JOURNAL OF BACTERIOLOGY, Feb. 2006, p. 1466–1472
0021-9193/06/$08.00⫹0 doi:10.1128/JB.188.4.1466–1472.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 188, No. 4

Roles of Charged Residues of Rotor and Stator in Flagellar Rotation:
Comparative Study using H⫹-Driven and Na⫹-Driven
Motors in Escherichia coli
Toshiharu Yakushi,1 Junghoon Yang,2 Hajime Fukuoka,1 Michio Homma,1* and David F. Blair2*
Graduate School of Biological Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan,1and Department of Biology, University of Utah, Salt Lake City, Utah 841122
Received 15 September 2005/Accepted 23 November 2005

In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions
between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues areconserved in the Naⴙ-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus
suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E.
coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor
might rely on similar interactions but incorporate additional features that make it more robust against
mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but
engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V.
alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteinsfunctioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double
mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in
E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additionalcharged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor
and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus
may be enhanced by the proteins MotX and MotY.
The flagellar motor of bacteria uses energy from either a H⫹
or Na⫹ ion gradient to drive rotation of the flagellar filament.
Bacterial flagella contain about two dozen different proteins,most of which fulfill structural roles. Torque generation appears to involve only five proteins: the MotA and MotB proteins that form the stator and the FliG, FliM, and FliN proteinsthat form the switch complex on the rotor (see Fig. 1). The
stator proteins go by different names in different motors. Thosein the polar Na⫹-driven motor of Vibrio alginolyticus are called
PomA and PomB (1), those in a Na⫹-driven variant of the B.
subtilis motor are called MotP and MotS (12), and some variants that appear unique to Pseudomonas spp. and whose ion
specificity is not yet certain are called MotC and MotD (9, 25).
In spite of the different names, the proteins are closely related
in sequence and are believed to carry out essentially similarfunctions. In addition to PomA and PomB, rotation of thepolar flagellum of V. alginolyticus also requires the proteins
MotX and MotY, which are in the outer membrane and are
not closely related to the other Mot or Pom proteins (21).
The stator proteins function in complexes with composition
A4B2 (15, 22). Several such complexes are arrayed in the membrane surrounding the basal body (7, 13), where they function to
conduct ions and couple ion flow to rotation (4, 5, 8). The MotB

and PomB proteins contain an invariant Asp residue that is essential for function and is believed to participate directly in ion
conduction (30). Our working hypothesis is that ion binding/dissociation at this site drives conformational changes in the statorthat work on the rotor to drive rotation (6, 14).
Proton-utilizing motors can rotate as fast as 300 revolutions
per second (rps) (18), while some Na⫹-driven motors havebeen clocked at 1,700 rps (19). The H⫹- and Na⫹-fueled motors appear to operate by fundamentally similar mechanisms.
Orthologs of the rotor proteins FliG, FliM, and FliN occur inNa⫹-utilizing motors, and the C-terminal domain of FliG is toa large extent interchangeable between species. A FliG chimera with N-terminal domain from Escherichia coli and Cterminal domain from V. alginolyticus can function well in theE. coli motor, and the complementary construct is functional in
V. alginolyticus (26). The same is true of the stator components.
PomA and an appropriately engineered PomB variant canfunction in the E. coli motor, where they use sodium ions andsupport rotation at speeds higher than normal (2). This hybridNa⫹-utilizing motor requires neither MotX nor MotY, in contrast to the motor of V. alginolyticus. Its Na⫹ dependence has
been exploited to allow detection of discrete stepping events inthe motor (24). MotA and MotB can function in the motors ofVibrio spp., where they use protons and support relatively slow
motility (2, 11).
Both the rotor protein FliG and the stator protein MotA
contain well-conserved charged residues. Mutational studies inE. coli showed that these residues are important for function(16, 28) and that the charged residues of FliG interact withthose of MotA (29) (Fig. 1). The precise function of theseelectrostatic interactions is not known. They could provide a* Corresponding author. Mailing address for Michio Homma: Graduate School of Biological Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan. Phone: 81 52 789 2991. Fax: 81 52 789 3001.
E-mail: g44416@nucc.cc.nagoya-u.ac.jp. Mailing address for David F.
Blair: Department of Biology, University of Utah, Salt Lake City, UT
84112. Phone: (801) 585-3709. Fax: (801) 581-4668. E-mail: blair@bioscience.utah.edu.
1466

VOL. 188, 2006

H⫹- AND Na⫹-DRIVEN MOTORS IN E. COLI

1467

FIG. 1. (A) Arrangement of proteins that function in rotation in the flagellar motor of E. coli. (B) Topology of the stator proteins MotA and
MotB and functionally important residues of the rotor and stator. The indicated charged residues of MotA and FliG were shown to engage infunctionally important interactions in the flagellar motor of E. coli (16, 28, 29). Asp32 of MotB is essential for rotation and has been implicatedin proton conduction (30). PBD, peptidoglycan binding domain.

physical linkage between MotA and FliG to ensure that conformational movements in the stator are effectively transmitted
to the rotor, or they might serve to signal the rotor position to
the stator so that ion-gating events in the stator are appropriately timed (6, 14). Whatever their roles, the rotor-stator interactions are fairly robust, because mutations that neutralized
any one of the charges had little effect on function. Mutations
that reversed charge, or that neutralized two charges, werenecessary to disrupt swarming ability in E. coli (16, 28, 29).
Mutational studies of charged residues in the motor of V.
alginolyticus have given somewhat different results. Although
the charged residues that are functionally important in E. coli
are conserved in the V. alginolyticus FliG and PomA proteins,most mutations in them had little effect on V. alginolyticus
motility in swarming or swimming assays (26, 27). Althoughcertain combinations of mutations did impair motility, thecharge alterations needed to impair motility were even greaterthan in E. coli, and because multiple mutations were necessary
to give a strong motility impairment, the functional defects
might conceivably have been due to alterations in protein
structure rather than loss of functionally important charges per
se. Thus, the possibility arose that electrostatic interactions
between the rotor and stator do not occur in the V. alginolyticus
motor or are relatively unimportant for function. Alternatively,
such interactions might occur and make a significant contribution to motor function but be augmented by other features of
the V. alginolyticus motor that make the rotor-stator interface
more resilient. One might expect the rotor-stator interface tobe more robust in the V. alginolyticus motor, because it rotates
about five times faster than that of E. coli and the viscous load
is borne by a single filament rather than a bundle.
Here, we have undertaken comparative studies of the E. coli
and V. alginolyticus proteins that form the rotor-stator interface to understand better the differences noted in previous
mutational studies. We engineered flagellar motors that wereresident in E. coli cells but contained rotor components, stator
components, or both stator and rotor components from V.
alginolyticus. In each type of motor, mutations were made in
the conserved charged residues and effects were measured inswarming and swimming assays. The results indicate that the V.
alginolyticus proteins engage in electrostatic interactions thatare similar to those occurring in E. coli. The V. alginolyticus
motor must incorporate some additional features that makethe rotor-stator interface more resistant to mutation. Some ofthis robustness appears due to additional charged residues thatare present in PomA but not MotA, but most is due to factors
besides the MotA (PomA) and FliG proteins themselves. The
motor in E. coli remained relatively sensitive to mutation of thecharged residues even when it used both rotor and stator
elements from V. alginolyticus, implying that other factors, possibly including the MotX and MotY proteins unique to thesodium-driven motor, must contribute to the resiliency of therotor-stator interface in the V. alginolyticus motor.
MATERIALS AND METHODS
Strains, plasmids, and mutagenesis. Strains and plasmids are listed in Table 1.
MotA and its mutant variants were expressed from plasmid pDFB45. The chimeric FliG protein FliGEV consists of residues 1 to 241 of E. coli FliG fused toresidues 262 to 351 of V. alginolyticus FliG. It was expressed from the arabinose
promoter in plasmid pTY402. Mutations in FliGEV were made using the
QuikChange procedure (Stratagene). Mutations in E. coli FliG were made using
the Altered Sites procedure (Promega), as described previously (17).
PomA and its variants were expressed from either pYS3 or pYS13, dependingon whether ampicillin or chloramphenicol resistance was needed. These plasmids
also encode PotB, a fusion protein consisting of residues 1 to 50 of V. alginolyticus
PomB fused to residues 59 to 308 of E. coli MotB. PotB allows PomA to functionin E. coli, whereas PomB does not (2). Mutations in PomA were made by using
the QuikChange procedure.
Motility assays. Assays of swarming in soft agar used TB (1% tryptone, 0.5%NaCl) and 0.27% Bacto agar. When needed, chloramphenicol was used at 12.5
␮g/ml and ampicillin at 50 ␮g/ml. When cells contained pYS3 or pTY402 (ormutant variants), 1 mM arabinose was included to induce expression of thechimeric fliG gene. Single colonies of fresh transformants were picked onto
swarm plates, plates were incubated at 32°C, and swarm diameters were measured at regular intervals. Swarm rates were calculated from linear fits to the data
and are reported relative to wild-type controls included in the experiments.
To assay motility in liquid medium, a single colony of a fresh transformant was
inoculated into TB plus appropriate antibiotics and cultured overnight withshaking at 32°C. Overnight cultures were diluted 100-fold into fresh TB andincubated for 4.5 h at 32°C. Motility was scored visually under a phase-contrast
microscope.

RESULTS
Motor with stator from E. coli and FliG C-terminal domainfrom V. alginolyticus. Charged residues in the C-terminal do-

1468

YAKUSHI ET AL.

J. BACTERIOL.
TABLE 1. Strains and plasmids

Strain orplasmid

Description

Source or reference

DFB245pAlter-1
pBAD24
pBAD33
pDFB45
pDFB97
pMMB206
pSL27
pTY402
pYA25
pYS3
pYS13

motA⌬fliG
Tcr; defective Apr; vector for mutagenesis
pBR322-derived, araBAD promoter, Apr
pACYC184-derived, araBAD promoter, Cmr
motA motB behind trp promoter
E. coli fliG behind ara promoter; Cmr
lacI, tac promoter, Cmr
pAlter-1 derivative encoding E. coli fliG
fliGEV gene in pBAD33
pBAD24 derivative with inverted multiple cloning site
pomA potB in pYA25
pomA potB in pMMB206

30
Promega
J. Beckwith, Harvard Medical School
J. Beckwith
4
D. Blair
20
17
26
Y. Asai, Nagoya University
Y. Sowa, Nagoya University
Y. Sowa, Nagoya University

main of FliG were found to be important for motor rotation in
E. coli (16) but less important in V. alginolyticus (26). A chimeric FliG protein (FliGEV) consisting of residues 1 to 241 of
E. coli FliG fused to residues 262 to 351 of V. alginolyticus FliG
functions well in the motor of E. coli (26). To examine the roleof electrostatic interactions in the motor using the chimericrotor protein, we mutated charged residues in the C-terminal,
V. alginolyticus-derived domain of FliGEV and measured theeffects on swarming and swimming. Results are summarized inTable 2.
In E. coli, three charged residues of FliG (Arg281, Asp288,
and Asp289) were found to be of primary importance formotor rotation, while two others (Lys264 and Arg 297) madesecondary contributions. The corresponding residues in V.
alginolyticus FliG are Arg301, Asp308, and Asp309 (primary)and Lys 284 and Arg317 (secondary). In the E. coli motor using
FliGEV, most mutations in these residues caused severe motility defects. The charge-reversing mutations R301D and
D309K in FliGEV eliminated swarming in soft agar, and thecells were immotile under the microscope. Swarming was severely reduced by the charge-neutralizing mutation R301A.
The charge reversals K284E and R317D also caused significant
motility reductions (to about 40% and 30% of wild type, respectively); only the reversal D308K had no effect. Thus, al-though most of these mutations had only mild effects in the V.
alginolyticus motor (an exception was R317D, which caused a
severe motility impairment) (26), the charged residues of V.
alginolyticus FliG were important for rotation when the domain was made to function in the E. coli motor.
Next, we examined the effects of mutations in charged residues of MotA in the motors using FliGEV. In the native E. coli
motor, rotation is prevented by mutations that neutralize both
Arg90 and Glu98 of MotA or that reverse the charge of either(28). (The conserved charged residue Glu150 was shown tomake a secondary contribution to rotation in E. coli and isimportant for rotation in Sinorhizobium meliloti [3], but Glu150
mutations were not tested here.) Arg90 and Glu98 also proved
essential for rotation in the motor using FliGEV. Swarming andswimming were eliminated by mutations that neutralized both
Arg90 and Glu98 or that reversed the charge of either residue(Table 2). The single charge-neutralizing mutations had stronger effects in the motor using FliGEV than in the native E. coli
motor; swarming was prevented by the E98Q mutation anddecreased to less than half the wild-type rate by the R90A
mutation. Thus, the stator residues important for rotation in
the native E. coli motor are also important in the motor using
FliGEV.

TABLE 2. Effects of charged-residue mutations in a motor using the E. coli stator and the chimeric rotor protein FliGEV
Swarming rate for MotA mutation(s)a

FliGEV mutation

wt
K284A
K284E
R301A
R301D
D308A
D308K
D309A
D309K
R317A
R317D

wt

R90A

R90E

E98Q

R90A/E98Q

E98K

1.00
0.61
0.41
0.13
0.0 (imm)
0.88
1.04
0.84
0.0 (imm, t)
0.38
0.32

0.44
0.0 (imm)
0.0 (imm)
0.0 (imm)
0.0.14
0.0 (imm)
0.36
0.27
0.0 (imm)
0.0 (imm)

0.0 (imm)

0.0 (w,f)
0
0
0
0
0
0
0
0
0
0

0.0 (imm)

0.0 (imm)

0.22 (f)aSwarming rates in soft agar relative to controls expressing wild-type (wt) MotA and FliGEV proteins. The measurements used cells of the E. coli strain DFB245(fliG motA) transformed with a plasmid expressing MotA and a plasmid expressing the chimeric FliG protein FliGEV, each with the mutations indicated. Numbers in
FliGEV correspond to numbers of the residues in the V. alginolyticus FliG protein to allow direct comparison with the V. alginolyticus FliG sequence; actual residue
positions within the chimeric protein sequence are lower by 20. Instances of strong synergism are indicated in boldface and suppression by italics. Swimming behavior
in liquid is indicated in parentheses: imm, immotile; w, weak motility; f, only a few cells were motile; t, trails seen in swarm plates indicating the rare occurrence of
motile cells.

VOL. 188, 2006

FIG. 2. Mutual suppression of charge-reversing mutations in therotor and stator in the motor that uses the chimeric rotor protein
FliGEV. E. coli strain DFB245 (fliG motA) was transformed with one
plasmid that expresses MotA and a second plasmid that expresses the
chimeric FliG protein FliGEV, each with the mutations indicated. The
residue numbers given for FliGEV correspond to numbers in the V.
alginolyticus FliG protein. Actual residue positions within the chimeric
protein sequence are less, by 20. Fresh transformants were picked ontoa plate containing TB and 0.28% agar. The plate was incubated at 32°C
for 24 h.

Synergism and suppression in the motor using FliGEV. Evidence for electrostatic interactions in the E. coli motor camefrom instances of synergism and suppression in MotA/FliG
double mutants (29). To test for synergism and suppression in
the motor using FliGEV, we expressed various combinationsof charged-residue mutations in MotA and FliGEV in the
motAfliG double mutant strain and measured the effects on
swarming and swimming.
In several cases, mutations in MotA and FliGEV were tolerated singly but not when paired. The R90A mutation in MotA
acted synergistically with mutations in four charged residues of
FliGEV (Table 2). Each of the residues showing synergism inthe present experiments showed similar behavior in the previous study of the E. coli motor (29). Synergism involving Glu98
of MotA was also reported for the native E. coli motor (29) but
could not be observed in the present case because the E98Q
mutation alone was sufficient to prevent swarming of the cells
using FliGEV.
Instances of mutational suppression were also observed. The
charge-reversing mutation D309K in FliGEV prevented motility when the stator was the wild type, but some swarming andswimming were restored when the R90A or R90E mutation
was present in MotA (Table 2 and Fig. 2). The suppression
seen here in the motor using FliGEV was not as strong as thatreported previously in E. coli but was similar in involving thesame pair of charged residues (28).
Chimeric motor with stator from V. alginolyticus and rotor
from E. coli. Results above indicate that rotation of the motorusing FliGEV depends upon electrostatic interactions similar to
those occurring in the native E. coli motor. Although the motorusing FliGEV contains a rotor element from V. alginolyticus, it
did not exhibit the resiliency of the V. alginolyticus motor but
was somewhat more sensitive to mutation than the motor of E.
coli. To determine whether the stator from V. alginolyticus canconfer greater resistance to mutation, we examined a motorusing the stator proteins from V. alginolyticus and the nativeH⫹- AND Na⫹-DRIVEN MOTORS IN E. COLI

1469

E. coli rotor. The V. alginolyticus stator proteins have previously been shown to function well in E. coli, provided that the
C-terminal part of PomB is replaced with the corresponding
part of MotB (2). The C-terminal domain of MotB is locatedin the periplasm and has not been implicated in direct rotorstator interactions. We studied the effects of charged-residue
mutations in PomA, coexpressed with the appropriate PomBMotB fusion, termed PotB. Results of swarming and swimming
assays are summarized in Table 3.
The relevant charged residues of PomA are Arg88 and
Glu96, which correspond to residues Arg90 and Glu98 of
MotA. In the V. alginolyticus motor, mutations of Arg88 or
Glu96 had relatively weak effects (27). When PomA was resident in the E. coli motor, Arg88 and Glu96 were important forfunction. Swarming was eliminated by mutations that neutralized both residues (R88A/E96Q) or reversed the charge ofeither (R88E, E96K) (Table 3). Cells of the R88E and E96K
mutants were immotile, and cells of the R88A/E96Q mutant
showed only weak motility under the microscope. This weak
motility was eliminated by the additional neutralization ofother residues nearby in the sequence (K89, E97, and E99).
Thus, although the PomA protein resident in E. coli motors isslightly more resistant to mutation than MotA (the R90A/
E98Q mutant is immotile in E. coli [28]), Arg88 and Glu96 areclearly important for rotation.
Next, we examined the effects of mutations in FliG in the
motor using the V. alginolyticus stator. Mutations in the
charged residues of primary importance (Arg281, Asp288, andAsp289 [16]) caused severe defects in the motors using PomA.
Swarming was prevented by mutations that neutralized any twoof these residues or that reversed the charge of Arg281 or
Asp289 (Table 3). Swimming in liquid was either eliminated or
greatly weakened. Thus, the same charged residues in FliG are
essential for rotation whether the stator is from E. coli or V.
alginolyticus.

TABLE 3. Effects of charged-residue mutations in a motor using
stator components from V. alginolyticus and the rotor of E. coli
Mutation(s)Swarming ratea

PomA
R88A
E96Q
R88A/E96Q
R88A/K89A/E96Q/E97Q/E99Q
R88E
E96K

0.0 (w)
0.9
0.0 (w)
0.0 (imm)
0.0 (f)0.0 (f)

FliG
R281D
D288K
D289K
R281A/D288A
R281A/D289A
D288A/D289A
D288K/D289K

0.0 (imm)
0.2
0.0 (imm)b
0.0 (imm)
0.0 (imm)
0.0 (w)
0.0 (imm)aRelative to a control strain expressing wild-type PomA and FliG. The measurements used cells of the E. coli strain DFB245 (fliG motA) transformed witha plasmid expressing PomA and PotB, and another plasmid expressing E. coli
FliG, each with the mutations indicated. Swimming behavior in liquid medium isindicated in parentheses: imm, immotile; f, a few cells show weak motility; w,
weakly motile.
b
Becomes motile in the presence of attractant.

1470

YAKUSHI ET AL.

J. BACTERIOL.

TABLE 4. Effects of charged-residue mutations in a motor using both stator and rotor elements from V. alginolyticusa
PomA mutation(s)FliGEVmutation

wtb
wt
K284A
K284E
R301A
R301D
D308A
D308K
D308K/E311Q
D309A
D309K
R317A
R317D

wtb

wt

R88A

1.0
1.0
1.0
0.55
0.2
0.23
0.7
0.33
0.4
0.0 (imm)
1.1
0.98
0.9
0.78
NM
1.08
1.2
0.98
1.1
0.16 (f)0.6
0.12
0.0 (imm) 0.0 (f)

K89A

E96Q

E97Q

1.0
1.1
1.1
1.1
0.36
0.85
0.97
1.0
0.0 (w) 0.0 (w) 0.0 (w) 0.48

E99Q R88A/E96Q

0.9
1.0
0.62

0.0 (f)0.0 (f)0.0 (f)0.0 (m) 0.1

0.55

1.11

0.91

1.18

1.18

0.19

0.38

0.07

0.91

1.04

0.0 (f)0.0 (w) 0.0 (w) 0.06

0.22

1.1
0.0 (w)

R88A/K89A/E96Q/
E97Q/E99Q

R88E

K89E

1.0
0.0 (imm)

NM
0.0 (w)

NM
0.75

NM
0.0 (NM)
0.04
0.08
0.09
0.04
0.05

0.0 (imm) 0.0 (imm)
0.0 (imm) 0.0 (imm)
0.76
0.0 (imm)
0.85
0.0 (imm)
0.68
0.0 (imm)
0.12
0.0 (imm)
0.0 (w)
0.0 (imm)

E96K

NM
0.0 (imm)a
Swarming rates of mutants relative to controls expressing wild-type (wt) PomA and FliGEV proteins. The measurements used cells of the E. coli strain DFB245(motA fliG) transformed with a plasmid expressing PomA and PotB and a second plasmid expressing the chimeric rotor protein FliGEV, each with the mutations
indicated. Instances of strong synergism are indicated in boldface and suppression by italics. Swimming behavior in liquid is indicated in parentheses: m, motile; w,
weakly motile; f, a few cells showed weak motility; imm, immotile; NM, not measured.
b
Swarming rates of FliG mutants and of PomA mutants in the native Vibrio motor are from references 26 and 27, respectively.

E. coli motor that uses both stator and rotor components
from V. alginolyticus. The E. coli motor using either rotor or
stator elements from V. alginolyticus remained relatively sensitive to mutation of the charged residues. To determine
whether the V. alginolyticus stator and rotor components together can confer greater resistance to mutation, we examined
a motor that uses both stator and rotor components from V.
alginolyticus. PomA, PotB, and FliGEV were expressed in theE. coli motAfliG strain, and the effects of various single or
double mutations were studied in swarming and swimming
assays as before. Results are summarized in Tables 4 and 5.
The motor using both rotor and stator components from V.
alginolyticus showed mutational defects that were generally
similar to the E. coli motor. Swarming was prevented bycharge-reversing mutations in Arg88 or Glu96 of PomA or
residue Arg301 or Asp308 of FliGEV. Synergistic effects werealso similar: although most single mutations were tolerated,swarming was prevented by several pairs of charge-neutralizing
mutations, involving two residues of PomA or a residue of
PomA in combination with a residue of FliGEV (Table 4 andFig. 3). Suppression was observed for the residue pair Arg88/
Asp309, which correspond to Arg90/Asp289 in the E. coli proteins, and also for the pair Arg88/Asp308 (Table 4 and Fig. 4).

Additional charged residues in PomA contribute to function. The slightly greater resiliency of the motor using the V.
alginolyticus stator suggests that PomA might contain one ormore functionally important charged residues not found in
MotA. Lys89 of PomA, in particular, was found to be important for optimal function of the motor that used V. alginolyticus
components, and instances of synergism suggest that it mightinteract with a charged residue(s) of the rotor (Table 4). To
identify other, potentially important charged residues present
in PomA but not MotA, we aligned sequences of MotA fromseveral H⫹-utilizing motors and PomA proteins from several

TABLE 5. Effects of double charge-neutralization mutations in
PomA in the motor using both rotor and stator
components from V. alginolyticusa
First PomA mutation

Second PomA
mutation

wt

R88A

K89A

E96Q

E97Q

E99Q

wt
R88A
K89A
E96Q
E97Q
E99Q

1.0
0.36
0.85
0.97
1.00
1.00

0.36

0.85
0.0

0.97
0.0
0.40

1.00
0.17
0.80
NM

1.00
0.35
0.93
NM
NM

a
Swarming rates relative to wild-type (wt) controls. Boldface indicates cases ofsynergism in the double mutants. NM, not measured.

FIG. 3. Synergistic action of two charge-neutralizing mutations inthe stator (A) or a charge-neutralizing mutation in the stator andanother in the rotor (B) in the motor that uses both stator and rotor
elements from V. alginolyticus. E. coli strain DFB245 (fliGmotA) wastransformed with a plasmid that expresses PomA and PotB and asecond plasmid that expresses FliGEV, each with the mutations indicated. The residue number for FliGEV is the position in the V. alginolyticus FliG protein. Transformants were cultured in liquid medium
overnight, and aliquots were spotted onto a plate containing TB and
0.27% agar. Plates were incubated at 30°C for 10 h (most strains) or
10.8 h (the E96Q/D309A mutant). w.t., wild type.

H⫹- AND Na⫹-DRIVEN MOTORS IN E. COLI

VOL. 188, 2006

FIG. 4. Suppression of a charge-reversing mutation in the stator bya charge-reversing mutation in the rotor in the motor that uses bothstator and rotor elements from V. alginolyticus. PomA, with the mutation R88E, and PotB were expressed from one plasmid, and FliGEV
with the mutation D309K was expressed from a second plasmid. Procedures were as described in the legend to Fig. 2, except that the plates
were incubated for 28 h.

(putatively) Na⫹-using motors. Mutations were made in candidate charged residues that occur in PomA but not MotA, andfunction was assayed both in motors using E. coli FliG andmotors using the chimeric rotor protein FliGEV. Sixteen positions in PomA were tested. Motility was eliminated or greatly
reduced by charge-reversing mutations in any of the six residues Asp114, Arg135, Lys203, Arg215, Asp220, and Arg232(Table 6).
DISCUSSION
Because most mutations in the charged residues of rotor andstator components in V. alginolyticus had little or no effect on
motility (10, 26, 27), the possibility arose that these charged
residues, although conserved, do not make a critical contribution to function in the polar flagellar motor of V. alginolyticus.
Alternatively, the residues might have important roles in bothtypes of motor, but their action could be augmented by additional features of the V. alginolyticus motor that make its function more resistant to mutation. The present results argue thatcharged residues at the rotor-stator interface play similar rolesin the flagellar motors of V. alginolyticus and E. coli. When therotor and stator components of V. alginolyticus are made tofunction in an otherwise E. coli-based motor, mutations in the
key charged residues cause motility defects that are similar to
those seen in E. coli. Patterns of synergism and suppression inrotor/stator double mutants are also similar, indicating that theinteractions between rotor and stator are basically the same
whether the proteins come from E. coli or V. alginolyticus.
The previous mutational studies of PomA (27) and FliG (26)gave some indication that charged residues contribute to function of the V. alginolyticus motor, albeit to different extents
than in the E. coli motor. The charge-reversing mutation
R317D in V. alginolyticus FliG impairs motility strongly (moreso than the corresponding mutation in E. coli) (26). Double
mutations in PomA that neutralize one charge and reverse
another charge cause a strong motility impairment (27); i.e., achange of 3 charge units in the V. alginolyticus stator had effects

1471

roughly equivalent to a 2-unit change in E. coli. Thus, we may
suggest that electrostatic interactions between the rotor andstator contribute to motor rotation in both species and that theV. alginolyticus motor incorporates additional features thatstrengthen the rotor-stator interaction and make it more resistant to mutation.
The robustness of the rotor-stator interaction in V. alginolyticus appears due in part to the presence of additionalcharged residues in PomA. These include residue Lys89, which
is near the previously identified residues of importance, residues Asp114 and Arg135 in other segments of the first cytoplasmic domain, and residues Lys203, Arg215, Asp220, andArg232 in the second cytoplasmic domain. We do not knowwhether all of these charged residues of PomA contribute to asingle functionally important site on the protein, nor have wedetermined whether they interact with charged residues of
FliG. Additional charged residues cannot be responsible for allof the robustness of the V. alginolyticus motor, however, because an E. coli motor using both rotor and stator components
from V. alginolyticus remained sensitive to mutation. We have
not yet identified all of the factors contributing to the robustness of the V. alginolyticus motor; among the possibilities are
the proteins MotX and MotY, which are essential for rotationof the V. alginolyticus motor and might modulate the rotorstator interaction so that it is more resistant to mutation.
A recent analysis of charged residues in the S. meliloti motor
casts additional light on the variation that can occur at therotor-stator interface (3). Unlike E. coli, which steers by meansof reversals in motor direction, S. meliloti directs its movements by modulating the speed of exclusively clockwise-rotating motors, a behavior termed chemokinesis (23). The charged
residues in MotA and most of those in FliG are conserved in
S. meliloti, and detailed mutational analysis confirms that they
are engaged in functionally important electrostatic interactions
at the rotor-stator interface (3). The topology of the interactions and the relative importance of the various residues

TABLE 6. Effects of mutations in other charged residues of PomAa
Swarming ratea
Mutation

E97K
E99K
K107E
D114K
D117K
D119K
R122E
D128K
D133K
R135E
H136D
K203E
D209K
R215E
D220K
R232E

FliG rotor

FliGEV rotor

1.0
1.0
1.0
0.0 (imm)
1.0
1.0
1.0
0.5
1.0
0.0 (imm)
1.0
0.0 (imm)
1.0
0.0 (imm)
0.0 (imm)
0.0 (f)

1.0
1.0
1.0
0.0 (imm)
1.0
1.0
1.0
1.0
1.0
0.0 (imm)
0.4
0.0 (imm)
1.0
0.0 (imm)
0.0 (imm)
0.0 (w)a
Rates relative to wild-type controls included on the plates. Positions 97
through 136 are in the first cytoplasmic domain of MotA, and positions 203 andhigher are in the second cytoplasmic domain (see topology in Fig. 1). Swimming
behavior in liquid is given in parentheses: imm, immotile; w, weakly motile; f, a
few cells showed weak motility.

1472

YAKUSHI ET AL.

were found to be somewhat different from either E. coli or V.
alginolyticus. Most notably, the pattern of mutational defects in
S. meliloti suggested that controlled modulation of the rotorstator interface might be the basis of chemokinesis (3). Thus, itappears that the rotor-stator interface can vary not only between species but also with circumstances.
A stronger rotor-stator interaction in the V. alginolyticus
motor may be an adaptation to the greater operational demands on the Na⫹-driven polar flagellum. Owing to its much(ca. fivefold) greater rotation speed, the polar flagellum of V.
alginolyticus is likely to operate against a greater viscous load
than that of E. coli. Closer tolerances at the interface, and/or alarger number of participating groups, might be necessary tomaintain efficient coupling between the rotor and stator in theface of this greater load.
In summary, electrostatic interactions between rotor andstator appear to be a general and functionally important feature of bacterial flagellar motors. The particular residues involved and the detailed relationship of the rotor and stator may
vary, but the protein domains present at the interface and theelectrostatic nature of the interaction seem to be conserved.
The precise role(s) of the electrostatic interactions remains to
be determined. Further comparative studies of the motors ofdifferent species should be helpful for addressing this question.
ACKNOWLEDGMENTS
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan,
from the Japan Science and Technology Corporation (to M.H. andT.Y.), from the Soft Nano-Machine Project of the Japan Science andTechnology Agency (to T.Y. and M.H.), from the Japan Foundation
Advanced Technology Institute (to T.Y.), and by grant R01-GM64664
from the U.S. National Institute of General Medical Sciences (toD.B.). The Protein-DNA core facility at the University of Utah receives support from the National Cancer Institute (5P30 CA42014).
We thank Yoshiyuki Sowa for technical assistance and discussions.
REFERENCES
1. Asai, Y., S. Kojima, H. Kato, N. Nishioka, I. Kawagishi, and M. Homma.
1997. Putative channel components for the fast-rotating sodium-driven
flagellar motor of a marine bacterium. J. Bacteriol. 179:5104–5110.
2. Asai, Y., T. Yakushi, I. Kawagishi, and M. Homma. 2003. Ion-coupling
determinants of Na(⫹)-driven and H(⫹)-driven flagellar motors. J. Mol.
Biol. 327:453–463.
3. Attmannspacher, U., B. Scharf, and R. Schmitt. 2005. Control of speed
modulation (chemokinesis) in the unidirectional rotary motor of Sinorhizobium meliloti. Mol. Microbiol. 56:708–718.
4. Blair, D. F., and H. C. Berg. 1990. The MotA protein of E. coli is a protonconducting component of the flagellar motor. Cell 60:439–449.
5. Blair, D. F., and H. C. Berg. 1991. Mutations in the MotA protein ofEscherichia coli reveal domains critical for proton conduction. J. Mol. Biol.
221:1433–1442.
6. Braun, T. F., S. Poulson, J. B. Gully, J. C. Empey, S. Van Way, A. Putnam,
and D. F. Blair. 1999. Function of proline residues of MotA in torquegeneration by the flagellar motor of Escherichia coli. J. Bacteriol. 181:3542–
3551.
7. Coulton, J. W., and R. G. E. Murray. 1978. Cell envelope associations ofAquaspirillum serpens flagella. J. Bacteriol. 136:1037–1049.

J. BACTERIOL.
8. Dean, G. E., R. M. Macnab, J. Stader, P. Matsumura, and C. Burke. 1984.
Gene sequence and predicted amino acid sequence of the MotA protein, amembrane-associated protein required for flagellar rotation in Escherichia
coli. J. Bacteriol. 143:991–999.
9. Doyle, T. B., A. C. Hawkins, and L. L. McCarter. 2004. The complex flagellar
torque generator of Pseudomonas aeruginosa. J. Bacteriol. 186:6341–6350.
10. Fukuoka, H., T. Yakushi, and M. Homma. 2004. Concerted effects of aminoacid substitution in conserved charged residues and other residues in thecytoplasmic domain of PomA, a stator component of Na⫹-driven flagella. J.
Bacteriol. 186:6749–6758.
11. Gosink, K. K., and C. C. Hase. 2000. Requirements for conversion of theNa⫹-driven flagellar motor of Vibrio cholerae to the H⫹-driven motor ofEscherichia coli. J. Bacteriol. 182:4234–4240.
12. Ito, M., D. B. Hicks, T. M. Henkin, A. A. Guffanti, B. D. Powers, L. Zvi, K.
Uematsu, and T. A. Krulwich. 2004. MotPS is the stator-force generator for
motility of alkaliphilic Bacillus, and its homologue is a second functional Mot
in Bacillus subtilis. Mol. Microbiol. 53:1035–1049.
13. Khan, S., M. Dapice, and T. S. Reese. 1988. Effects of mot gene expression
on the structure of the flagellar motor. J. Mol. Biol. 202:575–584.
14. Kojima, S., and D. F. Blair. 2001. Conformational change in the stator of thebacterial flagellar motor. Biochemistry 40:13041–13050.
15. Kojima, S., and D. F. Blair. 2004. Solubilization and purification of the
MotA/MotB complex of Escherichia coli. Biochemistry 43:26–34.
16. Lloyd, S. A., and D. F. Blair. 1997. Charged residues of the rotor protein
FliG essential for torque generation in the flagellar motor of Escherichia coli.
J. Mol. Biol. 266:733–744.
17. Lloyd, S. A., H. Tang, X. Wang, S. Billings, and D. F. Blair. 1996. Torquegeneration in the flagellar motor of Escherichia coli: evidence of a direct rolefor FliG but not for FliM or FliN. J. Bacteriol. 178:223–231.
18. Lowe, G., M. Meister, and H. C. Berg. 1987. Rapid rotation of flagellar
bundles in swimming bacteria. Nature 325:637–640.
19. Magariyama, Y., S. Sugiyama, K. Muramoto, Y. Maekawa, I. Kawagishi, Y.
Imae, and S. Kudo. 1994. Very fast flagellar rotation. Nature 371:752.
20. Morales, V. M., A. Backman, and M. Bagdasarian. 1991. A series of wide
host-range low-copy number vectors that allow direct screening for recombinants. Gene 97:39–47.
21. Okabe, M., T. Yakushi, and M. Homma. 2005. Interactions of MotX with
MotY and with the PomA/PomB sodium ion channel complex of the Vibrio
alginolyticus polar flagellum. J. Biol. Chem. 280:25659–25664.
22. Sato, K., and M. Homma. 2000. Functional reconstitution of the Na(⫹)driven polar flagellar motor component of Vibrio alginolyticus. J. Biol. Chem.
275:5718–5722.
23. Schmitt, R. 2002. Sinorhizobial chemotaxis: a departure from the enterobacterial paradigm. Microbiology 148:627–631.
24. Sowa, Y., A. D. Rowe, M. C. Leake, T. Yakushi, M. Homma, A. Ishijima, andR. M. Berry. 2005. Direct observation of steps in rotation of the bacterial
flagellar motor. Nature 437:916–919.
25. Toutain, C. M., M. E. Zegans, and G. A. O’Toole. 2005. Evidence for two
flagellar stators and their role in the motility of Pseudomonas aeruginosa. J.
Bacteriol. 187:771–777.
26. Yorimitsu, T., A. Mimaki, T. Yakushi, and M. Homma. 2003. The conserved
charged residues of the C-terminal region of FliG, a rotor comonent of theNa⫹-driven flagellar motor. J. Mol. Biol. 334:567–583.
27. Yorimitsu, T., Y. Sowa, A. Ishijima, T. Yakushi, and T. Homma. 2002. The
systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na⫹-driven flagellar motor component PomA. J. Mol. Biol.
320:403–413.
28. Zhou, J., and D. F. Blair. 1997. Residues of the cytoplasmic domain of MotA
essential for torque generation in the bacterial flagellar motor. J. Mol. Biol.
273:428–439.
29. Zhou, J., S. A. Lloyd, and D. F. Blair. 1998. Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc. Natl. Acad. Sci.
USA 95:6436–6441.
30. Zhou, J., L. L. Sharp, H. L. Tang, S. A. Lloyd, S. Billings, T. F. Braun, andD. F. Blair. 1998. Function of protonatable residues in the flagellar motor ofEscherichia coli: a critical role for Asp 32 of MotB. J. Bacteriol. 180:2729–
2735.