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

Vol. 275, No. 40, Issue of October 6, pp. 31387–31391, 2000
Printed in U.S.A.

Intermolecular Cross-linking between the Periplasmic Loop3– 4
Regions of PomA, a Component of the Naⴙ-driven Flagellar
Motor of Vibrio alginolyticus*
Received for publication, February 3, 2000, and in revised form, July 17, 2000
Published, JBC Papers in Press, July 18, 2000, DOI 10.1074/jbc.M000848200

Tomohiro Yorimitsu, Yukako Asai, Ken Sato, and Michio Homma‡
From the Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan

PomA and PomB form a complex that conducts sodium ions and generates the torque for the Naⴙ-driven
polar flagellar motor of Vibrio alginolyticus. PomA has
four transmembrane segments. One periplasmic loop
(loop1–2) connects segments 1 and 2, and another (loop3–4), in which cysteine-scanning mutagenesis had been
carried out, connects segments 3 and 4. When PomA
with an introduced Cys residue (Cys-PomA) in the Cterminal periplasmic loop (loop3– 4) was examined without exposure to a reducing reagent, a 43-kDa band wasobserved, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band wasenhanced in most mutants by the oxidizing reagent
CuCl2. The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely
reduced, and P172C showed a dominant-negative effect,
whereas substitution of Pro with Ala, Ile, or Ser at thisposition did not affect motility. In the presence of DTT,
the ability to swim was partially restored, and theamount of 43-kDa protein was reduced. These results
suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified with N-ethylmaleimide, only the 25-kDa PomA band
was labeled, demonstrating that the 43-kDa form is across-linked homodimer and suggesting that the loops3– 4of adjacent subunits of PomA are close to each other in
the assembled motor. We propose that this loop region isimportant for dimer formation and motor function.

Many bacteria rotate flagellar filaments to swim. The flagellar filament is attached via a flexible hook to a protein complex
termed the basal body, which is embedded in the cell surface.
The motor is composed of the basal body and the C ring (rotor),
whereas the multiple torque-generating units of the stator surround the rotor (1). The motor is driven by the flow of specific ions(H⫹ or Na⫹) through the stator, and mechanical force is presumably generated at the stator-rotor interface (2, 3).
Escherichia coli has H⫹-driven flagellar motors whose
torque-generating units consist of two proteins, MotA and MotB.
These two proteins are believed to form a H⫹ channel to permit
the H⫹ influx and to provide the energy for motor rotation* This work was supported in part by grants-in-aid for scientificresearch from the Ministry of Education, Science and Culture of Japan(to M. H. and K. S.) and from the Japan Society for the Promotion of
Science (to Y. A. and T. Y.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 81-52-7892991; Fax: 81-52-789-3001; E-mail: g44416a@nucc.cc.nagoya-u.ac.jp.
This paper is available on line at http://www.jbc.org(4 –7). In contrast, the polar flagellum of Vibrio spp., Vibrio
alginolyticus, Vibrio parahaemolyticus, and Vibrio cholerae isdriven by the Na⫹-motive force, and the products of four genes,
pomA, pomB, motX, and motY, are essential for Na⫹-driven
rotation (8 –15). Of these, PomA and PomB are similar to MotA
and MotB, respectively. It has been inferred that PomA and
PomB also form an ion channel (8). This inference was supported by the observation that mutations conferring resistanceto phenamil, which is a known Na⫹-channel inhibitor andspecifically and strongly inhibits the Na⫹-driven motor,
mapped to pomA and pomB (14, 16, 17). The mutations occurred at Asp-148 of PomA and Pro-16 of PomB, which are near
the cytoplasmic ends of putative transmembrane segments.
The D148Y and P16S mutations combined to produce a synergistic increase in resistance to phenamil and impaired motor
function much more severely than the individual mutations in
the absence of inhibitor (17). We have also shown that PomA
and PomB physically interact with each other (18), and thatNa⫹ influx can be detected in reconstituted proteoliposomes
containing purified PomA/PomB complex (19). The molar ratioof the isolated complex is calculated to be 2PomA/1PomB, andPomA seems to form a stable dimer.
To characterize the molecular architecture of PomA, we carried out Cys-scanning mutagenesis (20). This technique is useful for determining the structure and function of many proteins(21–24). In PomA, periplasmic loops are predicted for residues
Val-21 through Leu-36 between transmembrane segments 1
and 2 (loop1–2) and for residues Ser-167 through Ala-180 between transmembrane segments 3 and 4 (loop3– 4) (Fig. 1). Each
residue in these loops has been substituted with Cys. Except for
A174C, all Cys substitutions from M169C to M179C in loop3– 4
impaired motility, whereas only the D31C substitutions inloop1–2 significantly impaired motility. The motility of someCys mutants was inhibited by the thiol-modifying reagents
5,5⬘-dithiobis(nitrobenzoic acid) and NEM.1 The inhibition profiles of these reagents were consistent with the membrane
topology predicted from the hydrophobicity profiles (20). The
accessibility of loop3– 4 to the periplasm was confirmed by labeling with biotin maleimide; however, none of the Cys residues ofloop1–2 were labeled by the reagent. These results suggest thatthe environments of the two loops are different (20).
In the present study, we analyzed PomA mutants with Cys
substitutions in loop3– 4. A cross-linked form of these Cys-PomA
proteins was detected under non-reducing conditions. We thinkthat the cross-linked form is a homodimer and that the loops of
PomA subunits are adjacent in the motor. We discuss thefunction and the structural topology of loop3– 4 of PomA.
1
The abbreviations used are: NEM, N-ethylmaleimide; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

31387

31388Cross-linking between the Periplasmic Loops of PomA

FIG. 1. Putative secondary structure of PomA. This secondary
structure is based on the hydrophobic profiles (8) and the reactivity ofCys-substituted proteins with SH-modifying reagents (16). PomA has
two putative periplasmic loops, loop1–2 and loop3– 4. The amino acids inloop3– 4 are shown explicitly. The putative transmembrane (TM) regions
are depicted as rectangles. Numbers indicate residue positions within
PomA.
MATERIALS AND METHODS

Bacterial Strains, Plasmids, Growth Conditions, and Media—V. alginolyticus strains VIO5 (Rifr, Pof⫹, Laf⫺), VIO586 (Rifr, Pof⫹, Laf⫺,
pomA), and NMB188 (Rifr, Pof⫹, Laf⫺, Che⫺, pomA) were used (8, 11).
E. coli strain DH5␣ (F⫺␭- recA1 hsdR17 endA1 supE44 thi-1 relA1
gyrA96 ⌬(argF-lacZYA) U169 ␾80dlacZ⌬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). E. coli
cells were cultured at 37 °C in LB medium. When necessary, kanamycin
was added to a final concentration of 100 ␮g/ml for V. alginolyticus cells
or 50 ␮g/ml for E. coli cells. Plasmid pYA301, a pSU41-based plasmid,
carries the pomA gene under the control of the lac promoter (17).
Site-directed Mutagenesis—To introduce the P172A, P172I, andP172S substitutions into PomA, we used a two-step polymerase chain
reaction method described previously (17). We synthesized pairs ofmutant primers homologous to either the sense or antisense strand of
the pomA gene, with a 1–3-base mismatch at the mutation site. In
addition to these primers, we used end primers and amplified the fullgene. This fragment was cloned into pSU41, and the identity of the total
insert was confirmed by DNA sequencing.
Swarm Assay—An overnight culture in VC medium was spotted onto
VPG plates containing 0.25% agar and 100 ␮g/ml kanamycin and incubated at 30 °C. Dithiothreitol (DTT) was added to a final concentrationof 1 mM, as needed.
Measurement of Swimming Speed—Cells were harvested at late logarithmic phase and suspended in V-buffer (25 mM Tris-HCl, pH 7.5, 10mM MgCl2, 300 mM NaCl). The cell suspension was diluted 100-fold in
V-buffer, and motility of the cells were observed at room temperature
under a dark field microscope and recorded on video tape. Swimming
speed was determined as described previously (25). If necessary, DTT
was added to V-buffer at a final concentration of 1 mM.
Detection of Cys-PomA—VIO586 (PomA⫺) cells producing wild-type
or various Cys-PomA mutant proteins were cultured in VPG medium
and collected by centrifugation at mid-log phase. Cells were washedwith V-buffer and then resuspended in the same buffer or in V-buffer
containing 10 mM DTT or 1 mM CuCl2. After 30 min of incubation atroom temperature, the cells were collected, washed with V-buffer containing 2 mM N-ethylmaleimide (NEM), suspended in the same buffer,and then incubated at room temperature for 10 min. Next, an equalvolume of 15% (w/v) trichloroacetic acid was added to the cell suspension, and the resulting precipitate was washed once with acetone, dried,
and then dissolved in SDS sample buffer without reducing reagent.
This sample was subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody
against PomA, as described previously (18).
[14C]NEM Modification—VIO586 cells producing wild-type or P172C
PomA were grown in VPG medium to mid-log phase. The culture (80 ml)
was centrifuged, and the cells were washed and suspended in V-buffer
to an optical density at 660 nm of 10. After the cells were sonicated, theFIG. 2. Detection of Cys mutant PomA proteins. VIO586 (pomA)
cells harboring pYA301 (pomA⫹) and mutant derivatives were incubated in V-buffer (B) and buffer containing 10 mM DTT (A) or 1 mM
CuCl2 (C). After the cells were treated with 2 mM NEM, proteins wereprecipitated by trichloroacetic acid, washed with acetone, and separated by SDS-PAGE without reducing reagent. PomA was detected byimmunoblotting with anti-PomA antibody. The PomA residues substituted with Cys are indicated. Numbers with arrows on the right side are
the estimated molecular masses of the indicated bands.
cell debris was removed by centrifugation at 10,000 ⫻ g for 10 min at
4 °C, and the supernatant was then centrifuged at 100,000 ⫻ g for 60
min at 4 °C. The resulting precipitate (membrane fraction) was resuspended in V-buffer containing 10 mM DTT, and this suspension was
incubated at room temperature. After 30 min, it was centrifuged at
100,000 ⫻ g for 60 min at 4 °C, the precipitate was resuspended in 500
␮l of V-buffer containing 0.5 mM [14C]NEM (20 ␮Ci/ml; NEN Life
Science Products), and the resuspended material was incubated for 60
min at room temperature. The membrane fraction was recovered bycentrifugation at 100,000 ⫻ g, solubilized with TNET buffer (50 mM
Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, and 1% (w/v) Triton
X-100), and incubated for 60 min at 4 °C. Immunoprecipitation with
anti-PomA antibody was carried out as described previously (18). The
resulting precipitates were subjected to SDS-PAGE, followed byfluorography.
RESULTS

Detection of Cys-PomA Protein—Asai et al. (20) carried out
Cys-scanning mutagenesis of the two periplasmic loops (loop1–2
and loop3– 4) of PomA and characterized the mutant proteins.
This work showed that Cys replacements in loop3– 4 affectedmotility more severely than those in loop1–2, and we therefore
focused on the replacements in loop3– 4. When we examined
Cys-PomA proteins in loop3– 4 by immunoblotting in the absence of reducing reagent, we observed, in addition to the
25-kDa PomA band, a 43-kDa band with Cys replacements at
residues 171–174 and at residue 177 (Fig. 2B). The intensity of
the 43-kDa band was greatest with the P172C protein. Whencells were treated with DTT and immunoblotting was carriedout, only the 25-kDa PomA band was observed (Fig. 2A). Whenthe cells were treated with CuCl2, the intensity of the 43-kDa
band was greatly enhanced with the Cys mutant proteins, andthe intensity of the 25-kDa PomA band was correspondinglyCross-linking between the Periplasmic Loops of PomA

FIG. 3. Mutants with residue substitutions at PomA Pro-172. A,
swarming abilities conferred by the mutant PomA proteins. Overnight
cultures of VIO586 cells producing wild-type PomA (wt) and PomA
P172C, P172S, or P172A were spotted onto 0.25% agar VPG plates
containing 100 ␮g/ml kanamycin and incubated at 30 °C for 3.5 h. B,
immunoblotting of mutant PomA proteins. VIO586 cells producing
PomA as in A were treated and immunoblotted as described in thelegend to Fig. 2.

reduced (Fig. 2C). The 43-kDa band was never observed withwild-type PomA, which contains no cysteine residues.
To examine whether the 43-kDa band results from the Cys
substitution and to test whether formation of the 43-kDa protein affects motor function, we constructed three additional
Pro-172 mutants, P172A, P172I, and P172S. The motility of apomA null mutant expressing any of these three PomA proteins
was the same as that seen with wild-type PomA (Fig. 3A). In all
three mutants, the 43-kDa band observed with P172C PomA
was absent under all conditions tested (Fig. 3B).
Effect of DTT on Motility—Motility was more affected by Cys
substitutions near residue Pro-172 than at other positions in
the loop regions (20). The P172C mutant produced swarms only
after prolonged incubation, and motile cells were seldom observed in liquid culture (data not shown). We predicted that the
inhibition of motility is caused by disulfide-bound formation
between PomA subunits. The residual motility is probably due
to incomplete cross-linking (see below).
To examine this possibility, motility was assessed on swarm
plates in the presence or absence of DTT (Fig. 4A). DTT did notaffect swarming of cells expressing wild-type PomA, but cells
producing P172C PomA swarmed well only in the presence ofDTT (Fig. 4A). This result suggests that motility is restored by
reductive cleavage of the disulfide cross-link between CysPomA molecules. We next tested for dominant-negative effectsof the cross-linked form of PomA. When P172C PomA wasproduced in wild-type cells (VIO5) on agar plates without DTT,
motility was significantly reduced (Fig. 4B). However, motility
was restored in the presence of DTT. These results suggest thatthe cross-linked forms can be assembled into the motor
complex.
To examine the relationship between recovery of motility andreduction of the cross-linked PomA further, we measured theswimming speed of cells in the presence of DTT and the amountof the cross-linked PomA in the cells (Fig. 5). The swimming
speed of the cells with wild-type PomA did not change in thepresence or absence of DTT, showing a result consistent withthe swarming assay. The cells producing PomA P172C did not

31389

FIG. 4. Effect of DTT on motility. A, overnight cultures of VIO586
cells without PomA (vector) or producing wild-type PomA (wt) or PomA
P172C were spotted onto 0.25% agar VPG plates containing 100 ␮g/ml
kanamycin in the absence (left) or the presence of 1 mM DTT (right) andincubated at 30 °C for 3.5 h. B, overnight cultures of wild-type VIO5
cells producing plasmid-encoded wild-type PomA (wt) or P172C PomA
were spotted onto 0.25% agar VPG plates containing 100 ␮g/ml kanamycin, as in A.

FIG. 5. Recovery of motility and reduction of the cross-linked
PomA. A, swimming speed was measured in the presence or absence of
1 mM DTT as indicated under “Materials and Methods.” Each filled
symbol indicates swimming speeds of cells (strain NMB188) producing
wild-type PomA in the presence (triangles) or absence (inverted triangles) of DTT, or PomA P172C in the presence (squares) or absence(circles) of DTT. Open symbols connected by dotted lines indicate relative amounts of 43-kDa PomA P172C dimer to total PomA P172C
protein in the presence (squares) or absence (circles) of DTT. The
amounts were estimated from the data shown in B. B, cells of strain
NMB188 producing PomA P172C were collected at late logarithmic
phase and suspended in V-buffer in the presence (left) or absence (right)of DTT. At the times indicated, cells were collected, and PomA wasdetected as described in the legend to Fig. 2.

swim in the absence of DTT. On the other hand, in the presenceof DTT, the swimming speed of the cells gradually increased
with time. After 30 min, the swimming speed of the cells
producing PomA P172C recovered to about 40% compared withthat of the cells with wild-type PomA (Fig. 5A). We observed aconcomitant decrease in the amount of cross-linked PomA andan increase in the amount of monomeric PomA protein with

31390

Cross-linking between the Periplasmic Loops of PomA

FIG. 6. Formation of cross-links. Cells producing wild-type PomA(wt) or PomA P172C, K173C, or I175C were incubated in V-buffer withor without 10 mM DTT, then incubated in buffer with or without 2 mM
NEM, and finally incubated in buffer with 1 mM CuCl2. PomA separated
by SDS-PAGE was detected as described in the legend to Fig. 2. The
symbols ⫹ or ⫺ above the lanes indicate incubation with or without DTTor NEM. Numbers on the right side are the estimated molecular masses
corresponding to the bands indicated.

time. Immediately after DTT was added, the amount of thecross-linked PomA relative to total PomA in the cell was estimated to be about 80%. However, after 30 min in the presenceof DTT, the amount of the cross-linked PomA decreased to only
25% of the total. Thus, the defect in motor function correlatedwith the amount of cross-linked PomA in the cell.
Extent of Cross-linking of the Cys Mutant PomA in Whole
Cells—We sought to eliminate the possibility that cross-linking
occurs during the isolation of proteins after cell lysis. NEM,
which is a thiol group-specific reagent whose reaction is irreversible, was used to quench disulfide bond formation. Cells
producing Cys-PomA were treated with DTT, then with NEM,
and finally with CuCl2, and immunoblotting was carried out
with anti-PomA antibody (Fig. 6). Without DTT treatment, adecrease of the 43-kDa band in the P172C and K173C mutants
was observed after the addition of NEM, and the band disappeared completely in the I175C mutant. When the cells weretreated with both DTT and NEM, the 43-kDa band was significantly reduced in the P172C mutant and disappeared completely in the other two mutants. These results suggest that thedisulfide bond is cleaved with DTT and that the cleaved Cys
residue is modified with NEM, so that formation of the 43-kDa
band upon oxidation with CuCl2 is inhibited.
To examine whether P172C PomA is modified directly by
NEM, the membrane fraction of cells producing either wildtype or mutant PomA was prepared and treated with DTT
followed by [14C]NEM treatment. Labeled 25-kDa PomA, immunoprecipitated with anti-PomA antibody, was detected with
the P172C mutant but not with the wild type (data not shown).
This finding demonstrates that NEM directly modifies the thiolgroup of Cys in the P172C PomA protein.
Cross-linking of Cys-PomA Monomer—We wanted to establish that the 43-kDa protein is a cross-linked product of two
Cys-PomA molecules and not PomA cross-linked to anotherprotein. Membrane fractions were prepared from cells producing wild-type or P172C PomA and lysed with 1% (w/v) Triton
X-100. Proteins were immunoprecipitated with anti-PomA antibody, and the precipitated proteins were separated by SDSPAGE carried out with or without DTT and stained with Coomassie Brilliant Blue (Fig. 7). In the absence of DTT, the
25-kDa band or the 43-kDa band was detected with wild-typeor P172C PomA, respectively. These bands were not detected ina control in which PomA was not produced although an unknown band above the 43-kDa band was detected. In the presence of DTT, only the 25-kDa form was detected with either
wild-type or P172C PomA and the intensities of the bands weresimilar to the corresponding bands in the absence of DTT. In alllanes in the presence of DTT, the bands with a molecular massof about 50 kDa are the heavy chains of IgG. These results

FIG. 7. Protein composition of the 43-kDa band. Membrane fractions prepared from VIO586 cells producing wild-type (wt) or P172C
PomA were solubilized with 1% (w/v) Triton X-100, and immunoprecipitation was carried out with anti-PomA antibody. The precipitatedproteins were separated by SDS-PAGE in the absence (left) or presence(right) of 10 mM DTT and stained with Coomassie Brilliant Blue. The
antibody proteins were also stained by Coomassie Brilliant Blue in thegels. The numbers on the right side correspond to the positions ofmolecular size markers.

suggest that the 43-kDa band is composed entirely of crosslinked dimers between two Cys-PomA molecules.
DISCUSSION

The PomA protein of the Na⫹-driven flagellar motor of Vibrio
alginolyticus is predicted to have four transmembrane segments, whereas the PomB, MotX, and MotY proteins have onlya single transmembrane segment (8 –11). From the predicted
topology of PomA, the N- and C-terminal regions and the large
loop between transmembrane segments 2 and 3 are located in
the cytoplasm. Loop1–2 and loop3– 4, between transmembranesegments 1 and 2 and segments 3 and 4, respectively, arethought to be exposed to the periplasmic space (8). When theloops were reacted with biotin maleimide, the labeling pattern
was different; substitutions in loop3– 4 were biotinylated consistently with the membrane topology, whereas none of residues in loop1–2 was labeled (20). It has been proposed thatloop1–2 is associated with other proteins, such as the motor
proteins PomB, MotX, or MotY, or that it is embedded into thepore region of the channel, as is predicted for the extracellular
loops of many ion channels. It has been shown that the negative charge Asp-31 of loop1–2 contributes to optimal speed
and/or efficiency of the motor, although the charge is not essential (26). By random mutagenesis of the pomA gene, it was
shown that loop3– 4 and transmembrane segments 3 and 4 haveresidues more crucial for function than loop1–2 or transmembrane segments 1 and 2 (27).
In the present study, we focused on periplasmic loop3– 4. We
found that the P172C mutant protein of PomA is not functional
and inhibits the motility of the wild-type cell in the absence of
reducing reagents, although motility is restored by DTT. This
result suggests that a cross-link formed between two moleculesof Cys-PomA via loop3– 4 inhibits the function of the torquegenerating units. The interaction between cross-linked PomA
dimer and PomB is apparently not disrupted, since PomB appears to be co-precipitated about as well with the cross-linked
PomA dimer as with the wild-type PomA (data not shown). It isinferred that two molecules of native PomA interact with eachother intimately in a torque-generating unit of the motor. Recently, PomA alone was purified as a stable homodimer even if
PomB was absent (19). The purified PomA/PomB complex has
been reconstituted into proteoliposomes and has been shown tocatalyze Na⫹ influx. Furthermore, a tandem PomA dimer produced as a single polypeptide is functional. Inactivation ofeither half of the dimer results in complete loss of PomA func-

Cross-linking between the Periplasmic Loops of PomA
tion. When a phenamil-resistant mutation was introduced intoeither the first or second half of the tandem PomA dimer, theresistant phenotype was identical to the phenotype of the tandem dimer in which the both halves carried the mutation.
Thus, the two halves of the PomA dimer appear to function
together (28).
The H⫹-driven flagellar motor contains MotA and MotB,
which are homologues to PomA and PomB, respectively. It isthought that the MotA/MotB complex converts H⫹ influx into
the rotation of the flagellar motor (4, 6, 29). From the results of
Trp-scanning mutagenesis, a structural model of MotA/MotB
complex has been proposed in which a single transmembranesegment of one MotB protein assembles with four transmembrane segments of one MotA protein at a tilt relative to them(5). If this model holds for the H⫹-driven motor, the existence ofa heteromultimeric ion channel complex might be specific for
the Na⫹-driven flagellar motor. On the other hand, estimates ofthe MotA and MotB protein levels in the membrane of E. colisuggest that the ratio of MotA to MotB is about 4:1 (30). Thisfinding is consistent with the possibility that MotA and MotB
do not make 1:1 complex.
The MotA protein of the H⫹-driven flagellar motor of
Rhodobacter sphaeroides can generate torque in response to a
sodium-ion flux in a pomA mutant of V. alginolyticus (31).
MotA of R. sphaeroides may work in the Na⫹-driven motor
because the torque generators in the H⫹-and Na⫹-driven motors have similar structures, i.e. they are heteromultimer complexes. In any case, clarification of the structure of the torquegenerator is clearly important for an understanding of themechanism of coupling ion flow to flagellar rotation.
Two additional components, MotX and MotY, are required
for the rotation of the Na⫹-driven motor of V. alginolyticus, but
homologous proteins are not found in the H⫹-driven motor(9 –12). The functions of MotX and MotY are not clear, although
it is thought that they may be involved in ion recognition. MotX
and MotY do not co-purify with the PomA/PomB complex (19).
This observation may mean that MotX and MotY are not associated with the PomA/PomB complex, that the association is
weak, or that MotX and MotY are unstable during purification.
It will be interesting to determine the structural difference
between the torque generators of the Na⫹- and H⫹-driven
flagellar motors.
It has been proposed that the rotor is surrounded with multiple torque-generating units, approximately eight units in theH⫹-driven flagellar motor (32) or five to nine in the Na⫹-driven
motor (33). We can envision two models to explain the crosslinking of PomA molecules. One is an intra-torque-generator
model, in which the cross-links form between two PomA molecules in the same unit. In this argument, loop3– 4 must face into
the unit. The other possibility is an inter-torque-generator
model, in which the cross-link occurs between two PomA molecules in different units, so that the loop3– 4 faces outward. The
former model is supported by following criteria; the distance
between two generators may be too great for disulfide crosslink to form, PomA forms a stable dimer in the cell, and genetically connected tandem PomA is functional. We need moreexperiments to elucidate which model is correct.

31391

Residue Pro-172 by itself does not seem to be essential for
motor function, since motility is normal in PomA mutants inwhich Pro-172 was replaced by three amino acids other thanCys. Additionally, we observed recovery of swimming ability
when the cross-linked dimer was reduced in cells with P172C
PomA by DTT. We suggest that the flexibility of loop3– 4 isrestricted by the cross-linking, thereby impairing motor function. There are at least three possible mechanisms for thisinhibition of motor function. (i) Na⫹ influx is inhibited bydisruption of the channel structure. (ii) Conversion of the Na⫹
influx to conformational changes in the torque-generating stator units is inhibited. (iii) Interactions between the stator andthe rotor are inhibited. Understanding of the inhibitory mechanism might lead us to an understanding of the energy transduction mechanism in the flagellar motor.
Acknowledgment—We thank R. M. Macnab for critically reading themanuscript.
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