doi:10.1016/S0022-2836(02)00426-6 available online at http://www.idealibrary.com onw
B

J. Mol. Biol. (2002) 320, 403–413

The Systematic Substitutions Around the Conserved
Charged Residues of the Cytoplasmic Loop of
Na1-driven Flagellar Motor Component PomA
Tomohiro Yorimitsu1, Yoshiyuki Sowa2, Akihiko Ishijima3
Toshiharu Yakushi1 and Michio Homma1*
1

Division of Biological Science
Graduate School of Science
Nagoya University
Chikusa-Ku, Nagoya 464-8602
Japan
2

Department of BiophysicalEngineering, Osaka University
Toyonaka, Osaka 560-8531
Japan
3

Department of Applied Physics
School of Engineering
Nagoya University
Chikusa-Ku, Nagoya 464-8603
Japan

PomA, a homolog of MotA in the Hþ-driven flagellar motor, is an essential
component for torque generation in the Naþ-driven flagellar motor.
Previous studies suggested that two charged residues, R90 and E98,
which are in the single cytoplasmic loop of MotA, are directly involvedin this process. These residues are conserved in PomA of Vibrio
alginolyticus as R88 and E96, respectively. To explore the role of thesecharged residues in the Naþ-driven motor, we replaced them with otheramino acids. However, unlike in the Hþ-driven motor, both of the single
and the double PomA mutants were functional. Several other positively
and negatively charged residues near R88 and E96, namely K89, E97 andE99, were neutralized. Motility was retained in a strain producing
the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The
swimming speed of the AAQQQ strain was as fast as that of the wildtype PomA strain, but the direction of motor rotation was abnormallycounterclockwise-biased. We could, however, isolate non-motile or poorly
motile mutants when certain charged residues in PomA were reversed or
neutralized. The charged residues at positions 88 – 99 of PomA may not
be essential for torque generation in the Naþ-driven motor and mightplay a role in motor function different from that of the equivalent residuesof the Hþ-driven motor.
q 2002 Elsevier Science Ltd. All rights reserved*Corresponding author

Keywords: energy transduction; flagella; motor protein; sodium-driven
motor; Vibrio alginolyticus

Introduction
Bacterial flagella derive their energy for rotationfrom the electrochemical potential of a specificion, either Hþ or Naþ, across the cell membrane.1,2This energy is harnessed by a motor complex
located at the base of the flagellum. From studies
of Salmonella typhimurium and Escherichia coli,
which have Hþ-driven flagellar motors, threeproteins, MotA, MotB, and FliG, are thought tobe involved directly in the energy conversion to
generate the rotation from the ion flux.3 – 6
MotA has four transmembrane segments and asingle large cytoplasmic loop,7 and MotB has asingle transmembrane segment.8 The C-terminal
Abbreviations used: CCW, counterclockwise;
CW, clockwise.
E-mail address of the corresponding author:
g44416a@nucc.cc.nagoya-u.ac.jp

domain of MotB appears to be bound to peptidoglycan, and the MotA/B complex is proposed toact as a stator.8 – 11 The MotA/B complex functions
as a Hþ channel, converting the Hþ flux intomechanical power for flagellar rotation.12 – 16 It
is speculated that conformational changes are
induced by ion flow and are transferred to thecytoplasmic region of MotA, and that interactions
between this region of MotA and FliG of the rotor
complex drive rotation. FliG is part of the switch
complex, which includes the FliM and FliN
proteins, which together make up the C-ring.6,17,18
Unlike E. coli and S. typhimurium, some Vibrio
species, including Vibrio alginolyticus, V. parahaemolyticus, and V. cholerae, and alkalophilic Bacillus
species are known to have Naþ-driven flagella.19,20
In Vibrio, PomA and PomB, which are homologsof MotA and MotB, respectively, are essential forthe function of the Naþ-driven motor. Like MotA
and MotB, PomA and PomB have been shown to0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

404

Charged Residues in the Cytoplasmic Loop of PomA

Figure 1. Alignment of the sequences comprising the cytoplasmic loops of the stator components, PomA of
V. alginolyticus (VaPomA) and MotA of E. coli (EcMotA). White letters in black boxes indicate the conserved residues
between PomA and MotA. The charged amino acid residues mutagenized in this study are indicated by single arrowheads (positively charged) and double arrowheads (negatively charged). Filled arrowheads show the conserved
charged residues, and open arrowheads show the non-conserved charged residues. The numbers over the arrowheads
are the positions of the amino acid residues in V. alginolyticus PomA.

be functional partners.21,22 Additional components,
MotX and MotY, are needed for torque generationby the Naþ-type motor.23 – 26 These two components
are specific to the Naþ-motor, but their precise
function is still unclear.

Figure 2. Effect on swarm formation of single substitutions of charged residues in the cytoplasmic loop of
PomA. (a) Swarms of NMB190 cells containing vector,
wild-type (wt), or single-substituted PomA (R88A,
K89A, E96Q, E97Q, and E99Q). (b) Swarms of NMB190
cells containing vector, wild-type (wt), or double-substituted PomA on soft agar plates containing 513 mM (left)
or 50 mM (right) NaCl. The pattern in which the various
strains were inoculated is indicated in the square above
the photograph.

When MotA of Rhodobacter sphaeroides, which
has a Hþ-type flagellar motor, is expressed in
V. alginolyticus cells defective for PomA, the cells
display Naþ-driven motility.27 However, MotB
from R. sphaeroides cannot functionally substitutefor PomB, strongly suggesting that ion specificity
is not mediated by the MotA/PomA component.
In the MotB/PomB components of Naþ and Hþ
motors, a single negatively charged residue isconserved in the putative transmembrane segment.
This charged residue is believed to be an ionbinding site.28 PomB/MotB chimeras were produced to explore the mechanism of ion specificity.
Surprisingly, in Vibrio cells, a chimeric protein
composed of the periplasmic domain of PomB andthe transmembrane segment of MotB functions asa Naþ-type motor, though the reverse chimera
does not function at all.29
The PomA/PomB complex was purified andreconstituted into proteoliposomes. Naþ uptake
activity was observed in the absence of MotX andMotY when the PomA/B complex was present,
but not with PomA alone. It has been suggested
that PomA and PomB form a complex as
oligomers. By gel-filtration chromatography, themolecular mass of the PomA/B complex was estimated to be 175 kDa. PomA alone forms a stable
dimer, and a genetically fused tandem PomA
dimer functions in the Naþ-type motor. The
molecular mass of the tandem dimer was estimated by gel-filtration chromatography to be thesame as that of the wild-type PomA.30 Moreover,when either one or both of the PomA subunits in
the homodimer is mutated, the resulting complex
is completely defective in motor function.31 Fromthese lines of evidence, PomA is inferred to function as at least a dimer in the PomA/B complex.
This inference is supported by cross-linking
experiments using Cys-substituted mutants of
PomA.32 Many Cys residues were introduced into
the two periplasmic loops, with the result that thetwo PomA proteins were cross-linked at certain
sites. Function was lost upon cross-linking andrestored by treatment with reducing reagent. Thisfinding suggests that a specific interaction of two
PomA subunits is important for function.
A model has been presented in which electrostatic interactions between charged residues in
MotA and FliG, is involved in torque generation.33

Charged Residues in the Cytoplasmic Loop of PomA

405

Figure 3. Effect on swarm formation of neutralizing both of thepositively charged residues and all
three of the negatively charged
residues, separately and in combination. (a) Swarms of NMB190
cells containing vector, wild-type(wt) PomA, or PomA in which
both of the charged residues and/or all charged residues were neutralized. Plates contained 513 mM(above) or 50 mM (below) NaCl.
(b) Histogram showing the swarm
sizes of NMB190 cells expressing
mutant PomA proteins. The values
shown are given relative to that ofcells expressing wild-type PomA
proteins at 513 mM NaCl (filledbar) or 50 mM NaCl (open bar).

The charged residues in the cytoplasmic region of
MotA and FliG were analyzed by mutagenesis,
and the residues crucial for torque generation
were proposed to be R88 and E96 in MotA and
R281, E288 and E289 in FliG.34,35 When either R90
or E98 of E. coli MotA was substituted by alanine
residue, motor function was preserved. However,when both of R90 and E98 were converted to Ala,or either residue was replaced with an oppositely
charged amino acid, the motor was unable torotate.
PomA shares those two conserved charged
residues in the cytoplasmic loop with MotA,
which correspond to R88 and E96 in PomA of
V. alginolyticus. Other charged residues, namely
K89, E97 and E99, are located near R88 and E96 of
V. alginolyticus PomA. Substitutions at these sites
have not been evaluated. Here, we examine bymutational analysis whether these residues are
involved in the function of the Naþ-driven flagellar
motor.

Results
Neutralizing the charged residues in thecytoplasmic loop of PomA
To study the role of the conserved R88 and E96
charged residues in the function of the Naþ-driven
motor of V. alginolyticus, we replaced them withneutral amino acid residues (Figure 1). R88 was
replaced with Ala (R88A) and E96 with Gln(E96Q). Both mutant proteins were expressed in astrain of V. alginolyticus that is defective for lateral
flagella, and motility was examined by the swarm
assay in a soft agar plate. As had been shown for
MotA of the Hþ-driven motor,35 neither single
mutation affected motility (Figure 2(a)). However,replacement of both of the charged residues eliminated the motor function of MotA completely. On
the other hand, Vibrio cells expressing the R88A/
E96Q double mutant PomA were still fully motile(Figure 2(b), left panel). This result suggests thatthese two conserved, charged residues of PomA
are not essential for Naþ-driven torque generation.
In the primary sequence of PomA, there areother charged residues around R88 and E96: K89,
E97, and E99 (Figure 1). These residues were
replaced as above. Cells producing PomA containing either K89A, E97Q or E99Q were motile (Figure2(a)). Mutants containing all possible combinationsof two of the substitutions (positive and negative)
in R88A, K89Q, E96Q, E97Q and E99Q weresimilarly functional (Figure 2(b), left panel).
Next, the two positively charged residues were
neutralized, R88A/K89A (AAEEE), all threenegatively charged residues were neutralized,
E96Q/E97Q/E99Q (RKQQQ), and all five charged
residues were neutralized, R88A/K89A/E96Q/
E97Q/E99Q (AAQQQ). The swarm assay was
carried out for cells expressing each of these406

Charged Residues in the Cytoplasmic Loop of PomA

Figure 4. Effect of various combinations of the charge-substituting mutations on swarm behavior. Swarm diametersof NMB190 cells containing the various PomA mutants are shown in the histogram as a fraction of the diametersof swarms formed by cells expressing wild-type PomA at 513 mM NaCl (filled bar) or 50 mM NaCl (open bar).
(a) Swarm behavior of the charge-neutralizing mutants. (b) Swarm behavior of the mutants reversing the charge of
positive or negative residues.

mutant proteins. It was shown that even cells containing all five of the replacements swarmed as
effectively as cells with the wild-type PomA
(Figure 3(a), upper panel).
Effect of NaCl concentration on the
motor function
The soft agar plate used normally for the swarm
assay contains 513 mM (3%, w/v) NaCl. To test
whether swarming is affected by the extracellularNaþ concentration, we performed the swarm

assay using soft agar plates containing only
50 mM NaCl (Figures 2(b), right panel, and 3(a),lower panel). The AAEEE and RKQQQ mutants
seemed to function as well as wild-type PomA.
However, the quintuple mutants of PomA formed
swarms with only 50% of the diameter of thewild-type cells at the decreased Naþ concentration(Figure 3(b), filled versus open bars). This decrease
was not observed with any combinations of double
mutation shown in Figure 2(b) (right panel).
To determine whether the AAQQQ mutation isdominant, we expressed this mutant PomA in

407

Charged Residues in the Cytoplasmic Loop of PomA

Figure 5. Effect of Naþ concentration on the motility ofcells expressing AAQQQ mutant PomA. (a) The relativeswarm sizes compared with the diameters of NMB190
cells expressing AAQQQ PomA to those of cells expressing wild-type PomA are plotted as a function of NaCl
concentration. (b) Cells expressing wild-type (circles) or
AAQQQ mutant PomA (squares) were cultured in
medium containing 50 mM NaCl (broken lines with
open symbols) or 513 mM NaCl (continuous lines withfilled symbols), collected at late logarithmic phase, andsuspended in TMN medium. The swimming speeds
were measured at various concentrations of NaCl as
described in Materials and Methods.

VIO5 cells, which is wild-type for motility. In
swarm agar containing either 513 mM or 50 mM
NaCl, the cells were equally motile, indicating that
this mutant PomA is recessive with respect to its
motility phenotype at reduced concentrations ofNaþ (data not shown).
Table 1. Direction of flagellar rotation in cells with wildtype (wt) or AAQQQ mutant PomA
Dwell time (s)CCW[NaCl]
(mM)
50
300

CW

Wt

AAQQQ

Wt

AAQQQ

1.18 ^ 0.85
1.19 ^ 0.84

3.25 ^ 2.34
2.33 ^ 1.14

0.55 ^ 0.20
0.50 ^ 0.22

0.64 ^ 0.38
0.66 ^ 0.30

We further analyzed the motility phenotypes ofcells expressing mutant PomA proteins containing
various combinations of amino acid substitutions(Figure 4(a)). First, one of the two positively
charged residues, R88 or K89, was neutralized
together with all three negatively charged residues(E96Q/E97Q/E99Q), to generate either AKQQQ
or RAQQQ PomA. Second, one of the threenegatively charged residues, E96, E97 or E99, wasneutralized together with both positive residues(R88A/K89A), generating AAQEE, AAEQE, andAAEEQ PomA. Finally, pairs of two of threenegatively charged residues were neutralized in
combination with the R88A/K89A mutation,
generating AAQQE, AAQEQ, and AAEQQ PomA.
Cells producing either of these mutant PomA proteins swarmed as well as the wild-type cells athigh concentrations of Naþ (filled bars). Amongthem, the AAQEE protein showed a little decreasein swarming at 50 mM Naþ and the AAQQE
protein showed a decrease in swarming at low
concentrations of Naþ, to almost the same extent
as the quintuple-mutant AAQQQ protein (openbars). These results demonstrate that only somecombinations of charge loss at residues R88, K89,
E96 and E97 inhibit swarming activity by areduced concentration of Naþ.
We measured the swarm size of cells with the
AAQQQ mutant PomA at various concentrationsof Naþ (Figure 5(a)). The relative swarm size
decreased slowly from 513 mM to 100 mM NaCl,below which concentration the size decreased
dramatically. To examine whether the swarming
deficiency is caused by deficiency in torque generation, we measured the swimming speed of thecells (Figure 5(b)). Swimming speed profiles did
not differ between wild-type and AAQQQ cells,
and the cell growth is not different in these concentrations of NaCl. We therefore tested the possibility
that the mutant PomA is not installed into themotor correctly when the cells are grown at a low
concentration of Naþ. We cultured cells in medium
containing 50 mM NaCl and measured the swimming speed (Figure 5(b), broken line). However,the swimming speed was not reduced. These
results suggest that the decreased swarming ability
witnessed in the AAQQQ PomA mutant is not due
to a decrease in the torque generation.
Single flagellar rotation
Previous studies showed that pomA-deletedVibrio cells expressing MotA of R. sphaeroides,
whose flagellum is powered by Hþ, show
Naþ-dependent motility but exhibit a decreased
tumble frequency.27 This behavior led us to speculate that the AAQQQ PomA might inhibit
CW – CCW switching. To investigate this possibility, we attached a bead to a single polar flagellar
filament and monitored the direction of motor
rotation (Table 1). The motor containing the
AAQQQ mutant PomA rotated counterclockwise(CCW) for longer periods than motors containing

408

Charged Residues in the Cytoplasmic Loop of PomA

Figure 6. Properties of non-functional PomA mutant proteins
created by combinations of sign
reversal and neutralization of
charged residues. (a) Swarms of
NMB190 cells containing vector,
wild-type PomA (wt), or the indicated PomA mutant proteins in
the presence of 513 mM NaCl.
(b) Immunoblot of PomA proteinsin membrane fractions. Membrane
vesicles (each containing 5 mg of
total protein) from NMB190 cells
harboring the vector plasmid, aplasmid encoding wild-type PomA,
or a plasmid encoring the indicated
PomA mutant protein, were subjected to SDS-PAGE and detectedwith anti-PomA antibody.

wild-type PomA, although the duration of theintervals of rotation were not different in the twostrains. The dwell time of CCW rotation was about
twice as long at 300 mM NaCl and about threetimes longer at 50 mM NaCl for the AAQQQ
mutant PomA compared to wild-type PomA.
When we measured the tumble frequency of theswimming cells in various media, it also decreased
to about 51% of the wild-type frequency in 50 mM
NaCl and 57% of wild-type frequency at 300 mM.
Thus, the decrease in swarming ability in semisolid
agar containing low concentrations of salt may be aconsequence of those effects. The AAQQQ mutant
PomA biases the motor toward CCW rotation at
any concentration of salt, but this decrease intumbling may impair swarming only when thecells are swimming more slowly at low speeds(i.e. at low concentrations of NaCl) but have little
effect on swarming when the cells swim at higher
speeds. The rotation speeds of the 1.0 mm beads
were not significantly different between the wildtype and the mutant motor under the high or low
concentrations of NaCl, suggesting the generating
torques in high load also are similar (data notshown).

NaCl (filled bars). As seen with cells expressing
the AAQQQ mutant PomA, the swarming abilityof cells expressing these reversed-charge mutants
was severely affected at low concentrations(50 mM) of NaCl (open bars). These results indicate that these charges are not essential for torquegeneration in the Naþ motor.
To analyze these charged residues further, either
R88 or K89 in the RKRRQ mutant and either E96
or E97 in the EEEEQ mutant were neutralized,
resulting in the AKRRQ, RARRQ, EEQEQ andEEEQQ proteins. In the presence of 513 mM NaCl,the swarm size of the RARRQ and EEEQQ mutants
was less than half that of wild-type PomA,
whereas the swarms of cells containing the
AKRRQ and EEQEQ proteins were very small(Figure 4(b)). At 50 mM NaCl, the diameters of theswarms of cells expressing the RARRQ andEEEQQ proteins decreased even more, to about
16% and 36%, respectively, of those containing
wild-type PomA, whereas the AKRRQ and
EEQEQ mutants did not swarm and no motile
cells were observed under the microscope. We
speculate that positive charge at R88 and K89 or
negative charge at E96 and E97 might be required
to compensate for any charge reversal.

Reversing the charges
Previous studies showed that reversing thecharge of either residue R90 or E98 of MotA completely abolishes motility of the E. coli Hþ-driven
motor.35 We replaced the R88 and K89 residues
with Glu, or the E96 and E97 residues with Arg to
generate R88E/K89E/E99Q (EEEEQ) PomA or
E96R/E97R/E99Q (RKRRQ) PomA (Figure 4(b)).
Cells expressing either of those mutant proteinsswarmed as well as wild-type cells at 513 mM

Non-functional PomA protein
We examined the combination of chargereversing and charge-neutralizing mutations byconstructing the following PomA mutants: R88E/
K89E/E96Q/E97Q/E99Q (EEQQQ) and R88A/
K89A/E96R/E97R/E99Q (AARRQ). Neither protein supported any motor function (Figure 6(a))
although we could detect normal amounts ofthese mutant PomA proteins in the membraneCharged Residues in the Cytoplasmic Loop of PomA

(Figure 6(b)). When these proteins were expressedin VIO5 cells, swarming was inhibited about
20– 30%. When the extracellular concentration ofNaþ was decreased to about 50 mM, this dominant-negative effect was enhanced to be 40% (datanot shown). As shown above, reversing only the
positive or negative charges (EEEEQ or RKRRQ)
did not affect swarming significantly. However,when both of the charges were reversed simultaneously to generate the EERRQ mutant protein(R88E/K89E/E96R/E97R/E99Q), cells displayed
no motility (Figure 6(a)) and no swimming cells
were observed under the microscope (data notshown). These proteins were also present innormal amounts in membrane (Figure 6(b)). These
results support our idea that the effect caused byreversing charges is not seen when some of theoriginal residues with opposite charge are retained
but are observed when these additional charged
residues are neutralized or substituted by residuesof opposite charge.

Discussion
In the bacterial flagellar motor, the stator androtor cooperate to generate torque from ion
influx, resulting in motor rotation. Studies of theHþ-driven flagellar motor of E. coli led to a model
for force generation in which the cytoplasmic
region of MotA, a stator component, and the
C-terminal region of FliG part of the rotor, interact
electrostatically in the cytoplasm via specificcharged residues; R90 and E98 in MotA and
R281, E288, and E289 in FliG.33 Similar functional charged residues have been identified in
Sinorhizobium meliloti, whose flagellar motor isHþ-driven (R. Schmitt, personal communication).
PomA, the MotA homolog in the Naþ-driven
flagellar motor of V. alginolyticus, also has these
conserved charged residues, R88 and E96. In thiswork, we focused on the role of these charged residues in torque generation. In the Hþ-driven motor,
when R90 and E98 in MotA are changed to Ala
and Gln, respectively, MotA is not functional,
although MotA with either single change still
functions.35 On the other hand, the same doublesubstitution of R88 and E96 in PomA does notaffect motor function significantly. This result
suggests that these charged residues themselves
may not mediate the electrostatic interactionthat generates force in the Naþ-driven motor of
V. alginolyticus. Therefore, we suggest that R88 andE96 of PomA are less critical than the corresponding residues of the Hþ-driven motor, because there
are other charged residues nearby in PomA,
namely K89, E97, and E99. Since these residues
might mediate electrostatic interaction, we
additionally neutralized one negative and one
positive residue in all possible combinations, but
none of these mutations impaired motility. We
thought that more than one positively and negatively charged residue may be used to generate409
the torque in the Naþ motor, though a single positively charged residue and a single negatively
charged one are enough to generate torque in theHþ motor. To test this hypothesis, we neutralized thepositive charges R88 and K89 and/or the negative
charges E96, E97 and E99. PomA proteins with either
R88A/K89A-double or E96Q/E97Q/E99Q-triple
mutations did not affect the motility of the cells. The
Naþ-driven motor, which rotates about five times
faster than Hþ-driven motor, may have additionalcharged residues essential for energy coupling.
In order to further analyze the role of thecharged residues of the Naþ motor PomA, we
substituted the charged residues in the region ofinterest with residues of opposite charge. We
expected to see more severe effects on the functionof PomA than with substitutions of neutral aminoacid residues. In MotA of E. coli, reversal of thecharge of either R90 or E98 abolished the motor
function completely.35 In contrast, reversing any
single positive or negative charge never completely
disrupted motor function in the Naþ-driven
flagellar motor, suggesting that these charged
residues might not be very important in theNaþ-driven motor. Some charge-reversal mutations
conferred reduced swarming at low concentrationsof Naþ, as seen with the neutralized R88A/K89A/
E96Q/E97Q/E99Q (AAQQQ) mutant PomA. These
reversed-charge PomA proteins might interact lessefficiently with FliG to change the direction of
the motor rotation. We went on to combine themutations that reverse and neutralize the charged
residues of PomA. Motor function was lost completely in cells containing the EEQQQ andAARRQ PomA proteins, even though proteins
were present in the membrane. Reversing thecharges of R88, K89, E96 and E97 (EERRQ) alsoabolished motor rotation. However, EEEQQ or
RARRQ PomA proteins support some motility,
suggesting that the function can be maintained
when one of the original charged residues remains,in which there is E96 or R88, respectively. Thesemight indicate that E96 and R88 are as importantas the corresponding residues of E. coli, though it
is hard to interpret the effect of the charges that
can affect the conformations indirectly.
When all five charges were neutralized in the
AAQQQ mutant, swarming ability was impaired
when the concentration of NaCl in the soft agar
was lowered from 513 mM (3%) to 50 mM. On thebasis of this result, we thought that the AAQQQ
mutant PomA generates torque less efficiently
than wild-type PomA. To examine this, wemeasured swimming speed in liquid medium
containing various concentrations of Naþ.
However, the swimming speed in response to Naþconcentration was similar in cells containing thewild-type or the AAQQQ mutant PomA. This
result shows that the neutralizing mutations do
not block force generation. Secondly, we tested thepossibility that the AAQQQ mutant PomA is notincorporated efficiently into the motor at thedecreased concentration of Naþ. However, there

410

was no difference between swimming speeds ofcells with wild-type and AAQQQ mutant PomA
cultured in medium containing high (513 mM) orlow (50 mM) concentrations of Naþ, indicatingthat motor assembly is not affected by low
concentrations of Naþ.
In a previous study, Asai et al. demonstrated that
Vibrio cells expressing MotA of the Hþ-driven
motor of R. sphaeroides use Naþ to rotate theflagellar motor.27 Cells with this hybrid motor
could swarm, although very poorly, and theswarm size did not correspond to the swimming
speed of the cells. This discrepancy arises presumably because the hybrid motor is CCW(smooth)-biased and rarely rotates clockwise(CW), even if the cells are stimulated with achemical repellent. If MotA in the stator interacts
with FliG in the rotor to rotate the motor, and thisinteraction is abnormal in the hybrid motor due toa poor match between the cytoplasmic loop of
MotA of R. sphaeroides and FliG of V. alginolyticus,
a directional bias could result. To examine whether
this phenomenon occurs with the AAQQQ PomA
mutant in bias, we monitored flagellar rotation
directly by attaching a bead to a single filament.
The flagella of cells containing AAQQQ PomA
had a longer CCW-rotating dwell time than did
cells containing wild-type PomA, a result consistent with the lower tumble frequency of cells
containing AAQQQ PomA. Although there is no
significant difference between the CW-rotatingtime of cells with wild-type and AAQQQ mutant
PomA, the CCW-rotating time of the motor withthe mutant PomA was longer than that containing
wild-type PomA. In other words, the CW mode israre in the motor but, once changed to CW, it canmaintain the direction stably. Garza et al. reported
that E. coli MotA containing a single residue
substitution in its periplasmic region conferred a
CW-bias on the motor.36 As far as we know, theAAQQQ form of PomA represents the first example
of an alternation in the cytoplasmic region of a
stator component that displays an altered CCW/CW ratio. The swarming ability of the most severe
mutants, the reversed-charge mutants, was reduced
at low concentrations of NaCl. This may suggestthat the reversed charges affect the interactions
between PomA and FliG at low rotation speed.
It is known that the direction of flagellar rotation
is ruled by CheY in E. coli. Chemotactic stimuli aretransduced into the cell, CheA is activated, theactivated CheA phospholylates CheY,37,38 and thephospho-CheY binds to the C-ring of the flagellar
motor.39 Consequently, the conformation of therotor probably changes and the direction of theflagellar motor changes from CCW to CW.40 It has
been speculated that the conformation of FliG
changes reversibly between CCW and CW statesof the motor, although there is little direct supporting evidence for this proposition. One model has
been proposed to describe how a conformationalchange in FliG could change the direction of
motor rotation. FliG is involved in three processes:

Charged Residues in the Cytoplasmic Loop of PomA

assembly of the flagella; rotational switching; andtorque generation.41 In the Hþ motor, multiple
copies of FliG compose a ring structure with FliM
and FliN.18,42,43 Each C-terminal region of theassembled FliG might interact with the cytoplasmic region of MotA in the stator. Lloyd et al.
obtained a high-resolution structure of the
C-terminal domain of FliG (FliG-C) from thehyperthermophilic bacterium Thermotoga maritima,
which has a Hþ-driven motor.44 FliG-C of
T. maritima contains the conserved charged residues essential for torque generation in E. coli. Two
subsets of charged residues of MotA might beaccessed differently upon conformational changesof FliG-C and used to switch from CCW to CW
rotation. Each arrangement of these importantcharged residues might be involved in interactingwith residues in the stator subunit, MotA.
The fliG gene of the Naþ-driven motor has beencharacterized in V. alginolyticus (unpublishedresults), V. cholerae,45 and V. parahaemolyticus,46
which are closely related. The charged residues
functionally important in FliG-C of the Hþ-driven
motor are conserved in the FliG proteins of theVibrio species. If the model MotA – FliG interactions in the Hþ-driven motor applies to theNaþ-driven motor, the charges in the cytoplasmic
loops of PomA should interact with charged residues in FliG in either of two conformations. It
may seem surprising that the charge-neutralized
mutant AAQQQ PomA can support rotation ineither the CCW or CW direction, although thefrequency of motor switching is lower. In theNaþ-driven motor, these charge interactions are
not likely to determine the direction of rotation
against the Hþ-driven motor model. On the other
hand, a chimeric FliG protein composed of the C
terminus of E. coli FliG and the N terminus ofV. cholerae FliG is functional in V. cholerae, although
full-length E. coli FliG does not function in thesecells.35 V. cholerae has a Naþ-driven motor similar
to that of V. alginolyticus47,48 and PomA is functionally interchangeable between the two species(unpublished results). Similarly, a FliG chimera
composed of the C terminus of V. cholerae FliG and
the N terminus of E. coli FliG was functional in
E. coli.45 According to this evidence, the rotor-stator
interface cannot be very different between theNaþ-driven and the Hþ-driven motors. However,
PomA of the Naþ-driven motor may haveadditional essential charged residues necessary
for the high-speed rotation. To understand themechanism of force generation, it will be necessary
to model the interface between the stator and therotor in the two types of flagellar motors.

Materials and Methods
Bacterial strains, plasmids, growth conditions
and media
V. alginolyticus strains VIO5 (Rifr, Pofþ, Laf2), NMB190(Rifr, Pofþ, Laf2, DpomA), and NMB194 (Rifr, Pofþ, Laf2,

411

Charged Residues in the Cytoplasmic Loop of PomA

Che2 DpomA) were used.8,11 E. coli strain JM109(recA1, endA1, gyrA96, thi 2, hsdR17, relA1, supE44,
l2,D(lac-proAB); (F 0, traD36, proAB, lacI q, lacZDM15 ))was used for DNA manipulations. V. alginolyticus cells
were cultured at 30 8C in VC medium (0.5% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.4% (w/v) K2HPO4,
3% (w/v) NaCl, 0.2% (w/v) glucose); or VPG medium(1% (w/v) polypeptone, 0.4% (w/v) K2HPO4, 3% (w/v)
NaCl, 0.5% (w/v) glycerol). For the swarm, overnight
cultures of cells were spotted onto 0.25% agar-VPG
plates containing 100 mg/ml of kanamycin and incubated at 30 8C. If the Naþ concentration was changed ina VPG agar, the total salt concentration was held at
513 mM by the addition of a suitable amount of KCl.
E. coli cells were cultured at 37 8C in LB medium. Whennecessary, kanamycin was added to a final concentrationof 100 mg/ml for V. alginolyticus cells or 50 mg/ml forE. coli. Plasmid pYA301, a pSU41-based plasmid, carries
the pomA gene under the control of the lac promoter.49

Site-directed mutagenesis
To introduce substitutions into PomA, we used a PCR
method described elsewhere.49 We synthesized pairs ofmutant primers homologous to either the sense or antisense strand of the pomA gene, with a 1 –3 base mismatchat the mutation site. We amplified the full plasmid
sequence containing the pomA gene. The presence of thepomA mutations was confirmed by DNA sequencing.

Measurement of swimming speed
Cells were harvested at late logarithmic phase andsuspended in TMN buffer (50 mM Tris – HCl (pH 7.5),5 mM MgCl2, 50 mM NaCl, 250 mM KCl). The cell suspension was diluted 100-fold in TMN buffer, and cells
were observed at room temperature under a dark-fieldmicroscope and recorded on videotape. Their swimming
speed was determined as described.50

Measurement of flagellar rotation
Cells were prepared as for measurements of swimming speed, and 1.0 mm latex beads (Molecular Probes)
coated with poly-L -lysine (15 – 30 kDa; Sigma ChemicalCo.) was mixed with cells and placed on a glass coverslip. Rotation of the bead spontaneously attached to thefilament of a cell that was immobilized to the glass wasmeasured by projecting the phase contrast image of thebead onto a quadrant photodiode through a 60 £ oil
immersion objective lens. The outputs from the quadrant
photodiode were recorded on a digital data recorder(TEAC, RD-125T).

Detection of PomA proteins
NMB190 (DpomA ) cells producing wild-type orvarious mutant PomA proteins were cultured in VPG
medium and collected by centrifugation at mid-log
phase. Membranes were prepared from sonicated cells
as described.32 The membranes were solubilized with
SDS-sample buffer and subjected to SDS/12% polyacrylamide gel electrophoresis (SDS-PAGE) followed
by immunoblotting with antibody against PomA, as
described.32

Acknowledgments
We thank Dr M. Manson for invaluable discussions
and critically reading the manuscript, and Dr R. Schmitt
for discussions and supplying the unpublished results.
This work was supported, in part, by Grants-in-aid for
Scientific Research from the Ministry of Education,
Science and Culture of Japan (to A.I., T.Y. and M.H.)
and from the Japan Society for the Promotion of Science(to T.Y. and Y.S.).

References
1. Blair, D. F. (1995). How bacteria sense and swim.
Annu. Rev. Microbiol. 49, 489– 522.
2. Imae, Y. & Atsumi, T. (1989). Naþ-driven bacterial
flagellar motors. J. Bioenerg. Biomembr. 21, 705– 716.
3. Dean, G. D., Macnab, R. M., Stader, J., Matsumura, P.
& Burks, C. (1984). Gene sequence and predicted
amino acid sequence of the MotA protein, amembrane-associated protein required for flagellar
rotation in Escherichia coli. J. Bacteriol. 159, 991– 999.
4. Stader, J., Matsumura, P., Vacante, D., Dean, G. E. &Macnab, R. M. (1986). Nucleotide sequence of the
Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.
J. Bacteriol. 166, 244– 252.
5. Lloyd, S. A., Tang, H., Wang, X., Billings, S. & Blair,
D. F. (1996). Torque generation in the flagellar motorof Escherichia coli: evidence of a direct role for FliG
but not for FliM or FliN. J. Bacteriol. 178, 223– 231.
6. Tang, H., Braun, T. F. & Blair, D. F. (1996). Motility
protein complexes in the bacterial flagellar motor.
J. Mol. Biol. 261, 209– 221.
7. Zhou, J. D., Fazzio, R. T. & Blair, D. F. (1995). Membrane topology of the MotA protein of Escherichia
coli. J. Mol. Biol. 251, 237– 242.
8. Chun, S. Y. & Parkinson, J. S. (1988). Bacterial
motility: membrane topology of the Escherichia coli
MotB protein. Science, 239, 276–278.
9. Blair, D. F., Kim, D. Y. & Berg, H. C. (1991). Mutant
MotB proteins in Escherichia coli. J. Bacteriol. 173,4049 –4055.
10. De Mot, R. & Vanderleyden, J. (1994). The C-terminal
sequence conservation between OmpA-related outermembrane proteins and MotB suggests a common
function in both Gram-positive and Gram-negative
bacteria, possibly in the interaction of these domains
with peptidoglycan. Mol. Microbiol. 12, 333– 334.
11. Koebnik, R. (1995). Proposal for a peptidoglycanassociating alpha-helical motif in the C-terminal
regions of some bacterial cell-surface proteins. Mol.
Microbiol. 16, 1269– 1270.
12. Blair, D. F. & Berg, H. C. (1990). The MotA proteinof E. coli is a proton-conducting component of theflagellar motor. Cell, 60, 439– 449.
13. Stolz, B. & Berg, H. C. (1991). Evidence for interactions between MotA and MotB, torque-generating
elements of the flagellar motor of Escherichia coli.
J. Bacteriol. 173, 7033– 7037.
14. Garza, A. G., Harrishaller, L. W., Stoebner, R. A. &Manson, M. D. (1995). Motility protein interactionsin the bacterial flagellar motor. Proc. Natl Acad. Sci.
USA, 92, 1970– 1974.
15. Sharp, L. L., Zhou, J. D. & Blair, D. F. (1995). Tryptophan-scanning mutagenesis of MotB, an integral
membrane protein essential for flagellar rotation in
Escherichia coli. Biochemistry, 34, 9166– 9171.

412

16. Sharp, L. L., Zhou, J. D. & Blair, D. F. (1995). Featuresof MotA proton channel structure revealed bytryptophan-scanning mutagenesis. Proc. Natl Acad.
Sci. USA, 92, 7946– 7950.
17. Yamaguchi, S., Aizawa, S., Kihara, M., Isomura, M.,
Jones, C. J. & Macnab, R. M. (1986). Genetic evidence
for a switching and energy-transducing complex in
the flagellar motor of Salmonella typhimurium.
J. Bacteriol. 168, 1172– 1179.
18. Francis, N. R., Sosinsky, G. E., Thomas, D. & Derosier,
D. J. (1994). Isolation, characterization and structureof bacterial flagellar motors containing the switch
complex. J. Mol. Biol. 235, 1261– 1270.
19. Yorimitsu, T. & Homma, M. (2001). Naþ-driven
flagellar motor of Vibrio. Biochim. Biophys. Acta, 1505,
82 –93.
20. McCarter, L. L. (2001). Polar flagellar motility of theVibrionaceae. Microbiol. Mol. Biol. Rev. 65, 445– 462.
21. Asai, Y., Kojima, S., Kato, H., Nishioka, N., Kawagishi,
I. & Homma, M. (1997). Putative channel components for the fast-rotating sodium-driven flagellar
motor of a marine bacterium. J. Bacteriol. 179,
5104– 5110.
22. Yorimitsu, T., Sato, K., Asai, Y., Kawagishi, I. &Homma, M. (1999). Functional interaction between
PomA and PomB, the Naþ-driven flagellar motor
components of Vibrio alginolyticus. J. Bacteriol. 181,5103– 5106.
23. McCarter, L. L. (1994). MotY, a component of thesodium-type flagellar motor. J. Bacteriol. 176,
4219– 4225.
24. McCarter, L. L. (1994). MotX, the channel componentof the sodium-type flagellar motor. J. Bacteriol. 176,
5988– 5998.
25. Okunishi, I., Kawagishi, I. & Homma, M. (1996).
Cloning and characterization of motY, a gene coding
for a component of the sodium-driven flagellar
motor in Vibrio alginolyticus. J. Bacteriol. 178,
2409– 2415.
26. Okabe, M., Yakushi, T., Asai, Y. & Homma, M. (2001).
Cloning and characterization of motX, a Vibrio
alginolyticus sodium-driven flagellar motor gene.
J. Biochem. 130, 879– 884.
27. Asai, Y., Kawagishi, I., Sockett, E. & Homma, M.
(1999). Hybrid motor with the Hþ- and Naþ-driven
components can rotate Vibrio polar flagella by using
sodium ions. J. Bacteriol. 181, 6322– 6338.
28. Zhou, J., Sharp, L. L., Tang, H. L., Lloyd, S. A.,
Billings, S., Braun, T. F. & Blair, D. F. (1998). Functionof protonatable residues in the flagellar motor ofEscherichia coli: a critical role for Asp 32 of MotB.
J. Bacteriol. 180, 2729– 2735.
29. Asai, Y., Sockett, R. E., Kawagishi, I. & Homma, M.
(2000). Coupling ion specificity of chimeras between
Hþ- and Naþ-driven motor proteins, MotB and
PomB, in Vibrio polar flagella. EMBO J. 19,
3639– 3648.
30. Sato, K. & Homma, M. (2000). Functional reconstitution of the Naþ-driven polar flagellar motor component of Vibrio alginolyticus. J. Biol. Chem. 275,
5718– 5722.
31. Sato, K. & Homma, M. (2000). Multimeric structureof PomA, the Naþ-driven polar flagellar motor component of Vibrio alginolyticus. J. Biol. Chem. 275,
20223– 20228.
32. Yorimitsu, T., Sato, K., Asai, Y. & Homma, M. (2000).
Intermolecular cross-linking between the periplasmic
loop3-4 regions of PomA, a component of theCharged Residues in the Cytoplasmic Loop of PomA

Naþ-driven flagellar motor of Vibrio alginolyticus.
J. Biol. Chem. 275, 31387– 31391.
33. Zhou, J. D., Lloyd, S. A. & Blair, D. F. (1998). Electrostatic interactions between rotor and stator in thebacterial flagellar motor. Proc. Natl Acad. Sci. USA,95, 6436– 6441.
34. Lloyd, S. A. & Blair, D. F. (1997). Charged residues of
the rotor protein FliG essential for torque generationin the flagellar motor of Escherichia coli. J. Mol. Biol.
266, 733– 744.
35. Zhou, J. D. & Blair, D. F. (1997). Residues of thecytoplasmic domain of MotA essential for torquegeneration in the bacterial flagellar motor. J. Mol.
Biol. 273, 428– 439.
36. Garza, A. G., Biran, R., Wohlschlegel, J. A. &Manson, M. D. (1996). Mutations in motB suppressible by changes in stator or rotor components of thebacterial flagellar motor. J. Mol. Biol. 258, 270– 285.
37. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I.
(1988). Phosphorylation of three proteins in thesignalling pathway of bacterial chemotaxis. Cell, 53,
79 –87.
38. Borkovich, K. A., Kaplan, N., Hess, J. F. & Simon,
M. I. (1989). Transmembrane signal transduction
in bacterial chemotaxis involves ligand-dependent
activation of phosphate group transfer. Proc. Natl
Acad. Sci. USA, 86, 1208– 1212.
39. Welch, M., Oosawa, K., Aizawa, S. I. & Eisenbach, M.
(1993). Phosphorylation-dependent binding of asignal molecule to the flagellar switch of bacteria.
Proc. Natl Acad. Sci. USA, 90, 8787 –8791.
40. Barak, R. & Eisenbach, M. (1992). Correlation
between phosphorylation of the chemotaxis protein
CheY and its activity at the flagellar motor. Biochemistry, 31, 1821– 1826.
41. Irikura, V. M., Kihara, M., Yamaguchi, S., Sockett, H.
& Macnab, R. M. (1993). Salmonella typhimurium fliGand fliN mutations causing defects in assembly,
rotation, and switching of the flagellar motor.
J. Bacteriol. 175, 802– 810.
42. Zhao, R. H., Pathak, N., Jaffe, H., Reese, T. S. & Khan,
S. (1996). FliN is a major structural protein of the
C-ring in the Salmonella typhimurium flagellar basal
body. J. Mol. Biol. 261, 195–208.
43. Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S.
(1990). Stoichimetric analysis of the flagellar hook(basal-body) complex of Salmonella typhimurium.
J. Mol. Biol. 212, 377–387.
44. Lloyd, S. A., Whitby, F. G., Blair, D. F. & Hill, C. P.
(1999). Structure of the C-terminal domain of FliG, acomponent of the rotor in the bacterial flagellar
motor. Nature, 400, 472– 475.
45. Gosink, K. K. & Häse, C. C. (2000). Requirements forconversion of the Naþ-driven flagellar motor of
Vibrio cholerae to the Hþ-driven motor of Escherichia
coli. J. Bacteriol. 182, 4234– 4240.
46. Boles, B. R. & McCarter, L. L. (2000). Insertional
inactivation of genes encoding components of thesodium-type flagellar motor and switch of Vibrio
parahaemolyticus. J. Bacteriol. 182, 1035– 1045.
47. Kojima, S., Yamamoto, K., Kawagishi, I. & Homma,
M. (1999). The polar flagella motor of Vibrio cholerae
is driven by an Naþ motive force. J. Bacteriol. 181,
1927– 1930.
48. Häse, C. C. & Mekalanos, J. J. (1999). Effects ofchanges in membrane sodium flux on virulencegene expression in Vibrio cholerae. Proc. Natl Acad.
Sci. USA, 96, 3183– 3187.

Charged Residues in the Cytoplasmic Loop of PomA

49. Kojima, S., Asai, Y., Atsumi, T., Kawagishi, I. &Homma, M. (1999). Naþ-driven flagellar motor
resistant to phenamil, an amiloride analog, caused
by mutations of putative channel components.
J. Mol. Biol. 285, 1537 –1547.

413

50. Atsumi, T., Maekawa, Y., Yamada, T., Kawagishi, I.,
Imae, Y. & Homma, M. (1996). Effect of viscosity on
swimming by the lateral and polar flagella of Vibrio
alginolyticus. J. Bacteriol. 178, 5024– 5026.

Edited by J. Karn
(Received 20 February 2002; received in revised form 25 April 2002; accepted 30 April 2002)