The EMBO Journal Vol. 19 No. 14 pp. 3639±3648, 2000

Coupling ion speci®city of chimeras between
H+- and Na+-driven motor proteins, MotB and PomB,in Vibrio polar ¯agella

Yukako Asai, Ikuro Kawagishi,
R.Elizabeth Sockett1 and Michio Homma2
Division of Biological Science, Graduate School of Science,
Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan and
1
Genetics Division, Clinical Laboratory Sciences, Nottingham
University, Queen's Medical Centre, Nottingham NG7 2UH, UK
2
Corresponding authore-mail: g44416a@nucc.cc.nagoya-u.ac.jp

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodiumtype Vibrio alginolyticus PomB, MotX and MotY, canwork as a sodium-driven motor in Vibrio cells. In thisstudy, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembraneregion. Rhodobacter sphaeroides MotB did not workwith either MotA or PomA in Vibrio cells. Therefore,
we constructed chimeric proteins (MomB), which had
N-terminal MotB and C-terminal PomB. MomB
proteins, with the entire transmembrane region
derived from the H+-type MotB, gave rise to an Na+
motor with MotA. The other two MomB proteins, inwhich the junction sites were within the transmembrane region, also formed Na+ motors with PomA, butwere changed for Na+ or Li+ speci®city. These results
show that the channel part consisting of the transmembrane regions from the A and B subunits caninterchange Na+- and H+-type subunits and this canaffect the ion speci®city. This is the ®rst report to havechanged the speci®city of the coupling ions in a bacterial ¯agellar motor.
Keywords: coupling ion speci®city/¯agella/motor
proteins/Vibrio

Introduction
Bacterial cells have two kinds of rotary machine: the F0F1ATPase and the ¯agellar motor (Khan, 1997). Both motors
use the electrochemical potential of a speci®c ion (a proton
or a sodium ion, depending on the organism) for the energy
source of rotation. The F0F1-ATPase exists in bacteria as
well as mitochondria and chloroplasts, and produces ATP
by the ion ¯ux. The ¯agellar motor is only present inbacteria and converts the ion ¯ux to the locomotive forceof the cell. These are all the rotary motors that are known
in nature.
In the case of the F0F1-ATPase, rotation of the watersoluble F1 part as well as of the membrane-embedded F0part was observed (Noji et al., 1997; Sambongi et al.,1999). The F1 part is composed of three a and three b, g, dand e subunits, with the b subunits having ATPase activity.
On the other hand, the membrane-embedded F0 partconsists of an a, two b and 10±12 c subunits (Dimroth,ã European Molecular Biology Organization

1997; Fillingame, 1997). It has been found that a hybrid
enzyme composed of the F0 part from the sodium type andthe F1 part from the proton type shows a sodium-type
phenotype (Laubinger et al., 1990; Kaim and Dimroth,
1993, 1994). Thus, the F0 part seems to determine thecoupling ion. It has been suggested that the a and c subunitsof F0 are involved in the ion translocation, because
mutation of either subunit changes the coupling ion
speci®city (Kaim et al., 1997, 1998; Valiyaveetil andFillingame, 1997). In the c subunits, some residues that are
involved in ion recognition have been identi®ed, andinclude the conserved dicyclohexylcarbodiimide (DCCD)reactive acidic residue (Zhang and Fillingame, 1995; Kaim
et al., 1997). Recently, the three-dimensional structure(Dmitriev et al., 1999) and the conformational changes
between protonated and deprotonated forms (Rastogi andGirvin, 1999) of Escherichia coli c subunits have been
resolved.
Other rotary machines, namely bacterial ¯agellar
motors, rotate helical ¯agella and propel cells towardfavorable stimuli. Among various bacteria, the protondriven motor of E.coli is most studied in terms of structure,
genetics, biochemistry and biophysics (Blair, 1995;
DeRosier, 1998; Berry and Armitage, 1999). The innerpart of the motor consists of FliG, FliM and FliN, and iscalled C-ring or switch complex. On the other hand, thestator part is composed of MotA and MotB in protondriven motors (DeRosier, 1998), or PomA and PomB in
sodium-driven motors (Asai et al., 1997; Jaques et al.,1999). In these sodium motors there are two additional
stator subunits: MotX and MotY (McCarter, 1994a,b;Okunishi et al., 1996).
The A subunits, MotA and PomA, have four transmembrane regions and a large cytoplasmic loop. It was
proposed that some charged residues in this loop in
E.coli MotA interact with oppositely charged residues of
FliG during torque generation (Zhou et al., 1998a). The
B subunits, MotB and PomB, have a single transmembraneregion in the N-terminus, including an important Asp
residue thought to be involved in ion transfer (Sharp et al.,
1995; Zhou et al., 1998b). A peptidoglycan-binding motif
is conserved in the C-terminus of B subunits, which plays arole in anchoring the motor to the cell wall. The region
between the transmembrane and the C-terminal region was
thought to be a linker. This was also supported by analysis
of a series of 10 amino acid deletions in Salmonella MotB
(Muramoto and Macnab, 1998). MotA and MotB interact
with each other via their transmembrane regions and arethought to form ion-conducting channels (Blair and Berg,
1990; Stolz and Berg, 1991; Blair, 1995). PomA and
PomB of Vibrio alginolyticus have been shown to interact
functionally with each other (Yorimitsu et al., 1999).
Recently, it was reported that when PomA and PomB werepuri®ed and reconstituted into proteoliposomes, they3639

Y.Asai et al.

catalyzed 22Na+ uptake activity (Sato and Homma, 2000).
In addition, mutations conferring resistance to phenamil, a
speci®c inhibitor of sodium-driven motors or sodium
channels (Atsumi et al., 1990), were found in both pomAand pomB in V.alginolyticus (Kojima et al., 1999), and intheir homologs in Vibrio parahaemolyticus (Jaques et al.,1999). These data strongly support the hypothesis that
PomA and PomB form a sodium-conducting channel.
The proteins MotX and MotY have single transmembrane regions and are unique components of the sodiumdriven motor. They do not have similarity to the proton
motor components or any other proteins, although a
peptidoglycan-binding motif is in the C-terminal region of
MotY (McCarter, 1994a,b). MotX is inferred to be a
sodium channel component (McCarter, 1994a). Theirfunctions are rather unclear.
It should be possible to investigate the couplingmechanisms of proton or sodium ion ¯ux for force
generation in ¯agella. As reported previously, we wereable to produce and study a hybrid motor made of the
proton and sodium types (Asai et al., 1999). The
components from Rhodobacter sphaeroides (for the protontype) and from V.alginolyticus (for the sodium type) wereused, because both A and B subunits of R.sphaeroides
were found to be most similar to those of V.alginolyticus(Shah and Sockett, 1995; Shah et al., 1995; Asai et al.,1997). The photosynthetic bacterium R.sphaeroides has asingle ¯agellum extending from the center of the cell body.
The ¯agellar motor of R.sphaeroides rotates unidirectionally in the clockwise direction, and stops and restarts
periodically (Armitage and Macnab, 1987). Both RsMotA(MotA of R.sphaeroides) and VaPomA (PomA of
V.alginolyticus) are 253-amino-acid proteins, and they
have ~40% identity over their entire length. A PomAde®cient Vibrio strain expressing RsMotA from plasmids
can swim using sodium ion ¯ux. This showed that
A subunits do not determine the ion speci®city. In thisstudy, we tried to clarify whether the B subunits wereinvolved in coupling ion recognition and whether thenature of the B subunit transmembrane region affected theion speci®city. We constructed and characterized bacterial
strains with motors containing either RsMotA or VaPomA,
each with one of a series of chimeric B subunits formed
between RsMotB and VaPomB.

Results
Introduction of R.sphaeroides motB into the pom
mutants of V.alginolyticus

While this study was in progress we found that the
published sequence of R.sphaeroides motB gene containedsome errors in the 3¢ region. We determined the nucleotide
sequence of the whole motB gene again. Figure 1A shows
the corrected amino acid sequence. It was found that
RsMotB has a peptidoglycan-binding motif in the
C-terminal region, like all members of the MotB/PomB
family. The linker region of RsMotB between thetransmembrane region and the peptidoglycan-binding
motif is longer than those of the B subunits from otherspecies. The transmembrane region of the proton-type
R.sphaeroides MotB (RsMotB) is very similar to thetransmembrane region of the sodium-type PomB ofV.alginolyticus.
3640

As shown in Figure 1B, the pomAB and RsmotAB genes
could be controlled from the lac promoter in pYA303,
pYA3031, pYA703 and pYA7031. We put a SpeI site
between the stop codon of the A subunit gene and theShine±Dalgarno sequence for the B subunit gene of
pYA303 and pYA703, to give pYA3031 and pYA7031,
respectively. This allowed us to switch the SpeI±SacI
fragments of pYA3031 and pYA7031, to give pYA3032(pomA and motB) and pYA7032 (motA and pomB). These
plasmids were introduced into the pomAB mutant
NMB191. Figure 2A shows the motility of the transformants. The pomAB mutant was restored to motility by
pYA303, pYA3031 and pYA7032. All of these plasmids
contain the pomB gene. On the other hand, pomAB mutant
cells carrying pYA703, pYA7031 (data not shown) or
pYA3032 were not motile. Thus, the whole motB gene
could not complement the pomB defect of Vibrio polar¯agella even when PomA or MotA was co-expressed with
it. We tested whether the proteins were expressed in nonswarming cells by immunoblotting (Figure 3). In all of theswarming strains as well as non-swarming strains, both the
A and B subunits were detected. For example, in pomAB
mutant cells containing pYA3032, bands of PomA and
MotB were detected (Figure 3A and D). Thus, weconcluded that non-swarming cells also expressed bothsubunits, and that MotB can not function in Vibrio cells.
Construction of chimeric proteins between MotB
and PomB

As we had found that the intact MotB protein can not workin the polar ¯agellar motors of V.alginolyticus, we wishedto determine whether differences between N- or
C-terminal regions of MotB and PomB were responsible
for this result. To do this we constructed two chimeric
proteins, named PotB0 and PotB1, consisting ofN-terminal regions of PomB and C-terminal regions of
MotB. The chimeric junctions are shown in Figure 1A(junction site 0 for PotB0 and site 1 for PotB1). Althoughmost of PotB0 and PotB1 is derived from PomB, neither ofthe chimeras was functional in the presence of either MotA
or PomA (data not shown). Accordingly, we constructed
six other chimeric proteins, named MomB, consisting ofN-terminal regions of MotB and C-terminal regions of
PomB. The swarming abilities of pomAB mutant cells coexpressing MotA with each different MomB are shown in
Figure 2B. The three chimeric proteins, MomB3, MomB5
and MomB6, expressed together with MotA, restored the
motility of the pomAB mutant. The swarm sizes of thestrains containing MomB5 and MomB6 were comparable
to that of control cells expressing MotA with PomB,
although the MomB3-expressing strain made a smallerswarm than the others. When expressed with PomA,
MomB5 and MomB6 were also functional in pomAB
mutant cells. Cells expressing MomB6 swarmed as fast as
cells expressing wild-type PomB. Unlike with MotA,
MomB3 did not restore the motility with PomA. The otherchimeric proteins, MomB1, MomB2 and MomB4, did notsupport swarming regardless of whether they were coexpressed with PomA or MotA. We checked the expression of the MomB proteins by immunoblotting using
antibodies raised against RsMotB and VaPomB peptides(data not shown). All of the MomB proteins wererecognized by both anti-RsMotB and anti-PomB

Chimeras between MotB and PomB

Fig. 1. (A) Amino acid alignments of V.alginolyticus PomB and R.sphaeroides MotB. White letters in black boxes and arrowheads show identicalresidues and the junction site of chimeric protein, respectively. Gray bars indicate putative transmembrane regions. (B) Restriction maps of plasmids.
White boxes and the direction of solid arrowheads in the boxes indicate the vector part of pSU41 and the direction of transcription from the lacpromoter, respectively. Inserted fragments from V.alginolyticus and R.sphaeroides, respectively, are indicated by a black or gray bold line. The
hatched part of pRED1 is the region of the native R.sphaeroides promoter. The open arrows show the coding regions of pomA, pomB, motA and motB.
VaPomB, V.alginolyticus PomB; RsMotB, R.sphaeroides MotB; H, HindIII; Xb, XbaI; B, BamHI; Sc, SacI; E, EcoRI; Sp, SpeI; Sh, SphI; X, XhoI;Sm, SmaI; Sl, SalI.

3641

Y.Asai et al.

Fig. 3. Detection of each protein by the anti-peptide antibody.
NMB191 cells expressing each protein were cultured to exponential
phase, harvested and suspended in distilled deionized water.
Immunoblottings were carried out using anti-VaPomA (A), antiVaPomB (B), anti-RsMotA (C) and anti-RsMotB antibody (D).

Fig. 2. Swarming abilities of transformants. Fresh colonies were
inoculated in 0.3% agar VPG plates containing 100 mg/ml kanamycin
and incubated at 30°C for 5 h. The pomAB mutant (NMB191) wasused as the host strain. Proteins expressed in each strain, various
combinations of A and B subunits of the proton-driven or sodiumdriven motors (A), each MomB protein with proton-type MotA (B)and each MomB protein with sodium-type PomA (C), are notedaround swarms.

antibodies. Most of the chimeric proteins were detected as
bands with apparent molecular masses similar to wild-type
PomB and MotB.
Characterization of the MomB proteinsco-expressed with PomA or MotA

We examined which ion is coupled to generate torque inthe motile cells expressing the MomB proteins. As shown
3642

in Table I, we tested whether cells could swim in Na+-freebuffer (TMK300 containing 300 mM K+) and whether
their motility was affected by a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The results
showed that none of the functional motors rotated in the
Na+-free buffer nor did the motor consisting of PomA and
PomB (pYA303/NMB191), which uses Na+ but not H+ asa coupling ion. Moreover, the former and the latter motors
did not stop in TMN300 (pH 8.5) containing 20 mM
CCCP, but stopped completely with the further addition of
20 mM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a
speci®c inhibitor of the primary Na+ pump (Tokuda andUnemoto, 1982). In alkaline Tris motility buffer, YM19
(Pof± Laf+) cells swam in the absence of CCCP, but
stopped completely in the presence of 20 mM CCCP,
because lateral ¯agella have proton-driven motors. We
concluded from these results that all of the MomB-based
motors are driven by Na+-motive force. We found a few

28 (2.0)
27 (1.7)
28 (1.7)
9.9 (1.5)0
4.4 (0.8)
53 (3.9)42 (3.6)
54 (1.7)
5 (2.2)0
L.F.
26 (2.5)
20 (1.9)31 (5.5)5 (1.0)05.5 (0.7)
20 (2.3)
14 (2.0)23 (2.2)
3 (1.7)0
L.F.b70 (4.7)
51 (4.8)
67 (7.9)
6 (0.9)08.5 (1.2)
11 (0.9)8 (1.3)10 (1.0)
3 (1.5)0
24 (2.9)

MomB3
MomB6

80 (3.8)a64 (6.9)80 (3.7)
10 (1.7)
035 (4.5)

Li+ coupling abilities of functional MomBs

bL.F., low motile fraction.

aSD is shown in parentheses.

K+
Li+

7.5

8.5
Na+

0
20
0
20
0
0

MomB5
PomAB (pYA303)

pH

CCCP (mM)

Fig. 4. Swimming speed of NMB191 cells expressing PomAB(pYA303), MomB5 with PomA or MomB6 with PomA. The cells weresuspended in Tris-motility buffer (TMN50) and diluted 100-fold into
buffers containing various concentration of Na+ (open squares) or Li+(closed squares) as coupling ions, and the swimming speeds were
measured.

motile cells among cultures expressing MomB1 or
MomB2 with MotA in TMN300. However, motile fractions of these strains were too low to measure theswimming speeds and to determine their coupling ions.

Salt (300 mM)

Table I. Characterization of chimeric proteins

Swimming speed (mm/s)

PomA+

MotA+

MomB5

MomB6

MotA +PomB(pYA7032)

YM19

20 (1.8)027 (1.4)
022 (2.4)
24 (2.2)

Chimeras between MotB and PomB

The wild-type polar ¯agellar motor of V.alginolyticus canuse Li+ as a coupling ion less ef®ciently than Na+. The
maximal swimming speed produced by Li+ is only ~30%of that of Na+ ions (Liu et al., 1990; Imae, 1991). To test
the ion speci®city of the MomB-based motors, wemeasured swimming speeds of cells expressing MomB
proteins in 300 mM Li+ (Table I). In 300 mM Li+, themotile fractions and the swimming speeds of the pomAB
strain expressing MomB3 or MomB6 with MotA were too
low for quantitative measurement. The motors consistingof PomA and MomB5 or MomB6 showed an interesting
property. Unlike cells expressing PomA and PomB, cells
expressing MomB5 with PomA swam faster in 300 mM
Li+ than in 300 mM Na+. Cells expressing MomB6 with
PomA swam much slower in 300 mM Li+ than thoseexpressing PomA and PomB, although in 300 mM Na+ theformer cells swam nearly as fast as the latter cells. We
examined the effect of salt concentration on swimming
speed (Figure 4). Cells of any strain did not swim in the3643

Y.Asai et al.

Fig. 5. Competition of Na+ with Li+ for swimming speed of eachstrain. NMB191 cells expressing PomAB(pYA303), PomA+MomB6,
RsMotA+PomB or RsMotA+MomB6 were suspended in buffers at
various concentrations of Na+ with 200 mM K+ (open squares) or
200 mM Li+ (closed diamonds) and their swimming speeds were
measured. The total salt concentration was adjusted to 300 mM
with K+.

Fig. 6. The swimming speeds of each strain were measured at various
concentrations of the speci®c inhibitor of sodium motor, phenamil, in
TMN50. The results of MomB chimeras with RsMotA (upper panel) orwith PomA (lower panel) are shown.

Resistance to inhibitors of hybrid motors
containing MomB

absence of Na+ or Li+, and their swimming speeds
increased with an increase in either salt concentration.
Cells expressing MomB5 with PomA swam faster with Li+
than with Na+ at any ®xed concentration. The maximal
swimming speed of cells expressing PomA and PomB in
Li+ was approximately one-third of that in Na+, as reported
before. On the other hand, the maximal speed of cells
expressing MomB6 with PomA in Li+ is one-tenth of thatin Na+. Next, we examined the competitive effect of Li+against Na+, because it is thought that Li+ shares the same
binding site(s) in the motor as Na+. We measured theswimming speed in Tris motility buffer, which contained
various concentrations (from 0 to 100 mM) of Na+ with orwithout 200 mM Li+. The total salt concentration of every
condition was constantly adjusted to 300 mM with K+(Figure 5). The addition of Li+ reduced the maximum
speed of cells expressing PomA and PomB from 80 to
65 mm/s, suggesting that Na+ and Li+ compete for the same
binding site(s) in the motor. In the absence of Li+, cells
expressing a motor consisting of MomB6 with PomA
showed a similar swimming speed to that of cells
expressing PomA and PomB. However, Li+ had alarger effect on the PomA and MomB6 motor than the
PomA and PomB motor: the maximum speeds in thepresence of Li+ decreased to 45 and 65 mm/s, respectively.
Cells expressing MomB6 with MotA gave similar results
to those with PomA, although the speed of the former
motor was always lower than the latter. These results may
suggest that Li+ binds to motors containing MomB6, but is
not conducted, or that Li+ ¯ux is not coupled to rotation inthese motors.
3644

If the ion speci®city was changed in motors consisting of
MomB and PomA, the effects of phenamil and amiloride,
speci®c inhibitors of the sodium-driven motor, may also bechanged. As shown in Figure 6, cells expressing MomB5
or MomB6 with either MotA or PomA showed phenamilresistant phenotypes. For MomB6 motors, the swimming
speed slowed initially on phenamil addition, but thenreached a phenamil-resistant plateau of 20±40 mm/s. It isnoteworthy that cells expressing MomB5 with either
MotA or PomA were hardly inhibited even by 50 mM
phenamil, the highest concentration tested, which completely inhibits the wild-type motor. The IC50 value, theconcentration of inhibitor required for 50% inhibition of
motility, for phenamil in cells expressing MomB3 with
MotA was comparable to that for cells expressing PomA
and PomB. This suggests that changing motor conformation alters phenamil-binding sites, yielding MomB strains
with resistance to phenamil. On the other hand, all thestrains tested showed the same sensitivity phenotypes toamiloride as cells expressing PomA and PomB (data notshown). The noteworthy features of all the chimeric
B proteins are summarized in Figure 7.

Discussion
Recently, we reported that a hybrid motor consisting of
R.sphaeroides MotA, V.alginolyticus PomB, MotX and
MotY, can work as a sodium-driven motor in Vibrio cells(Asai et al., 1999). This showed that the A subunits do not
determine the ion speci®city and raises the question: which
components decide the ion selectivity? The most likely
candidate seemed to be the B subunits. The PomB proteinsChimeras between MotB and PomB

Fig. 7. Summary of the chimeric protein features. Black and grayboxes show regions that are derived from VaPomB and RsMotB,
respectively. TM, transmembrane region; PGB, peptidoglycan-binding
motif; W.T., wild type; MpaS, motility that is phenamil sensitive; N.F.,
non-functional; Mpar, motility that is phenamil resistant; Na+, motility
coupling to sodium ion; Li+, motility coupling to lithium ion;¯, impaired motility.

are known to form a sodium channel together with the
PomA proteins (Sato and Homma, 2000), and contain aconserved Asp residue, which is located in the transmembrane region and seems to be essential for the ion transfer.
In this study, we examined whether speci®c regions of the
B subunits are involved in the ion speci®city of the motor.
Initially, we constructed plasmids encoding various
combinations of A and B subunits of the proton-type(R. sphaeroides) or the sodium-type motors (V.alginolyticus),
i.e. MotA and PomB, or PomA and MotB. These plasmids
were introduced into the pomAB mutant of Vibrio cells. As
shown in Figure 2A, MotB could not work with either
MotA or PomA in the pomAB mutant. In spite of the higher
GC content of the motB gene compared with the Vibrio
pomB gene, the MotB protein seemed to be expressed atsuf®cient levels to give function. A possible reason for thelack of complementation is that the peptidoglycan-binding
domain of MotB can not associate with the Vibrio
peptidoglycan layer. This speculation was supported by
the evidence that the chimeric proteins, PotB, between the
N-terminal region of PomB and the C-terminal (peptidoglycan-binding) region of MotB, did not function at all(data not shown) and that the mutation of the highly
conserved Ser residue (but not conserved in RsMotB) in
the peptidoglycan-binding domain caused a defect of the
PomB function in Vibrio NMB161 (Asai et al., 1997; ourunpublished data).
Next, we constructed the chimeric protein series, named
MomB, consisting of N-terminal regions of MotB andC-terminal regions of PomB. The features of all chimeras
are shown in Figure 7. MomB1, MomB2, MomB3 andMomB4 have junction sites in a non-conserved `linker'
region, which connects the transmembrane region and the
peptidoglycan-binding motif. The other chimeric proteins,
MomB5 and MomB6, have junction points within thetransmembrane region that are highly conserved. These six

MomB proteins were expressed with either MotA or
PomA in the pomAB mutant. Out of all the MomB
chimeras, only MomB5 and MomB6 functioned as
sodium-driven motor components. However, the speci®cities for Na+ and Li+ were changed in the motors with
MomB5 and MomB6. The MomB6-containing motor was
driven by Na+ as effectively as the wild-type motor, butmotility could barely be driven by Li+. The MomB5containing motor was driven slowly by Na+, but rotation
was coupled to Li+ more effectively than to Na+. Structuralchanges to the channel in the transmembrane regions of
the A and B subunits may affect the ef®ciency of the iontransfer and/or the energy coupling for Li+ or Na+. We
speculate that the differing ion speci®cities for rotation
shown by MomB5- and MomB6-containing motors may
correlate with ion size. This may be because the MomB5
protein, whose transmembrane region derived mostly fromthe proton-type protein MotB, works better with thesmaller ion Li+, and because MomB6, whose transmembrane region derived mostly from the sodium-type protein
PomB, works better with the larger Na+ ion. There are few
non-conserved amino acids between the MotB and PomB
proteins in the region between the MomB6 and MomB5
junction points. While these small differences may
account for ion-speci®city changes, it is more likely thatthe periplasmic region of PomB proximal to the surface ofthe membrane (which is present in MomB6 but not in
MomB5) plays a role in ion speci®city or interaction withother sodium-motor proteins. Thus, the ion size discrimination of channels containing MomB6 is different to thatof channels containing MomB5. This idea is further
supported by the MomB3 data.
MomB3 retains the sequence of the sodium-type
B subunit from the linker region to the C-terminus, andMomB3 was also functional in a pomAB strain with MotA,
although the swimming ability was poor. However,
MomB3 did not function with PomA. The region aroundthe junction site 3 may need to interact with A subunits
precisely. Moreover, from the evidence that the motor
consisting of MotA and MomB3 is sodium type, it seems
that the transmembrane regions of the B subunit as well as
the A subunit do not determine the ion speci®city directly.
MomB1, MomB2 and MomB4 expressed with either
PomA or MotA were non-functional. The MomB4 junction site may be more important for maintaining thestructure of an ion-speci®c channel composed of thetransmembrane regions of the A and B subunits. Garzaet al. (1996) also suggested that in E.coli the MotA
periplasmic region interacts with the linker region of
MotB, or that this region helps the transmembrane domain
to position properly to interact with the transmembraneregions of MotA. We have investigated the PomA periplasmic region (loop1±2 and loop3±4) by cysteine scanningmutagenesis, and it is inferred that loop1±2 may be covered
with other proteins or may be embedded in the pore regionof the channel (Asai et al., 2000). We speculate thatloop1±2 interacts with the linker region of the B subunit.
`Where' and `how' do the motor proteins recognize thecoupling ion? Many mutations that alter the ion speci®city
are known in various ion channels and transporters (Hamaand Wilson, 1993; Heginbotham et al., 1994; Kellenberger
et al., 1999). However, it is unclear how these residues
recognize the different ions. In the other rotary motor of3645

Y.Asai et al.

Table II. Bacterial strains and plasmids
Strain or plasmid
V.alginolyticus strains
VIO5
VIO586
NMB191
YM19
E.coli strains
DH5a
Plasmids
pSU41
pRED1
pYA303
pYA3031
pYA3032
pYA701
pYA703
pYA7031
pYA7032

Genotype or description

Reference or source

VIK4 laf (Rif r Pof+ Laf±)
VIO5 pomA (Rif r Laf± Pom±)
VIO5 DpomAB (0.94 bp DraI±HpaI fragment deletion) (Rif r Laf± Pom±)
138-2 pof (Pof±)

Okunishi et al. (1996)
Asai et al. (1997)
Yorimitsu et al. (1999)
Kawagishi et al. (1995)

F± l±recA1 hsdR17 endA1 supE44 thi-1 relA1 gyrA96 D(argF-lacZYA)U169 f80dlacZDM15

Grant et al. (1990)

kan (Kmr) PlaclacZ a
pUC19 2.5 kb SphI±SalI fragment (motAB+)
pSU41 1.9 kb BamHI±SacI fragment (pomAB+)
pSU41 0.8 kb BamHI±SpeI fragment (pomA+) and 1.1 kb SpeI±SacI fragment (pomB+)
pSU41 0.8 kb BamHI±SpeI fragment (pomA+) and 1.1 kb SpeI±SacI fragment (motB+)
pSU41 0.8 kb BamHI±EcoRI fragment (motA+)
pSU41 1.9 kb BamHI±SacI fragment (motAB+)
pSU41 0.8 kb BamHI±SpeI fragment (motA+) and 1.1 kb SpeI±SacI fragment (motB+)
pSU41 0.8 kb BamHI±SpeI fragment (pomA+) and 1.1 kb SpeI±SacI fragment (pomB+)

Bertolome et al. (1991)
Shah and Sockett (1995)
Kojima et al. (1999)
this work
this work
Asai et al. (1999)
this work
this work
this work

Kmr, kanamycin resistant; Mpar, motility resistant to phenamil; Rifr, rifampicin resistant; Plac, lac promoter.

F0F1-ATPase, a hybrid enzyme composed of the F0 partfrom the sodium type and the F1 part from the proton typefunctioned as a sodium-type enzyme (Laubinger et al.,
1990; Kaim and Dimroth, 1993, 1994). The residues
involved in ion recognition were identi®ed in the a andc subunit and the ions should interact with the subunits.
The ion-binding residue, which is the DCCD-reactive
residue, was postulated as Glu65 for Propionigenium
modestum c subunit and as Asp61 for E.coli c subunit. In
the case of sodium-type F0F1-ATPase c subunits, ®veresidues that are involved in the ion recognition have been
identi®ed, and include the DCCD-reactive Glu65 residue,
Ser66 and Gln32 (Kaim et al., 1997). The corresponding
residues in proton-type c subunits are Asp61, Ala62 andIle28, respectively. The substitution of Gln32 of sodiumtype to Ile abolished only the property of Na+ coupling.
The substitution of Ser66 to Ala abolished the property of
Na+ and Li+ coupling, and retained H+ coupling ability.
From these results, it was proposed that the side chains ofthese residues coordinated an ion. Those side chains ofcorresponding residues in the proton-type c subunit wereshown to face each other from the three-dimensional
structure (Dmitriev et al., 1999). Another important
example is the KcsA potassium channel, for which the
crystal structure has been solved and the mechanism of ion
recognition has been revealed (Doyle et al., 1998).
Mutations at some residues in the pore region change theability of the channel to discriminate K+ from Na+(Heginbotham et al., 1994). The side chains of theseresidues face the outside of the pore and K+ ions interact
with the oxygen atoms of these peptide backbones. The
selective ®lter formed by four oxygen atoms ®ts andstabilizes a dehydrated K+ electrostatically. However,smaller Na+ ions can not ®t into the ®lter and therefore failto overcome electrostatic destabilization in the pore.
In the ¯agellar motor, Asp32 of E.coli MotB is
speculated to function proximal to the surface of themembrane in conveying protons (Sharp et al., 1995; Zhou
et al., 1998b) and the acidic residue is conserved inV.alginolyticus PomB and R.sphaeroides MotB. If theacidic residue has a similar function to the DCCD-reactive
3646

residue of F0, a few residues necessary for Na+ or Li+
binding might exist in the transmembrane region of the A
and B subunits and the other components. It would beinteresting to know whether the mechanism of ion
recognition is common to the two kinds of ion-driven
rotary motor. Otherwise, by the KscA analogy, thetransmembrane regions of the A and B subunits may
form a selective pore for ion recognition. The selective
pore may be modulated by a speci®c part of the linker
region of the B subunits or the other motor proteins, MotX
and/or MotY. From our results with the motor consistingof MotA and MomB3 showing that the transmembraneregion of the B subunit does not decide the ion speci®city,
the linker region might be required for the precise
arrangement of the transmembrane regions. Accordingly,
the linker regions of the proton- and sodium-type subunits
might force the transmembrane regions to form small andlarge pores, which accommodate protons and sodium ions,
respectively. Alternatively, MotX and/or MotY mightrecognize the ions and pass the ions to the PomA±PomB
complex. FliG is also a candidate protein for determining
the ion speci®city, because it was implied that the charged
residues at the MotA±FliG interface might form a pathway
for the conduction of protons (Zhou et al., 1998a). It is
possible that all of these proteins are essential for the
precise ion speci®city in the ¯agellar motor.

Materials and methods
Bacterial strains, plasmids, growth conditions and media
The strains and plasmids used are shown in Table II. Vibrio alginolyticus
cells were cultured at 30°C in VC medium (0.5% Polypepton, 0.5% yeast
extract, 0.4% K2HPO4, 3% NaCl, 0.2% glucose) or VPG medium (1%Polypepton, 0.4% K2HPO4, 3% NaCl, 0.5% glycerol). When necessary,
kanamycin was added to a ®nal concentration of 100 mg/ml. Escherichia
coli cells were cultured at 37°C in LB medium (1% bactotryptone, 0.5%
yeast extract, 0.5% NaCl). For E.coli, kanamycin was added to a ®nal
concentration of 50 mg/ml. The swimming speed was measured in Trismotility buffers, TMN300 (300 mM NaCl, 50 mM Tris±HCl pH 7.5,5 mM MgCl2, 5 mM glucose), TMK300 (300 mM KCl, 50 mM Tris±HCl
pH 7.5, 5 mM MgCl2, 5 mM glucose) or mixtures of TMN300 and
TMK300 to vary the sodium concentration.

Chimeras between MotB and PomB
DNA manipulations and sequencing
Routine DNA manipulations were carried out according to standard
procedures (Sambrook et al., 1989). Restriction endonucleases and otherenzymes for DNA manipulations were purchased from Takara Shuzo(Kusatsu, Japan) and New England Biolabs (Beverly, MA). Nucleotide
sequences were determined by using the dye terminator cycle sequencing
kit (Perkin Elmer Co.) and an ABI PRISM sequencer Model 377 (PEApplied Biosystems).
Plasmid construction and site-directed mutagenesis
We constructed a plasmid, pYA703, including the PCR-ampli®ed motAB
fragment from 40 bp upstream of the start codon to 8 bp downstream ofthe stop codon. The PCR ampli®cation was performed as described
previously (Kojima et al., 1999). In the reaction, we used the end primers,
RsmotA.B1 and RsmotB.Z3, and pRED1 as templates. RsmotA.B1 has a
BamHI site and is the sense primer anealing the upstream RsmotA gene,
and was used when we constructed pYA701 (Asai et al., 1999).
RsmotB.Z3, including a SacI and EcoRI site, is the antisense primer
annealing the downstream of the gene, 5¢-CGGAATTCGAGCTCGGACAAGGTCAGTCCT-3¢. This PCR-ampli®ed fragment was inserted into
pSU41 (Bartolome et al., 1991) between the BamHI site and the EcoRI
site. The motAB gene of pYA703 is under the control of the lac promoter
directly and does not have the native promoter.
Site-directed mutagenesis was performed by a two-step PCR reaction
as described previously (Kojima et al., 1999). To introduce a SpeI site
between the stop codon of the A subunit and Shine±Dalgarno sequence of
the B subunit in pYA703 or pYA303, we used the pairs of mutant primers,
RsmotA.spe1 and RsmotA.spe2 or VapomA.spe1 and VapomA.spe2. The
sequences of the sense strand of RsmotA.speI and VapomA.speI were
5¢-GGCGGCGTGACTAGTGAGGCCAGGCCATG-3¢ and 5¢-ACGAGTAACTAGTGGAGAGTCG-3¢, respectively. We also constructed momB
genes using the two-step PCR. The sense strand sequences of the mutant
primers were as follows: MomB1, 5¢-AGAAGGCGCTCGCTGGTGGCACAAAACGT-3¢; MomB2, 5¢-AAGTCGGACACGATGCAGCAAACCATG-3¢; MomB3, 5¢-TTCCATTCGATCCTTGAAGTGAAAGAC-3¢;
MomB4, 5¢-GCTTCGCGAAGATGGATGTACTGAAGTTC-3¢; MomB5, 5¢-ATGGCCTTCTTCGTTCTTCTGCTCTCG-3¢; MomB6; 5¢-CTTGCGACCTTTGCAGATTTGATGTCG-3¢.
Transformation of Vibrio cells
Transformation of Vibrio cells was carried out by electroporation as
described previously (Kawagishi et al., 1994). Cells were subjected toosmotic shock and washed thoroughly with 20 mM MgSO4.
Electroporation was carried out with the Gene Pulser electroporation
apparatus (Japan Bio-Rad Laboratories, Tokyo, Japan) at an electric ®eldstrength between 5.0 and 7.5 kV/cm.
Immunoblotting
Preparation of cells and immunoblotting were carried out as described
previously (Yorimitsu et al., 1999). For the ®rst antibody of
immunoblotting,weused
anti-peptide
antibodies
againstV.alginolyticus PomA (VaPomA), VaPomB, R.sphaeroides MotA
(RsMotA) or RsMotB. PomA91, PomB93 and RsMotA253 antibodies
were used as anti-VaPomA, anti-VaPomB and anti-RsMotA antibodies,
respectively (Asai et al., 1999; Yorimitsu et al., 1999). The production
and af®nity puri®cation of anti-RsMotB antibodies (RsMotB2) werecarried out by Sawady technology (Tokyo, Japan). The selected peptide
fragment was synthesized arti®cially, with the sequence NH2SAKPKVIRFQPPVPDDDEGEC-COOH, which is located at the
N-terminus of RsMotB. A cysteine was added to the C-terminus andthe synthesized fragment was conjugated to keyhole limpet hemocyanin(KLH). Rabbits were immunized with the conjugated KLH. For thesecond antibody, we used an alkaline phosphatase-conjugated goat antirabbit IgG antibody.
Measurement of swimming speed
An overnight culture in VC medium was inoculated into fresh VPG
medium at a 100-fold dilution and grown at 30°C to exponential phase.
Cells were harvested by centrifugation and suspended in TMN50 (50 mM
NaCl, 250 mM KCl, 50 mM Tris±HCl pH 7.5, 5 mM MgCl2, 5 mM
glucose). The cell suspension was diluted ~100-fold into Tris motility
buffers containing various concentrations of NaCl, and 20 mM serine tosuppress the directional change of swimming. Motility of the cells wasobserved under a dark-®eld microscope and recorded on videotape.
Swimming speed was determined as described previously (Atsumi et al.,1996).

Acknowledgements
We thank Dr Deepan Shah for critically reading the manuscript. This
work was supported in part by grants-in-aid for scienti®c research from
the Ministry of Education, Science and Culture of Japan (to I.K. andM.H.), the Japan Society for the Promotion of Science (to Y.A.), and theRoyal Society and BBSRC (to R.E.S.).

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Received April 25, 2000; revised and accepted May 30, 2000