Microbiology (2004), 150, 911–920

DOI 10.1099/mic.0.26577-0

Isolation of Vibrio alginolyticus sodium-driven
flagellar motor complex composed of PomA and
PomB solubilized by sucrose monocaprate
Toshiharu Yakushi, Masaru Kojima and Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan

Correspondence
Michio Homma
g44416a@cc.nagoya-u.ac.jp

Received 18 June 2003
Revised 17 November 2003
Accepted 18 December 2003

The polar flagella of Vibrio alginolyticus have sodium-driven motors, and four membraneproteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor. PomA
and PomB are believed to form a sodium-conducting channel. This paper reports the purificationof the motor complex by using sucrose monocaprate, a non-ionic detergent, to solubilize thecomplex. Plasmid pKJ301, which encodes intact PomA, and PomB tagged with a C-terminal
hexahistidine that does not interfere with PomB function, was constructed. The membrane fractionof cells transformed with pKJ301 was solubilized with sucrose monocaprate, and the solubilizedmaterials were applied to a Ni-NTA column. The imidazole eluate contained both PomA and PomB,
which were further purified by anion-exchange chromatography. Gel-filtration chromatography
was used to investigate the apparent molecular size of the complex; the PomA/PomB complex
was eluted as approx. 900 kDa and PomB alone was eluted as approx. 260 kDa. Thesefindings suggest that the motor complex may have a larger structure than previously assumed.

INTRODUCTION
Many bacteria swim by rotating helical flagella in liquid
environments. The torque of flagella is generated by areversible rotary motor embedded in the cytoplasmic
membrane at the base of each flagellar filament (Blair,
1995; Macnab, 1999). The motor is composed of the statorand rotor parts. The energy for torque generation comesfrom the transmembrane electrochemical potential of specificions, sodium ions or protons. The flagellar motors can beclassified into two types: proton-driven and sodium-driven,
which are dependent on different coupling ions (McCarter,
2001; Yorimitsu & Homma, 2001). Although the specificmechanism is unknown, ions are thought to pass throughthe stator complex while the ion flux generates the rotational force between the stator and rotor.
Escherichia coli and Salmonella typhimurium have protondriven motors that have been extensively studied (Berry &Armitage, 1999; Blair, 1995; Derosier, 1998). The stator
complex consists of the cytoplasmic membrane proteins
MotA and MotB, which are essential for torque generation.
MotA has four transmembrane segments and a large cytoplasmic loop (Zhou et al., 1995). MotB has a single transmembrane domain and a peptidoglycan-binding motif(Chun & Parkinson, 1988); there is an Asp residue in theputative transmembrane region, which is speculated to be
protonated during torque generation. The protonation ofAbbreviation: b-octylglucoside, n-octyl b-D-glucopyranoside.

0002-6577 G 2004 SGM

Printed in Great Britain

the Asp residue induces a conformational change of the
motor protein (Kojima & Blair, 2001). MotA and MotB
together have been suggested to form a proton channel,
based on extensive genetic and physiological evidence(Blair & Berg, 1990; Garza et al., 1995, 1996; Sharp et al.,1995a; Stolz & Berg, 1991).
Vibrio alginolyticus has two types of flagella in one cell: alateral flagellum with a proton-driven motor and a polar
flagellum with a sodium-driven motor (Atsumi et al., 1992;
Kawagishi et al., 1995). In the polar flagellar motor, thefollowing four proteins have been identified and found tobe essential for torque generation: PomA, PomB, MotX
and MotY (Asai et al., 1997; Furuno et al., 1999; Okabe
et al., 2001; Okunishi et al., 1996). PomA and PomB are
homologous to MotA and MotB, respectively, of the protondriven motor. Thus it is thought that PomA and PomB
form a complex and have similar function to MotA and
MotB. The direct interaction between PomA and PomB
has been demonstrated (Yorimitsu et al., 1999). PomA was
required for the stability of PomB. PomB also has an
essential charged residue, Asp, which is highly conservedin the transmembrane region of the motor protein. MotX
and MotY were first identified in Vibrio parahaemolyticus(McCarter, 1994a, b), and homologous genes have been
identified only in Vibrio species. We suggest that MotX
and MotY mutually stabilize each other and that MotX
is more directly involved in motor function than MotY
(Okabe et al., 2001). MotY has a peptidoglycan-binding
motif at the C-terminal region. The motif is also observed
911

T. Yakushi, M. Kojima and M. Hommain MotB and PomB. The roles of MotX and MotY havenot always been clear. However, these proteins are locatedin the outer membrane (Okabe et al., 2002) and may beessential for sodium recognition in the motor and/or for
the fast rotation speed of flagella in Vibrio cells (Asai et al.,2003). The maximal rotational speed of the sodium-driven
motor is 1700 revolutions per second, whereas the maximal
rotational speed of the proton-driven E. coli motor is 300
revolutions per second (Yorimitsu & Homma, 2001). The
sodium-driven motor has advantages for the study of motor
function because sodium-motive force can be easily manipulated. Moreover, the specific sodium channel inhibitors,
amiloride and phenamil, can be used to study the mechanism of torque generation in this system (Atsumi et al., 1990;
Sugiyama et al., 1988). Phenamil-resistant mutations were
mapped near the cytoplasmic ends of the putative transmembrane segment of PomA and PomB (Jaques et al., 1999;
Kojima et al., 1999). It is inferred that the high-affinity
phenamil-binding site is located around the interface ofthe cytoplasmic ends of PomA and PomB.
We previously purified the components of the torquegenerating unit, PomA and PomB, which were solubilized
by the non-ionic detergent b-octylglucoside. We showedthat the PomA/PomB complex exhibits significant sodium
uptake activity (Sato & Homma, 2000a). From further
experiments, however, we found that the complex dissociates easily with b-octylglucoside. In the present study,
the membrane-associated motor proteins were solubilized
and isolated by using sucrose monocaprate, another nonionic detergent. We partly characterized the motor complex composed of PomA and PomB, which appears to bemuch larger than previously believed.

METHODS
Bacterial strains, plasmids, growth conditions and media.

The strains and plasmids used are shown in Table 1. V. alginolyticus
cells were cultured at 30 uC 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 theswarm assay, VPG-0?3 % agar plates were used. When necessary,
kanamycin was added to the final concentration of 100 mg ml21 for
V. alginolyticus.
Plasmid construction. Plasmid pKJ201, a pSU41-based plasmid,

was constructed to carry pomB-his6 under the control of the lacpromoter. A 0?9 kb DNA fragment including the pomB open reading
frame with a 39 attachment of 59-GAAGAAGTGCCGGTAATTCAACATCACCATCACCATCACTAAGAGCTCAAG-39 (stop as His6end; the underlining indicates the created SacI restriction sites) was
prepared by PCR. The fragment was cloned into pSK603 (pSU41
carrying the wild-type pomB gene; Kojima et al., 1999) digested by
HpaI and SacI. The total inserts were confirmed by DNA sequencing. The fragment of pKJ201, digested by HpaI and SacI, wasreplaced with the fragment of pYA303 carrying pomA and pomB
under lac promoter control. The resultant plasmid pKJ301, carrying
pomA and pomB-his6, was constructed. Other plasmids used arelisted in Table 1.
Association of PomA and PomB. Cells were harvested andwashed with V-buffer (25 mM Tris/HCl, pH 7?5, 10 mM MgSO4,
300 mM NaCl). The washed cells were suspended in 400 ml
20TMPD (20 mM Tris/HCl, pH 8?0, 5 mM MgSO4, 0?5 mM PMSF
and 1 mM DTT) and mixed with a homogenizer (Polytron, type
PT3000), with the rotation speed set at 10 000 r.p.m. for 2 min.
After adding DNase I to 20 mg ml21, membrane vesicles were prepared by subjecting the suspension to a single passage through a
French press (5501-M, Ohtake Works) at 500 kg cm22 at 4 uC.
Undisrupted cells were removed by low-speed centrifugation(10 000 g for 20 min at 4 uC) and the membrane fraction was recovered from the supernatant by centrifugation at 200 000 g for 2 h at

Table 1. Bacterial strains and plasmids
Strain or plasmid
V. alginolyticus
NMB190NMB191NMB192
Plasmids
pSU41
pKS101
pKS102
pSK603
pKJ201
pYA303
pKJ301
pMMB206

Genotype or description*

Reference

Rif r Pof+ Laf 2, DpomA
Rif r Pof+ Laf 2, DpomAB
Rif r Pof+ Laf 2, DpomB

Asai et al. (1999)
Yorimitsu & Homma (2001)
Asai et al. (2003)

kan(Kmr) Plac lacZa
pSU41 0?8 kb BamHI–BamHI (his6-pomA)
pSU41 1?9 kb BamHI–SacI (his6-pomA/pomB)
pSU41 1?1 kb BamHI–SacI (pomB)
pSU41 1?1 kb BamHI–SacI (pomB-his6)
pSU41 1?9 kb BamHI–SacI (pomAB)
pSU41 1?9 kb BamHI–SacI (pomA/pomB-his6)cat(Cmr) IncQ lacIq Dbla Ptac-lac lacZa

Bartolome et al. (1991)
Sato & Homma (2000a)
Sato & Homma (2000a)
Kojima et al. (1999)
This study
Kojima et al. (1999)
This study
Morales et al. (1991)*Cmr, chloramphenicol-resistant; Kmr, kanamycin-resistant; Plac, lac promoter; Ptac-lac, tandemly located tac
and lac promoters; Rif r, rifampicin-resistant; Pof+, polar flagella formation; Laf 2, lateral flagellar formation
defect.
912

Microbiology 150

Purification of the motor complex
4 uC. The membrane pellet was suspended in 20TNPDG (20 mM
Tris/HCl, pH 8?0, 0?15 M NaCl, 0?5 mM PMSF, 1 mM DTT and
10 %, w/v, glycerol). Sucrose monocaprate or b-octylglucoside wasadded to the membrane fraction suspended in 20TNPDG to give
final concentrations of 2?5 % and 10 mg membrane protein ml21.
Two millilitres of the suspension was mixed for 30 min at 4 uC andcentrifuged for 30 min at 100 000 g. Imidazole was added to eachsupernatant at 5 mM final concentration, then the solubilized membrane preparation was mixed with 25 ml Ni-NTA agarose (QIAGEN)
for 1 h at 4 uC. The loaded resin was extensively washed with
20TNPDG containing 5 mM imidazole and 0?5 % sucrose monocaprate or 1?25 % b-octylglucoside. Bound proteins were eluted with
100 ml 20TNPDG containing 400 mM imidazole and 0?5 % sucrose
monocaprate or 1?25 % b-octylglucoside.

resuspended in 100 ml dilution buffer, frozen in dry ice/ethanol andstored at 280 uC. For assays, the suspension was thawed at room
temperature.
The standard incubation mixtures contained the following, in 500 ml,
at 30 uC: (i) 20 mM Tris/HCl, pH 8?0, 200 mM choline chloride,
1 mM 22NaCl (0?4 mCi ml21; 14?8 kBq ml21), and (ii) proteoliposomes loaded with 200 mM KCl (50 ml). Components (i) and (ii) wereseparately incubated at 30 uC for 3 min; they were then mixed andvalinomycin was added at the final concentration of 2 mM to form
a diffusion membrane potential. At intervals, 90 ml of the reaction
mixture was passed through Dowex 50WX8-100 (Sigma) prewashedwith 20 mM Tris/HCl buffer, pH 8?0, containing 200 mM choline
chloride, to trap the unincorporated 22Na+. The radioactivity of theflow-through fraction was determined by a c-counter.

Preparation of the motor complex. A culture of bacteria wasgrown to stationary phase in 18 litres of VC medium. The membrane fraction was prepared as above and suspended in 20TNPDG.
This suspension was homogenized with a Polytron homogenizer andsucrose monocaprate was added to 2 % final concentration. The
suspension was mixed on ice and centrifuged for 20 min at 10 000 g.
After addition of imidazole to 10 mM final concentration, theclarified extract was mixed with Ni-NTA agarose (QIAGEN), incubated at 4 uC for 1 h with gentle mixing, and then packed into thecolumn. The loaded resin was washed with 20TNPDG containing
0?5 % sucrose monocaprate and 10 mM imidazole. Elution was conducted with 20TNPDG containing 0?5 % sucrose monocaprate and
400 mM imidazole. The eluate from the Ni-NTA column was
diluted twofold with 20TPDG (20 mM Tris/HCl, pH 8?0, 0?5 mM
PMSF, 1 mM DTT and 10 %, w/v, glycerol) and applied to a
Q-Sepharose HiTrap Q HP column (Pharmacia) equilibrated with
20TPDG containing 75 mM NaCl. The column was washed with
20TPDG containing 75 mM NaCl and bound material was eluted
with a 75–700 mM linear gradient of NaCl in 20TPDG.
Gel-filtration chromatography. A Superose 6 PC 3.2/30 gel-filtration column (Pharmacia) was equilibrated with 20TNPDG
containing 0?5 % sucrose monocaprate. A 50 ml sample was applied
to the Superose 6 column and eluted with the same buffer as used
for the equilibration, at 30 ml min21 flow. The sample was recovered
as 60 ml fractions 25 min after the sample injection.
Sucrose density-gradient centrifugation. The suspension was

layered on 5–30 % linear sucrose gradient in 20TNPDG containing
0?5 % sucrose monocaprate. Centrifugation was carried out in a
Beckman SW41Ti rotor for 22 h at 24 000 r.p.m. and 6 uC.
SDS-PAGE and immunoblotting. These were performed as pre-viously described (Okabe et al., 2001). The antipeptide antibodies
against PomA and PomB, which are referred to as PomA91 andPomB93, respectively, were prepared as previously described(Yorimitsu et al., 1999).
Amino acid sequence of the N terminus. Proteins separated by

SDS-PAGE were transferred to a PVDF membrane and stained by
Coomassie blue R250. The protein band corresponding to PomB
was excised and analysed with a peptide sequencer (analysis done by
APRO Science Co., Tokushima, Japan).
Na+

22

uptake by proteoliposomes. Proteoliposomes werereconstituted by the detergent dilution method as follows. A sample
was mixed with 5?0 mg E. coli phospholipids (Avanti Polar Lipids)in 20 mM Tris/HCl, pH 8?0, 200 mM KCl, 0?5 %, w/v, sucrose
monocaprate, 10 %, w/v, glycerol. The mixture (100 ml) was sonicated briefly and incubated on ice for 20 min and rapidly diluted(40-fold) into the dilution buffer (20 mM Tris/HCl, pH 8?0,
200 mM KCl). After 15 min at 4 uC with gentle shaking, proteoliposomes formed were recovered by centrifugation at 200 000 g for 1 h,

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RESULTS
Association of PomA with PomB in different
detergentsThe membrane fractions of NMB190 transformed with
pKS101 and NMB191 transformed with pKS102 weresolubilized with b-octylglucoside or sucrose monocaprate.
It seemed that the membrane was more effectively solubilized by sucrose monocaprate because the amount of PomA
and PomB protein detected was greater with this detergent.
The solubilized membrane proteins were passed through a
Ni-NTA agarose column and the column was washed.
Bound proteins were eluted with buffer containing imidazole (Fig. 1, lanes 5). The PomA protein was detected byimmunoblotting after solubilization by detergent. However,the PomB protein was only detected after sucrose monocaprate solubilization, even when both proteins were overproduced from pKS102. This indicates that the complexof PomA and PomB was dissociated by b-octylglucoside.
The amount of PomA recovered was greater with sucrose
monocaprate. This may reflect the efficiency of the solubilization from the membrane fraction.
Construction of PomB-His6 and its function
The binding efficiency of His6-PomA to Ni-NTA agarose
was not good enough to allow suitable recovery of PomA/
PomB complexes, so we tried to make a His-tagged PomB
protein. The hexahistidine sequence was inserted at the
C terminus of PomB, where it was predicted to localizein the periplasmic space or bind to the peptidoglycan. We
constructed plasmid pKJ201, which encodes PomB with anattached hexahistidine sequence (PomB-His6) at the C
terminus. pKJ201 complemented the pomB null mutant;the complementation profile was similar to that observedwith pSK603, which encodes wild-type PomB (Fig. 2). In
addition, the swimming speed of cells harbouring pKJ201
was similar to that of wild-type cells. Thus, the attachmentof the hexahistidine tag does not interfere with PomB
function. This hexahistidine tag was transferred to theplasmid pYA303 carrying pomA and pomB; the resultant
plasmid was named pKJ301. The motility of the pomB
mutant (NMB192) and the pomA pomB mutant (NMB191)
was restored by pKJ301.
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T. Yakushi, M. Kojima and M. Homma

Fig. 1. Stability of the complex solubilized by detergents.
Membrane fractions (lanes 1) of NMB190 transformed with
pKS101 (his6-pomA) and NMB191 transformed with pKS102(his6-pomA pomB) were prepared. Sucrose monocaprate (SC)and b-octylglucoside (OG) were added to the membrane fraction and centrifuged for 30 min at 100 000 g and the unsolublized pellet (lanes 2) and solubilized supernatant (lanes 3)
were recovered. The solubilized membrane was mixed with NiNTA agarose and the loaded resin was washed with 20TNPDG
containing 5 mM imidazole and 0?5 % sucrose monocaprate or
1?25 % b-octylglucoside (lanes 4). Bound proteins were eluted
with 20TNPDG containing 400 mM imidazole and 0?5 %sucrose monocaprate or 1?25 % b-octylglucoside (lanes 5).
Proteins in each elute were separated by SDS-PAGE andimmunoblotting was performed with anti-PomA antibody (a) andanti-PomB antibody (b).

Separation of the motor complex composed of
PomA and PomB
The membrane fractions of NMB191 transformed with
pKJ301 were prepared, solubilized with sucrose monocaprate and applied to a Ni-NTA agarose column. The
agarose column was washed and bound proteins were
eluted with buffer containing imidazole. The PomB protein was detected by staining with Coomassie brilliant
blue but the PomA protein was not. The N-terminal fiveamino acids (MDDED) of the stained protein matched thepredicted sequence of PomB. Both PomA and PomB weredetected by immunoblotting (data not shown).
The eluate from the Ni-NTA agarose resin was applied toan anion-exchange column and eluted with a NaCl gradient. Each fraction was analysed by SDS-PAGE followed
by staining with Coomassie brilliant blue (Fig. 3a) and by
914

Fig. 2. (a) Predicted membrane topology of PomA and PomB.
Thick black lines indicate the predicted transmembrane regions.
The dotted line at the C-terminal region of PomB indicates theadded hexahistidine. (b) Swarming ability by cells encoding
PomB-His6. Swarming abilities of NMB192 (DpomB) cells,
which were co-transformed with pMMB206 (lacIq) plus pSU41(vector), pSK603 (pomB+), or pKJ201 (pomB-his6+) were
examined. Overnight cultures were spotted on VPG-0?3 % agar
plates containing kanamycin, chloramphenicol with (lower
photograph) or without (upper photograph) IPTG and incubatedat 30 6C for 4 h.
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Purification of the motor complex

Fig. 3. Isolation of the PomA/PomB complex. The membrane fraction from NMB191 transformed with pKJ301 (pomA pomBhis6+) was solubilized with sucrose monocaprate and subjected to Ni-NTA chromatography. The eluate was applied to a Q
Sepharose HP anion-exchange column and eluted with a linear gradient from 75 to 700 mM NaCl. The proteins of eachfraction were separated by SDS-PAGE and were detected by Coomassie brilliant blue (a) or by immunoblotting with the antiPomA and the anti-PomB antibodies (b). Fractions 9, 11 and 13 from the anion-exchange chromatography were analysed on
a Superose 6 column, and PomA and PomB in each fraction were detected by immunoblotting with anti-PomA and the antiPomB antibodies (c).
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915

T. Yakushi, M. Kojima and M. Homma

immunoblots using antibodies generated against PomA
and PomB (Fig. 3b). PomB was efficiently recovered. PomA
and PomB mostly co-eluted and the main peaks were at
approximately 0?3 M NaCl. However, the elution profilesof PomA and PomB, at the peak fractions 11 and 10,
respectively, did not completely correspond with each other.
Size-separation of the motor complex
Some of the fractions from anion-exchange chromatography were also analysed by gel-filtration chromatographyusing a Superose 6 column, which has a separation range
from 5 kDa to 5000 kDa (Fig. 3c). A PomA/PomB complex
of high molecular size was recovered when ion-exchange
fraction 11 or 13 was applied to the gel-filtration column.
However, fraction 9, which was eluted from the anionexchange column at a lower concentration of NaCl,contained very little high molecular size complexes. PomA
eluted around fraction 10 of the gel-filtration column.
PomB alone seemed to be eluted at peak fraction 14 in gelfiltration chromatography. Elution profiles of UV absorbance for the gel-filtration chromatography are shown in
Fig. 4. The peaks of the molecular size of fractions 10
and 13 were estimated as about 900 kDa and 260 kDa,
respectively, when soluble globular proteins were used as

Fig. 4. Elution profiles on gel-filtration chromatography. Fraction 11 from the anion-exchange chromatography was mixed
with blue dextran, and applied to the Superose 6 column; theA280 was measured in the eluate. Similarly, samples consistingof pooled fractions 10 and 11 or fractions 13 and 14 from thegel-filtration column used to separate fraction 11 of anionexchange column were analysed again by the same Superose
column without blue dextran. The numbers on the scale above
the UV absorbance trace correspond to the gel-filtration
chromatography fractions. A standard curve was made from theelution time of molecular size standards: ferritin (440 kDa),
catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa) andribonuclease A (14 kDa). The molecular size of the peaks wasestimated as shown by arrows.
916

Fig. 5. Separation of the purified PomA/PomB complex by gelfiltration chromatography and sucrose density-gradient centrifugation. Samples consisting of pooled fractions 10 and 11 (a)or pooled fractions 13 and 14 (b) of the gel-filtration column
used to separate fraction 11 of the anion-exchange column
were analysed again on the same gel-filtration column. (c)Purified PomA/PomB complex from fraction 10 of (a) was analysed by sucrose density-gradient centrifugation (5–30 %). The
gradient was divided into 23 fractions from the top; the arrow
indicates the fraction in which the thyroglobulin (669 kDa) peak
was detected. PomA and PomB of each fraction, which wereseparated by SDS-PAGE, were detected by immunoblottingwith the anti-PomA and the anti-PomB antibodies.
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Purification of the motor complex

standards. The ion-exchange fraction 11 was applied to thegel-filtration column and the resulting fractions 10 and 11
or fractions 13 and 14 of the gel-filtration column werepooled. Each pooled fraction was separated again by thesame Superose column (Figs 4 and 5). PomA and PomB
were separated at similar fraction positions as before,suggesting that the complex is fairly stable. These experiments show that the structures composed of PomA and/or
PomB separated by gel-filtration chromatography havedifferent molecular sizes. The size of the huge complex of
PomA and PomB was confirmed by sucrose densitygradient centrifugation (Fig. 5c). The PomA/PomB complex
isolated by the Ni-NTA and by gel-filtration chromatography was reconstituted into proteoliposomes. However,
we could not detect sodium uptake activity in any samples
used (data not shown).
Fractions from gel-filtration chromatography wereanalysed by SDS-PAGE without the reducing reagent
2-mercaptoethanol (Fig. 6). The peak fraction 14 fromthe gel-filtration chromatography mainly contained the
PomB dimer connected by disulfide cross-linking.
Effects of SDS and DTT on the PomA/PomB
complex
We performed gel-filtration chromatography after the addition of SDS or DTT (Fig. 7). The approximately 900 kDa
complex seemed to be dissociated by the SDS treatment.
PomA eluted at fraction 17, which corresponded to approximately 60 kDa. On the other hand, PomB of the highmolecular size complex disappeared and the approximately
260 kDa complex remained as a result of this SDS treatment. The PomA complex appeared to be unaffected by the
DTT treatment. PomB continued to be eluted at fraction 9

Fig. 7. Treatment with SDS or DTT for the PomA/PomB complex. (a) A 20 % SDS solution was added at 1/20 to fraction
11 of anion-exchange chromatography and the mixture wasincubated at 65 6C for 5 min. (b) A 0?5 M DTT solution wasadded at 1/10 to fraction 11 of anion-exchange chromatography. The SDS- (1 %) or DTT- (50 mM) treated sample
was analysed on a Superose 6 column. PomA and PomB in
each fraction were separated by SDS-PAGE and detected byimmunoblotting.

or 10, corresponding to approximately 900 kDa. However,
the amount of PomB at this size was reduced. The main
peak of PomB corresponded to approximately 260 kDa
and shifted one fraction later, which corresponded to about
half of the original size.

DISCUSSION

Fig. 6. SDS-PAGE profiles without reducing reagent. Fraction
11 from anion-exchange chromatography was analysed on a
Superose 6 column. Proteins in each fraction were separated
by SDS-PAGE without 2-mercaptoethanol and PomA and
PomB were detected by immunoblotting.
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The flagellar motor consists of the stator and rotor. The
stator is composed of MotA and MotB for the protondriven motors or of PomA, PomB, MotX and MotY forthe sodium-driven motors. The essential components arethought to be similar in the two systems, based on thefollowing evidence. The structure of the basal body issimilar (Bakkeva et al., 1986). The proton-type MotA
functions like the sodium-type PomA (Asai et al., 1999).
MotA and MotB can function as a proton motor with the917

T. Yakushi, M. Kojima and M. Homma

rotor of the Vibrio polar motor, which is originally of thesodium type (Gosink & Häse, 2000; Asai et al., 2003). The
structure and composition of the rotor part of the proton
motor have been studied extensively by electron microscopic and biochemical analyses. At the early stage of the
motor study, the rotor part (MS-ring) was thought tofunction as the source of force generation (Homma et al.,1987). However, we now know that it is just a passiverotating ring of the motor (DeRosier, 1998). A C-ring
structure has been found under the MS-ring when isolated
by a mild condition (Driks & DeRosier, 1990). Even in mild
conditions, the stator parts or the Mot proteins were notassociated with the rotor structure. We can assume that theinteraction between the rotor and stator is not so strong
because they have to move relative to one another to produce torque. It has been shown by a co-isolation experimentthat MotA binds to FliG and to FliM (Tang et al., 1996). Thissuggests that the rotor and stator interact under certain
conditions so that a force is generated between them. We
purified the stator complex or the PomA/PomB complexof V. alginolyticus. The complex did not contain MotX
and MotY, or the rotor proteins FliG, FliM and FliN (thisstudy; Sato & Homma, 2000a). We demonstrated sodium
uptake activity in proteoliposomes reconstituted with the
PomA/PomB complex, which was purified by using boctylglucoside. The amount of protein of the complex
recovered by this purification procedure was too low toproceed to further experiments for structural or biochemical analyses.
The purification procedure of the PomA/PomB motor
complex was improved in this study. The detergent sucrose
monocaprate allowed better solubilization of membrane
fractions associated with the PomA/PomB complex; however, MotX or MotY was still not associated with thiscomplex. We changed the position of the attached hexahistidine tag to motor components. Previously, the tag was
fused to the N terminus of PomA. The binding efficiency of
His6-PomA to Ni-NTA agarose was not adequate. Therefore, we made a His-tagged PomB protein. The hexahistidine sequence was inserted at the C terminus of PomB,
where it was predicted to localize in the periplasmic spaceor bind to peptidoglycans. PomB-His6 functions like thewild-type PomB. Interaction with the rotor has been shown
to be necessary to adjust the structure to a functional formof the stator, which is composed of A and B subunits (Garzaet al., 1996). We speculate that a small fraction of the motor
complex in the overproduced condition was activated byinteraction with the rotor because the functional rotors
were not overproduced. Therefore, cells can survive in the
overproduced condition. Similarly, the overproduction of
MotA and MotB did not severely affect cell growth (Wilson& Macnab, 1990)
A complex composed of MotA and MotB is thought to be
the force-generating unit. There is no direct evidence of
this; however, the molar ratio of MotA and MotB is thought
to be 1 : 1 and an ion-conducting pore forms with the918transmembrane regions of each protein (Sharp et al., 1995a,b). In E. coli, a systematic Cys-substitution against a single
transmembrane segment of MotB was recently performed.
Periodic formation of cross-links on one face of its a-helix
with or without the co-expression of MotA was found(Braun & Blair, 2001). It has been proposed that the twocopies of MotB form a dimer in the MotA/MotB complex
and the two Asp residues are displayed on separate faces.
A new topological model was presented from the assumption that the stoichiometry of the force-generating unit isa 4 : 2 complex of MotA and MotB (Blair, 2003). Furthermore, this stoichiometry of PomA and PomB was reported
in one of our recent papers (Yorimitsu et al., 2004). Thismodel is consistent with the biochemical evidence that theratio of PomA and PomB is 2 : 1. This has been determinedfrom the intensity of the Coomassie blue staining. Also, thesize of the complex estimated by gel filtration assay is
175 kDa (Sato & Homma, 2000a). This size corresponds tothe complex of four PomA and two PomB proteins. It has
been shown that PomA forms a stable homodimer and both
halves of the dimer seem to function together to conduct
sodium ions (Sato & Homma, 2000a, b).
The molecular size of the PomA/PomB-His6 complex has
been estimated to be greater than 900 kDa when solubilized
and separated by the detergent sucrose monocaprate. Whengel-filtration chromatography was used, PomB was eluted
as an approximately 260 kDa complex, which is based on
the analysis of fraction 9 by anion-exchange chromatography. The size of 260 kDa is much larger than themonomer size of PomB (37 kDa). In the presence of 50 mM
DTT, the position of the main PomB eluted fraction was
shifted to one corresponding to about half the size. Since
PomB has three cysteine residues, the most probableexplanation is that PomB forms a disulfide bridge between
PomB molecules (Yorimitsu et al., 2004). MotB is predicted to form a dimer with an estimated size of 74 kDa
based on cross-linking experiments (Braun & Blair, 2001).
However, the size of approximately 260 kDa is still muchlarger than the dimer size of PomB. The reliability of theestimated size of the membrane protein in the presence ofthe detergent is unknown. However, the size of the hugecomplex of PomA and PomB was confirmed by sucrose
density-gradient centrifugation. On the other hand, thestructure of MotB or PomB is expected to be very extended.
Therefore, estimation of the molecular mass may be very
difficult when globular proteins are used as standard
proteins. Even with treatments of SDS and DTT, PomB
was eluted at a size greater than 100 kDa as a globular
protein. Currently, we cannot confirm that PomB still forms
a complex.
Apparently, most of PomB does not associate with PomA.
PomA and PomB proteins were co-purified in chromatography fractions with the highest molecular size (approx.
900 kDa), which suggests that they form the 900 kDa
complex. This might not be an intact force-generating unit
because the amount of PomA is smaller than that of PomB
Microbiology 150

Purification of the motor complex

in the high molecular size complex. Specifically, the PomB
band was clearly detected by Coomassie blue R250 while the
PomA band was not. This may suggest that PomA interacts
with multiple PomB proteins or PomB complexes. In thiscondition, the association of PomA and PomB was probably
destroyed. Even though the amount recovered improved,
the sodium uptake activity was lost in this procedure. We
suspect that the native PomA/PomB complex does not haveion-conducting activity, rather the disordered complex
treated with certain detergents such as b-octyoglucosidedoes. We need to further clarify the biochemical characterof the membrane components, PomA and PomB, or thecomplex, and to investigate improved conditions or detergents that maintain both association and activity.

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ACKNOWLEDGEMENTS

Mutations in motB suppressible by changes in stator or rotor
components of the bacterial flagellar motor. J Mol Biol 258, 270–285.

We thank Tatsuo Atsumi for critically reading this manuscript. This
work was supported in part by grants-in-aid for scientific research from
the Ministry of Education, Science and Culture of Japan (to T. Y. and
M. H.) and from Soft Nano-Machine Project of Japan Science andTechnology Agency (to T. Y. and M. H.).

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