doi:10.1016/S0022-2836(03)00096-2

J. Mol. Biol. (2003) 327, 453–463

Ion-coupling Determinants of Na1-driven andH1-driven Flagellar Motors
Yukako Asai, Toshiharu Yakushi, Ikuro Kawagishi andMichio Homma*
Division of Biological Science
Graduate School of Science
Nagoya University
Chikusa-Ku
Nagoya 464-8602, JapanThe bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of Hþ or Naþ ions to drive flagellar rotation. In protondriven motors, the membrane proteins MotA and MotB interact via theirtransmembrane regions to form a proton channel. The sodium-driven
motors that power the polar flagellum of Vibrio species contain homologsof MotA and MotB, called PomA and PomB. They require the uniqueproteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia
coli MotA/B restore motility in DpomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain containmutations in motB. We constructed proteins in which segments of MotB
were fused to complementary portions of PomB. A chimera joining the N
terminus of PomB to the periplasmic C terminus of MotB (PotB7E) functioned with PomA as the stator of a sodium motor, with or without
MotX/Y. This stator (PomA/PotB7E) supported sodium-driven motility
in motA or motB E. coli cells, and the swimming speed was even higher
than with the original stator of E. coli MotA/B. We conclude that thecytoplasmic and transmembrane domains of PomA/B are sufficientfor sodium-driven motility. However, MotA expressed with a B subunit
containing the N terminus of MotB fused to the periplasmic domain of
PomB (MomB7E) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is notnecessary for sodium-driven motility in a PomA/B motor, it can convert
a MotA/B proton motor into a sodium motor.
q 2003 Elsevier Science Ltd. All rights reserved*Corresponding author

Keywords: flagella; motor proteins; ion channels; ion selectivity; Vibrio

Introduction
Many species of bacteria monitor their chemical
environment and swim toward favorable conditions by rotating helical organelles known as
flagella.1 – 3 The energy source for flagellar rotationis the electrochemical potential of a specific ion(Hþ or Naþ) across the cytoplasmic membrane.
The most commonly studied bacteria, e.g. Escherichia
coli, Salmonella typhimurium and Bacillus subtilis,
have proton-driven motors. The polar flagellum of
Present address: Y. Asai, Laboratory of Sensory
Neuroscience, The Rockefeller University, 1230 York Ave.
314, New York, 10021-6399, USA.
Abbreviations used: TM, transmembrane segment;
Pof, polar flagellum.
E-mail address of the corresponding author:
g44416a@cc.nagoya-u.ac.jp

Vibrio spp. (V. alginolyticus, V. parahaemolyticus andV. cholerae) and the peritrichous flagella of alkalophilic Bacillus species, in contrast, have sodiumdriven motors.4,5The pomA, pomB, motX and motY genes, whose
protein products are membrane-associated proteins that are essential for sodium-driven torquegeneration, have been cloned.6 – 10 PomA and
PomB are homologous to MotA and MotB, respectively, of the proton-driven motor.10 MotX andMotY are unique to the sodium-driven motor. Little
is known about the function of MotX and MotY,
except that the C terminus of MotY contains a
consensus peptidoglycan-binding motif7,9 and thatboth proteins localize to the outer membrane.11
MotA and PomA each have four membranespanning regions, a large cytoplasmic loop and acytoplasmic C-terminal tail. MotB and PomB eachhave a single membrane-spanning region.10 PomA

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

454

Ion Selectivity of the Flagellar Motor

Figure 1. (a) Restriction mapsof plasmids. Inserted fragmentsof each plasmid are indicated ashatched bars. Filled arrows show
PCR primers. The inverted triangle shows the location of thepromoter of the E. coli mocha
operon.
The
openarrowsdelineate the coding region of
motA, motB and cheA. (b) and(c) Complementation of pom
mutants by E. coli MotA and/or
MotB.
(b)
Statorproteins
expressed are: 1, MotA/B; 2,
MotA/B; 3, MotA; 4, MotA/B;
5, MotB. The host strains are:
1, NMB191 (DpomAB mutant); 2
and 3, NMB190 ( pomA mutant);
4 and 5, NMB192 ( pomB mutant).
(c) Stator proteins expressed are:1, 4 and 7, MotA/B; 2 and 5,
none; 3 and 6, PomA/B. The
host strains are: 1, NMB191(DpomAB mutant); 2– 4, NMB119(motX mutant); 5 – 7, VIO542(motY mutant).

and PomB interact with each other.12 A reconstituted PomA/B complex is capable of supporting22
Naþ uptake into proteoliposomes.13 These results
suggest that the PomA/B complex functions as a
sodium-ion channel. MotB and PomB both havea highly conserved acidic residue that faces into
the transmembrane channel. This residue is presumably crucial both for ion flux and for torquegeneration. Protonation of the acidic residue may
induce conformational changes in the stator thatdrive rotation of the rotor. These conformationalchanges were detected by the differential proteasesensitivities of MotA in membrane-bound MotA/B
complexes in which the acidic residue was replacedwith neutral or positively charged residues.14
The C-terminal regions of MotB and PomB
contain a predicted peptidoglycan-binding motif10
that has been implicated in anchoring the stator tothe cell wall. The region between the membranespanning and peptidoglycan-binding domains ispoorly conserved among B subunits (Figure 3)
and is largely dispensable for function.15 It may
serve as a flexible linker.
It has been proposed that two charged residues
in the cytoplasmic region of MotA interact with
oppositely charged residues of FliG, a componentof the rotor, and that these interactions are important for flagellar rotation.16 On the other hand,
it has been shown that these residues, which
are conserved in sodium-driven motors, may not
be essential for their function.17 Thus, there arelikely to be some fundamental differences in theoperation of sodium-driven and proton-driven
motors.

The Naþ-binding site of the motor was identified by mapping pomA and pomB mutations
that confer resistance to phenamil,18 an analog ofthe sodium-channel blocker amiloride. The mutations targeted residues near the cytoplasmic
ends of the transmembrane segments of PomA or
PomB, supporting the proposal that PomA and
PomB combine to form a Naþ channel.19,20 The
overlapping distributions of pomA and motA
missense mutations that interfere with motor
function suggest that PomA and MotA have similar structures and roles.21 Periplasimic loop1 – 2 of
PomA, which connects transmembrane helices 1
and 2, might be in contact with other proteins, or
it may interact with the pore region of the channel
to contribute to the efficient transport of ions.22 – 24
Crosslinking studies indicate that periplasmic
loop3 – 4, which joins transmembrane helices 3
and 4, of one PomA subunit is close to loop3 – 4of an adjacent PomA subunit within the stator
complex.24,25
We have shown that MotA/B from the protondriven motor of Rhodobacter sphaeroides do notcomplement a DpomAB mutant of V. alginolyticus,26,27
nor do PomA/B support flagellar rotation without
MotX and MotY. However, a DpomABmotXY strainof V. cholerae can swim using E. coli MotA/B andproton motive force.28 In this study, we haveaddressed the question of what differences exist
between PomA/B and E. coli MotA/B by determining whether hybrid stators that contain different combinations of elements from these
proteins can support rotation of the sodium-driven
polar flagellum of V. alginolyticus.

455

Ion Selectivity of the Flagellar Motor

motor without MotX or MotY in the V. alginolyticus
polar flagellar motor, as previously reported for
V. cholerae.28
When the pomAB mutant expressing MotA/B
from plasmid pYA6022 was incubated on semisolid agar plates for a few days, some fasterswarming flares appeared. Thirty-eight of 45 isolates from independent flares contained plasmidborne mutations that conferred hypermotility, andall of these hypermotile isolates formed swarms
that were as large as those formed by wild-type
E. coli. (Figure 2). The motA and motB genesof plasmids prepared from each strain weresequenced to find the mutations. Seventeen uniqueplasmid-borne mutations were found (Figure 3).
All of them were located in motB, althoughboth motA and motB are present in pYA6022. The
mutations were distributed widely and somemutations were clustered in the periplasmic region
near the putative transmembrane segment (TM).
Most strains, except Hp14 and Hp26, which seemto be less motile than the others, showed similar
sizes of swarming.
Properties of PomB/MotB chimeras

Figure 2. Complementation by the hyper-motile
mutants. Fresh colonies of (a) the V. alginolytucus pomAB
mutant or (b) the E. coli motB mutant carrying eachplasmid, pYA303 (PomA/B), pSU41 (none), pYA6022(MotA/B), and the hyper-motile mutant plasmids (HP)from pYA6022, were inoculated and incubated on 0.3%
VPG (pH7.0) agar for 15 hours (for Vibrio cells) or on T
broth agar for six hours (for E. coli cells) at 30 8C.

Results
Complementation of a V. alginolyticus
DpomAB mutation by E. coli motAB
To examine whether E. coli motA and/or motB
can complement pomA, pomB and pomAB nullmutations in V. alginolyticus, we constructed plasmids containing motAB (pYA6022), motA (pYA601)
and motB (pYA6001), as shown in Figure 1(a). The
motA and/or motB genes were transcribed fromthe lac promoter, and the native sigma-28 promoterof the mocha operon was excluded from the plasmids. All of the pom mutants that expressed
MotA/B were motile, although they formed verysmall swarms (Figure 1(b)). MotA alone did notrestore motility to a pomA mutant, nor did MotB
alone restore motility to a pomB mutant. Furthermore, the strains swarmed significantly when theE. coli MotA/B proteins were expressed in a motX
or a motY mutant (Figure 1(c)). Motility was notrestored by overexpression of PomA/B in either
the motX or motY mutant. This result indicatedthat E. coli MotA/B functions as the proton-driven

In a previous study, we constructed fusions
between R. sphaeroides MotB and V. alginolyticus
PomB.27 One of these chimeras (MomB3R) contains
an N-terminal portion of R. sphaeroides MotB (fromthe first residue through the transmembraneregion) and a C-terminal portion of PomB (theextracellular region from the end of the transmembrane region through the last residue). The
fusion joint is at junction site 3 (Figure 3).
MomB3R functioned together with R. sphaeroides
MotA in Vibrio, whereas intact R. sphaeroides MotB
did not. MomB4R, named in the same fashion forjunction site 4, did not support swarming in combination with either R. sphaeroides MotA or PomA.
Thus, the integrity of the region around junction
site 4 may be important for the function of the B
subunit.
For these reasons, and because of the strong
similarity in the amino acid sequences of PomB,
R. sphaeroides MotB and E. coli MotB around thissite (Figure 3), we joined the extracellular domainsof V. alginolyticus PomB and E. coli MotB at site 7.
This chimeric B protein was named PotB7E(Figure 4). Similarly, we constructed MomB7E andMomB7R. These chimeric B subunits were coexpressed with A subunits corresponding to thespecies that contributed the transmembrane regionof the B subunits. All three chimeric stators function with the Vibrio rotor (Figure 5(a)). The
PomA/PotB7E stator supported the best swarming,
which was much better than that of a motor
containing the E. coli MotA/B stator. The reciprocal
stator, MotAE/MomB7E, also functioned well.
We examined whether each B-subunit chimera
could function in the absence of MotX or MotY.
Although the MotAE/MomB7E and MotAR/
MomB7R stators (data not shown) did not restore

456

Ion Selectivity of the Flagellar Motor

Figure 3. Amino acid alignments of V. alginolyticus PomB (Va), R. sphaeroides MotB (Rs), and E. coli MotB (Ec), andlocation of hypermotile mutations and junction sites of chimeric proteins. White letters in black boxes and arrows
show identical residues and junction sites of chimeric proteins, respectively. Gray and hatched bars indicate putativetransmembrane (TM) regions and peptidoglycan binding motif (PGB), respectively. Hypermotile mutations and strain
numbers are indicated under the alignment.

motility to motX or motY mutants, which have
MotY or MotX in addition to PomA and PomB,
the PomA/PotB7E stator allowed both mutants toswarm (Figure 5(a)). This result demonstrated that
the PomA/PotB7E stator functions without MotX
or MotY. Furthermore, the PomA/PotB7E stator
conferred motility to a motA or motB mutant of
E. coli (Figure 5(b)). On the other hand, neither the
MotAE/MomB7E stator nor the MotAR/MomB7R
stator functioned in E. coli.

Determination of the coupling ion in
hybrid motors
PomA/PotB7E worked without MotX and MotY
in either V. alginolyticus or E. coli. We therefore
examined whether hydrogen or sodium ions drive
motor rotation in Vibrio and E. coli cells containing
such hybrid stators (Figure 6). Vibrio cells can produce both a sodium-driven polar flagellum (Pof)and proton-driven lateral flagella (Laf) in the same

Ion Selectivity of the Flagellar Motor

457

Figure 4. Diagram of the properties of each chimeric B subunit.
White, gray and hatched bars show
the contributions of PomB or MotB
from V. alginolyticus, R. sphaeroides
and E. coli, respectively. TM, transmembrane region; PGB, peptidoglycan-binding motif. The stator
proteins
were
expressedinV. alginolyticus or E. coli. R. sphaeroides
MotA/MotB do not functionin E. coli or V. alginolyticus, andPomA/PomB function only in
V. alginolyticus. MotBE and PotB7E
function in E. coli and V. alginolyticus.
The properties of motors containing
either protein are essentially thesame in both species. Stators containing MomB7E or MomB7R function only in V. alginolyticus.

Figure 5. Complementation of(a) V. alginolyticus and (b) E. coli
mutants by expression of chimeric
proteins. Mutant cells expressing
the indicated proteins were inoculated into 0.3% VPG (pH 7.0) agar
and incubated at 30 8C for 15 hours(for V. alginolyticus) or 0.3% tryptone agar for six hours (for E. coli).
(a) Stator proteins expressed fromplasmids are: 1, PomA/B; 2, none;
3, MotAE/BE; 4, none; 5, PomA/
PotB7E;
6,
PomA/PotB7E;
7,8,
MotAE/
MotAR/MomB7R;
MomB7E; 9, PomA/PotB7E. The
host strains are: 1 – 3, and 7 – 9,
NMB191 (DpomAB mutant); 4,
YM19 ( pof laf þ); 5, NMB119 (motX
mutant); 6, VIO542 (motY mutant).
(b) Stator proteins expressed are: 1
and 2, MotAE/BE; 3 and 4, MotAE/
MomB7E; 5 and 6, PomA/PotB7E; 7
and 8, none. The host strains are:1, 3, 5, and 7, M254 (E. coli motB
mutant); 2, 4, 6, and 8, M256 (E. coli
motA mutant).

458

Ion Selectivity of the Flagellar Motor

Figure 6. Effect of CCCP on
swarming of V. alginolyticus. Freshcells were inoculated into 0.3%
VPG (pH8.5) agar (a) without or(b) with CCCP and incubated at
30 8C for 15 hours. Stator proteins
expressed are: 1, PomA/B; 2, none;
3, MotAE/BE; 4, none; 5, MotAR/
MomB7R; 6, MotAE/MomB7E; 7,
PomA/PotB7E. Strain NMB191(DpomAB mutant) was used as host
except in inoculum 4, for which
strain YM19 ( pof laf þ) was used.

cell.29,30 The Pof2 Lafþ strain YM19 (Figure 6-4)
did not swarm in the presence of CCCP, anHþ-shuttling uncoupler that specifically dissipates
the proton motive force while leaving the sodium
motive force intact. The Pofþ Laf2 strain PomA/
B/NMB191 (Figure 6-1), in contrast, was unaffected by CCCP because V. alginolyticus can produce a sodium motive force directly in an alkaline
environment.31 The motility of V. alginolyticus cells
expressing E. coli MotA/B was sensitive to CCCP
like the Pof2 Lafþ strain (Figure 6-3).
Available evidence suggests that E. coli MotA/B
function as an Hþ-conducting channel in the
proton-driven flagellar motor. Thus, substitutionof PomA/B by E. coli MotA/B converted thesodium-driven polar motor of V. alginolyticus
into a proton-driven polar motor. However,V. alginolyticus DpomAB mutants expressing any
one of the chimeric stators MotAR/MomB7R,
MotAE/MomB7E or PomA/PotB7E were motile in
the presence of CCCP at alkaline pH (Figures
6b-5, 6b-6 and 6b-7). Therefore, the polar motors
in these strains must be driven by sodium motive
force. We had confirmed that they did not movein a buffer containing no Naþ.
A current of Li ions can drive rotation of
the wild-type polar flagellum of V. alginolyticus,
although the maximal swimming speed in thepresence of Liþ is only one-third that observed in
the presence of Naþ.27 Kþ cannot be utilized by
the polar flagellum. E. coli motB cells expressing
MotA/B swam equally well in Tris motility buffer
containing 100 mM KCl, NaCl or LiCl; these proton-driven motors are not affected by the presence

Figure 7. Swimming speed of E. coli mot-cells having MotA/B or PomA/PotB7E. The swimming speeds of the(a) E. coli motA and (b) E. coli motB cells expressing E. coli MotA/B (open circles) or PomA/PotB7E (filled circles) were
measured at various concentration of NaCl as described in Materials and Methods.

Ion Selectivity of the Flagellar Motorof absence of these ions or by phenamil, a specificinhibitor for the sodium-driven motor. However,no motility was observed in the motB cells expressing the PomA/PotB7E stator when only 100 mM
Kþ was present, although these cells did swim in
buffer containing 100 mM Naþ or Liþ.
Next, we examined the dependency for Naþ
concentration in E. coli mot mutants expressing the
MotA/B or the PomA/PotB7E stator (Figure 7).
The swimming speeds with the MotA/B stator
did not change with the concentration of NaCl,at ca. 20 –25 mm s21. On the other hand, theswimming speed with the PomA/PotB7E stator
increased with an increase of NaCl concentration
and was saturated at about 50 mM NaCl. The
speed profiles dependent on the external concentration of Naþ were similar to those of
V. alginolyticus.26 The maximal swimming speedof cells expressing PomA/PotB7E was at ca.
40– 50 mm s21.
Those results demonstrate that an E. coli rotor
equipped with PomA and PotB7E stator elementsfunctions as a sodium-driven, rather than aproton-driven, motor. It shows also that a sodiumdriven motor can function without MotX and
MotY, although the PomA/B-containing polar
flagellum of V. alginolyticus absolutely requires
both of those proteins for its sodium-dependent
motility.

Discussion
The proton-driven motor of E. coli contains the
MotA and MotB proteins in its stator complex,
whereas the stator complex of the sodium-driven
polar flagellum of V. alginolyticus consists of four
components, including MotX and MotY in addition
to the MotA/B homologs PomA and PomB.
We showed previously that MotA and MotB of
R. sphaeroides do not restore motility to a DpomABnull mutant of V. alginolyticus, whereas MotA
together with an R. sphaeroides/V. alginolyticus
chimeric MotB protein could function with MotX
and MotY in the sodium motor.26,27 It has beenreported that a DpomABmotXY strain of V. cholerae
could swim using a proton flux if E. coli MotA/B
were supplied, although the swimming ability of
the cells was very poor.28
Here, we have demonstrated that E. coli MotA/B
can support rotation of the polar flagellum of
V. alginolyticus in the absence of MotX and MotY.
This motor is driven by a proton current, forcing
the conclusion that the E. coli MotA/B proteinsswitch the Vibrio polar flagellum from thesodium-driven type to the proton-driven type.
Although MotX and MotY were thought to be
essential for operation of a sodium-driven motor,
pomA þB þ motX or motY cells containing the motAB
plasmid swarmed more poorly than did DpomAB
motX þ motY þ cells containing this plasmid. The
likely reason is that MotA/B compete with chromosomally encoded PomA/B for insertion into459
the motor in a pomA þB þ motX or pomA þB þ motY
strain. In the presence of MotX and MotY, PomA/
B are probably incorporated more efficiently into
the polar motor than MotA/B are, because motility
was not impaired when E. coli MotA/B wereexpressed in wild-type V. alginolyticus strain VIO5
(data not shown).
Segregants with improved motility (hypermotile strains) could be isolated easily from aV. alginolyticus DpomAB mutant containing plasmid
YA6022, which encodes E. coli MotA/B. Thirtyeight of 45 mutations causing hypermotility werelocated within the motB gene on the plasmid,
but the remaining mutations presumably map to
the V. alginolyticus chromosome. The mutations of
motB were distributed widely and some mutations
were clustered in the periplasmic region near
the putative transmembrane segment (TM). Thismight suggest that the periplasmic region of MotB
forms a structure to support the function, such as
the interaction with the other motor components
or peptidoglycan. Garza et al. reported that a missense mutation that causes a residue substitution
near the consensus peptidoglycan-binding motifof MotB could be suppressed by mutations in
the gene encoding the intracellular rotor protein
FliG.32 They proposed that the original motB
mutations misaligned the stator relative to therotor, and that the suppressing residue substitutions in FliG caused compensating realignments.
Thus, in DpomAB V. alginolyticus expressing E. coli
MotA/B, mutations affecting the extracellular portion of MotB might improve motility by correcting
a suboptimal interaction at the stator-rotor interface. If so, at least some of the chromosomal
mutations that enhance motility might be locatedin the V. alginolyticus fliG gene.
We constructed various chimeric proteins whose
extracellular domains contain different segmentsof E. coli or R. sphaeroides MotB combined with thecomplementing segment from PomB. The PomA/
PotB7E, MotAE/MomB7E and MotAR/MomB7R
chimeric stator complexes all function as components of sodium-driven motors when they are
expressed in the DpomAB mutant NMB191. The
PomA/PotB7E stator can function in a motX or
motY mutant, meaning that this motor does notneed MotX and MotY to function.
E. coli mot cells expressing PomA/B are notmotile. However, if the extracellular domain of
PomB is exchanged with that of MotB, the resultant
stator (PomA/PotB7E) functions with the E. coli
rotor. This motor is driven by a sodium ion current.
These results make it clear that there is no intrinsic
necessity for MotX or MotY to be present fora flagellum to be powered by sodium ions,although they are essential for function, at leastin V. alginolyticus.
The swarming ability of E. coli mot mutants
expressing E. coli MotA/B was better than thoseexpressing the PomA/PotB7E stator. However, themaximal swimming speed of E. coli mot mutants
expressing the PomA/PotB7E stator was more

460

Figure 8. Diagrams for the summary of this study. The
hatched and the open parts are derived from the components of the proton-driven (E. coli) and the sodiumdriven (Vibrio) components, respectively. The dottedboxes and the columns indicate the cytoplasmic membrane and the transmembrane regions, respectively.
PGB; peptidoglycan binding motif. The (a) MotA/B
and (c) PomA/PotB7E stator function in either E. coli or
V. alginolyticus as the proton-driven motor and thesodium-driven motor, respectively. The (b) PomA/B
and (d) MotA/MomB7 stator function in V. alginolyticus
as the sodium-driven motor but they do not function in
E. coli, though MotX and MotY are not co-expressed.

than twice that of those expressing the originalE. coli MotA/B stator. This discrepancy is often
observed in the che mutants, which cannot regulate
the direction of the motor rotation, suggestingthat the switching of clockwise/counterclockwise
rotation was affected or was not suitable for the
faster swimming speed. To investigate the reason
for the faster swimming of the sodium-driven
E. coli cells, the torque-speed relationship shouldbe measured and the sodium motive force inE. coli cells should be estimated.
Our findings are summarized in Figure 8. In thenative V. alginolyticus polar flagellum, the sodiumdriven motor requires PomA, PomB, MotX and
MotY. Motors containing E. coli MotA/B function
as proton motors. If the extracellular region of
MotB is exchanged with the corresponding region
from PomB, as in the functional MotA/MomB
hybrid stators, the hybrid motor is driven bysodium, and both MotX and MotY are essential for
its function in Vibrio, as shown in this study andby our previous results.27 MomB is not functional

Ion Selectivity of the Flagellar Motorin E. coli under the present condition that MotX
and MotY are not co-expressed. It will be interesting to determine whether the presence of MotX/Y
in E. coli will allow PomA to function as part of aNaþ motor in conjunction with PomB or MomB.
Moreover, it will be informative to determine
whether the sodium-driven flagella of Grampositive alkalophilic Bacillus species require MotX
and MotY. The PomA/PotB7E stator, in contrast,
can function in a sodium-driven motor without
MotX and MotY in either E. coli or Vibrio. It thus
appears that MotX and MotY are needed whenever
the extracellular domain of the B subunit comesfrom PomB and some feature of the extracellulardomain of E. coli MotB allows it to function in a
sodium-ion motor in the absence of MotX and
MotY. Although there is no direct evidence forinteraction between PomA/B and MotX/Y, it may
be that MotX and/or MotY stabilize the attachmentof the extracellular domain of PomB to the cellwall. MotY has a peptidoglycan-binding motif in
its C-terminal region similar to that of PomB, andMotX and MotY interact with each other.6,8 Furthermore, we found recently that MotX and MotY
appear to be outer membrane proteins.11
The discovery that a sodium-driven motor canfunction without MotX and MotY opens up new
possibilities for investigating flagellar motors. For
example, we can compare proton-driven motors
containing MotA/B and sodium-driven motors
containing PomA/PotB7E in E. coli cells without
the complication of having to supply MotX and
MotY. The results presented here emphasize boththe complexity of ion selectivity and the intrinsic
similarity of proton-powered and Naþ-powered
motors. The PomA/PotB7E stator will be an
excellent subject for research into the mechanismsof ion selectivity, energy coupling and torquegeneration by bacterial flagella.

Materials and Methods
Bacterial strains, plasmids, growth conditions
and media
The strains and plasmids used in this study are listed
in Table 1. The mutant and chimeric plasmids aredescribed in the text. V. alginolyticus was cultured at
30 8C in VC medium or VPG medium, as described.27 To
adjust the pH of the media, HCl (for pH 7.0) and K2CO3(for pH 8.5) were used. For culturing E. coli cells, LB
broth (1% (w/v) Bactotryptone, 0.5% (v/v) yeast extract,
0.5% (w/v) NaCl) or tryptone broth (1% (w/v) tryptone,
0.5% NaCl) was used. When necessary, kanamycin
was added to final concentrations of 100 mg/ml for
V. alginolyticus and 50 mg/ml for E. coli. Cells examined
for swimming behavior were prepared as described.27

DNA manipulations and sequencing
Routine DNA manipulations were carried out asdescribed.27

461

Ion Selectivity of the Flagellar Motor

Table 1. Bacterial strains and plasmids used in this study
Strain or plasmid

Genotype or description

Reference or source

A. V. alginolyticus strains
VIO5
NMB191YM19
NMB190NMB192
NMB119
VIO542

VIK4 laf (Rifr Pofþ Laf2)
VIO5 DpomAB (Rifr Laf2 Pom2)
138-2 pof (Pof2)
VIO5 DpomA (Rifr Laf2 Pom2)
VIO5 DpomB (Rifr Laf2 Pom2)
YM4 motX (Rifr Laf2 Pom2)
VIO5 motY (Rifr Laf2 Pom2)

9
12
29
26
This work
89B. E. coli strains
DH5a
M254
M256

AW330 motB
AW330 motA

33
34
34

C. Plasmids
pSU41
pYA303pGD2
pMN1
pYA600pYA6001
pYA602
pYA6022
pYA703kan (Kmr) Plac lac Za
pSU41 1.9 kb Bam HI-Sac I ( pomAB þ)
pBR322 3.2 kb Eco RI-Pst I (motAB þ)
pUC118 3.2 kb Eco RI-Pst I (motAB þ)
pSU41 2.6 kb Sph I-Sal I (motAB þ)
pSU41 1.6 kb NspI-Sal I (motB þ)
pSU41 0.9 kb Sal I-Eco RI (motA þ)
pSU41 2.4 kb Sal I-Sal I (motAB þ)
pSU41 1.9 kb Bam HI-Sac I (motAB þ)

35
21
36
This work
This work
This work
This work
This work
27

Kmr, kanamycin-resistant; Rifr, rifampicin-resistant; Plac, lac promoter.

Plasmid construction and site-directed mutagenesis
Plasmid pYA602 contains a PCR-amplified fragmentof motA that extends from upstream of the start codon
to downstream of the stop codon. PCR amplification
was done as described.20 We used plasmid pMN1,
whose mot genes are derived from plasmid pGD2, as
the template. The end primers were EcmotA.S1 andEcmotA.E2. EcmotA.S1 has a SalI site and was the sense
primer that annealed with the sequence upstream of
motA. EcmotA.E2 includes an EcoRI site and was theantisense primer that annealed downstream of the gene.
This fragment was inserted into plasmid pSU41 between
the SalI site and the EcoRI site. The other plasmids
were constructed as noted in Table 1. Two-step PCR for
construction of chimeric genes was performed as
described.20
Construction of a pomB null mutant
A pomB-deletion strain was constructed by aprocedure similar to that described.26 The 0.2 kb
HpaI-SnaBI region in the pomB gene was deleted fromplasmid pYA303, and the fragment encompassing intact
pomA and pomB containing the deletion was moved
onto a suicide vector. The deleted pomB gene wasthen introduced via homologous recombination into thechromosome of strainVIO5, which produces a wild-type
polar flagellum.
Transformation of Vibrio cells
Transformation of Vibrio cells was carried out byelectroporation, as described.37
Measurement of swimming speed
An overnight culture of E. coli cells grown in LB at
30 8C was inoculated into TG medium (1% Bactotryptone, 0.5% NaCl, 0.5% (w/v) glycerol) at 1/50

dilution. Cells were harvested at late logarithmic phase,
washed with Kpi buffer (10 mM potassium phosphate(pH 7.5), 10 mM DL -lactate), and suspended in Kpi
buffer. The cell suspension was diluted 50-fold into Kpi
buffer containing various concentrations of NaCl, andcells were observed at room temperature under a darkfield microscope and recorded on videotape. Theirswimming speed was determined as described.38

Acknowledgements
We thank Drs David F. Blair and Michael
D. Manson for critically reading the manuscript,
and we thank Dr Robert M. Macnab for invaluable
discussions. This work was supported, in part,by grants-in-aid for scientific research from the
Ministry of Education, Science and Culture ofJapan (to T.Y., I.K. and M.H.), and the Japan Society
for the Promotion of Science (to Y.A.).

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Edited by J. Karn
(Received 23 September 2002; received in revised form 13 January 2003; accepted 16 January 2003)