoration of torquein defective flagellar motors. Science, 242, 1678–1681.
36. Block, S. M. & Berg, H. C. (1984). Successive
incorporation of force-generating units in the bacterial
rotary motor. Nature, 309, 470–472.
37. Yorimitsu, T., Mimaki, A., Yakushi, T. & Homma, M.
(2003). The conserved charged residues of the
C-terminal region of FliG, a rotor component of the
NaC-driven flagellar motor. J. Mol. Biol. 334, 567–583.
38. Yakushi, T., Hattori, N. & Homma, M. (2005). Deletion
analysis of the carboxyl-terminal region of the PomB
component of the Vibrio alginolyticus polar flagellar
motor. J. Bacteriol. 187, 778–784.
39. Driks, A. & DeRosier, D. J. (1990). Additional
structures associated with bacterial flagellar basal
body. J. Mol. Biol. 211, 669–672.
40. VanWay, S. M., Hosking, E. R., Braun, T. F. & Manson,
M. D. (2000). Mot protein assembly into the bacterial
flagellum: a model based on mutational analysis of
the motB gene. J. Mol. Biol. 297, 7–24.
41. Okunishi, I., Kawagishi, I. & Homma, M. (1996).
Cloning and characterization of motY, a gene coding
for a component of the sodium-driven flagellar motorin Vibrio alginolyticus. J. Bacteriol. 178, 2409–2415.

Assembly of Stator Components in Flagellar Motor

42. Okabe, M., Yakushi, T., Asai, Y. & Homma, M. (2001).
Cloning and characterization of motX, a Vibrio
alginolyticus sodium-driven flagellar motor gene. J.
Biochem. 130, 879–884.
43. Okabe, M., Yakushi, T., Kojima, M. & Homma, M.
(2002). MotX and MotY, specific components of thesodium-driven flagellar motor, colocalize to the outermembrane in Vibrio alginolyticus. Mol. Microbiol. 46,
125–134.
44. Okabe, M., Yakushi, T. & Homma, M. (2005).
Interactions of MotX with MotY and with the
PomA/PomB sodium ion channel complex of theVibrio alginolyticus polar flagellum. J. Biol. Chem, 280,
25659–25664.
45. Shapiro, L., McAdams, H. H. & Losick, R. (2002).
Generating and exploiting polarity in bacteria.
Science, 298, 1942–1946.
46. Lybarger, S. R. & Maddock, J. R. (2001). Polarity inaction: asymmetric protein localization in bacteria. J.
Bacteriol. 183, 3261–3267.
47. Homma, M., Shiomi, D., Homma, M. & Kawagishi, I.
(2004). Attractant binding alters arrangement ofchemoreceptor dimers within its cluster at a cellpole. Proc. Natl Acad. Sci. USA, 101, 3462–3467.
48. Banno, S., Shiomi, D., Homma, M. & Kawagishi, I.
(2004). Targeting of the chemotaxis methylesterase/
deamidase CheB to the polar receptor-kinase cluster
in an Escherichia coli cell. Mol. Microbiol. 53, 1051–1063.
49. Robbins, J. R., Monack, D., McCallum, S. J., Vegas, A.,
Pham, E., Goldberg, M. B. & Theriot, J. A. (2001). The

717
making of a gradient: IcsA (VirG) polarity in Shigellaflexneri. Mol. Microbiol. 41, 861–872.
50. Yorimitsu, T., Sato, K., Asai, Y., Kawagishi, I. &Homma, M. (1999). Functional interaction between
PomA and PomB, the NaC-driven flagellar motor
components of Vibrio alginolyticus. J. Bacteriol. 181,5103–5106.
51. Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I. &Homma, M. (1999). NaC-driven flagellar motor
resistant to phenamil, an amiloride analog, caused
by mutations of putative channel components. J. Mol.
Biol. 285, 1537–1547.
52. Asai, Y., Kawagishi, I., Sockett, E. & Homma, M.
(1999). Hybrid motor with the HC-and NaC-driven
components can rotate Vibrio polar flagella by using
sodium ions. J. Bacteriol. 181, 6322–6338.
53. Bartolome, B., Jubete, Y., Martı́nez, E. & de la Cruz, F.
(1991). Construction and properties of a family ofpACYC184-derived cloning vectors compatible with
pBR322 and its derivatives. Gene, 102, 75–78.
54. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J.
(1995). Tight regulation, modulation, and high-level
expression by vectors containing the arabinose pBAD
promoter. J. Bacteriol. 177, 4121–4130.
55. Kojima, S., Kuroda, M., Kawagishi, I. & Homma, M.
(1999). Random mutagenesis of the pomA geneencoding the putative channel component of the
NaC-driven polar flagellar motor of Vibrio
alginolyticus. Microbiology, 145, 1759–1767.

Edited by J. Karn
(Received 4 May 2005; received in revised form 13 June 2005; accepted 15 June 2005)
Available online 29 June 2005