THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 26, Issue of June 30, pp. 20223–20228, 2000
Printed in U.S.A.

Multimeric Structure of PomA, a Component of the Naⴙ-driven
Polar Flagellar Motor of Vibrio alginolyticus*
Received for publication, March 17, 2000, and in revised form, April 23, 2000
Published, JBC Papers in Press, April 26, 2000, DOI 10.1074/jbc.M002236200

Ken Sato‡ and Michio Homma
From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan

Four integral membrane proteins, PomA, PomB,
MotX, and MotY, are thought to be directly involved intorque generation of the Naⴙ-driven polar flagellar motor of Vibrio alginolyticus. Our previous study showedthat PomA and PomB form a complex, which catalyzes
sodium influx in response to a potassium diffusion potential. PomA forms a stable dimer when expressed in a
PomB null mutant. To explore the possible functional
dependence of PomA domains in adjacent subunits, weprepared a series of PomA dimer fusions containing different combinations of wild-type or mutant subunits.
Introduction of the mutation P199L, which completely
inactivates flagellar rotation, into either the first or thesecond half of the dimer abolished motility. The P199L
mutation in monomeric PomA also altered the PomAPomB interaction. PomA dimer with the P199L mutation even in one subunit also had no ability to interact
with PomB, indicating that the both subunits in the
dimer are required for the functional interaction between PomA and PomB. Flagellar rotation by wild-type
PomA dimer was completely inactivated by phenamil, asodium channel blocker. However, activity was retainedin the presence of phenamil when either half of the
dimer was replaced with a phenamil-resistant subunit,
indicating that both subunits must bind phenamil for
motility to be fully inhibited. These observations demonstrate that both halves of the PomA dimer function
together to generate the torque for flagellar rotation.

Bacterial flagella are the organelles responsible for motility.
Flagellar rotation is driven by a reversible rotary motor embedded in the cytoplasmic membrane at the base of each flagellar filament (1–3). Energy for rotation derives from the transmembrane electrochemical potential of specific ions. Two typesof motors, proton-driven (2) and sodium-driven (4), have been
investigated. The proton-driven motors of Escherichia coli and
Salmonella typhimurium have been studied intensively. The
stator part of the torque generator consists of two cytoplasmic
membrane proteins, MotA and MotB, which contain four and
one transmembrane-spanning segments, respectively (5– 8).
There exists much genetic and physiological evidence that suggests that MotA and MotB together form a proton channel(9 –12), surrounding the rotor (13), and this complex is believed
to be anchored to the cell wall via a peptidoglycan-binding
domain of MotB (8, 14, 15). It is thought that ions passing* This study was supported by grants from Ministry of Education,
Science, Sports and Culture of Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 81-52-7892992; Fax: 81-52-789-3001.
This paper is available on line at http://www.jbc.org

through those proteins somehow generate torque (16), which istransmitted to the rotor part of the motor, the FliG protein (17,18). Together with FliM and FliN (19, 20), FliG forms theswitch complex, which is essential for torque generation, flagellar assembly, and controlling the direction of motor rotation(21–25).
Bacteria such as alkalophilic Bacillus and Vibrio species use
the electrochemical gradient of sodium to drive flagellar rotation (4). The sodium-driven motor has advantages for studying
the motor function, because sodium-motive force can be easily
manipulated, and amiloride and phenamil work as specificinhibitors of their function (26, 27). Four proteins essential for
torque generation, PomA, PomB, MotX, and MotY, were recently identified in the polar flagellar motor of Vibrio alginolyticus (28, 29). PomA and PomB are homologous to MotA and
MotB and contain four and one transmembrane segments,
respectively. Both MotX and MotY, which were first identified
in Vibrio parahaemolyticus (30, 31), have a putative single
transmembrane segment; they are unique to the sodium-type
motor, and their function is unknown so far. We recently
showed that PomA and PomB functionally interact with eachother in a molar stoichiometric ratio of 2 PomA:1 PomB andtogether form a sodium-conducting channel (32). Furthermore,
PomA forms a stable homodimer when expressed in the absence of PomB, implying that this may be the absolute numberof PomA subunits in the functional complex.
In this study, we describe a fusion protein that contains
different combinations of wild-type PomA or mutant (P199L or
D148Y) subunits that we have used to evaluate whether adjacent PomA domains may be required for torque generation. Our
results demonstrate that PomA functions as an even number ofsubunits in a single functional complex and that both subunits
contribute to PomA-PomB assembly.
MATERIALS AND METHODS

Bacterial Strains, Plasmids, Growth Conditions, and Media—V. alginolyticus strains NMB190 (Rifr, Pof⫹, Laf⫺, ⌬pomA) (33) and VIO5
(Rifr, Pof⫹, Laf⫺) were used. E. coli strain JM109 (recA1, endA1, gyrA96,
thi-, hsdR17, relA1, supE44, ␭-, ⌬(lac-proAB); (F⬘, traD36, proAB, lacIq,
lacZ⌬M15)) was used for DNA manipulations. V. alginolyticus cells
were cultured at 30 °C in VC medium (0.5% (w/v) polypeptone, 0.5%(w/v) yeast extract, 0.4% (w/v) K2HPO4, 3% (w/v) NaCl, 0.2% (w/v)
glucose) or VPG medium (1% (w/v) polypeptone, 0.4% (w/v) K2HPO4, 3%(w/v) NaCl, 0.5% (w/v) glycerol). For swarm assay, a VPG-0.3% agar
plate was used. E. coli cells were cultured at 37 °C in LB medium. Whennecessary, kanamycin was added to a final concentration of 100 ␮g/ml
for Vibrio cells or 25 ␮g/ml for E. coli cells. Plasmid pKS101, a pSU41based plasmid, was constructed to carry his6-pomA under the lac promoter control. A 0.8-kb1 DNA fragment including the pomA open reading frame with a 5⬘ attachment of 5⬘-ATTGGATCCATGCATCACCATCACCATCACATGGATTTAGCAACCCTATTA-3⬘ (initiates as Met-1
The abbreviations used are: kb, kilobase pair(s); NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis.

20223

20224Multimeric Structure of PomA

His6; the underline indicates the created (BamHI and EcoT22I)
restriction sites) was originally prepared by polymerase chain reaction
and cloned into BamHI-digested pSU41. Plasmid pKS105, encoding thetandem PomA with a six-histidine linker, was generated by engineering
an EcoT22I site at the 3⬘-end of the pomA clone and then linked to the5⬘-end of his6-pomA via EcoT22I. pKS106 (PomA-PomA), a pSU41based plasmid with a kanamycin resistance marker, encoded the tandem PomA under lac promoter control. An oligonucleotide with thesequence 5⬘-ATGCATTATCCGTACGATGTTCCAGATTATGCAATAGAAGGTCGCATGCAT-3⬘ was inserted into the EcoT22I site of pKS105,
allowing an in frame insertion of the sequence MHYPYDVPDYAIEGR
into the 3⬘-end of the pomA clone. The final linking sequence was
MHYPYDVPDYAIEGRM-His6, i.e. a hemagglutinin tag and six histidines. Plasmid pKS107 (PomA-P199L) was prepared by the insertion of
a 0.8-kb DraI fragment of pKS106 into the corresponding site ofpMK101-P199LKm. pMK101-P199LKm was constructed by the insertion of a 0.8-kb BamHI fragment of pMK101-P199L (34) into the corresponding site of pSU41. A 1.0-kb HindIII fragment of pKS106 wasligated with pMK101-P199LKm that had been partially digested with
HindIII, creating pKS108 (P199L-PomA). Plasmid pKS109 (P199LP199L) was constructed by the insertion of a 0.8-kb SphI fragment of
pKS108 into the corresponding site of pKS107. pKS111 carrying theHis6-PomA P199L gene was created by insertion of a 0.6-kb DraI–EcoRI
fragment of pMK101-P199LKm into the corresponding site of pKS101.
Plasmid pKS113 (PomA-D148Y) was constructed by the insertion of a
0.8-kb DraI fragment of pKS106 into the corresponding site of pYA301D148Y (35). A 1.0-kb HindIII fragment of pYA301-D148Y was ligatedwith pKS106 that had been partially digested with HindIII, creating
pKS114 (D148Y-PomA). pKS115 (D148Y-D148Y) was constructed by
the insertion of a 0.8-kb SphI fragment of pKS114 into the corresponding site of pKS113. All inserts were confirmed by DNA sequencing.
Measurement of Swimming Speed—Cells were harvested at late logarithmic phase and resuspended in an equal volume of TMN medium
containing 50 mM Tris-Cl (pH 7.5), 5 mM MgCl2, 5 mM glucose, 50 mM
NaCl, 250 mM KCl. The cell culture was diluted 100-fold into TMN
medium containing various concentrations of NaCl, and then motility of
the cells was observed at room temperature under a dark field microscope and recorded on video recorder. Swimming speed was determined
as described (36). The average swimming speed was obtained by measuring at least 20 swimming tracks.
Co-elution Assay—Cells of NMB190 expressing His6-tagged PomA
derivative were cultured at 30 °C under strong aeration in VPG medium, harvested, washed with buffer (20 mM Tris-Cl, pH 8.0, 5 mM
MgSO4, 10% (w/v) sucrose), and resuspended (0.2 g, wet weight/ml) in
20 mM Tris-Cl, pH 8.0, containing 1 mM dithiothreitol, 5 mM MgSO4, 30
␮g/ml DNase I, and 0.5 mM phenylmethylsulfonyl fluoride. Membrane
vesicles were prepared by subjecting the suspension to a single passage
through a French press (5501-M Ohtake Works) at 4000 p.s.i. at 4 °C.
Undisrupted cells were removed by low speed centrifugation (10,000 ⫻g for 20 min at 4 °C), and the membrane fraction was recovered from thesupernatant by centrifugation at 200,000 ⫻ g for 2 h. The membranepellet was suspended in buffer A (20 mM Tris-Cl, pH 8.0, 150 mM NaCl,
1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20% (w/v)
glycerol) containing 5 mM imidazole, pH 8.0, and 2.5% (w/v) ␤-octyl
glucoside. The suspension was stirred for 30 min at room temperatureand centrifuged for 20 min at 10,000 ⫻ g. The clarified extract was
mixed with Ni2⫹-NTA-agarose (QIAGEN) prewashed with the same
buffer, incubated at 4 °C for 1 h with gentle mixing, and then packed
into the column. The loaded resin was washed with buffer A containing
40 mM imidazole, pH 8.0, and 1.25% (w/v) ␤-octyl glucoside. Elution was
conducted with buffer A, containing 500 mM imidazole, pH 8.0, and1.25% (w/v) ␤-octyl glucoside. The Ni2⫹-NTA-purified material was
analyzed by SDS-PAGE followed by immunoblotting.
RESULTS

Construction and Expression of PomA Dimers—Our previous
studies suggested that the native PomA protein alone forms astable dimer in detergent extract (32). The possibility that the
dimerization of PomA subunits may be required for torquegeneration was approached by constructing a series of tandem
dimer PomA proteins. The construction was performed by linking two pomA open reading frames in frame with 22 residues asa linker (Fig. 1A). The resulting plasmid encodes a 528-residue
polypeptide (PomA-PomA) with an N-terminal subunit continuous with a C-terminal subunit. As shown in Fig. 1B, NMB190(⌬pomA) cells expressing the tandem PomA gained the ability

FIG. 1. Membrane topology and function of dimeric PomA fusion protein. A, schematic diagram of a PomA-PomA dimer. Two
PomA monomers joined tail-to-head in tandem are expressed as a single
polypeptide. The arrow indicates the position of the connecting site; thelinking sequence is MHYPYDVPDYAIEGRM-His6 (22 amino acids). B,
swarming abilities of NMB190 (⌬pomA) cells expressing nothing (vector), His6-PomA (PomA), or tandem PomA (PomA-PomA). Overnight
cultures were spotted on VPG-0.3% agar plate containing kanamycin
and incubated at 30 °C for 5 h. a.a., amino acid.

to swarm on a soft agar plate. Compared with wild-type monomeric PomA, tandem PomA slightly reduced the swarming
ability. This is also shown in Fig. 6B, where NMB190 cells withtandem PomA had significantly reduced swimming speed compared with cells expressing wild-type monomeric PomA. Since
the C terminus of Salmonella MotA, the PomA homolog, hasbeen shown to be important for function (37), diminishedswarm ability may be due to restricted flexibility around the C
terminus of the N-terminal half of tandem PomA. To demonstrate that swarming was due to the activity of full-length
tandem PomA and not to monomeric PomA resulting frompartial translation or proteolytic cleavage of the PomA fusion,
an immunoblotting was carried out with membranes from
NMB190 expressing the tandem fusion protein. As shown in
Fig. 2B, tandem PomA migrates around 50 kDa, as do SDSresistant dimeric aggregates of monomer PomA (Fig. 2B, lane2) that we have observed routinely (38). No other immunoreactive species is observed on the immunoblots compared withwild-type endogenous PomA (lanes 7–9), particularly around 25
kDa, where monomeric PomA migrates. The immunoreactive
materials at slightly larger than the monomer size (Fig. 2B,
lanes 3 and 6) are the nonspecific degradation products during
membrane preparation.
Function of PomA Dimers Containing Different Combinations of Wild-type or Mutant Subunits—In addition to wild-type
PomA dimer, the following mutant dimers were created: PomAP199L, P199L-PomA, and P199L-P199L (Fig. 2A). As a measure of function, the ability of PomA dimer and mutant dimers
to swarm in a soft agar plate was assayed in V. alginolyticus
NMB190 (⌬pomA). Fig. 3A shows the swarm abilities conferredby PomA dimer (PomA-PomA) and dimers containing mutation
P199L. Previous mutational studies have shown that a conserved proline residue in PomA (Pro199 in the protein fromV. alginolyticus and Pro222 in E. coli MotA), which is importantfor function, since mutations of this residue completely prevent
flagellar rotation and showed strong negative dominance (34).
When this inactivating mutation was introduced into the N-

Multimeric Structure of PomA

FIG. 2. Expression of tandem PomA. A, schematic diagrams ofvarious PomA-PomA dimers. Two PomA monomers joined tail-to-headin tandem are expressed as a single polypeptide. The arrows indicate
the positions where site-specific substitutions were introduced. B (upper panel), membrane vesicles (each 10 ␮g of protein) of NMB190(⌬pomA) harboring a vector plasmid (lane 1), a plasmid encoding His6PomA (lane 2), tandem PomA dimer (lane 3), or tandem PomA dimer
mutants (lanes 4 – 6) were subjected to SDS-PAGE and immunoblottingwith anti-PomA antibody. B (lower panel), membrane vesicles (each 20
␮g of protein) of NMB190 (⌬pomA) harboring a vector plasmid (lane 7),
VIO5 (PomA⫹) harboring a vector plasmid (lane 8), or NMB190(⌬pomA) harboring a plasmid encoding tandem PomA dimer (lane 9)
were subjected to SDS-PAGE and analyzed with immunoblotting as
above.

terminal (P199L-PomA) or the C-terminal (PomA-P199L) halfof the dimer, swarm activity was completely diminished. PomA
dimer with the mutation in both halves (P199L-P199L) was
also completely inactive (Fig. 3A). Under the dark field microscope (39), no flagellar rotation was observed with cells producing P199L-containing dimers (data not shown).
We also investigated for dominant-negative effects of themutant PomA dimers. The plasmids encoding PomA dimer ormutant dimers were introduced into wild-type cells, VIO5
(PomA⫹), and the transformants were inoculated on a soft agar
plate. As shown in Fig. 3B, all PomA dimers containing theP199L mutation in either half of the dimer reduced the swarming ability of VIO5 (PomA⫹) cells, whereas PomA homodimer
resulted in no significant reduction in swarm size comparedwith wild-type cells. These results suggest that PomA dimers
with the P199L mutation compete with the wild-type PomA for
occupying the functional site of the torque-generating unit.
Taking the results together, it is highly possible that PomA
dimers are inserted in the membrane at the functional site.
Furthermore, the inactivation of half of the dimer interfereswith the function of the other half, implying that the two halvesof the dimer function together.
Interaction of PomA Dimer and PomB—Our previous studies
showed that PomA and PomB functionally interact with eachother (38). To investigate whether PomA dimers have the ability to interact with PomB, a co-elution assay was performed(Fig. 4A). Membrane fractions of NMB190 (⌬pomA) expressing

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FIG. 3. Functions of PomA dimer mutants. A, swarming abilitiesof the PomA dimer mutants. Four independent clones of NMB190(⌬pomA) each carrying the PomA dimers were cultured overnight,
spotted on a VPG-0.3% agar plate containing kanamycin, and incubatedat 30 °C for 5 h. vector, NMB190/pSU41 used as a negative control. B,
dominant-negative effects of the mutant PomA dimers. Overnight cultures of four independent clones of VIO5 (PomA⫹) each expressing
PomA dimers from a multicopy plasmid were spotted on a VPG-0.3%
agar plate containing kanamycin and incubated at 30 °C for 5 h.

various PomA dimers were prepared, solubilized with ␤-octyl
glucoside, and passed through a Ni2⫹-NTA-agarose column.
The column was washed, bound protein was eluted with buffer
containing imidazole, and the eluate was analyzed by immunoblotting using antibodies against PomA and PomB (Fig. 4A).
Although only His6-PomA dimer is overexpressed in the cells,
the protein complex contains only endogenous PomB, whichshould therefore reflect the native interaction between PomA
and PomB. PomB co-eluted with PomA dimer (PomA-PomA;
lane 1), whereas no PomB band was observed with PomA
dimers containing the P199L mutation in either or both halves(P199L-PomA, PomA-P199L, or P199L-P199L; lanes 2– 4).
Although Pro222 of E. coli MotA has been proposed to function as the regulator for the conformational changes of the
MotA-MotB complex (40), its specific role still remains unclear.
To examine the interaction between monomeric PomA P199L
and PomB, a His6 tag was attached to the N terminus of PomA
and P199L mutant monomer, and the co-elution assay was
performed as above (Fig. 4B). When the eluates were analyzed
by immunoblotting, PomB was found to have co-isolated withwild-type His6-PomA (lane 6), but no detectable PomB was
co-sedimented with His6-P199L (lane 7). The results describedabove suggest that as well as the Pro199 of PomA being essential for PomA-PomB interaction, probably affecting the C-terminal tail of PomA, both halves of the PomA dimer contributeto form the functional PomA-PomB complex.

20226

Multimeric Structure of PomA

FIG. 4. Co-elution analysis of PomA dimer. Upper panel, detergent extract from membrane vesicles of NMB190 (⌬pomA) expressing
either PomA-PomA (lane 1), PomA-P199L (lane 2), P199L-PomA (lane3), or P199L-P199L (lane 4) were incubated with Ni2⫹-NTA-agarose
resin as described under “Materials and Methods,” and eluates wereprecipitated with trichloroacetic acid and analyzed by SDS-PAGE andimmunoblotting with anti-PomB93 or anti-PomA1312 antibody (38).
Lower panel, solubilized membrane vesicles of NMB190 (⌬pomA) expressing either nothing (lane 5), His6-PomA (lane 6), or His6-P199L(lane 7) were mixed with Ni2⫹-NTA resin, and eluates were analyzed
with SDS-PAGE and immunoblotting as above.

Torque Generation of PomA Dimers Containing Phenamilresistant Mutations—We further extended this approach toinvestigate the relationship between torque generation andsodium conductance. Our previous studies had shown that thesubstitution of Asp148 by Tyr (D148Y) in PomA results in resistance to phenamil, a specific inhibitor of the PomA-PomB
sodium channel (35). Here, we have used this mutant to determine whether sodium conductance of both halves of the PomA
dimer are required for torque generation. D148Y-containing
mutant dimers were expressed as a 50-kDa protein, and no
degradation products were observed (data not shown). Introduction of this mutation into either or both halves of the dimer
reduced swarm activity (Fig. 5A). This is consistent with ourprevious observation that mutation at Asp148 results in impaired motility (35). 50 ␮M phenamil, which completely blocks
sodium conductance of the PomA-PomB channel (35), completely inhibited motility driven by tandem PomA (PomAPomA) (Fig. 5B). In contrast, NMB190 cells expressing tandem
PomA with D148Y mutations in both halves (D148Y-D148Y)
showed phenamil resistance. Interestingly, PomA heterodimers with either the N-terminal or the C-terminal halfcontaining the D148Y mutation (PomA-D148Y or D148YPomA) retained activity in the presence of phenamil. To further
examine the effect of sodium channel blockage on torque generation, the swimming speeds of cells harboring mutant PomA
were measured in the presence or the absence of phenamil(Fig. 6A). In the absence of phenamil, NMB190 cells expressing
tandem PomA with both halves containing the D148Y mutation had swimming speed reduced to about 80% compared withthose expressing PomA-D148Y or D148Y-PomA. This is consistent with the control experiment shown in Fig. 6B, that the
D148Y mutation significantly reduced the swimming speed
compared with cells expressing wild-type monomeric PomA.
When 50 ␮M phenamil was added to the cells expressing the
PomA dimer with either or both halves of subunits containing
D148Y mutations, all of the mutants showed similar motility.

FIG. 5. Effect of phenamil on torque generation driven by
PomA dimer (PomA-PomA) or dimers with D148Y mutations(D148Y-PomA, PomA-D148Y, or D148Y-D148Y). Four independent
clones of NMB190 (⌬pomA) expressing the given PomA dimers were
cultured overnight and spotted on a VPG-0.3% agar plate (100 mM
NaCl) containing kanamycin (A) or both kanamycin and 50 ␮M phenamil (B) and incubated at 30 °C for 6 and 12 h, respectively. vector,
NMB190/pSU41 used as a negative control.

This suggests that both subunits of the dimer contribute to thephenamil binding. The results might also indicate that theindividual halves of the dimer do not function independently
but function together to conduct sodium ions.
DISCUSSION

We demonstrate here that an engineered fusion protein containing two PomA molecules can insert into the membrane andexhibit torque generation activity. Tandem PomA dimer provides a unique model system that permits coupled synthesis of
two PomA molecules, with the advantage that both halves ofthe dimer are present at the identical level. A dimer containing
wild-type PomA and mutant P199L was constructed, and theeffect of the ability to rotate flagella was assessed. Inactivationof either half of the dimer caused complete loss of activity,
indicating that both halves of the dimer are essential for torquegeneration. If the two halves of the dimer functioned independently, inactivation of either half would be expected to result in⬃50% decrease in activity. In other words, the two halves of the
PomA dimer appear to function together.
To examine the possibility that incorrect membrane insertion of either half of the dimer might occur, negative dominance
was tested. Strong dominant negative effects were observedwith both P199L-PomA and PomA-P199L. Although P199Lcontaining PomA dimers cannot bind to PomB (Fig. 4A), thisresult is quite reasonable, because it might be still able tointeract with MotX and/or MotY, which are also essential forthe rotation of the sodium-driven motor. If so, the mutant
PomA dimers could interfere with MotX and/or MotY function

Multimeric Structure of PomA

FIG. 6. Sensitivity to phenamil of PomA dimers. A, swimming
speeds were measured in TMN medium in the presence or the absenceof 50 ␮M phenamil as indicated under “Materials and Methods.” Cells of
NMB190 (⌬pomA) expressing PomA-D148Y (squares), D148Y-PomA(circles), or D148Y-D148Y (triangles) were collected at late logarithmic
phase and suspended in TMN medium (pH 7.5, 50 mM NaCl). The cellsuspension was diluted to 100-fold into TMN medium containing various concentrations of NaCl. Filled and open symbols indicate speeds inthe absence or the presence of 50 ␮M phenamil, respectively. Cells of
NMB190 (⌬pomA) expressing PomA-PomA (crosses) in the presence of
50 ␮M phenamil are also indicated. B, cells of NMB190 (⌬pomA) expressing PomA (circles), PomA-PomA (squares), or D148Y (triangles)
were collected at late logarithmic phase, and swimming speeds were
measured as above.

or sequester them away from the functional site. It is not clearthat the wild-type PomA in the hetero-fused dimer folds like itwould by itself or in the homo-fused dimer. Limited proteasesensitivity of the dimer-expressed inverted membrane vesicles
revealed that there were no differences of digestion pattern
between wild-type fused dimer and mutant-containing dimer(data not shown), although further investigations are required.
Can we exclude the possibility that a PomA dimer containing
one wild-type PomA (P199L-PomA or PomA-P199L) might support rotation when this wild-type half of the mixed dimer isactive, while other dimers impede rotation because the inactive(P199L) subunit is incorporated? This possibility is excluded
for the following reason. If some of the active (wild-type) subunits of the P199L-bearing PomA dimers are incorporated into
their correct sites in the motor to form a functional unit, those
active subunits should form a PomA-PomB complex. However,
we have shown here that all of the P199L-containing dimers
cannot interact with endogenous PomB in Fig. 4A, which indicates that there are no such species as functional PomA-PomB
complexes. Taken together, the results provide support for thecontention that PomA is functional as an even number of subunits, either a dimer or a multimer of dimers.
The Pro199 residue of PomA is highly conserved in MotA
homologs and is thought not to have a direct role in ion translocation but rather a structural role (34). Pro222 in E. coli MotA

20227

was investigated by intensive mutagenesis, leading to the conclusion that it might function to mediate conformationalchanges that couple the events occurring on the membrane andcytoplasmic domains during energy conversion (40, 41). We
showed here that Pro199 of PomA also plays a critical role for
PomA-PomB interaction. Monomeric PomA with the mutation
P199L showed reduced ability to interact with PomB. It isnoteworthy that this was also observed with PomA dimer containing the mutation in either half of the dimer. Suppose that
PomA is functional as a monomer, and each molecule interacts
with PomB. It is difficult then to imagine how the introductionof single P199L mutation into one half of the dimer could result
in complete loss of PomA-PomB interaction. Function as a
dimer is consistent with our previous estimate that the molar
ratio of isolated functional PomA-PomB complex is 2 PomA:1
PomB (32). We therefore conclude that both subunits in PomA
dimer contribute to the PomA-PomB interaction.
One more approach was used to investigate the functional
impact of PomA dimer. Tandem PomA was constructed containing wild-type PomA and D148Y mutated PomA, and theeffect of phenamil on torque generation was measured. The
results indicate that the introduction of D148Y mutation to one
half of the dimer results in no significant decrease in bothswarm activity and swimming speed compared with dimer
where both halves carry the phenamil-resistant mutation, suggesting the possibility that the individual halves of the dimer
do not conduct sodium ions by themselves. Although it is notclear how many phenamils bind to this dimer molecule, it ishighly probable that both halves of the PomA dimer function
together to conduct sodium ions, and so both subunits cooperatively form a phenamil binding site.
Some integral membrane permeases such as the ribose
transporter RbsC (42) form homodimers for their function. The
FhuB, the ferric hydroxamate transporter component, functions as a single polypeptide containing two homologous repeats and is still active when those repeats are separated (43).
Oligomerization of PomA may be an analogous process.
In the absence of high resolution images of the torque-generating unit, subunit stoichiometry can be tentatively assessedusing biochemical methods. The results described above provide a framework for the subunit stoichiometry of the torquegenerating unit, but a definitive answer will require further
experimental documentation.
Acknowledgments—We thank Drs. Robert M. Macnab and Ikuro
Kawagishi for critically reading the manuscript.
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