JOURNAL OF BACTERIOLOGY, Oct. 2004, p. 6749–6758
0021-9193/04/$08.00⫹0 DOI: 10.1128/JB.186.20.6749–6758.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 186, No. 20

Concerted Effects of Amino Acid Substitutions in Conserved Charged
Residues and Other Residues in the Cytoplasmic Domain of
PomA, a Stator Component of Na⫹-Driven Flagella
Hajime Fukuoka, Toshiharu Yakushi, and Michio Homma*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan
Received 13 January 2004/Accepted 16 July 2004

PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motorin Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, arebelieved to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shownthat a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q,
and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replacedwith neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect thefunction of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M
substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility
phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype.
The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42°C and wasrestored to the original level approximately 10 min after the temperature was returned to 20°C. It is believedthat PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seemto be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic
boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA ischanged by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal
region is temperature sensitive.
determined at atomic resolution (14, 28). The structures
showed that the functionally important charged residues are
clustered along the ridges toward the outside of the FliG domain. On the other hand, the crystal structures of the stator
components are not yet available. The membrane topologies
have only been modeled as shown in Fig. 1. A model of thestator was generated based on the assumption that the forcegenerating unit is a 1:1 complex of MotA and MotB and thefive transmembrane helices comprise an ion channel. The
model was revised based on recent results showing the stoichiometry of the force-generating unit as a 4:2 complex of MotA
and MotB (11, 13, 24) and other previous results (39, 40, 49).
In the sodium-driven motor of Vibrio species, such as Vibrio
alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus, PomA,
PomB, MotX, and MotY have been identified as essential
components of the stator (2, 17, 33, 48). PomA and PomB,
which are homologs of E. coli MotA and MotB, are thought toform a complex as a sodium ion channel (39, 40, 51). MotX and
MotY, which are localized in the outer membrane (35), haveno homologous components in the proton-driven motor (31,32, 34, 36), and their function is currently unclear.
It is likely that the motors of the proton-driven and sodiumdriven flagella work essentially by the same mechanism. MotA
of Rhodobacter sphaeroides, whose motor is proton driven (38),can replace PomA and function with PomB, MotX, and MotY
in V. alginolyticus as the sodium motor component (1). MomB
is a chimeric protein between the N terminus of MotB from R.
sphaeroides and the C terminus of PomB. It functions as aNa⫹-driven motor component with MotX and MotY in V.
alginolyticus (3). PotB is a chimeric protein that has the transmembrane domain of the N terminus of PomB and theBacteria can swim by rotating helical flagellar filaments likea screw. The molecular motor is embedded in the membrane at
the base of the flagellar filament. It can rotate by using theelectrochemical potential of specific ions across the cytoplasmic membrane. Bacterial flagella are categorized based on the
coupling ion. The flagellar motor of Escherichia coli is a proton-driven motor (30), and the polar flagella of Vibrio species
are sodium driven (6, 18, 20, 26).
Based on studies of E. coli and Salmonella enterica serovar
Typhimurium, approximately 50 genes are known to be required for flagella assembly and function (9, 29). In the protondriven flagellar motor of E. coli, MotA, MotB, and FliG arecomponents required for flagellar rotation (11). MotA has fourtransmembrane segments and a relatively large cytoplasmic
domain between the second and third transmembrane segments (54). MotB has a single transmembrane segment and a
peptidoglycan-binding motif in the C-terminal domain (15, 16).
MotA and MotB form a complex and function as proton channels (12, 42). This complex is proposed to be the stator of theflagellar motor and surrounds the rotor structure (21). Genetic
studies demonstrated that FliG forms a switch complex with
FliN and FliM (47). FliG binds to both the C-ring proteins(FliM and FliN) and the MS-ring protein (FliF) (37, 44). However, the precise location of FliG is not known (43).
Partial structures of FliG from the thermophile Thermotoga
maritima, including the middle and C-terminal domains, were
* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan. Phone: 81-52-789-2991. Fax: 81-52-789-3001.
E-mail: g44416a@cc.nagoya-u.ac.jp.
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FIG. 1. (A) Alignments of the PomA cytoplasmic loop with homologous proteins of various species. VaPomA, V. alginolyticus PomA; Rs MotA,
R. sphaeroides MotA; BsProA, B. subtilis hypothetical protein A; BsMotA, B. subtilis MotA; VpLafT, V. parahaemolyticus LafT; EcMotA, E. coli
MotA; StMotA, S. enterica serovar Typhimurium MotA. The residues are shown by white letters in black boxes when more than four identicalresidues are present at the aligned position. Black arrows indicate the two conserved charged residues. White arrows indicate the charged residues
which were changed to neutral residues in this study. Black circles indicate the suppressor mutations of the TS PomA mutant. The paired black
triangles and black or white inverted triangles show the sites of the previously isolated double mutations. (B) Mutations mapped in a schematic
diagram of motor. E, substitutions of AAQQQ (R88A, K89A, E96Q, E97Q, and E99Q); F, nonmotile mutations; ‚, substitutions required for the
TS phenotype in addition to the AAQQQ substitutions; 䊐, suppressor mutations of the TS mutant.

periplasmic domain of the C terminus of E. coli MotB. Surprisingly, PotB functions with PomA as a sodium motor component not only in V. alginolyticus but also in E. coli (4). MotA
and MotB of E. coli can function in Vibrio cells as the proton
motor components without MotX and/or MotY. These results
indicate that the Vibrio sodium motor is converted into a proton motor and also that the E. coli proton motor can bechanged into a sodium motor (4, 17).
Site-directed mutagenesis of the proton-driven motor of E.
coli suggested that the charged residues R90 and E98 in thecytoplasmic domain of MotA have electrostatic interactions
with five charged residues in the FliG C-terminal domain:
K264, R281, D288, D289, and R297. These interactions arethought to be critical for flagellar rotation (27, 53, 55). The twocharged residues in MotA are conserved in the cytoplasmic
domain of PomA (R88 and E96). Several charged residues(K89, E97, and E99) are located near R88 and E96 of PomA.
It was recently reported that a PomA mutant with neutral
substitutions at all of these charged residues (R88A, K89A,
E96Q, E97Q, E99Q; AAQQQ) is functional (52). The fiveVOL. 186, 2004

CONSERVED CHARGED RESIDUES OF PomATABLE 1. Bacterial strains and plasmids
Descriptiona

Strain or plasmid

V. alginolyticus strain
VIO5
NMB190NMB191
E. coli strain
JM109
Plasmid
pSU41
pYA301
pYA303aRifr, Pof⫹, Laf⫺
VIO5 ⌬pomA (211-bp deletion)
VIO5 ⌬pomAB

Reference

36
1
51
50

r

kan (Km ), Plac lacZ␣
pSU41 0.8 kb BamHI-BamHI (pomA⫹)
pSU41 1.9 kb BamHI-SacI (pomAB⫹)

7
22
22

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

charged residues of E. coli FliG, K264, R281, D288, D289, andR297, are conserved in V. alginolyticus FliG (K284, R301,
D308, D309, and R317, respectively). We have introduced
single or multiple substitutions for the charged residues. Most
proteins with substitutions at these five conserved positions in
FliG still conferred significant motility. All of the triple-substitution proteins constructed in that study (which correspond to
those that resulted in the loss of motor function in E. coli) also
allowed motility (50). These lines of evidence suggest that
other charged residues are required for flagellar rotation in thesodium-driven motor.
In the present study, we first tried to identify other important charged residues of the cytoplasmic domain in PomA.
Next, we examined the cumulative effects of the conserved
charged residues and other mutations in the cytoplasmic domain. We found that the combination of AAQQQ and L131F
or T132M substitutions (AAQQQ⫹L131F and AAQQQ⫹
T132M PomA mutants) conferred a temperature-sensitive
phenotype (TS phenotype) for motility. As far as we know, thisis the first study to report TS mutations in any of the mot genes.
Our results suggest that the cytoplasmic interface of PomA andthe cytoplasmic boundaries of the second and third transmembrane segments of PomA might act synergistically in force
generation.

6751

and recorded on videotape. Swimming speed was determined as previously
described (5). The average swimming speed was obtained by measuring at least
20 swimming tracks from the two independent experiments.
Detection of PomA proteins in Vibrio cells. Vibrio cells that harbored vector
only or plasmids carrying wild-type or mutant pomA genes were cultured in
VPG500 medium at various temperatures. The cells were harvested at an opticaldensity at 660 nm of 1.0 and suspended in distilled water. The same volume ofsodium dodecyl sulfate (SDS) loading buffer was added to cell suspensions,which were boiled at 100°C for 5 min. These samples were separated by SDSpolyacrylamide gel (12%) electrophoresis (PAGE), and immunoblotting was
performed using anti-PomA antibody (PomA91).
Detection of PomA-PomB interaction. Vibrio cells that harbored plasmids
carrying His-tagged wild-type or mutant pomA genes were cultured in VC medium at 30°C for 3 h. The cells were harvested by centrifugation, washed with V
buffer (25 mM Tris-Cl [pH 7.5], 300 mM NaCl, 10 mM MgCl2), and resuspended
in 20TMPD (20 mM Tris-Cl [pH 8.0], 5 mM MgSO4, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT]) containing 5 ␮g of DNase
I/ml. The cell suspension was passed through a French press twice at 500 kg/cm2.
After unbroken cells were removed by low-speed centrifugation (10,000 ⫻ g for
5 min at 4°C), the membrane was recovered by ultracentrifugation (150,000 ⫻ g
for 1 h at 4°C). The membrane pellet was homogenized with 20TPD (20 mM
Tris-Cl [pH 8.0], 0.5 mM PMSF, 1 mM DTT) containing 20% (wt/vol) glyceroland stored at ⫺80°C. Total cell membrane (10 mg/ml of protein) was solubilized
in 20TNPD (20 mM Tris-Cl [pH 8.0], 0.15 M NaCl, 0.5 mM PMSF, 1 mM DTT)
containing 10% (wt/vol) glycerol, 5 mM imidazole, and 2.5% (wt/vol) CHAPS{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} and incubatedfor 10 min on ice. Solubilized samples were centrifuged at 150,000 ⫻ g for 30 min
at 4°C. Ni-nitrilotriacetic acid (Ni-NTA) agarose was added to the solubilized
fraction, and the sample solution was incubated for 1 h at 4°C. The supernatant
was removed by centrifugation, and the Ni-NTA pellet was washed three times
with 20TNPD containing 10% (wt/vol) glycerol, 5 mM imidazole, and 1% (wt/vol) CHAPS at 20°C or 42°C. PomB and His-tagged PomA proteins were elutedby 20TNPD that contained 10% (wt/vol) glycerol, 200 mM imidazole, and 1%(wt/vol) CHAPS at 20°C or 42°C. The same volume of SDS loading buffer wasadded to the eluted solution, and the samples were boiled at 100°C for 5 min.
These samples were separated by SDS-PAGE as described above. PomA and
PomB proteins were detected by immunoblotting with the anti-PomA (PomA91)
and anti-PomB (PomB93) antibodies.
Isolation of motile suppressors from the TS mutant. Single colonies of the TS
PomA mutant were inoculated on VPG500 swarm plates by toothpick andincubated at 37°C for 2 days. The revertant cells, which recovered swarming
ability, were picked from the edge of the swarm ring, and single colonies wereisolated. After the swarming ability of the single colonies was checked, plasmids
were extracted from the revertant cells. The plasmids were reintroduced into
NMB190, the pomA mutant, to confirm the mutations.

MATERIALS AND METHODSRESULTS

Bacterial strains, plasmids, growth conditions, and media. The strains andplasmids used are listed in Table 1. 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 VPG500 medium (1%[wt/vol] polypeptone, 0.4% [wt/vol] K2HPO4, 500 mM NaCl, 0.5% [wt/vol] glycerol). E. coli was cultured in Luria-Bertani broth (1% [wt/vol] tryptone peptone,
0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl). Kanamycin was added to a final
concentration of 100 ␮g/ml for V. alginolyticus and 50 ␮g/ml for E. coli. For theswarming assay, overnight cultures were spotted on 0.25% agar–VPG500 plates
that contained 100 ␮g of kanamycin/ml.
Site-directed mutagenesis. We used a PCR method as previously described(22) to introduce the substitutions into PomA. A pair of sense and antisense
primers that contained mismatches at the mutation site was synthesized, and thefull-length plasmid that contained the pomA gene was amplified by using theprimers. The presence of the mutations was confirmed by DNA sequencing.
Measurement of swimming speed. Overnight cultures in VC medium were
inoculated into VPG500 medium at 100-fold dilution and cultivated for 3 h at
30°C. Cells were harvested by centrifugation and suspended in TMN500 buffer(50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM glucose, 500 mM NaCl). The cellsuspensions were incubated for 10 min at the temperature setting for measurement. Suspensions were diluted 100-fold into the same buffer containing 20 mM
serine, which was used to suppress the directional change of swimming, andincubated at the same temperature. Cell motility was observed with a dark-fieldmicroscope on a microwarm plate, which was used to control the temperature,

Charged residues of V. alginolyticus PomA. Based on analignment with E. coli MotA, two charged residues (R90 andE98 of MotA) are well-conserved not only in V. alginolyticus
PomA but also in other bacterial species (Fig. 1). In the cytoplasmic domain between the second and third transmembranesegments in V. alginolyticus PomA, there are 14 positively
charged residues and 17 negatively charged residues. Some ofthese residues were conserved in E. coli MotA, as well as inproteins from other species (Fig. 1A). To identify the charged
residues that are required for flagellar rotation in the cytoplasmic domain of PomA, K60, D75, D128, D133, or E134 wereindividually substituted with neutral amino acid residues as thesixth mutation (K60A, D75N, D128N, D133N, and E134Q) in
the AAQQQ PomA protein. These PomA variants were expressed from plasmids in ⌬pomA strain NMB190. Based on theswarming assay, motility was not affected by the additional
substitutions tested in this study. The swimming speeds ofthese mutants were as fast as those of the wild type or the
AAQQQ mutant (data not shown).

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FIG. 2. Nonfunctional PomA variants. (A) Swarms of NMB190
cells that contain vector, wild-type PomA (WT), or single or doubleamino acid-substituted PomA. The cells were grown at 30°C for 5.5 h.
(B) Immunoblot of PomA proteins in whole-cell extracts. NMB190
cells harboring the vector only, plasmid containing wild-type PomA, ormutant PomA were harvested and suspended in distilled water. Thissuspension was subjected to SDS-PAGE and detected with anti-PomA
antibody.

Mutations in the cytoplasmic domain of PomA. We could
not find functionally critical residues for flagellar rotation in
PomA in a homology search with E. coli MotA or other homologous proteins. To get clues for studying the cytoplasmic
domain of PomA, we explored the mutations in this region. It
was previously reported that double substitutions in the cytoplasmic region, A87V and H136Y, L131F and T132M, and
R135Q and T169I (transmembrane segment 3), abolished the
motility of V. alginolyticus (25). Each substitution was introduced individually into wild-type PomA to determine which
substitution(s) affected flagellar rotation. Vibrio cells expressing each of the mutant proteins, PomA with an A87V substitution(PomA-A87V), PomA-L131F, or PomA-T132M, swarmed aswell as cells with wild-type PomA (Fig. 2A and 3A). On the other
hand, Vibrio cells expressing PomA-R135Q or PomA-H136Y did
not swarm at all (Fig. 2A). In addition, we confirmed under the
microscope that strains carrying these two PomA variants wereunable to swim (data not shown). These PomA variants weredetected at levels similar to that of wild-type PomA, and the
PomA bands were doublets as previously published (52) (Fig.
2B). Furthermore, PomA-H136A or PomA-H136D conferred
the ability to swim, but PomA-R135A, PomA-R135K, PomA-J. BACTERIOL.

R135D, and PomA-R135E did not (data not shown). These results suggest that the charged residue R135, which is not conserved in E. coli, is critical for the motor rotation of V. alginolyticus(Fig. 1).
Mutations in the motor protein causing temperature sensitivity. As mentioned above, cells with L131F and T132M substitutions in PomA have swarming abilities similar to those of cellswith wild-type PomA. When we introduced these substitutions
into AAQQQ PomA as the sixth substitution (AAQQQ⫹L131F
and AAQQQ⫹T132M), the additional change significantly reduced swarming ability at 30°C (Fig. 3A). The swarming abilitiesof strains carrying the AAQQQ⫹L131F and AAQQQ⫹T132M
PomA proteins were examined at various temperatures. We detected a clear difference in swarming abilities at 20°C and 37°C.
The mutant proteins did not allow swarming at 37°C, but thestrain with the wild-type PomA swarmed well at this temperature.
At 20°C, the strains with mutant and wild-type PomA proteinsdemonstrated similar swarming abilities (Fig. 3B). The swarming
abilities of strains with a single PomA substitution (L131F or
T132M) were similar to those of strains with wild-type PomA at
37°C (data not shown). When cells were grown at 20°C and 37°C,
there was almost no difference in the amount of mutant or wildtype PomA protein (Fig. 3C).
Next, we measured the swimming speeds at various temperatures to investigate the motor function of AAQQQ⫹L131F
PomA. The swimming speed of the wild-type strain was 87
␮m/s at 42°C (Table 2). AAQQQ⫹L131F PomA did not confer swimming at this temperature. At 30°C, cells with
AAQQQ⫹L131F PomA could swim; however, the swimming
speed (34 ␮m/s) was slower than that of the wild type (62 ␮m/s).
At 20°C, the wild type and the mutant carrying AAQQQ⫹L131F
PomA swam at similar speeds (Table 2). These results indicatethat the motor function of this form of PomA is comparable tothat of wild-type PomA at 20°C.
When PomB was coexpressed with AAQQQ⫹L131F PomA
from the same plasmid in the ⌬pomAB strain (NMB191), thefraction of swimming cells was higher than that in the strain
expressing AAQQQ⫹L131F PomA alone at various temperatures (Fig. 4A). In the strain that expressed TS PomA alone,
20% of the cells could swim at 30°C. On the other hand, at
30°C the fraction of swimming cells was similar to that of thewild-type in the strain coexpressing PomB and TS PomA. The
TS phenotype was alleviated by coexpression of PomB; however, cells were completely nonmotile at 42°C. This resultshows that the TS phenotype is alleviated by an increase in theamount of PomB.
We investigated the dominant effect of TS PomA against
wild-type PomA. Strain VIO5 has wild-type polar flagella.
VIO5 cells expressing the TS PomA protein swarmed as well ascells expressing wild-type PomA at 20°C. On the other hand, atthe restrictive temperature (42°C), VIO5 cells expressing TS
PomA had reduced swarming ability (Fig. 4B).
Recovery of swimming ability of TS PomA mutant cells. To
investigate whether or not the TS phenotype was reversible,
the recovery of swimming was measured at low temperature
after treatment at high temperature. In these experiments,
chloramphenicol (25 ␮g/ml) was added to the buffer to preventde novo synthesis of proteins. This concentration corresponds
to 20 times the MIC of chloramphenicol for V. alginolyticus(data not shown). Approximately the same fractions of cells

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FIG. 3. Temperature-sensitive mutants of the motor protein. (A) Swarms of NMB190 cells expressing wild-type (WT) PomA or PomA-L131F,
PomA-T132M, AAQQQ PomA, AAQQQ⫹L131F PomA, or AAQQQ⫹T132M PomA. The cells were grown at 30°C for 5.5 h. (B) Swarms of
NMB190 cells expressing AAQQQ⫹L131F and AAQQQ⫹T132M PomA. The cells were grown at 37°C for 5.5 h or at 20°C for 10 h.
(C) Immunoblot of PomA proteins in whole-cell extracts. The cells were grown to an optical density at 660 nm of 1.0 at 20°C or 37°C in VPG500
medium. NMB190 cells harboring vector only or the plasmid containing wild-type PomA or cells containing mutant PomA were harvested andsuspended in distilled water. This suspension was subjected to SDS-PAGE, and PomA was detected with anti-PomA antibody.

expressing wild-type PomA were capable of swimming at 20
and 42°C. On the other hand, the swimming fraction of cells
expressing AAQQQ⫹L131F PomA immediately dropped tozero when the temperature was increased from 20 to 42°C.
When the temperature was returned to 20°C, the fraction ofswimming cells gradually increased and was restored to its
original value in approximately 10 min (Fig. 5).
Interaction of TS PomA with PomB. It has been shown that
PomA interacts with PomB to form a complex (39, 40, 46, 49,
51). We speculated that the mutations causing temperature

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TABLE 2. Swimming speeds of cells expressing TS PomA
Temp (°C)

42
30
20
a

Swimming speed (␮m/s) ⫾ SD of
NMB190 expressing:WT PomA

AAQQQ ⫹ L131F PomA

87 ⫾ 1762 ⫾ 1046 ⫾ 9NAa
34 ⫾ 942 ⫾ 9NA, not applicable because motile cells were not observed.

sensitivity might affect the PomA-PomB interaction at hightemperatures. A His tag was fused to the N terminus of TS
PomA, and the fusion protein was expressed in NMB190. This
His-tagged TS PomA protein conferred the same TS pheno-

type as the original TS PomA. After the membrane fraction
was isolated and solubilized with CHAPS, which can solubilize
the membrane with minimal dissociation of the PomA/PomB
complex (T. Atsumi, personal communication), the His-tagged
TS PomA proteins were bound to Ni-NTA agarose. Afterelution, the His-tagged TS PomA and PomB were detected byimmunoblotting. We detected similar amounts of TS PomA
and wild-type PomA at each elution temperature. In addition,
no differences were detected in the amount of coeluted PomBwith TS PomA or wild-type PomA (Fig. 6). Based on thisresult, it appears that temperature does not affect the interaction of TS PomA with PomB.
Suppressors of the TS PomA mutation. Motile suppressorsof TS PomA were obtained by long incubation (1 to 2 days) on

FIG. 4. (A) Swimming ability of cells expressing TS PomA. The fractions of swimming cells expressing AAQQQ⫹L131F PomA alone in NMB190(squares) or with PomB in NMB191 (circles) from the plasmid are shown. (B) Effect on swarm formation of the TS PomA mutation in VIO5 cells. Shownare swarms of VIO5 cells that harbor the vector only, a plasmid encoding wild-type PomA, and plasmids encoding AAQQQ⫹L131F or
AAQQQ⫹T132M PomA. The cells were grown at 20°C for 10 h or 42°C for 2.5 h. vector*, NMB190 cells harboring vector (nonmotile control).

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FIG. 6. Interaction of TS PomA with PomB. The His-tagged
PomA/PomB complex was isolated as described in Materials andMethods. PomA and PomB proteins were detected with anti-PomA(A) and anti-PomB (B) antibodies. M, total membrane; S, solubilized
fraction; T, flowthrough; W, washed fraction; E, eluted fraction.

FIG. 5. Recovery of swimming ability of the TS PomA mutants.
The fractions of swimming NMB190 cells expressing AAQQQ⫹L131F
PomA (circles) and wild-type PomA (squares) are shown. The cellsuspension was prepared in a manner similar to that for the measurement of swimming speed, except that it contained 25 ␮g of chloramphenicol/ml in TMN500 buffer. This cell suspension was incubated at
20°C for 10 min, then placed at 42°C, and returned to 20°C. The
number of swimming cells was counted at the incubation temperature
on a microwarm plate. The value was the average of results from two
independent experiments.

swarm plates at 37°C. Eleven motile suppressors were obtained, including five revertants and six pseudorevertants. In all
five revertants, the L131F substitution was returned to leucine.
Four of the five revertants had the original codon (CTT), andin one revertant, TTT was changed to TTG. In the six pseudorevertants, the following additional substitutions were foundin four positions: A62V (two isolates), A70V (single isolate),
V142I (two isolates), and A150T (single isolate) (Fig. 7). The
newly substituted residues were larger than the original residues and were clustered around the boundaries of the cytoplasmic domain and the second or third transmembrane segment (Fig. 1).
Minimum requirements for amino acid substitutions conferring the temperature-sensitive phenotype. To identify which of
the five substitutions in AAQQQ PomA affects the TS phenotype, three PomA variants, PomA-R88A⫹L131F, PomA-E96Q⫹
L131F, and PomA-R88A/E96Q⫹L131F (PomA with the triple
substitution of R88A and E96Q [the conserved charged residues]with L131F), were constructed (Fig. 8). PomA-R88A⫹L131F and
PomA-E96Q⫹L131F conferred motilities at 20°C and 30°C similar to that of wild-type PomA. However, the swarming ability ofcells expressing these two PomA variants was remarkably reduced
at temperatures over 37°C. PomA-R88A/E96Q⫹L131F and wildtype PomA conferred similar motilities at 20°C, but the swarming

ability of the mutant cells was reduced at 30°C. AAQQQ⫹L131F
PomA and PomA-R88A/E96Q⫹L131F did not confer the ability
to swarm at temperatures over 37°C. When each of the AAQQQ
substitutions in TS PomA (AAQQQ⫹L131F PomA) was returned to the original charged residue, only those proteins with
R88 or E96 resulted in a strongly reduced TS phenotype, but theother substitutions had no effect (data not shown). These results
may suggest that R88A and E96Q are important mainly for the
TS phenotype with L131F and that the other substitutions may
also have a slight effect on the TS phenotype. Without the L131F
substitution, the R88A and/or E96Q substitutions did not affectswarming ability at any temperature. It is worth noting that the

FIG. 7. Motile suppressors of the TS PomA mutants. Shown are
swarms of NMB190 cells expressing no PomA (vector), wild-type
PomA (WT), AAQQQ⫹L131F PomA, or the suppressor mutants (TSPomA-V142I, TS PomA-A150T, TS PomA-A70V, or TS PomAA62V). The plasmids were extracted from the revertant cells andreintroduced into the pomA mutant (NMB190), and single colonies
were inoculated into a semisolid agar plate. The cells were grown at
37°C for 2.5 h.

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FIG. 8. Minimum amino acid substitutions required to confer the
TS phenotype. NMB190 cells expressing no PomA (vector), wild-type
PomA (WT), and the PomA mutants (PomA-R88A, PomA-E96Q,
PomA-R88A/E96Q, AAQQQ PomA, PomA-R88A⫹L131F, PomAE96Q⫹L131F, PomA-R88A/E96Q⫹L131F, or AAQQQ⫹L131F
PomA) were grown at 20°C (for 10 h), 30°C (for 5 h), 37°C (for 3.5 h),and 42°C (for 3.5 h).

swarming ability of the strain expressing the AAQQQ PomA
mutant was slightly reduced at 37 and 42°C.
DISCUSSION
In bacterial flagellar movement, the stator and rotor cooperate to generate torque from the ion influx, which results inmotor rotation. In the proton-driven flagellar motor of E. coli,
the charged residues R90 and E98 in the cytoplasmic domainof the stator protein MotA are critical for flagellar rotation.
However, these conserved charged residues in PomA are apparently not essential for flagellar rotation in Vibrio (52). In thepresent study, the secondary conserved charged residues (Fig.
1A) were replaced with neutral residues in the AAQQQ PomA
mutant to identify additional charged residues in the cytoplasmic domain of PomA that may be required for motor rotation.
However, all of the sextet mutants were functional. The substitution of D128 in PomA, which corresponds to E150 of

J. BACTERIOL.

MotA (a moderately critical residue in E. coli) (11, 53), did notaffect function. This result may suggest that the conserved
charged residues in PomA are a subset of residues essential for
sodium-driven rotation. Additional residues other than theconserved or the secondary conserved charged residues may be
involved in electrostatic interactions with FliG. Alternatively,common residues required for specific electrostatic interactions may not be present in the stator components of theH⫹-driven and Na⫹-driven motors. The R135 residue may bea candidate for an important charged residue required for therotation of the Na⫹-driven motor of V. alginolyticus. This Arg
residue is not conserved in E. coli and S. enterica serovar
Ttyphimurium; however, it is conserved in Vibrio species, R.
sphaeroides, and Bacillus subtilis (Fig. 1).
We herein reported that the AAQQQ⫹L131F andAAQQQ⫹T132M PomA proteins exhibited a temperaturesensitive phenotype for motor function (Fig. 3). The strain
expressing the AAQQQ⫹L131F PomA protein was nonmotile
at temperatures over 37°C, and the swimming ability of thisstrain was restored at 20°C even though protein synthesis wasinhibited (Fig. 5). Thus, the de novo synthesis of PomA is not
required for restoration of motility. The same levels of PomB
were associated with TS PomA and wild-type PomA underboth permissive and restrictive temperatures (Fig. 6). This
result suggests that PomB and TS PomA can interact with eachother to maintain the proper complexes even at restrictive
temperatures. The TS phenotype caused by the AAQQQ⫹
L131F mutations was suppressed by mutations that were clustered at the border of the cytoplasmic domain and in thetransmembrane segment. These results suggest that the cytoplasmic boundaries of the second and third transmembranesegments are critical for the function of PomA to sustain the
structure of the cytoplasmic domain. The cytoplasmic boundaries of PomA and its cytoplasmic interface to FliG mightsynergistically act in force generation. Taking this evidence
together, we speculated that PomA mutations in the cytoplasmic domain might affect the structure of PomA or the assemblyof the stator complex to the rotor and could result in theprevention of interactions between PomA and FliG to cooperatively generate torque. At this time, however, we do nothave evidence to indicate whether or not the regions around
L131F or AAQQQ interact directly with FliG in wild-type
PomA.
Strains carrying PomA variants with the double substitutions
R88A or E96Q and L131F exhibited a TS phenotype above
37°C. Furthermore, the triple substitution of R88A and E96Q
(the conserved charged residues) with L131F appeared to be
sufficient for conferring the TS phenotype (Fig. 8). These results may suggest that the region near these two conserved
charged residues has an important role in flagellar rotation.
The other substitutions seem to affect the TS phenotype
slightly. This is not surprising because it is easy to imagine thatthe region around the conserved residues is affected by othernearby mutations.
The TS phenotype was alleviated by coexpressing PomB
from the plasmid (Fig. 4). This effect may be explained by anincrease in the number of TS PomA/PomB complexes. Probably both functional and nonfunctional TS PomA/PomB complexes exist in the cell at 30°C. If the total number of PomA/
PomB complexes is increased by the overproduction of bothVOL. 186, 2004

CONSERVED CHARGED RESIDUES OF PomA

PomA and PomB from the plasmid, the number of the functional complexes should be increased. Thus, the possibility of
the assembly of a functional TS PomA/PomB complex with therotor is higher when PomB is provided from a plasmid thanwhen it is provided from the chromosome.
As far as we know, other TS mot mutants have not been
isolated; however, temperature-sensitive Mot⫺ fliG and Mot⫺fliN mutants were reported for S. enterica serovar Typhimurium (45). The mutants are capable of assembling a complete flagellum but are nonmotile at restrictive temperatures.
Mutations in the switch protein genes fliG, fliM, and fliN gave
three phenotypes: Mot⫺, Che⫺, and Fla⫺. The amino acidsubstitutions of these TS FliG mutants were mapped to F236L,
D244Y, and L259R in the C-terminal domain of S. enterica
serovar Typhimurium FliG (19). The other Mot mutants thatmapped to the fliG gene are clustered in this region. The
crystal structure of the middle and C-terminal regions of T.
maritima FliG has been reported (14). The middle and Cterminal regions of the T. maritima FliG molecule consist of
two compact globular domains linked by an ␣-helix and an
extended segment that contains a well-conserved Gly-Gly motif. The C-terminal region has a ridge containing the charged
residues that are believed to interact with the charged residuesof the MotA cytoplasmic domain (11, 14, 28). The Mot⫺ region
is located in the middle of the C-terminal region that is between the ridge surface region and the well-conserved Gly-Gly
motif. Mutations in the Mot⫺ region may affect the ridge
region that is the interface to the cytoplasmic domain of MotA.
The interactions between the cytoplasmic region of PomA or
MotA and the C-terminal region of FliG are thought to be
essential for force generation. In PomA, the region from L131
to H136 may contribute to torque generation together with theregion from R88 to E99, which is the conserved charged region. In the proton-driven motor, it seems that the protonationand deprotonation of an Asp residue in the MotB transmembrane segment induces a conformational change of the MotA
cytoplasmic region to generate the rotational force (23). The
force-generating unit was inferred to be a 4:2 complex of
PomA (MotA) and PomB (MotB) (11, 13, 24, 39, 40, 49).
Recent data suggest that the motor complex may have a larger
structure than previously assumed or that the minimal forcegenerating unit is not a 4:2 complex (46). Presently, however,
we do not know the mechanism of generation of rotational
force, although many models have been presented (8, 10, 41).
We anticipate that our present data will assist in the understanding of the mechanism once high-resolution data on thestator complex are available.
ACKNOWLEDGMENTS
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan;
the Japan Science and Technology Corporation (to M.H. and T.Y.);and the Soft Nano-Machine Project of Japan Science and Technology
Agency (to T.Y. and M.H.).
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