JOURNAL OF BACTERIOLOGY, May 2008, p. 3565–3571
0021-9193/08/$08.00⫹0 doi:10.1128/JB.00849-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 190, No. 10

Roles of Charged Residues in the C-Terminal Region of PomA, a
Stator Component of the Na⫹-Driven Flagellar Motor䌤
Madoka Obara, Toshiharu Yakushi,† Seiji Kojima, and Michio Homma*
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho, Chikusa-Ku, Nagoya 464-8602, Japan
Received 1 June 2007/Accepted 22 February 2008

lecular mass of the PomA/PomB complex has been estimated to
be 175 kDa by gel filtration chromatography, suggesting that it
forms a complex as oligomers, which may consist of four PomA
molecules and two PomB molecules (24). PomA alone forms astable dimer, and a genetically fused tandem PomA dimer functions in the Na⫹-type motor. PomA may function as at least a
dimer in the PomA/PomB complex (24, 25, 31). It has also beenreported that MotA and MotB oligomerize as a complex of four
MotA molecules and two MotB molecules in the H⫹-driven
flagellar motor of E. coli (6, 15).
In E. coli, mutational studies have shown that some crucial
charged residues of MotA and FliG are involved in torquegeneration; these residues are R90 and E98 in the second
cytoplasmic region of MotA and K264, R281, D288, D289, andR297 in the C-terminal region of FliG (17, 33). It has beensuggested that these charged residues in MotA and FliG might
engage in electrostatic interactions with each other (36). The
charged residues of MotA, R90 and E98, are conserved in
PomA as R88 and E96, and the charged residues of FliG in E.
coli, K264, R281, D288, D289, and R297, are conserved in V.
alginolyticus FliG as K284, R301, D308, D309, and R317. Although the coupling ions are different, it was expected thatelectrostatic interactions between charged residues in PomA
and FliG in V. alginolyticus should be important for motor
rotation, as found in E. coli. However, replacement of thesecharged residues, as well as residues adjacent to them in PomA
and FliG, had little effect on motility based on swimming assays
as well as swarming assays (30, 33). Electrostatic interactions
among these charged residues may not be important. Thus, thepossibility that other charged residues may be involved inflagellar rotation in V. alginolyticus was considered.
Recently, to clarify how ion selectivity is determined in proton and sodium motors, chimeric motors between the H⫹ typeof E. coli or Rhodobacter sphaeroides and the Na⫹ type ofVibrio species were constructed (1). For the rotor, the N ter-

Many bacteria can swim by rotating helical flagellar filaments like a screw. At the base of each filament, a rotary motor
is embedded in the cytoplasmic membrane. The fuel for rotation is the membrane gradient of ions, either H⫹ or Na⫹. The
flagellar motor has been extensively studied in Escherichia coliand Salmonella enterica serovar Typhimurium, which have H⫹driven motors (5), and it is known that approximately 50 genes
are required for flagellar assembly and rotation (4, 19). Proteins encoded by three of these genes, MotA, MotB, and FliG,
are essential components for rotation (5). MotA has fourtransmembrane segments and a relatively large cytoplasmic
domain between the second and third transmembrane segments (35). MotB has one transmembrane segment and a
peptidoglycan-binding motif in its C-terminal region (7, 8).
MotA and MotB form a complex and work as H⫹ channels(27). They are thought to function as the stator part of the
motor (11, 13, 34). In the rotor part of the motor, FliM and
FliN are the components of the C ring structure, the MS ringis composed of FliF, and FliG is thought to form a ring structure between the MS ring and the C ring to connect them. It isthought that FliM and FliG are involved mainly in switching
the rotation direction of the motor (26, 28) and in generating
the torque of the motor (18), respectively.
In Vibrio alginolyticus, PomA, PomB, MotX, and MotY havebeen identified as components that are essential for torque generation in the Na⫹-driven polar flagellum (20, 21, 22, 23). PomA
(27 kDa) and PomB (35 kDa), which are homologous to MotA
and MotB, respectively, form the stator complex (32). The mo* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho,
Chikusa-Ku, Nagoya 464-8602, Japan. Phone: 81-52-789-2991. Fax: 8152-789-3001. E-mail: g44416a@cc.nagoya-u.ac.jp.
† Present address: Applied Molecular Bioscience, Graduate Schoolof Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
䌤
Published ahead of print on 7 March 2008.
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Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that theelectrostatic interactions between charged residues of the stator and rotor proteins are important for rotationin Escherichia coli. Mutational studies have indicated that the Naⴙ-driven motor of Vibrio alginolyticus may
incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between
the rotor and stator proteins may occur in the Naⴙ-driven motor. Thus, we investigated the C-terminal charged
residues of the stator protein, PomA, in the Naⴙ-driven motor. Three of eight charge-reversing mutations,
PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Naⴙ-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant
proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus.
The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E.
coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus
but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA
interactions or PomA intramolecular interactions to form the stator complex.

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OBARA ET AL.

MATERIALS AND METHODS
Strains, plasmids, and mutagenesis. Strains and plasmids are listed in Table 1.
Mutations were introduced into PomA using the QuikChange procedure asreported previously (29). In Vibrio, PomA and its variants were produced fromplasmid derivatives of pYA303, which contains pomA and pomB. Plasmids wereselected or maintained in the presence of 100 ␮g ml⫺1 kanamycin.
In E. coli, the PomA proteins were produced from plasmid derivatives of
pYS3, which contains pomA and potB. Plasmids were selected or maintained in
the presence of 25 ␮g ml⫺1 chloramphenicol. PotB is a chimeric protein composed of residues 1 to 50 of V. alginolyticus PomB and residues 59 to 308 of E.
coli MotB. FliGEV is a chimeric protein with residues 1 to 241 of E. coli FliG
fused to residues 262 to 351 of V. alginolyticus FliG. These chimeric proteins were
produced from plasmid derivatives of pTY402 following addition of 1 mM
arabinose.
Culture of cells. V. alginolyticus was cultured at 30°C in VC medium (0.5%[wt/vol] polypeptone, 0.5% [wt/vol] yeast extract, 0.4% [wt/vol] K2HPO4, 3%[wt/vol] NaCl, 0.2% [wt/vol] glucose) or in VPG500 medium (1% [wt/vol]polypeptone, 0.4% [wt/vol] K2HPO4, 500 mM NaCl, 0.5% [wt/vol] glycerol). E.
coli was cultured at 37°C in LB broth (1% [wt/vol] tryptone peptone, 0.5%
[wt/vol] yeast extract, 0.5% [wt/vol] NaCl) or in T broth (1% [wt/vol] Bacto
tryptone, 0.5% [wt/vol] NaCl). For swarming assays, overnight cultures werespotted on VPG500 medium-0.25% agar plates containing 100 ␮g ml⫺1 kanamycin for Vibrio. For E. coli, overnight cells were cultured in T broth at 30°C for
4 h. When cultures reached the exponential growth phase, 1 mM arabinose wasadded, and incubation was continued for an additional 4 h. The cultured cells
were diluted appropriately and inoculated onto T broth-0.27% agar plates containing 12.5 ␮g ml⫺1 chloramphenicol, 50 ␮g ml⫺1 ampicillin, and 1 mM arabinose.
Detection of proteins. Vibrio and E. coli cells were cultured at 30°C for 4 h in
VPG500 medium and for 4.5 h in T broth. The cells from 25-␮l portions ofcultures (optical density at 660 nm, 10) were suspended in sodium dodecyl sulfate(SDS) loading buffer containing 5% (vol/vol) ␤-mercaptoethanol and boiled at

TABLE 1. Strains and plasmids
Strain or plasmid

Genotype or descriptiona

Reference orsourceV. alginolyticus
strains
VIO5
NMB191

Laf⫺ Pof⫹ Rifr
VIO5 ⌬pomAB

23
32

E. coli DFB245

motA ⌬fliG

36

Kmr Plac lacZ␣
pSU41, 1.9-kb BamHI-SacI(pomAB⫹)
His6-pomA/pomB in pSU41
pBR322 derived, PBAD AmprpACYC184 derived, PBAD Cmr
fliGEV gene in pBAD33
pBAD24 derivative withinverted multiple cloning
sites
pomA potB in pYA25

3
14

Plasmids
pSU41
pYA303
pJN152
pBAD24
pBAD33
pTY402
pYA25
pYS3

H. Fukuoka
11
11
30
29
29

a
Kmr, kanamycin resistant; Rifr, rifampin resistant; Ampr, ampicillin resistant;Cmr, chloramphenicol resistant; PBAD, araBAD promoter; Plac, lac promoter.

100°C for 5 min. SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed as described previously. Antibodies against PomA
(PomA91 for samples prepared from Vibrio and PomA1312 for samples prepared from E. coli), PomB (PomB93), and MotB were used as previously described (32).
Coelution assay. Cells of Vibrio strain NMB191 (⌬pomAB) expressing a His6tagged PomA derivative were cultured in VC medium at 30°C for 4 h, harvested
by centrifugation, washed with V buffer (50 mM Tris-HCl [pH 7.5], 300 mM
NaCl, 5 mM MgCl2), resuspended in TMPD (20 mM Tris-HCl [pH 8.0], 5 mM
MgSO4, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol[DTT]) containing 5 ␮g ml⫺1 of DNase I, and then sonicated. After undisrupted
cells were removed by low-speed centrifugation (4,000 ⫻ g for 5 min at 4°C), themembrane fraction was recovered by ultracentrifugation (110,000 ⫻ g for 1 h at4°C). The pellet was homogenized with TPD (20 mM Tris-HCl [pH 8.0], 0.5 mM
PMSF, 1 mM DTT, 20% [wt/vol] glycerol). The total cell membrane (5 mg ml⫺1of protein) was suspended in TNPD (20 mM Tris-HCl[pH 8.0], 0.5 mM PMSF,
0.15 M NaCl, 1 mM DTT, 10% [wt/vol] glycerol) containing 5 mM imidazole and2.5% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS) for solubilization, incubated on ice for 10 min, and centrifuged at
150,000 ⫻ g for 30 min at 4°C. The supernatant was mixed with Ni-nitrilotriacetic
acid (Ni-NTA) agarose (Qiagen) and incubated for 1 h at 4°C with gentle mixing.
The Ni-NTA beads were washed three times with TNPD containing 5 mM
imidazole and 2.5% (wt/vol) CHAPS. TNPD containing 200 mM imidazole and2.5% (wt/vol) CHAPS was added to the beads to elute His-tagged PomA and
PomB. Eluates and other samples were analyzed by SDS-PAGE following immunoblotting as described above.

RESULTS
Mutagenesis of PomA. As previous studies showed, it islikely that electrostatic interactions of the conserved charged
residues between the rotor and stator (MotA or PomA and
FliG) contribute to the rotation of the motor (28, 36). However, the V. alginolyticus motor seemed to contain other important charged residues in addition to the conserved residues
because it was more resistant to mutations in the conserved
residues than the E. coli motor. We first aligned the aminoacids of the C-terminal domains of the PomA orthologues invarious bacteria to find specific charged residues that are conserved in most Vibrio species but not in E. coli MotA; thissearch resulted in identification of PomA K203, D209, E211,

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minus of FliG from E. coli and the C terminus from V. alginolyticus were joined to create the chimeric protein FliGEV. This
protein functioned as a proton-driven motor in E. coli (10, 30).
Furthermore, chimeric protein PotB, which combines the Nterminal domain of PomB containing the transmembrane segment and the C-terminal domain of MotB containing the peptidoglycan-binding motif, was constructed. When PomA and
PotB were coexpressed, they functioned not only in V. alginolyticus but also in E. coli as an Na⫹-driven motor (2). The
Na⫹-driven motor of Vibrio can be converted into an H⫹driven motor in E. coli without MotX and MotY, which areessential for the wild-type Vibrio wild-type sodium motor.
Using this chimeric motor system, a comparative study was
carried out with E. coli (29). Neutral or charge reversal mutations were introduced into conserved charged residues in thecytoplasmic domain of PomA and the C terminus of FliG fromV. alginolyticus. Patterns of synergism and suppression in rotor/
stator double mutants indicated that proteins in V. alginolyticus
appeared to engage in electrostatic interactions in essentiallythe same way that they do in E. coli. However, the motor in V.
alginolyticus was relatively more robust in response to the mutations than the motor in E. coli, which means that there may
be specific electrostatic interactions between the rotor and thestator for the Na⫹-driven motor.
In this study, we focused on the conserved charged residues
in the C terminus of PomA in Vibrio species, which has an
Na⫹-driven motor, that are not present in MotA of E. coli,
which has the proton-driven motor. We made eight charge
reversal mutants and measured their effects on swarming in V.
alginolyticus. Furthermore, the mutant proteins were coexpressed with PotB in E. coli cells as an Na⫹-type motor, andthe effects on swarming were investigated.

J. BACTERIOL.

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ROLES OF CHARGED RESIDUES IN PomA

3567

R215, D220, R232, and D235 (Fig. 1). To examine whether
these residues, which are not conserved in the E. coli H⫹utilizing motor, are involved in the function of the motor, they
were individually replaced by amino acids with opposite
charges, as follows: K203E, D209K, E211K, R215E, D220K,
R232E, and D235K. Additionally, PomA R207 was also replaced by an amino acid with the opposite charge to obtain
PomA(R207E). This charged residue is conserved in MotA
and might be a common important charged residue.
Mutant profiles in Vibrio cells. The mutant pomA gene wasintroduced along with pomB into the ⌬pomAB strain NMB191,
and each transformant was inoculated onto a semisolid agar
plate. We measured the diameter of the swarming ring at
various times. Figure 2A shows the relative expansion values
for the swarming rings of PomA variants compared with that of

the wild type. The PomA(D209K), PomA(E211K), PomA
(R232E), and PomA(D235K) mutants retained swarming ability comparable to that of wild-type PomA strain, but motility
was completely eliminated by a mutation in residue K203,
R215, or D220. The swarming ability of the R207E mutant was
reduced moderately. The swimming speed was ca. 5 ␮m s⫺1,while the fraction of the swimming cells was ca. 22%. The
swimming speed of the wild type was ca. 63 ␮m s⫺1.
To detect mutant PomA and PomB proteins, we carried out
an immunoblot analysis of whole-cell extracts. When the proteins were overexpressed from plasmids, we could detect
PomA and PomB bands for all of the mutants, and somedifferences in the amounts of proteins among the mutants wereobserved (Fig. 2B). Although PomA(D235K) could restore themotility to almost wild-type levels, the amount of PomA(D235K)

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FIG. 1. (A) C-terminal amino acid alignment of PomA, PomA homologues, and E. coli MotA. A black background indicates residues identical
to V. alginolyticus PomA residues, and a gray background indicates residues having the same charge as V. alginolyticus PomA residues. Abbreviations: VaPomA, V. alginolyticus PomA; VpMotA, Vibrio parahaemolyticus MotA; VvMotA, Vibrio vulnificus MotA; VcPomA, Vibrio cholerae
PomA; PpMotA, Photobacterium profundum MotA; RsMotA, R. sphaeroides MotA; BsMotP, Bacillus subtilis MotP; BmMotP, Bacillus megaterium
MotP; BpMotP, Bacillus pseudofirmus MotP; BhMotP, Bacillus halodurans MotP; OiMotP, Oceanobacillus iheyensis MotP; PaMotA, Pseudomonas
aeruginosa MotA; EcMotA, E. coli MotA. (B) Mutations mapped in a schematic diagram of PomA. Black and gray circles show the residues that
we individually replaced with residues with the opposite charge in this study; dominant mutations are indicated by black circles, and the othermutations are indicated by gray circles. R215E suppressor mutations are indicated by black squares. Temperature-sensitive mutations, as described
previously (9), are indicated by gray diamonds. The suppressor mutation of the temperature-sensitive mutant is indicated by a black diamond.

3568

OBARA ET AL.

J. BACTERIOL.

FIG. 2. (A) Relative colony expansion values for Vibrio PomA mutants. The relative colony expansion of NMB191 cells expressing no
PomA (vector), wild-type PomA (WT), and PomA mutants with PomB
was determined. Swarming assays were carried out at least three times,
and the diameter of the swarming ring was measured at each time. The
relative expansion value was calculated by normalizing the approximate swarm expansion rates of swarming rings of the mutants to thatof the wild type (NMB191/pYA303). Cells expressing PomA(K203E),
PomA(R215E), PomA(D220K), or PomA(R232E) did not form any
swarming rings, like the cells possessed only the vector. (B) Immunoblotting of Vibrio cells. The cells described above and wild-type V.
alginolyticus cells (VIO5) were harvested and suspended in distilledwater. Each suspension was subjected to SDS-PAGE and immunoblotting with anti-PomA antibody and anti-PomB antibody.

expressed from the plasmid was significantly less than theamounts of the other PomA mutants and the wild-type PomA.
Several bands with higher molecular masses than the PomA
band were observed, and they may represent dimer forms
and/or different conformational forms, as suggested previously(32). The intensities of these bands were reduced forPomA(K203E), PomA(R207E), and PomA(D220K). The chromosomal PomA expressed in VIO5 (pomAB⫹) cells could not be
detected. Therefore, the level of PomA in wild-type cells is quite
low. The amount of PomB produced in the presence ofPomA(K203E) and PomA(R207E) was significantly reduced
even though the amount of PomA was not reduced. The K203E
and R207E mutations may affect the interaction with PomB because PomB seems to be unstable in the absence of PomA (32).
To test whether the mutations affect the PomA-PomB inter-action, a His tag was fused to the N terminus of PomA(K203E),
PomA(R207E), PomA(R215E), and PomA(D220K), and we performed a coelution assay. These PomA proteins were coexpressedwith PomB in NMB191 cells. The amounts of PomA and PomB
detected were similar to the amounts detected with cells producing tagless PomA and PomB (data not shown). The membraneproteins were solubilized with CHAPS, bound to Ni-NTA agarose, and eluted by using imidazole. The eluted proteins weredetected by immunoblotting using anti-PomA and anti-PomB
antibodies (Fig. 3). Almost the same amount of PomB protein
coeluted with PomA(R215E) and PomA(D220K) as with wildtype PomA. However, an almost undetectable amount of PomB
coeluted with the PomA(K203E) protein. Only a small amount of
PomB protein was detected with PomA(K203E) with the wholecell extract (Fig. 2B and data not shown). The level of eluted
PomA(R207E) appeared to be the same as the level of wild-type
PomA, but the amount of coeluted PomB was significantly reduced.
Mutant profiles in E. coli cells carrying a chimeric motor.
Recently, a chimeric system has been established, which meansthat we can investigate the roles of PomA and FliG from V.
alginolyticus in E. coli cells (2, 10, 30). To investigate the effectsof PomA mutants on the motility of E. coli with the chimericmotor, the same PomA mutants that were examined in V.
alginolyticus were made. Each of the PomA mutants was expressed together with PotB from pYS3 in motors possessing
the chimeric rotor protein, FliGEV. Motors of the transformants contained both MotB expressed from the chromosome
and PotB expressed from the plasmid. The function of the B
subunits of the transformants was thought to be due to PotB
because PomA/MotB is not functional in E. coli (2).
The swarming ability was eliminated completely when any of
the following four mutated PomA proteins was expressed: PomA

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FIG. 3. Coelution analysis of PomB and PomA. The solubilized membrane extracts from NMB191 cells expressing PomA, PomA(K203E),
PomA(R207E), PomA(R215E), or PomA(R220K) with PomB were incubated with Ni-NTA agarose and eluted by using imidazole as describedin Materials and Methods. The eluted fraction of cells expressing
PomA(R207E) was diluted 1:2 with SDS loading buffer, and the eluted
fraction of cells expressing wild-type PomA was diluted 1:4 with SDS
loading buffer. PomA and PomB proteins were detected by immunoblotting using anti-PomA and anti-PomB antibodies. WT, wild type.

VOL. 190, 2008

(K203E), PomA(R215E), PomA(D220K), or PomA(R232E)(Fig. 4A). These results are consistent with those obtainedwith Vibrio except for the PomA(R232E) mutant results.
PomA(D209K) and PomA(D235K) mutants restored the motility to the wild-type levels. Interestingly, unlike the results
obtained with Vibrio, the PomA(R207E) and PomA(E211K)
mutant proteins restored the swarming ability slightly. Although the R232E mutant had no swarming ability, we observed some swimming cells (the fraction of swimming cells
was ca. 20%) by dark-field microscopy. We have recently foundthat the torque at low speed was reduced by the R232E mutation (12). The PomA(R207E) mutation reduced the motilityof both V. alginolyticus and E. coli. Without arabinose, whichacts as an inducer, all of the strains remained completely immotile (data not shown).
The immunoblot analysis of whole-cell extracts of E. coli was
carried out using anti-PomA and anti-MotB antibodies (Fig.
4B). All PomA proteins expressed from plasmids were present
in the same amount as the wild-type protein except for the
PomA(E211K) protein. The E211K mutant, which could be
detected as a faint single band and caused reduced swarming
ability, may be rapidly degraded or may be produced at lower
levels. It is worth noting that the levels of the bands with

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FIG. 5. (A) Dominance of PomA mutants. Wild-type V. alginolyticus cells (VIO5) expressing no motor protein (vector), PomA(K203E),
PomA(R215E), or PomA(D220K) cells with PomB, and ⌬pomAB cells
expressing wild-type PomA with PomB were incubated on semisolid agarat 30°C for 4.5 h. (B) Motile suppressor of the R215E mutant. Swarms of
NMB191 cells expressing wild-type PomA (WT), PomA(R215E), or the
PomA(R215K), PomA(R215K/T5P), PomA(R215E/Q54L), or
PomA(R215E/G139D) suppressor mutant are shown. The plasmids
were extracted from the revertant cells and reintroduced into NMB191
cells. The retransformants were incubated overnight in the liquid cultures and inoculated onto semisolid agar plates. Cells were grown at
30°C for 6 h.

molecular masses higher than that of PomA were significantly
reduced in the K203E and R207E mutants. PotB, which is achimeric protein composed of PomB and MotB, was detectedusing anti-MotB antibody. PotB proteins from all PomA mutants were detected at almost the same level as wild-type
PomA at a higher position than MotB expressed from thechromosome.
Dominance of pomA mutations in Vibrio. We investigatedthe dominant-negative effects of the PomA(K203E), PomA
(R207E), PomA(R215E), and PomA(D220K) variants. These
PomA mutants were introduced into VIO5 (pomAB⫹) cells,
and each transformant was inoculated onto a semisolid agar
plate. Motility was greatly reduced by PomA(R215E) andPomA(D220K) even with the wild-type motor, indicating thatthe proteins were integrated into the Vibrio motor and were
important residues for rotation (Fig. 5A).
Motile suppressors of the R215E mutant. To investigate
potential residues that have the ability to interact with either
R215 or D220, we obtained motile suppressor mutants by using
long incubation on semisolid agar plates. The nucleotide

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FIG. 4. (A) Relative colony expansion values for E. coli PomA
mutants. The relative colony expansion values for DFB245 cells expressing no PomA (vector), wild-type PomA (WT), and PomA mutants
with PotB and FliGEV were obtained by using the same method that wasused for Vibrio cells (Fig. 2A). Cells expressing PomA(K203E), PomA
(R215E), PomA(D220K), or PomA(R232E) did not form any swarming rings, like the cells possessing only the vector. (B) Immunoblottingof E. coli cells. The whole cells described above were harvested andsubjected to SDS-PAGE and immunoblotting with anti-PomA antibody and anti-MotB antibody.

ROLES OF CHARGED RESIDUES IN PomA

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OBARA ET AL.

changes in pomAB for all of the suppressor mutants were
determined by sequencing. Eleven motile suppressors wereobtained from NMB191 with PomA(R215E) but not from
NMB191 with PomA(D220K). In 8 of the 11 revertants, R215E
substitutions were changed to lysine, and in each case GAA
was changed to AAA (the wild-type codon is CGC). In one
revertant, the R215E substitution was changed to lysine, andthe T5 residue of PomA was changed to proline. In two of therevertants, a Q54L mutation in PomA (single isolate) and a
G139D mutation in PomA (two isolates) were found in theplasmids in addition to the original R215E mutation (Fig. 1B
and 5B).
DISCUSSION

residues, including Q54L and G139D in PomA. Furthermore,
we found a suppressor mutant in which R215 and T5 were
replaced by lysine and proline, respectively. The mutations, inaddition to the original R215 mutation, occurred near theboundaries of the cytoplasmic and transmembrane domains(Fig. 1B and 5B). In particular, the suppressor mutation
G139D is interesting because the mutation at one charged
residue was suppressed by another residue that was changed toa charged residue. We speculate that this mutation mightchange the structure around this residue and rescue the interaction between the C-terminal region of PomA and the region
around G139.
It has been suggested that the charged residues in MotA
(R90 and E98) and FliG (K264, R281, D288, D289, and R297)
might engage in electrostatic interactions with each other andthat a charge-neutralized mutant of MotA was nonfunctional(36). However, a charge-neutralized mutant of PomA withresidues corresponding to the critical charged residues of
MotA was functional. From the charge-neutralized mutant of
PomA, we could isolate mutants with a temperature-sensitive
phenotype whose mutations were mapped to the L131 or T132
residue. Furthermore, a suppressor mutant with the temperature-sensitive phenotype was isolated and mapped to the V142
residue, which is close to G139 (9). This implies that an interaction between the critical charged residues and the C-terminal charged residues occurs, although it is not necessarily adirect interaction.
The mutations in K203 and R207 seem to affect dimer formation in E. coli, as well as in V. alginolyticus. This region may
be involved in PomA-PomA interactions or may confer proper
conformation of the protein. When PomA(K203E) andPomA(R207E) were individually coexpressed with PomB, theamount of PomB protein was significantly reduced only in
Vibrio cells. It has been reported that in Vibrio PomB is very
unstable without PomA (32). This may indicate a difference inthe protein interactions in Vibrio and E. coli, or it may simply
indicate that PotB is more stable than PomB. A coelution assay
showed that the motility defect of R215E and D220K mutants
is not due to the defect in the interaction between PomA andPomB.
We think that the C-terminal region of PomA may be partially involved in protein-protein interactions to form the stator
complex, or it may be important for protein conformation
rather than for the PomA-FliG interaction to generate torque.
We identified important charged residues in the C-terminal
region but do not know if these residues contribute to electrostatic interactions between the rotor and the stator in V. alginolyticus. Clarification of the precise role of the C terminus of
PomA would help define the mechanism of the rotor-stator
interaction that generates torque.

ACKNOWLEDGMENTS
We thank Ikuro Kawagishi and Hajime Fukuoka for invaluable
discussions and also thank John Frederick Morton for reading themanuscript.
This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (to M.H.
and S.K.); from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (to T.Y.); and from the Japan Science andTechnology Agency (to M.H.).

Downloaded from jb.asm.org at NAGOYA UNIVERSITY on October 8, 2008

In E. coli, which has proton-driven motors, it has beenthought that electrostatic interactions between charged residues in the rotor protein FliG and charged residues in thestator protein MotA are important for rotation (36). A mutational study of PomA and FliG provided an indication that inE. coli the interactions between the rotor and the stator
seemed to be electrostatic (29), but other mutational studies
have shown that the homologous charged residues in PomA
and FliG are apparently not essential for rotation in Vibrio (30,33). The possibility that the V. alginolyticus motor might incorporate interactions similar to those in the E. coli motor wasconsidered, as was the possibility that the other charged residues not homologous to E. coli residues are important and thatspecific electrostatic interactions between the rotor and stator
proteins might occur in the V. alginolyticus motor.
In this study, eight charged residues (K203, R207, D209,
E211, R215, D220, R232, and D235) present in the C terminusof PomA and its homologues, but not in E. coli MotA (except
for R207), were replaced with residues with the opposite
charge to search for the important charged residues. E211K
and R232E substitutions did not affect the swarming and swimming ability of only Vibrio cells; on the other hand, D209K and
D235K substitutions did not affect either Vibrio or E. coli cells.
The results may also suggest that the different charged residues
contribute to the motor function in the E. coli and Vibrio
motors. The amount of the PomA(D235K) protein was significantly reduced when this protein was expressed in Vibrio cells,suggesting that the mutation affects the protein stability but notits function. We could not detect the PomA protein expressedfrom the wild-type cells (pomAB⫹) by the same method. The
stator complex seems to function at a level that cannot be
detected by available methods, as previously shown (16).
The results for V. alginolyticus and E. coli cells showed that
K203, R215, and D220 appear to be important residues formotor function, such as torque generation or assembly. The
R215E and D220K mutations showed dominant-negative effects in Vibrio cells, suggesting that mutant proteins are individually inserted into the motor but do not function properly
and cause a motility defect. Mutations in M219 and D220,
which are adjacent to each other, were identified as dominant
double mutations (M219I/D220N) by random mutagenesis of
pomA (16). The strong dominant effect of the D220 mutation
may indicate that this region is very important for motor function but not for assembly or protein interactions.
The R215E mutation was suppressed by mutations in other

J. BACTERIOL.

VOL. 190, 2008

ROLES OF CHARGED RESIDUES IN PomA
REFERENCES

generation in the flagellar motor of Escherichia coli: evidence of a direct rolefor FliG but not for FliM or FliN. J. Bacteriol. 178:223–231.
19. Macnab, R. M. 1999. The bacterial flagellum: reversible rotary propellor andtype III export apparatus. J. Bacteriol. 181:7149–7153.
20. McCarter, L. L. 1994. MotX, the channel component of the sodium-typeflagellar motor. J. Bacteriol. 176:5988–5998.
21. McCarter, L. L. 1994. MotY, a component of the sodium-type flagellar
motor. J. Bacteriol. 176:4219–4225.
22. Okabe, M., T. Yakushi, Y. Asai, and M. Homma. 2001. Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor
gene. J. Biochem. (Tokyo) 130:879–884.
23. Okunishi, I., I. Kawagishi, and M. Homma. 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.
24. 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.
25. Sato, K., and M. Homma. 2000. Multimeric structure of PomA, the Na⫹driven polar flagellar motor component of Vibrio alginolyticus. J. Biol. Chem.
275:20223–20228.
26. Sockett, H., S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab.
1992. Molecular analysis of the flagellar switch protein FliM of Salmonella
typhimurium. J. Bacteriol. 174:793–806.
27. Stolz, B., and H. C. Berg. 1991. Evidence for interactions between MotA and
MotB, torque-generating elements of the flagellar motor of Escherichia coli.
J. Bacteriol. 173:7033–7037.
28. Welch, M., K. Oosawa, S. I. Aizawa, and M. Eisenbach. 1993. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc. Natl. Acad. Sci. USA 90:8787–8791.
29. Yakushi, T., J. Yang, H. Fukuoka, M. Homma, and D. F. Blair. 2006. Rolesof charged residues of rotor and stator in flagellar rotation: comparative
study using H⫹-driven and Na⫹-driven motors in Escherichia coli. J. Bacteriol. 188:1466–1472.
30. Yorimitsu, T., A. Mimaki, T. Yakushi, and M. Homma. 2003. The conserved
charged residues of the C-terminal region of FliG, a rotor component of theNa⫹-driven flagellar motor. J. Mol. Biol. 334:567–583.
31. Yorimitsu, T., K. Sato, Y. Asai, and M. Homma. 2000. Intermolecular crosslinking between the periplasmic loop3-4 regions of PomA, a component of
the Na⫹-driven flagellar motor of Vibrio alginolyticus. J. Biol. Chem. 275:
31387–31391.
32. Yorimitsu, T., K. Sato, Y. Asai, I. Kawagishi, and M. Homma. 1999. Functional interaction between PomA and PomB, the Na⫹-driven flagellar motor
components of Vibrio alginolyticus. J. Bacteriol. 181:5103–5106.
33. Yorimitsu, T., Y. Sowa, A. Ishijima, T. Yakushi, and M. 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.
34. 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.
35. Zhou, J., R. T. Fazzio, and D. F. Blair. 1995. Membrane topology of the
MotA protein of Escherichia coli. J. Mol. Biol. 251:237–242.
36. 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.

Downloaded from jb.asm.org at NAGOYA UNIVERSITY on October 8, 2008

1. Asai, Y., R. E. Sockett, I. Kawagishi, and M. Homma. 2000. Coupling ion
specificity of chimeras between H⫹- and Na⫹-driven motor proteins, MotB
and PomB, in Vibrio polar flagella. EMBO J. 19:3639–3648.
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. Bartolome, B., Y. Jubete, E. Martinez, and F. de la Cruz. 1991. Construction
and properties of a family of pACY184-derived cloning vectors compatible
with pBR322 and its derivatives. Gene 102:75–78.
4. Berg, H. C. 2003. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72:19–54.
5. Blair, D. F. 2003. Flagellar movement driven by proton translocation. FEBS
Lett. 545:86–95.
6. Braun, T. F., and D. F. Blair. 2001. Targeted disulfide cross-linking of the
MotB protein of Escherichia coli: evidence for two H⫹ channels in the stator
complex. Biochemistry 40:13051–13059.
7. Chun, S. Y., and J. S. Parkinson. 1988. Bacterial motility: membrane topology of the Escherichia coli MotB protein. Science 239:276–278.
8. De Mot, R., and J. Vanderleyden. 1994. The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a
common function in both gram-positive and gram-negative bacteria, possiblyin the interaction of these domains with peptidoglycan. Mol. Microbiol.
12:333–334.
9. Fukuoka, H., T. Yakushi, and M. Homma. 2004. Concerted effects of aminoacid substitutions in conserved charged residues and other residues in thecytoplasmic domain of PomA, a stator component of Na⫹-driven flagella. J.
Bacteriol. 186:6749–6758.
10. 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.
11. Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing thearabinose PBAD promoter. J. Bacteriol. 177:4121–4130.
12. Inoue, Y., C. J. Lo, H. Fukuoka, H. Takahashi, Y. Sowa, T. Pilizota, G. H.
Wadhams, M. Homma, R. M. Berry, and A. Ishijima. 2008. Torque-speed
relationships of Na⫹-driven chimeric flagellar motors in Escherichia coli. J.
Mol. Biol. 376:1251–1259.
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., Y. Asai, T. Atsumi, I. Kawagishi, and M. Homma. 1999. Na⫹driven flagellar motor resistant to phenamil, an amiloride analog, caused by
a mutation of putative channel component. J. Mol. Biol. 285:1537–1547.
15. Kojima, S., and D. F. Blair. 2004. Solubilization and purification of the
MotA/MotB complex of Escherichia coli. Biochemistry 43:26–34.
16. Kojima, S., M. Kuroda, I. Kawagishi, and M. Homma. 1999. Random mutagenesis of the pomA gene encoding a putative channel component of theNa⫹-driven polar flagellar motor of Vibrio alginolyticus. Microbiology 145:
1759–1767.
17. 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.
18. Lloyd, S. A., H. Tang, X. Wang, S. Billings, and D. F. Blair. 1996. Torque3571