THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 8, Issue of February 25, pp. 5718 –5722, 2000
Printed in U.S.A.

Functional Reconstitution of the Naⴙ-driven Polar Flagellar
Motor Component of Vibrio alginolyticus*(Received for publication, October 27, 1999, and in revised form, November 30, 1999)

Ken Sato‡ and Michio Homma
From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, JapanThe bacterial flagellar motor is a molecular machinethat couples the influx of specific ions to the generationof the force necessary to drive rotation of the flagellar
filament. Four integral membrane proteins, PomA,
PomB, MotX, and MotY, have been suggested to be directly involved in torque generation of the Naⴙ-driven
polar flagellar motor of Vibrio alginolyticus. In the present study, we report the isolation of the functional component of the torque-generating unit. The purified protein complex appears to consist of PomA and PomB andcontains neither MotX nor MotY. The PomA/B protein,reconstituted into proteoliposomes, catalyzed 22Naⴙ influx in response to a potassium diffusion potential. Sodium uptake was abolished by the presence of Liⴙ ionsand phenamil, a sodium channel blocker. This is the first
demonstration of a purification and functional reconstitution of the bacterial flagellar motor component involved in torque generation. In addition, this study demonstrates that the Naⴙ-driven motor component, PomA
and PomB, forms the Naⴙ-conducting channel.

Flagella are the filamentous organelles responsible for bacterial motility. Flagellar rotation is driven by a reversible rotary motor embedded in the cytoplasmic membrane at the baseof each flagellar filament (1–3). Energy for rotation of theflagellar motor comes from the transmembrane electrochemical
potential of specific ions. Two types of motors, proton-driven (2)and sodium-driven (4), dependent on different coupling ions,have been described. The proton-driven motors of Escherichia
coli and Salmonella typhimurium have been extensively studied, and the stator part of the torque generator consists of twocytoplasmic membrane proteins, MotA and MotB, which contain four transmembrane domains and one transmembrane
domain, respectively (5– 8). Much genetic and physiologicalevidence suggests that MotA and MotB together form a proton
channel (9 –13), and this complex is believed to be anchored tothe cell wall via the peptidoglycan-binding domain of MotB (8,14, 15). Ions passing through these proteins are thought togenerate torque (16), which is transmitted to the rotor part ofthe motor, the FliG protein (17, 18). FliG forms a complex
together with FliM and FliN (19, 20) called the “switch complex,” which is essential for torque generation, flagellar assembly, and control of the direction of motor rotation (21–23).
Bacteria such as alkalophilic Bacillus and Vibrio species use
an electrochemical gradient of sodium to drive flagellar rota-* This study was supported by grants from the Ministry of Education,
Science, Sports and Culture of Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Tel.: 81-52-7892992; Fax: 81-52-789-3001; E-mail: m47004a@nucc.cc.nagoya-u.ac.jp.

tion (4). The sodium-driven motor has advantages for the studyof motor function because sodium-motive force can be easily
manipulated. The specific sodium channel blockers, amiloride
and phenamil, are powerful tools for studying the mechanismof energy conversion in this system (24, 25). Four proteins
essential for torque generation, PomA, PomB, MotX, and MotY,
were recently identified in the polar flagellar motor of Vibrio
alginolyticus (26, 27). PomA and PomB are homologous to
MotA and MotB and contain four transmembrane segments
and one transmembrane segment, respectively. MotX and
MotY, first identified in Vibrio parahaemolyticus (28, 29), havea single putative transmembrane domain that is unique to thesodium-type motor. MotY has a peptidoglycan-binding motif at
the carboxyl-terminal region that is also observed in MotB and
PomB. Evidence suggests that MotX is part of the sodium
channel component of the motor. Overexpression of MotX is
lethal to E. coli in proportion to the external sodium ion concentration. Lethality is reversed in the presence of amiloride(29). Although the above four components are thought to forma sodium channel, no direct evidence has been demonstrated sofar.
In the present study, we report the isolation and reconstitution of the torque-generating unit of the Na⫹-driven polar
flagellar motor component of V. alginolyticus as a stage in its
biochemical characterization. The complex, purified via Histagged PomA, includes both PomA and PomB and has an
approximate molecular size of 175 kDa. In addition, reconstituted PomA/B proteoliposomes exhibit potassium diffusion potential-driven 22Na⫹ uptake that is blocked by Li⫹ ions andphenamil. These results suggest that the heteromultimericcomplex, consisting of PomA and PomB, acts as a sodium
channel that generates the torque to drive flagellar rotation.
MATERIALS AND METHODS

Bacterial Strains, Plasmids, Growth Conditions, and Media—V. alginolyticus strains NMB190 (Rifr, Pof⫹, Laf⫺, ⌬pomA) (30) and
NMB191 (Rifr, Pof⫹, Laf⫺, ⌬pomA, ⌬pomB) (31) were used and cultured
at 30 °C 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 VPG
medium (1% (w/v) polypeptone, 0.4% (w/v) K2HPO4, 3% (w/v) NaCl,
0.5% (w/v) glycerol). For the swarm assay, a VPG/0.3% agar plate wasused. E. coli strain JM109 (recA1, endA1, gyrA96, thi⫺, hsdR17, relA1,
supE44, ␭⫺, ⌬(lac-proAB); F⬘, traD36, proAB, lacIq, ⌬M15) was used for
DNA manipulations and cultured at 37 °C in LB medium. When necessary, kanamycin was added to a final concentration of 100 ␮g/ml for
Vibrio cells or 25 ␮g/ml for E. coli cells. Plasmid pKS101, a pSU41based plasmid, was constructed to carry his6-pomA under the lac promoter control. A 0.8-kilobase DNA fragment including the pomA open
reading frame with a 5⬘ attachment of 5⬘-ATTGGATCCATGCATCACCATCACCATCACATGGATTTAGCAACCCTATTA-3⬘ (initiates as MetHis6; the underline indicates the created (BamHI and EcoT22I) restriction sites) was originally prepared by polymerase chain reaction andcloned into BamHI-digested pSU41. Plasmid pKS105, encoding thetandem PomA, was generated by engineering an EcoT22I site at the 3⬘
end of the pomA clone, and then linked to the 5⬘ end of his6-pomA via
EcoT22I. The final linking sequence was Met-His6 (one methionine and5718

This paper is available on line at http://www.jbc.org

Reconstitution of the PomA/B Complex
six histidines), which was added at the junction. All the total inserts
were confirmed by DNA sequencing.
Purification of PomA/B Complex—Cells of NMB190 harboring
pKS101 were cultured at 30 °C under strong aeration in VPG medium.
Cells were harvested and washed with buffer (20 mM Tris-Cl, pH 8.0, 5mM MgSO4, 10% (w/v) sucrose) and stored at ⫺80 °C until use. The
frozen cells were thawed, resuspended (0.2 g/ml, wet weight) in 20 mM
Tris-Cl, pH 8.0, containing 1 mM dithiothreitol, 5 mM MgSO4, 30 ␮g/ml
DNase I, and 0.5 mM phenylmethylsulfonyl fluoride. Membrane vesicles
were prepared by subjecting the suspension to a single passage througha French press (5501-M Ohtake Works) at 4000 p.s.i. at 4 °C. Undisrupted cells were removed by low speed centrifugation (10,000 ⫻ g for
20 min at 4 °C), and the membrane fraction was recovered from thesupernatant by centrifugation at 200,000 ⫻ g for 2 h. The membranepellet was suspended in Buffer A (20 mM Tris-Cl, pH 8.0, 150 mM NaCl,
1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20% (w/v)
glycerol) containing 5 mM imidazole, pH 8.0, and 2.5% (w/v) ␤-octylglucoside. The suspension was stirred for 30 min at room temperatureand centrifuged for 20 min at 10,000 ⫻ g. The clarified extract was
mixed with Ni-NTA1-agarose (Qiagen) prewashed with the same buffer,
incubated at 4 °C for 1 h with gentle mixing, and then packed into thecolumn. The loaded resin was washed with Buffer A containing 40 mM
imidazole, pH 8.0, and 1.25% (w/v) ␤-octylglucoside. Elution was conducted with Buffer A containing 500 mM imidazole, pH 8.0, and 1.25%(w/v) ␤-octylglucoside. The Ni-NTA-purified material was diluted 5-foldwith Buffer B (20 mM Tris-Cl, pH 8.0, 20% (w/v) glycerol, 1.25% (w/v)
␤-octylglucoside) and applied to a MiniQ column (Amersham Pharmacia Biotech) equilibrated with Buffer B. The column was washed withBuffer B, and bound material was eluted with a 0 – 800 mM linear
gradient of KCl in Buffer B. Fractions were analyzed by immunoblotting with anti-PomB antibody. Peak fractions were combined and concentrated by a MiniQ column. PomA/B complex was eluted from thecolumn at a salt concentration of ⬃220 mM. The concentrated solution
was applied on a Superose 6 gel filtration column (Amersham Pharmacia Biotech) equilibrated with Buffer B containing 100 mM KCl. The
peak fractions containing both His6-PomA and PomB were pooled andstored at ⫺80 °C. Purification of His6-PomA alone from NMB191(⌬pomAB) strain harboring pKS101 was performed essentially as described above. Protein concentrations were assayed by the BCA protein
assay reagent (Pierce).
Reconstitution of Proteoliposomes—Proteoliposomes were reconstituted by the octylglucoside dilution method. A sample of purified
PomA/B complex (8.0 ␮g) or His6-PomA (4.7 ␮g) was mixed with 5.0 mg
of E. coli phospholipids (Avanti Polar Lipids) in 20 mM Tris-Cl, pH 8.0,
200 mM KCl, 1.25% (w/v) ␤-octylglucoside, 10% (w/v) glycerol. The
mixture (100 ␮l) was sonicated briefly, incubated on ice for 20 min, andrapidly diluted (40-fold) into the dilution buffer (20 mM Tris-Cl, pH 8.0,
200 mM KCl). After 15 min at room temperature with gentle shaking,the proteoliposomes formed were recovered by centrifugation at
200,000 ⫻ g for 1 h, resuspended in 100 ␮l of dilution buffer, frozen in
dry ice/ethanol, and stored at ⫺80 °C. When assayed, the suspension
was thawed at room temperature.
Orientation of the PomA/B Complex in the Proteoliposomes—Inverted membrane vesicles were prepared as described above. Preparation of spheroplasts of V. alginolyticus was performed as described
previously (44). Membranes (60 ␮l) were treated with 15 ␮l of Proteinase K at the indicated concentrations on ice for 30 min. The samples
were precipitated with trichloroacetic acid, washed with acetone, andthen analyzed by SDS-PAGE, followed by immunoblotting with antiPomB antibody (31).
Determination of 22Na⫹ Uptake by Proteoliposomes—The standard
incubation mixture contained 500 ␮l of the following at 30 °C: 20 mM
Tris-Cl, pH 8.0, 200 mM choline chloride, 0.5 mM 22NaCl (4 ␮Ci/ml), andproteoliposomes loaded with 200 mM KCl (50 ␮l). The mixture wasallowed to equilibrate for 5 min. Inhibitors were added, and after a
1-min incubation, a membrane potential was applied by adding 20 ␮Mvalinomycin. At intervals, 90 ␮l of the reaction mixture was filteredthrough a nitrocellulose filter (0.2-␮m pore size, Toyo Roshi Co. Ltd.)
with suction and washed three times with 1 ml of 20 mM Tris-Cl buffer,
pH 8.0, containing 200 mM choline chloride. The radioactivity trappedon the filter was determined by a ␥-counter.

1
The abbreviations used are: NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis.

5719

FIG. 1. Purification of the torque-generating unit. The protein
pattern at the different stages of the purification procedure is visualizedby 15% SDS-PAGE and by staining with Coomassie Brilliant Blue.
Lane 1, detergent extract (45 ␮g) of V. alginolyticus membrane; lane 2,
eluant resulting from the membrane fraction subjected to Ni-NTA chromatography (20 ␮g); lane 3, further purification by MiniQ anion exchange chromatography (11 ␮g); lane 4, combined eluant fractions
containing both PomA and PomB after Superose 6 chromatography(9 ␮g).

RESULTS

Isolation of the Torque-generating Unit of the Polar Flagellar
Motor from V. alginolyticus—We isolated the torque-generating unit of the polar flagellar motor from V. alginolyticus basedon the features characterized for PomA protein; at least it
forms a functional complex with PomB protein in the cytoplasmic membrane (31). We constructed a plasmid, pKS101, that
encodes PomA with an attached hexahistidine sequence on theamino terminus of the protein, His6-PomA. The plasmid,
pKS101, complemented a pomA null mutant; these results
were similar to those observed with pYA301, which encodeswild-type PomA. In addition, the swimming speed of the cells
harboring pKS101 was similar to that of wild-type cells (datanot shown). Thus, the attachment of the hexahistidine tag does
not interfere with PomA function. Membrane fractions of
NMB190 transformed with pKS101 were prepared, solubilizedwith ␤-octylglucoside (Fig. 1, lane 1), and passed through a
Ni-NTA-agarose column. The agarose column was washed, andbound protein was eluted with the buffer containing imidazole(Fig. 1, lane 2). The eluate from the Ni-NTA-agarose resin wasapplied to a MiniQ anion exchange column and eluted with asalt gradient. The eluate was analyzed by immunoblots using
antibodies generated against PomA and PomB, and fractions
that contained both PomA and PomB were collected (Fig. 1,
lane 3). Finally, using a Superose 6 column, most other proteins
were quantitatively removed, and PomA was eluted together
with PomB but not with either MotX or MotY (Fig. 1, lane 4).
Although His6-PomA is overexpressed in the cells, the protein
complex purified above contains only endogenous PomB. Coomassie Brilliant Blue staining intensities of PomA and PomB
bands of the complex separated by SDS-PAGE were measured
by densitometry. The ratio of bound dye was 1.0:0.55 (PomA:
PomB), and taking the molecular size of each protein into
account, a calculated ratio of PomA and PomB in the complex
was found to be 2.3:1.0 (PomA:PomB). Thus, it seems that theapparent molar ratio of PomA/B in the purified complex is 2

5720

Reconstitution of the PomA/B Complex

FIG. 2. Orientation of PomA/B in cells and liposomes. Treatment of spheroplasts, inverted membrane vesicles (IMV) from NMB190(⌬pomA) cells harboring pKS101, and proteoliposomes (PL) with Proteinase K was performed as described under “Materials and Methods.”
As controls, spheroplasts, inverted membrane vesicles, and proteoliposomes were lysed with Triton X-100 before the protease treatment.
Immunoblot was done with antibody generated against the carboxylterminal epitope of PomB. Samples that had not been treated withProteinase K were also examined.

PomA:1 PomB, although the exact estimation necessitates
more detailed investigation.
Reconstitution of the PomA/B Complex into Proteoliposomes—Proteoliposomes were prepared from purified PomA/B
and E. coli phospholipids by the detergent dilution method (32,33). To assess whether the PomA/B complex was properly reconstituted in the liposomes, we compared its topological orientation in liposomes to that of PomA/B in spheroplasts (Fig.
2). Treatment of spheroplasts with Proteinase K cleaved thecarboxyl-terminal epitope of PomB. The same cleavage occurred after purified PomA/B complex inserted into liposomes
was treated with Proteinase K. The carboxyl-terminal epitopeof PomB in inverted membrane vesicles was protected fromProteinase K digestion by the membranes. However, lysis by
Triton X-100 of spheroplasts, inverted membrane vesicles, orproteoliposomes resulted in carboxyl-terminal digestion by Proteinase K. A quantitative assessment of the PomB digestion in
spheroplasts and proteoliposomes indicates that at least 80 –90% of the purified PomA/B was inserted into liposomes withthe correct orientation (right side out). We conclude that the
purified PomA/B complexes that were incorporated into liposomes were asymmetrically inserted with the same relativeorientation as in the cytoplasmic membrane.
22
Na⫹ Transport into Reconstituted Proteoliposomes—The
kinetics of 22Na⫹ uptake into reconstituted proteoliposomes
are shown in Fig. 3. When valinomycin was added to potassium-loaded PomA/B liposomes, creating ⌬␺ in response to thepotassium gradient, 22Na⫹ was rapidly translocated to theinside of proteoliposomes. No significant 22Na⫹ uptake wasobserved in control liposomes without PomA/B (data notshown) or PomB, indicating that the PomA/B complex but notvalinomycin is responsible for 22Na⫹ translocation. If the driving force for sodium uptake consisted of only ⌬pNa⫹, the rate of22Na⫹ uptake was very low. These results are indicative of anelectrogenic Na⫹ transport by purified PomA/B complex. The
accumulation of 22Na⫹ ions was effectively prevented by thepresence of 50 ␮M phenamil, which is a specific inhibitor of thesodium-driven flagellar motor (25, 34).
The sodium-driven motor of V. alginolyticus has been shown
to function using lithium ion (35). Sodium translocation into
PomA/B proteoliposomes was effectively inhibited by Li⫹, indicating that Na⫹ and Li⫹ compete at a common binding site on
the protein. These results are the first clear evidence of abacterial flagellar motor component that acts directly as a
primary ion channel.
Molecular Properties of the PomA/B Complex—To determine
the apparent molecular size of the PomA/B complex, we performed gel permeation chromatography by loading purified
PomA/B complex along with several molecular mass standards
onto a Superose 6 column. The PomA/B complex was eluted
between catalase (240 kDa) and aldolase (158 kDa) (Fig. 4).

FIG. 3. Kinetics of 22Naⴙ uptake into reconstituted proteoliposomes containing PomA/B and effect of inhibitors. ⌬␺ was createdas a K⫹ diffusion potential by adding valinomycin to KCl-loaded proteoliposomes. The uptake of 22Na⫹ ions was determined in incubation
mixtures containing the proteoliposomes (2.5 mg of lipids) in a total
volume of 0.5 ml. The addition of valinomycin is marked by the arrow(filled circles). Parallel experiments were performed with incubation
mixtures containing 50 ␮M phenamil (open circles), 10 mM LiCl (opentriangles), and proteoliposomes containing His6-PomA (filled squares).
Also shown is a control without valinomycin (filled triangles). Uptake
experiments were carried out more than five times, and typical data areshown.

FIG. 4. Molecular size of PomA/B complex and PomA estimatedfrom Superose 6 chromatography. The purified PomA/B complex or
His6-PomA was loaded on a Superose 6 column equilibrated with 20 mM
Tris-Cl buffer, pH 8.0, containing 100 mM KCl and 20% (w/v) glyceroland eluted at a flow rate of 0.03 ml/min. The molecular size standards
used are ferritin (450 kDa), catalase (240 kDa), aldolase (158 kDa),
albumin (68 kDa), and cytochrome c (12.5 kDa). The arrows indicate theposition of the retention time of PomA/B (PomA/B) complex and His6PomA (PomA).

Considering the micellar size of ␤-octylglucoside (2.1 kDa), theapparent molecular size of the PomA/B complex was estimatedto be 175 kDa.
We further purified His6-PomA alone from a pomAB nullmutant strain (NMB191) harboring pKS101. When purified
His6-PomA was applied to the Superose 6 column as describedabove, His6-PomA was eluted faster than would be expected for
the PomA monomer (Fig. 4), which has a molecular size of 27
kDa. The estimated molecular size was 55 kDa. This result
suggests that PomA alone exists as a stable homodimer indetergent extract.
To further elucidate the dimeric structure of PomA, we con-

Reconstitution of the PomA/B Complex

5721

FIG. 6. Size exclusion chromatography of His6-PomA (PomA)
and tandem PomA (PomA-PomA) on a Superose 6 column. His6PomA and tandem PomA were briefly purified by a Ni-NTA column andthen applied to a Superose 6 column. The anti-PomA immunoblot
profile of each fraction is shown.
DISCUSSION

FIG. 5. Expression and function of tandem PomA. A, schematic
diagram of a PomA-PomA dimer. Two PomA monomers joined tail-tohead in tandem are expressed as a single polypeptide. The arrow
indicates the position of the connecting site, and the linking sequence is
Met-His6. B, membrane vesicles (10 ␮g of protein each) of NMB190(⌬pomA) harboring a vector plasmid (lane 1), a plasmid encoding His6PomA (lane 2), or tandem PomA (lane 3) were subjected to SDS-PAGE
and immunoblotting with anti-PomA antibody. C, swarming abilities of
NMB190 (⌬pomA) cells expressing no PomA (vector), His6-PomA(PomA), or tandem PomA (PomA-PomA). Overnight cultures were spotted on VPG/0.3% agar plates containing kanamycin and incubated at
30 °C.

structed a plasmid encoding tandems of two PomA subunits
expressed as a single polypeptide (Fig. 5A). Previous studies
suggested that PomA has four membrane spans and that theamino and carboxyl termini must be on the same side of themembrane (26). Hence a tandem complex of two covalently
linked PomA subunits might be expected to assemble into afunctional state. The construction was performed by linking
two pomA open reading frames in frame with six histidines asa linker. As shown in Fig. 5C, NMB190 (⌬pomA) cells expressing the tandem PomA did indeed show ability to swarm in asoft agar plate. Compared with wild-type PomA, NMB190 cells
expressing tandem PomA demonstrated a reduction in swarming ability. Since the carboxyl terminus of Salmonella MotA
has been shown to be important for its activity (36), diminishedswarm ability may be due to restricted flexibility around thecarboxyl terminus of the amino-terminal half of tandem PomA.
To demonstrate that the swarm activity observed is due to theactivity of full-length tandem PomA and not to monomeric
PomA, either from partial translation or proteolytic degradation of PomA fusion, immunoblots were prepared using membrane preparations from NMB190 expressing the tandem fusion protein. As shown in Fig. 5B, tandem PomA migrates ataround 50 kDa. It should be noted that no other immunoreactive species is observed on the immunoblots, particularlyaround 25 kDa, where the monomeric His6-PomA migrates.
The functional tandem fusion PomA was purified by Ni-NTA
resin from the NMB191 (⌬pomAB) strain that expresses tandem PomA by Ni-NTA resin. The molecular size was analyzed
by a Superose 6 column together with Ni-NTA-purified monomeric His6-PomA, and the fractions were examined by immunoblotting with anti-PomA antibody. The purified His6-PomA
was eluted in exactly the same fractions as tandem PomA (Fig.
6). These results strongly support the stable homodimerizationof native PomA.

We have reconstituted the flagellar motor component of V.
alginolyticus into proteoliposomes from pure phospholipids andpurified membrane protein. Our results indicate that the purified PomA/B complex forms a sodium-conducting channel,
whereas purified PomA alone does not. Although we have notexamined the ability of PomB alone to mediate 22Na⫹ translocation, PomB has been shown to be degraded in the absence ofPomA, and the simultaneous expression of both PomA and
PomB is required for the stability of PomB (31). MotX and
MotY, which are also essential components required for torquegeneration in vivo, are apparently not part of the complex. Ourdata, however, do not exclude the possibility of a further stimulatory effect on sodium transport by MotX and MotY, or MotX
and MotY might form a sodium channel independent of the
PomA/B complex that may even be essential for torque generation. The isolation of the MotX and MotY proteins is now inprogress and should help to clarify the function of those components. Can we exclude the possibility that minor contaminants contribute to or even cause the activities ascribed to the
PomA/B complex? On the basis of sodium uptake activity perunit amount of PomA/B complex, proteoliposomes reconstituted from the PomA/B complex, which was purified from thestrains overexpressing both His6-PomA and PomB, were as
active as those reconstituted from the His6-PomA-overproducing strain (data not shown), suggesting that PomA and PomB
were enough to catalyze sodium uptake.
Even though the functional partnership of PomA and PomB
proteins, as well as E. coli MotA and MotB, has been established, information concerning their interactions and molecular stoichiometry is very limited. Indeed, their physical interaction has been demonstrated so far only by a co-sedimentation
analysis (31, 37). An important finding in this study is that theNa⫹-driven flagellar motor components of V. alginolyticus,
PomA and PomB, are purified as a functional complex. The
subunit ratio of purified complex was estimated to be 2 PomA:1
PomB, which is about 89 kDa in size. Considering that theapparent molecular size of the PomA/B complex was 175 kDa,
the native functional complex might consist of four copies of
PomA and two copies of PomB as a single torque-generating
unit, although more detailed investigation is required. Whenthe purified His6-PomA alone was applied to a Superose 6column, His6-PomA was eluted as a stable homodimer, suggesting that an even number of PomA subunits is recruited perfunctional complex. Consistent with the presence of homodimeric PomA, when the purified PomA/B complex was analyzed by a Superose 6 column, the elution profile showed two
peaks corresponding to the PomA/B complex and to dimeric
PomA, which may be dissociated from the complex.
PomA/B reconstituted into liposomes facilitates Na⫹ uptake
in a phenamil-sensitive manner. This finding is consistent with
our previous observation that phenamil interacts directly with
PomA/B (34) and with the closely related bacterium V. parahaemolyticus (38). The present study also shows that Na⫹ uptake was abolished with the presence of Li⫹ ions. This result is5722

Reconstitution of the PomA/B Complex

in good agreement with previous results suggesting that lithium can substitute for sodium (35). These observations further
underscore the complexity of PomA/B pore selectivity. Interestingly, 22Na⫹ uptake appears to be directed by the membranepotential but not by the concentration gradient. This result
suggests that the transport of Na⫹ ions through the PomA/B
complex is an electrogenic event. At present, however, theimportance of this channel property in regard to torque generation remains unclear. The mechanism linking the translocation of ⌬␺-driven Na⫹ ions across the motor component to thegeneration of rotary torque for flagellar rotation may be similar
to the proposed voltage-generated torque generation of theNa⫹-translocating F1F0-ATPase of Propionigenium modestum(39).
Complex membrane events, such as solute transport (40),subreactions of oxidative phosphorylation, protein translocation in bacteria (41, 42) and the endoplasmic reticulum (43),and the membrane fusion step of trafficking (33), have beenfunctionally reconstituted into proteoliposomes, thus allowing
the direct investigation of fundamental cell biological mechanisms. This study demonstrates the availability of a pure,functional flagellar motor component, which can be reconstituted into proteoliposomes able to catalyze ion transport, as an
avenue toward the enzymological resolution of torquegeneration.
Acknowledgment—We thank Dr. Ikuro Kawagishi for critically reading the manuscript.
REFERENCES
1. Berg, H. C. (1995) Biophys. J. 68, (suppl.) 163–167
2. Blair, D. F. (1995) Annu. Rev. Microbiol. 49, 489 –522
3. DeRosier, D. J. (1998) Cell 93, 17–20
4. Imae, Y., and Atsumi, T. (1989) J. Bioenerg. Biomembr. 21, 705–716
5. Dean, G. E., Macnab, R. M., Stader, J., Matsumura, P., and Burks, C. (1984)
J. Bacteriol. 159, 991–999
6. Stader, J., Matsumura, P., Vacante, D., Dean, G. E., and Macnab, R. M. (1986)
J. Bacteriol. 166, 244 –252
7. Zhou, J. D., Fazzio, R. T., and Blair, D. F. (1995) J. Mol. Biol. 251, 237–242
8. Chun, S. Y., and Parkinson, J. S. (1988) Science 239, 276 –278
9. Blair, D. F., and Berg, H. C. (1990) Cell 60, 439 – 449
10. Stolz, B., and Berg, H. C. (1991) J. Bacteriol. 173, 7033–7037
11. Sharp, L. L., Zhou, J. D., and Blair, D. F. (1995) Biochemistry 34, 9166 –9171

12. Garza, A. G., Harrishaller, L. W., Stoebner, R. A., and Manson, M. D. (1995)
Proc. Natl. Acad. Sci. U. S. A. 92, 1970 –1974
13. Garza, A. G., Biran, R., Wohlschlegel, J. A., and Manson, M. D. (1996) J. Mol.
Biol. 258, 270 –285
14. Blair, D. F., Kim, D. Y., and Berg, H. C. (1991) J. Bacteriol. 173, 4049 – 4055
15. De Mot, R., and Vanderleyden, J. (1994) Mol. Microbiol. 12, 333–334
16. Meister, M., Lowe, G., and Berg, H. C. (1987) Cell 49, 643– 650
17. Lloyd, S. A., Tang, H., Wang, X., Billings, S., and Blair, D. F. (1996) J.
Bacteriol. 178, 223–231
18. Zhou, J. D., Lloyd, S. A., and Blair, D. F. (1998) Proc. Natl. Acad. Sci. U. S. A.
95, 6436 – 6441
19. Francis, N. R., Sosinsky, G. E., Thomas, D., and Derosier, D. J. (1994) J. Mol.
Biol. 235, 1261–1270
20. Oosawa, K., Ueno, T., and Aizawa, S. (1994) J. Bacteriol. 176, 3683–3691
21. Yamaguchi, S., Fujita, H., Ishihara, H., Aizawa, S., and Macnab, R. M. (1986)
J. Bacteriol. 166, 187–193
22. Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V., and Macnab, R. M. (1992)
J. Bacteriol. 174, 793– 806
23. Irikura, V. M., Kihara, M., Yamaguchi, S., Sockett, H., and Macnab, R. M.
(1993) J. Bacteriol. 175, 802– 810
24. Sugiyama, S., Cragoe, E. J., Jr., and Imae, Y. (1988) J. Biol. Chem. 263,
8215– 8219
25. Atsumi, T., Sugiyama, S., Cragoe, E. J., Jr., and Imae, Y. (1990) J. Bacteriol.
172, 1634 –1639
26. Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi, I., and Homma, M.
(1997) J. Bacteriol. 179, 5104 –5110
27. Okunishi, I., Kawagishi, I., and Homma, M. (1996) J. Bacteriol. 178,
2409 –2415
28. McCarter, L. L. (1994) J. Bacteriol. 176, 4219 – 4225
29. McCarter, L. L. (1994) J. Bacteriol. 176, 5988 –5998
30. Asai, Y., Kawagishi, I., Sockett, R. E., and Homma, M. (1999) J. Bacteriol. 181,
6332– 6338
31. Yorimitsu, T., Sato, K., Asai, Y., Kawagishi, I., and Homma, M. (1999) J.
Bacteriol. 181, 5103–5106
32. Racker, E., Violand, B., O’Neal, S., Alfonzo, M., and Telford, J. (1979) Arch.
Biochem. Biophys. 198, 470 – 477
33. Sato, K., and Wickner, W. (1998) Science 281, 700 –702
34. Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I., and Homma, M. (1999) J. Mol.
Biol. 285, 1537–1547
35. Liu, J. Z., Dapice, M., and Khan, S. (1990) J. Bacteriol. 172, 55236 –55244
36. Muramoto, K., and Macnab, R. M. (1998) Mol. Microbiol. 29, 1191–1202
37. Tang, H., Braun, T. F., and Blair, D. F. (1996) J. Mol. Biol. 261, 209 –221
38. Jaques, S., Kim, Y. K., and McCarter, L. L. (1999) Proc. Natl. Acad. Sci.
U. S. A. 96, 5740 –5745
39. Kaim, G., and Dimroth, P. (1998) EMBO J. 17, 5887–5895
40. Kaback, H. R. (1988) Annu. Rev. Physiol. 50, 243–256
41. Brundage, L., Hendrick, J. P., Schiebel, E., Driessen, A. J. M., and Wickner, W.
(1990) Cell 62, 649 – 657
42. Akimaru, J., Matsuyama, S., Tokuda, H., and Mizushima, S. (1991) Proc. Natl.
Acad. Sci. U. S. A. 88, 6545– 6549
43. Görlich, G., and Rapoport, T. A. (1993) Cell 75, 615– 630
44. Osborn, M. J., and Munson, R. (1974) Methods Enzymol. 31, 642– 653