Biochimica et Biophysica Acta 1505 (2001) 82^93www.bba-direct.com

ReviewNa -driven £agellar motor of Vibrio
Tomohiro Yorimitsu, Michio Homma *Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
Received 29 May 2000; received in revised form 18 October 2000; accepted 18 October 2000

Abstract
Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions acrossthe membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of thefilament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the couplingion : namely the H -driven motor and the Na -driven motor. Analysis of the flagellar motor at the molecular level is far
more advanced in the H -driven motor than in the Na -driven motor. Recently, the genes of the Na -driven motor havebeen cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In
this review, we summarize recent studies of the Na -driven flagellar motor. ß 2001 Elsevier Science B.V. All rights
reserved.
Keywords: Bacterial £agellar motor; Na -driven motor; Torque generation; Na channel blocker; Vibrio alginolyticus

1. Introduction
Flagella are the rotary machines that convert theion £ux to the locomotive force of the cell. Bacteria
have helical £agellar ¢laments extending from thecell body. They swim toward favored environments
in liquid media by reversibly rotating the £agellar¢laments, which function as propellers. Each £agellum is driven by a rotary motor embedded in thecytoplasmic membrane at its base. The £agellar basal
part (the rotor) in Gram-negative bacteria is composed of more than 10 protein components, and consists of the rod and the four ring structures, termed L
ring, P ring, MS ring, and C ring. It is speculated
that the C ring is surrounded by stator particles(force-generating units) (Fig. 1) [1^4]. However, the* Corresponding author. Fax: +81-52-789-3001;
E-mail: g44416a@nucc.cc.nagoya-u.ac.jp

C ring is not tightly bound to the basal part and iseasily lost in a puri¢cation procedure (Fig. 1c). The
rotation of the £agellar motor is powered by theelectrochemical gradient of speci¢c ions across thecytoplasmic membrane. In 1977, it was shown thatthe £agellar motors of Bacillus subtilis and Streptococcus spp. can be driven by an arti¢cial H gradient[5,6], indicating that the energy source of the motor
is the proton-motive force. It has been shown thatthe motors of other bacteria, such as Escherichia coliand Salmonella typhimurium, are also energized byH in£ux [3,7,8].
The force-generating units, which form the stator,
are composed of two proteins, MotA and MotB
[9,10]. MotA has four transmembrane segments andone large cytoplasmic loop [11]. MotB has a single
N-terminal transmembrane segment and a peptidoglycan binding motif at the C-terminus [12^14].
These components form a complex to function as aH channel [15^19]. It is inferred that their trans-

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Fig. 1. Structure of £agella. (a) Cartoon of a £agellar structureof Gram-negative bacteria. (b) The components and substructure of £agella. (c) Electron micrographs of a ¢lament^hook^basal body complex which is stained by phosphotungstate. The
switch complex (C ring), motor complex, and export apparatus
are missing.

membrane segments, which are predicted to form Khelices, make an ion channel. One of the most prominent hypothesis is that structural changes induced in
MotA generate the rotary power by coupling H
£ow to changes of the interaction with the rotor
component FliG, as discussed later [20^24]. The
mechanism of coupling between the H £ow andthe structural changes has not been resolved.
In some bacteria, the £agellar motor is powered by
the electrochemical gradient of Na [25]. Early studies of the Na -driven £agellar motor were carriedout using alkaliphilic Bacillus. Alkaliphilic Bacillus
optimally lives and is vigorously motile at pH 10^
11 [26]. Under alkaline conditions, the H -motive
force is quite small because the intracellular pH ofthese bacteria is maintained at pH 8^9 by the mechanism of homeostasis [27]. It was shown that the83

presence of Na in the medium was essential forthe motility of these bacteria [28] and it was provedthat the £agellar motor of these bacteria rotates by
the electrochemical potential of Na [29^31]. Some
Vibrio spp., V. alginolyticus, V. parahaemolyticus,
and V. cholerae, use the Na -motive force to rotate
polar £agellar motors [32^36]. It is known that V.
alginolyticus and V. parahaemolyticus also have H driven lateral £agella [34,37] (Fig. 2a). These marine
Vibrio species use the Na -driven polar £agella forswimming in liquids, such as sea water, and the H driven lateral £agella for swarming in viscous environments, such as on surfaces of ¢sh [38,39]. As
shown in Fig. 2b, the swimming speed generated by
the polar £agella decreases rapidly as the bu¡er viscosity is increased, and stops at a viscosity of 200 cP.
In contrast, the swimming speed by the lateral £agella increases with viscosity to about 5 cP of viscosity, then decreases gradually, but is still about 20 Wm/
s at 200 cP viscosity [40]. V. alginolyticus as well asother Vibrio spp. have the Na -translocating respiratory chain and Na /H antiporter, which extrudeNa and might generate the electrochemical potential for the Na -driven £agellar motor [41]. Thus
wild-type Na motility is resistant to the H conductor CCCP, but the motility of mutant cells with adefective Na pump is sensitive to CCCP (Fig. 2c)[42].
The Na -driven motor has advantages for the
study of its function because the Na -motive force
can be more easily manipulated, and the motor function can be inhibited by speci¢c Na channel blockers, which are also powerful tools for the study of theenergy-coupling mechanism of the motor.
2. Components essential for the torque generation of
the Na+-driven motor
At ¢rst, the Na -driven £agellar motor was predicted to contain motor components homologous to
MotA and MotB of the H -driven £agellar motor.
Using homology, it was attempted to clone the genes
from bacteria that have Na -driven £agella. However, the strategy was not successful. The Na -driven
motor genes were cloned using motility-defective mutants. The essential genes, motX and motY, for therotation of the Na -driven motor were identi¢ed andBBABIO 45030 28-2-01

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Fig. 2. Lateral £agella and polar £agellum of V. alginolyticus.
(a) Di¡erentiation of £agellar system of V. alginolyticus. (b)Functional di¡erence between Laf and Pof with regard to viscosity [40]. The swimming speed was measured in a bu¡er inwhich the viscosity was changed by addition of polyvinylpyrrolidone. (c) Circulation of Na and H under the di¡erent pH
conditions for the £agellar motors of V. alginolyticus. In V. alginolyticus, Na and H are pumped out by the respiratory
chain and the Na pumping is only active in the alkaline condition. The H gradient can be changed to a Na gradient by
antiporters.

cloned from V. parahaemolyticus [43,44]. The motX
and motY genes were also identi¢ed in V. alginolyticus, closely related to V. parahaemolyticus [45,46].
From the deduced amino acid sequences, it is assumed that these two components, MotX and
MotY, are membrane proteins with a single transmembrane segment, and indeed both of them areseparated in a membrane fraction. MotY has a peptidoglycan binding motif (which is also observed in
MotB, an essential component of the H -driven motor) in its C-terminal region [13,14]. In the H -driven
motor, it is suggested that the interaction with peptidoglycan and the motif is important for ¢xing the
MotA/MotB complex around the rotor. On the other
hand, MotX might form a part of the Na channel
because overexpression of MotX causes E. coli to be
defective in growth when the extracellular Na concentration is increased. The growth defect is rescuedby amiloride [44], which is known to be a potent
inhibitor of Na channels as well as the Na -driven£agellar motor [47]. Moreover, MotY failed to belocalized to the cytoplasmic membrane in the absence of MotX. MotA is required for the stabilityof MotB in E. coli cells [48]. From these lines ofevidence it was speculated that MotX and MotY
form a complex of force-generating unit similar tothe complex of MotA and MotB. However, overallsimilarities with MotX and MotY were not found in
the components of the H -driven motor. Therefore,
it had been assumed that the mechanisms of the energy conversion with ion £ux were very di¡erent between the H - and the Na -driven motors.
In 1997, two novel genes, pomA and pomB, which
are essential for rotating the Na -driven £agellar
motor in V. alginolyticus, were isolated [49]. The amino acid sequences of PomA and PomB are similar to
those of MotA and MotB, respectively. By randommutagenesis of the pomA gene, it has been suggested
that PomA and MotA are very similar in structureand function [50]. It is inferred that PomA has fourtransmembrane segments, and PomB has a single
transmembrane segment and a peptidoglycan binding
motif in the C-terminal region. PomA and PomB are
most likely to interact with each other because they
are co-precipitated with each other and the stabilityof PomB is dependent on the presence of PomA in
the cell [51]. The complex of PomA and PomB seems
to function as a Na channel. Recently, homologous

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Fig. 3. Gene productions for the H -driven motor and theNa -driven motor. Membrane-localized components for theH -driven motor of E. coli and the Na -driven and H -driven
motors of V. alginolyticus are summarized. The predicted membrane topology of the four motor gene products in the Na driven motor is illustrated as previously [49].

genes were isolated in V. parahaemolyticus [52]. Further, four homologous genes are found also in the
genome sequence of V. cholerae. These results suggest that the force-generating unit of the Na -driven£agellar motor is composed of four components,
PomA, PomB, MotX, and MotY (Fig. 3).
3. Rotation analysis of the Na+-driven £agellar motor
The rotation rate of the £agellar motor of V. alginolyticus was measured by laser dark-¢eld microscopy. Laser dark-¢eld microscopy is a powerful tool
because it enables the measurement of the rotation
speed of a single £agellar motor. In this method, acell is stuck and ¢xed on the glass surface and its

85

rotating £agellum is irradiated by a laser beam.
The intensity of scattered light is detected by a photon-counting system, and then the rotation rate of asingle £agellum is measured from the change of theintensity [53]. In V. alginolyticus, it was shown thatthe rotation rate of a £agellum was proportional to
the speed of a freely swimming cell [54]. The swimming speeds and the £agellar rotation rates of individual free-swimming cells were measured simultaneously, and a roughly linear relation between
swimming speed and £agellar rotation rate was observed [55]. In other words, the £agellar rotation rate
can be estimated from the swimming speed. The
average rotation rate of the £agellar motor is 1100
rps and the highest value is 1700 rps [56]. On theother hand, the rotation rates for the H -driven motor of E. coli and S. typhimurium were measured asca. 300 rps [57] and ca. 200 rps [53], respectively.
This remarkable di¡erence of the rotational rates
might re£ect the di¡erence of the e¤ciencies for theenergy coupling between the H - and Na -driven
motor. In Table 1, various motors including theATP-driven motors are compared. Many possible
mechanisms for ion-driven rotary motors have been
proposed, although without reliable supporting evidence [58]. The Na -driven £agellar motor is inferred
to be the fastest motor among those that we know[56]. The Vibrio Na -driven £agellar motor may be
adapted to high-speed rotation.
4. Speci¢c inhibitors of the Na+-driven £agellar motor
The functions of mammalian Na channels, Na /H exchangers and Na /Ca2 exchangers have been
analyzed using amiloride, one of the speci¢c inhibitors of Na -coupled systems [59]. Amiloride isTable 1
Statistics for various motors
Axis distance
Rotational speed
Linear speed
Torque/generator
ForceE¤ciency
Energy source

Flagella of E. coli

Polar £agella of V. alginolyticus F1 motor of F0 F1 -ATPase

Myosin of skeletal muscle

ca. 20 nm
300 rps
40 Wm/sca. 450 pN nm
ca. 25 pN
V100% ??
Hca. 20 nm
1700 rps
210 Wm/s
ca. 100 pN nm
ca. 5 pN
V100% ??
NaNA
NA
10 Wm/sNAca. 5^10 pN
V50% ??
ATPca. 1 nm
10 rps
60 nm/sca. 40 pN nm
ca. 40 pN
V100% ??
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Fig. 4. Characterization of motility mutants resistant to phenamil. (a) Swarming abilities of the mutants in the presence ofphenamil. 1, the Mpas (wild-type for the phenamil sensitivity)
strain; 2 and 3, the Mpar mutants (motility mutants resistantto phenamil). (b) Relationship between Na concentration of
the medium and swimming speed was examined for the Mpas
strain and the Mpar mutant. (c) Motility inhibition of theMpas strain and the Mpar mutant by phenamil was examined.
The data are quoted from [64].

6thought to bind to the Na binding site in the channel. It was found that amiloride also speci¢cally inhibits the function of the Na -driven £agellar motor,
without a¡ecting the membrane potential, the intra-

cellular pH homeostasis, or the ATP content of thecells and only slightly a¡ecting the Na -coupled amino acid transport system in alkaliphilic Bacillus [47].
The inhibition of cell motility by amiloride is reducedwith increasing Na concentration in the medium.
This result indicates that amiloride reversibly inhibits
the motor function by competing with Na .
6-Iodoamioride (6-IA), which is a photoreactive
amiloride analog, competitively and reversibly inhibits the rotation of the £agellar motor of alkaliphilic
Bacillus. However, 6-IA irreversibly inhibits the motility after irradiation with UV light. When tetheredcells of alkaliphilic Bacillus were irradiated with UVlight in the presence of 6-IA, the rotation rate gradually decreased in ¢ve to nine steps, suggesting that
there are multiple torque generators that function
independently in the Na motor [60]. Similar results
have been obtained in the H -driven motor [61,62].
When MotB was inducibly produced in a tethered
motB cell of E. coli, the rotation rate of the cellincreased stepwise for a maximum of eight steps.
Moreover, by electron microscopy, about 10 particles
which might be the torque generators were found in
the cytoplasmic membrane, and they are absent inmotor-defective mutants [63]. From the electron microscopic images, the basal body structures of theH - and Na -driven £agella are similar to each other(Fig. 1c). These results suggest that the number of
torque generators is around 10 in both the H - andNa -driven motors.
To study the Na binding site of the Na -driven£agellar motor of V. alginolyticus, Kojima et al. [64]isolated motility mutants resistant to phenamil, an
amiloride analog (Fig. 4a). The swimming speeds ofboth strains showed a similar dependence on the external Na concentration (Fig. 4b). The wild-type
cells for motility cannot swim at 50 WM of phenamil,

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Fig. 5. Model of the membrane topology of PomA and PomB. Positions substituted by Cys are shown as closed circles with the whiteletters of amino acids [66]. Relative swarm sizes in the Cys mutants are indicated by the shaded circles. Where no shaded circle is given, swarm size was similar to that of the wild-type cells. The shaded circles with the white letters (D148 of PomA and P16 of PomB)show the mutation sites for the phenamil resistance. The thick circle (D24 of PomB) shows a possible Na binding site.

while the mutant cells can swim at this concentration(Fig. 4c). Unlike amiloride, the e¡ect of phenamil on
the motor function is not dependent on the concentration of Na in the medium. The £uctuation of therotation rate of the motor, which is de¢ned as thestandard deviation divided by the average rotation
rate, was investigated by laser dark-¢eld microscopy.
It was shown that the rotation rate of the wild-type£agella greatly £uctuates in the presence of phenamil,
but that of the mutant resistant to phenamil doesnot. This work suggests that the motor has the phenamil-speci¢c binding site and the resistant phenotype is explained by increasing the dissociation rateof phenamil to the motor. Phenamil is likely to bindnear the Na binding site. The mutations providing
the phenamil-resistant motility are independently
mapped in pomA and pomB genes, and consist of
substitution of Asp148 to Tyr in PomA or Pro16 to
Ser in PomB (Fig. 5) [65]. From the predicted topology of PomA and PomB, it is predicted that eachresidue is located near the cytoplasmic ends of transmembrane segments (Fig. 5). The double mutation of
PomA-D148Y/PomB-P16S synergistically impairs
motility in the absence of phenamil and makes the

motility less sensitive to phenamil. Those lines ofevidence suggest that Asp148 of PomA and Pro16 of
PomB are proximally located, and that multiple residues of both PomA and PomB including Asp148 and
Pro16 , respectively, contribute to the formation of thehigh-a¤nity phenamil binding site. Analysis of motor function by Na channel inhibitors was also carried out to isolate amiloride- and phenamil-resistantmutants, and the mutation sites were characterized inV. parahaemolyticus [52]. In the PomA homolog in
V. parahaemolyticus, mutations at the same position(Asp148 ) provide the phenamil-resistant phenotype.
Mutations at Gly20 and Ala23 and the deletion of
Pro12 to Pro16 caused resistance to phenamil in the
PomB homolog. Phenamil resistance mutation sites
have been found only in PomA or PomB, not in any
other components for torque generation. In both
species, no amiloride-resistant mutations have been
assigned. The high-a¤nity binding site for phenamilis likely to be composed of the multiple residues of
PomA and PomB which form a Na channel. The
high-a¤nity site seems to be close to Asp24 of PomB
which is the candidate of the Na binding site (seebelow).

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5. The role of the periplasmic loop regions of PomA
There are two putative periplasmic loops, loop1ÿ2
and loop3ÿ4 , in PomA. The loops may help to maintain the arrangement of the membrane-spanning helices that form the ion channel, guide the Na ion orinteract with the ions, or with other proteins, such as
PomB. By Cys-scanning analysis, the periplasmic
loops of PomA were investigated for functional andtopological information (Fig. 5) [66]. From pro¢lesof labeling by biotin maleimide, the membrane topology of loop3ÿ4 was directly predicted, on the other
hand, the residues of loop1ÿ2 were not labeled. Thissuggests that the environments of the two loops arevery di¡erent. Loop1ÿ2 may be covered with a protein, such as PomB, MotX, or MotY, or may beembedded in the pore region of the channel. In
loop3ÿ4 , many Cys substitutions and the ¢ve successive mutations from M169 to K173 impaired motility, and only the M179C mutation abolished motility. From the evidence that the reactivity of D170C
to SH-modifying reagents changed with the Naconcentration, it is speculated that D170 faces towards the channel pore and interacts with Na , orthat Na changes the structure around D170. The
residues from M169 to K173 may not be essential
but play an important role for the motor function.
When PomA with an introduced Cys residue (CysPomA) in the C-terminal periplasmic loop (loop3ÿ4 )was examined without exposure to a reducing reagent, a 43-kDa band was observed in some mutants,
whereas only a 25-kDa band, which corresponds tomonomeric PomA, was observed under reducing
conditions [67]. The 43-kDa band was strongest in
the P172C mutant. The intensity of the 43-kDa band
was enhanced in most mutants by the oxidizing reagent CuCl2 . The P172C mutant protein of PomA
was not functional and inhibits the motility of thewild-type cell in the absence of reducing reagents,
although motility was restored by dithiothreitol.
These results suggest that a cross-link formed between two molecules of Cys-PomA via loop3ÿ4 inhibits the function of the torque-generating units. Residue P172 by itself does not seem to be essential for
motor function, since motility is normal in PomA
mutants in which P172 was replaced by various amino acids other than Cys. The £exibility of loop3ÿ4
seems to be restricted by the cross-linking, thereby

impairing motor function. There are at least threepossible mechanisms for this inhibition of motor
function: (i) Na in£ux is inhibited by disruptionof the channel structure; (ii) conversion of the Nain£ux to conformational changes in the torque-generating stator units is inhibited; (iii) interactions between the stator and the rotor are inhibited.
The mutant PomA D31C, which is located at the
N-terminal periplasmic loop, loop1ÿ2 , does not swim
at low concentrations of Na in the medium, whichmeans that a higher threshold is required for retaining the motility of this mutant [68]. Introduction of
positive charges (D31K and D31R) or large structural changes (D31A and D31Y) result in greater shiftsof the threshold than in the D31C mutant. On theother hand, replacement by a negatively charged residue (D31E) results in the same dependence on Na
as the wild-type PomA. The negative charge of position 31 in PomA probably has an important role forretaining the Na in£ux through the motor or e¡ective torque generation. It is speculated that the negative charge of Asp31 itself or the circumstance of theregion including Asp31 is possibly involved in theguidance of Na into the channel complex. Direct
measurement of the Na current is necessary to clarify the recruitment of ions at the loop region.
6. Recognition of Na+
Only two kinds of rotary motors, the £agellar motor and the F1 Fo -ATPase, are known in biologicalsystems. Both systems are classi¢ed into two types
according to the coupling ions, H -driven type andNa -driven type. F1 F0 -ATPase (also known as ATP
synthase) is composed of two parts. The F1 part issoluble and has ATP synthesis and ATP hydrolysis
activity. The F0 part is embedded in the membrane
and transports speci¢c ions. F0 consists of threekinds of subunits, a, b, and c [69]. It was foundthat a hybrid enzyme composed of the Na -type F0part of Propionigenium modestum and the H -type
F1 part of E. coli has Na -pumping activity, andH -pumping activity is observed only in the absenceof Na [70^72]. This suggests that the F0 part canessentially conduct Na as well as H . From mutation analysis it is inferred that an ion speci¢city site ispresent in subunits a and c of the F0 part [73,74].

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Fig. 6. Hybrid and chimeric motors with the Na - and the H driven components. MotA of R. sphaeroides or PomA of V. alginolyticus, and MotB of R. sphaeroides or PomB of V. alginolyticus or chimeras between PomB and MotB were expressed in
pom mutants of V. alginolyticus. Black lines and gray lines
show the regions of the Na - and the H -driven component,
respectively. The regions of the thick line show the transmembrane regions. The light gray boxes show the inner membrane.
In (a), (b), and (c), the motor functions as the Na -driven type.
On the other hand, the motors of (d), (e), and (f) do not workby Na or H . The schematic representations are illustrated
from the data in [75,76].

Analysis of the ion speci¢city of the torque generator has also been carried out in the bacterial £agellar motor (Fig. 6). MotA and MotB of Rhodobacter
sphaeroides show the largest sequence similarity to
PomA and PomB respectively of V. alginolyticus,

89

comparing all currently sequenced Mot proteins ofH £agellar motors. When the R. sphaeroides motA
gene is expressed in a V. alginolyticus pomA mutant,motility is restored. However, the motility of a pomB
mutant is not restored by expressing the motB gene.
The swimming speed of the pomA mutant cell with
MotA increases with increasing concentration ofNa in the medium. The motility is inhibited by
the Na channel inhibitors phenamil and amiloride,
but not by the H ionophore CCCP. It is concluded
that the hybrid motor composed of the H -type andNa -type components is functional, and MotA of R.
sphaeroides can generate torque by coupling with theNa £ux in place of PomA of V. alginolyticus [75].
The chimeric protein (MomB), which has its N-terminal cytoplasmic region and the entire transmembrane segment derived from MotB of R. sphaeroides,
and the remaining C-terminal periplasmic part from
PomB, functions as a Na motor when co-producedwith PomA or MotA [76]. These lines of evidenceshow that the channel part consisting of the transmembrane segments from the A and B subunits does
not discriminate the ions Na and H .
The ion speci¢city of the hybrid F0 F1 -ATPase wasdetermined by the F0 part. The F1 part is thought tohave only mechanical coupling with the F0 part andthe F1 part should not be expected to alter the ion
speci¢city of the F0 part. On the other hand, when
the channel part of MotA and MotB, whose specificity is H , was incorporated into the Na motor,
the speci¢city was changed to Na channel. E. coliH motor components MotA and MotB could function in place of the motor proteins of V. choleraethough the motility was severely impaired and the
hybrid motor seems to be driven by the H -motive
force [77]. It has been speculated that the other two
Na type-speci¢c components, MotX and MotY, areinvolved in the ion recognition of the motor. The ion
recognition systems between the F0 F1 -ATPase andthe £agellar motor appear to di¡er from each other.
In the H -driven motor of E. coli, MotB, Asp32
has a critical role for the motor function and is
speculated to convey H [78]. Asp32 is the only conserved acidic residue in all presently known sequences of MotB and its homologs. (The corresponding
residue in PomB of V. alginolyticus is Asp24 [49].)
When Asp32 is substituted with other residues, allmutants fail to be motile, except for the D32E mu-

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tant, which has very slow motility. It is known thatoverexpression of MotA with the N-terminal 60 residues of MotB, which include the putative transmembrane segment, impairs cell growth [15,16,79,80].
When Asp32 is substituted with any other aminoacid, the growth retardation is no longer observed.
The growth defect is interpreted as being due to increased H permeability of the cell membrane. Asp32
of MotB is a good candidate for an H binding site.
If this is true, the conserved aspartate residue must
be a candidate for the binding site of Na in PomB.
7. Composition and structure of the torque generator
The membrane topology of the H -driven motor
proteins, MotA and MotB, was investigated by Trpscanning mutagenesis [18,19]. The residues of thetransmembrane segments of MotA and MotB weresystematically replaced by Trp and the e¡ect on the
motor function was examined. Trp has a large sizeand hydrophobic character, so it is postulated that if
Trp substitution faces the lipid, it would be tolerated,
but if Trp faces inside the protein complex, it would
not be tolerated. The e¡ect on the function seems to
imply the e¡ect on the structure. This strategy suggested a hypothetical structure of the MotA/MotB
channel complex. The pattern of the e¡ect on
MotA is periodic along the four transmembrane segments in the way of K-helices. On the other hand,substitution of MotB is tolerated at positions close to
the periplasmic end of the transmembrane segment,
but not at positions close to the cytoplasmic end.
These results suggest that a single transmembranesegment of MotB is associated with the segments of
MotA at a tilt relative to them and its cytoplasmic
end is located in the channel complex. In this model,
Asp32 of MotB, which might be involved in conducting H , faces inwards in the complex.
In the case of the Na -driven £agellar motor, unlike that of the H -driven motor, little information
about the structure of the torque generator is known.
However, it seems to be clear that PomA and PomB
are directly associated together [51]. When the
PomA/PomB complex is puri¢ed, the subunit ratioof the complex is estimated to be two PomA to one
PomB. The predicted size of a complex of two PomA
and one PomB molecules is about 90 kDa [81]. How-ever, the PomA/PomB complex obtained by gel ¢ltration assay was found in fractions of higher molecular size than expected (estimated as 175 kDa).
When PomA alone is applied to the gel ¢ltration
column, it is eluted as a 55-kDa protein, which islikely to correspond to the homodimer complex of
PomA as the size expected for monomeric PomA is
27 kDa. Furthermore, a genetically fused dimer of
PomA was constructed. It was observed that thistandem PomA dimer is not only stably produced inthe Vibrio cell but also enables the motor to function.
By gel ¢ltration assay, the tandem PomA dimer isalso eluted as a 55-kDa protein. These results
strongly suggest that native PomA forms a stablehomodimer. Moreover, considering the molecularsize of the PomA/PomB complex described above,it is speculated that the PomA/PomB complex iscomposed of four copies of PomA and two copies
of PomB. It is the simplest assumption that the complex is a single force-generating unit. From mutation
study of the PomA dimer, it is inferred that both
halves of the PomA dimer function together to conduct sodium ions, and cooperatively form an ion
recognition site [82]. This suggests that the dimeror the multimer is the functional unit of PomA. At
present, less structural information is available forthe torque generator of the H -driven £agellar motor
than for the Na -driven motor. In order to elucidate
the mechanism of the £agellar motor, it is important
to obtain more information on the structure of thetorque generator.
8. 22 Na+ conductance of the PomA/B complex
The PomA/PomB protein complex reconstituted
into proteoliposomes electrogenically catalyzes
22Na in£ux in response to a K di¡usion potential[81]. However, PomA only reconstituted into theproteoliposome has no signi¢cant Na uptake activity. Catalysis of the Na uptake by the PomA/PomB
complex is e¡ectively inhibited in the presence ofphenamil, to be at the same level as that of the proteoliposome without the PomA/PomB complex. It is
known that the Na -driven £agellar motor of V.
alginolyticus is driven by Li with lower e¤ciency[83]. The accumulation of 22 Na is prevented byLi , indicating that Na and Li compete for a com-BBABIO 45030 28-2-01

T. Yorimitsu, M. Homma / Biochimica et Biophysica Acta 1505 (2001) 82^93

91

mon binding site of the channel complex. Using thistechnique, it would be expected that the physiological features of the slow motility and the molecularfeatures of the Na uptake of the torque generator
are directly linked and the relationship at the celllevel and the molecular level is discussed in moredetail.
9. Torque generation
It is thought that three components, MotA, MotB,
and FliG, are directly involved in the torque generation of the H -driven £agellar motor (Fig. 7). FliG
is a soluble protein and one of the components in therotor [20,84,85]. Mutational analysis provided identi¢cation of the important and conserved residues forthe torque generation of MotA and FliG and led tothe proposal of a model for the interaction between
MotA and FliG (Fig. 7) [21^24]. In this model, it issuggested that three residues, Arg281 , Asp288 andAsp289 , in the C-terminal K-helix of FliG and two
residues, Arg90 and Glu98 , in the cytoplasmic loop
region of MotA are important for function. Double
mutants with substitution of charged residues synergistically inhibited the motor function. So, it is hypothesized that electrostatic interactions essential forthe rotation of the motor occur between the rotor
component FliG and the stator component MotA
via these residues. The two functionally important
residues of MotA are also conserved in PomA of
V. alginolyticus and the PomA homolog of V. parahaemolyticus [49,52]. Recently, the Na -driven switch
genes including £iG have been isolated from V. parahaemolyticus and the functionally important charged
residues are all conserved in FliG of V. parahaemolyticus [86]. It was shown that the rotation of V.
cholerae £agella is powered by the Na in£ux[35,36]. There is an open reading frame with a sequence similar to FliG in the genome of V. cholerae(from the un¢nished genome database) and this open
reading frame also conserves those charged residues.
This seems to suggest that the rotor component andthe stator component electrostatically interact in theNa -driven £agellar motor to generate the rotary
power as proposed in the H -driven £agellar motor.
This is supported by the suggestion that MotA of R.
sphaeroides works with FliG of V. alginolyticus, us-Fig. 7. Rotor and stator in the £agellar motor. A model of a
stator unit, which consists of MotA and MotB, and a rotor
unit, which consists of FliG, FliM, and FliN, is presented on
the assumption that each unit is composed of a single moleculeof each component though it may not be true. The rotor units
are attached to the MS ring and form a ring structure, the C
ring. The C-terminal region of MotB interacts with peptidoglycan to anchor the stator to the cell wall. The charged residues
on the rotor^stator interface, which seem to be important forforce generation, are indicated by the residue number with anarrow. TM: transmembrane region. The model of this H -driven motor should be similar to that of the Na -driven motor because the components are exchangeable between the both motors [75^77].

ing the energy of Na , in pomA-defective Vibrio cells
expressing motA [75,76].
10. Summary
There are four transmembrane components,
PomA, PomB, MotX and MotY, required for rotating the Na -driven £agellar motor. From the analysis of MotA and MotB in the H -driven £agellar
motor, PomA and PomB are likely to contact theBBABIO 45030 28-2-01

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T. Yorimitsu, M. Homma / Biochimica et Biophysica Acta 1505 (2001) 82^93

rotor component directly and participate in torquegeneration. However, the function of the two Natype-speci¢c components, MotX and MotY, is unknown though they may be involved in the ion recognition of the motor. How are these components
involved in the generation of the torque? To understand the functional role of MotX and MotY it isimportant to understand the mechanism of the coupling between Na in£ux and rotary power, sincethese proteins are speci¢cally found in the Na -driven £agellar motor. Comparatively easy manipulationof Na will be greatly advantageous for the study of
MotX and MotY.
Two rotary motors are known in live organisms,
one is the bacterial £agellar motor and the other is
F1 F0 -ATPase. The latter motor has been well studied
and detailed models of the rotary mechanism havebeen advanced. We believe that resolving the crystal
structure is an important contribution to the developing study of the F1 F0 -ATPase. For not only theNa - but also the H -driven £agellar motor, morestructural knowledge of the torque generator is required for understanding the rotary mechanism.
Acknowledgements
We thank Drs. Richard M. Berry, Ikuro Kawagishi, and Toshiharu Yakushi for critically reading
the manuscript. The work in our laboratory was supported in part by Grants-in-Aid for Scienti¢c Research from the Ministry of Education, Science andCulture of Japan. T.Y. was supported by the Japan
Society for the Promotion of Science.
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