JOURNAL OF BACTERIOLOGY, Oct. 1999, p. 6332–6338
0021-9193/99/$04.00⫹0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 20

Hybrid Motor with H⫹- and Na⫹-Driven Components Can
Rotate Vibrio Polar Flagella by Using Sodium IonsYUKAKO ASAI,1 IKURO KAWAGISHI,1 R. ELIZABETH SOCKETT,2 AND MICHIO HOMMA1*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan,1and Genetics Division, Clinical Laboratory Sciences, Nottingham University, Queen’s Medical Centre,
Nottingham NG7 2UH, United Kingdom2
Received 1 March 1999/Accepted 2 August 1999

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into
flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine
bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX,
and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of
the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its
residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembraneregion of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA andpomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone andtransformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants fromboth strains showed restored motility, although the swimming speeds were low. On the other hand, pomB
mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions(proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer andwas inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus,
we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling withthe sodium ion flux in place of PomA of V. alginolyticus.
part is composed of MotA and MotB, which are similar to
those of the proton-driven motors (40, 41). The flagellar motorof R. sphaeroides is active in the absence of sodium ions and isinhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Amiloride, a specific inhibitor of thesodium-driven flagellar motor, inhibits motility; however, theeffect is nonspecific. These results indicate that the motor of R.
sphaeroides is the proton-driven type (37).
The marine bacterium Vibrio alginolyticus has two types offlagella, lateral (Laf) and polar (Pof). The lateral flagella,
which have proton-driven motors, are expressed when cells aretransferred to high-viscosity environments. The polar flagellahave sodium-driven motors and work better for swimming in
low-viscosity environments. The rotation of polar flagella isvery fast, about 1,700 rps in 300 mM NaCl at 35°C (29, 30, 35).
The sodium-driven motor consists of four components, PomA,
PomB, MotX, and MotY, all of which are essential for torquegeneration (2, 31, 32, 36). Of these, MotX and MotY, which
are predicted to be single transmembrane proteins, do nothave similarity to proton motor components or any other proteins, except for a C-terminal region of MotY which contains a
peptidoglycan-binding motif. MotY and MotX are thought tomake a complex in the inner membrane, and MotX is inferred
to be a sodium channel component (31, 32). On the other
hand, PomA and PomB are similar to MotA and MotB, respectively, of proton-type motor components which arethought to form a proton channel. It is proposed that PomA
has four transmembrane regions and a large cytoplasmic loop
and that PomB spans the membrane once near the N terminus
and has a conserved peptidoglycan-binding motif in the Cterminal region (2). Thus, we thought that PomA and PomB
may have similar structure and function to the proton-type
MotA and MotB from R. sphaeroides and that it might bepossible to compare the coupling mechanisms of the proton

Bacteria swim by rotating helical flagella toward favorable
stimuli in liquid environments. The motor complex generating
torque is embedded in the cytoplasmic membrane at the baseof the flagella, and it converts a specific ion flux (either a
proton or a sodium ion) to motor rotation (7).
Most studies have centered on the proton-driven motors ofEscherichia coli and Salmonella typhimurium. The rotor part ofthe motor is composed of FliG, FliM, and FliN (28, 47), which
form the C-ring structure on the cytoplasmic face of the MS
ring (13, 50). The C ring is also called the switch complex, andit determines the direction of flagellar rotation, counterclockwise (CCW) or clockwise (CW). On the other hand, the stator
complex consists of MotA and MotB, which interact via theirtransmembrane regions and function as a proton-conducting
channel (7, 8, 44). MotA has four transmembrane regions anda large cytoplasmic loop. Recently, it was proposed that somecharged residues in this loop interact with oppositely charged
residues in FliG when they generate torque (52). MotB has asingle transmembrane region including an important Asp,
which seems to be involved in proton transfer (43). Moreover,a peptidoglycan binding motif is conserved in the C-terminal
region of MotB and plays the role of anchoring the motor tothe cell wall (10, 11).
The photosynthetic bacterium Rhodobacter sphaeroides has asingle flagellum extending from the center of the cell body. The
cell is motile under a wide range of growth conditions, from pH
6 to 9 (37), and at speeds of up to 100 ␮m/s (38). The rotationof the R. sphaeroides flagellum is unidirectional in the CW
direction, and it stops and restarts periodically (1). The motor* 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@nucc.cc.nagoya-u.ac.jp.
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TABLE 1. Bacterial strains and plasmids used in this study
Genotype or descriptiona

Strain or plasmid

Reference or source

V. alginolyticus strains
VIO5
VIO586
NMB190
NMB152
NMB161
YM19

VIK4 laf (Rifr Pof⫹ Laf⫺)
VIO5 pomA (Rifr Laf⫺ Pom⫺)
VIO5 ⌬pomA (211-bp deletion) (Rifr Laf⫺ Pom⫺)
NMB201 pomB (Rifr Laf⫺ Pom⫺ Mpar)
NMB201 pomB (Rifr Laf⫺ Pom⫺ Mpar)
138-2 pof (Pof⫺)

36
2
This work2
25
23

E. coli strains
DH5␣
SM10␭pirF⫺ ␭⫺ recA1 hsdR17 endA1 supE44 thi-1 relA1 gyrA96 ⌬(argF-lacZYA)U169 ␾80dlacZ⌬M15
thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km ␭pirRK6

15
34

Plasmids
pSU41
pKY704
pYA301
pMK201-M2
pYA303pRED1pYA603
pYA6031
pYA701
pYA303-M2
pYA801kan (Kmr) Plac lacZ␣
Suicide vector (Cmr)
pSU41 0.8-kb BamHI-BamHI fragment (pomA⫹)
pSU21 0.6-kb BamHI-BamHI fragment (pomA); 211-bp deletion in pomA
pSU41 1.9-kb BamHI-SacI fragment (pomAB⫹)
pUC19 2.5-kb SphI-SalI fragment (motAB⫹)
pSU41 2.5-kb HindIII-EcoRI fragment from pRED1 (native promoter region and motAB⫹)
pSU41 1.6-kb HindIII-SmaI fragment (native promoter and motA⫹)
pSU41 0.8-kb BamHI-EcoRI fragment (motA⫹)
pSU41 1.7-kb BamHI-SacI fragment (pomA pomB⫹); 211-bp deletion in pomA
pKY704 1.7-kb XbaI-SacI fragment (pomA pomB⫹) from pYA303-M2

6
46
25
26
25
41
This work
This work
This work
This work
This workaKmr, kanamycin resistant; Cmr, chloramphenicol resistant; Mpar, motility resistant to phenamil; Rifr, rifampin resistant; Plac, lac promoter.

and sodium ion flux for force generation. In addition, mutations conferring resistance to phenamil, a specific inhibitor ofa sodium-driven motor or sodium channels (5), are found inboth pomA and pomB (25). The phenamil-resistant mutants
have also been isolated in V. parahaemolyticus, which is closely
related to V. alginolyticus, and the mutations have been
mapped in the homologous genes of either of pomA and pomB(17). These results strongly support the notion that PomA and
PomB form a sodium-conducting channel.
In the case of another rotary machine, FoF1 ATPase (theenzyme that couples ion flux to ATP synthesis), the couplingions can be proton or sodium, as with the bacterial flagellar
motor. The Fo part of the structure is embedded in the membrane and consists of a, b, and c subunits (12). It has beensuggested that the c subunits determine the ion specificity (19).
The proton-type c subunits of E. coli and the sodium-type
subunits of Propionigenium modestum have 25% identity (22).
It has been found that a hybrid enzyme composed of the Fopart from the sodium type and the F1 part from the proton typeis functional and shows different ion specificities depending on
the conditions (18, 20, 27). Moreover, the ion recognition sites
have been proposed based on comparison of amino acid sequences between the c subunits (49). Recently, it has beensuggested that the coupling ion selectivity of FoF1 ATPase also
involves the a subunit of the Fo part (21).
Ultimately, we hope to determine the torque-generating region or ion recognition sites of flagellar motors by comparison
between R. sphaeroides MotA and V. alginolyticus PomA. As aprecursor to this work, we sought to find the effects of introducing the entire R. sphaeroides motA and motB genes into
pomA or pomB mutants of V. alginolyticus, respectively.
MATERIALS AND METHODS
Bacterial strains, plasmids, growth conditions, and media. The strains andplasmids used are shown in Table 1. V. alginolyticus cells were cultured at 30°C
in VC medium (0.5% Polypepton, 0.5% yeast extract, 0.4% K2HPO4, 3% NaCl,
0.2% glucose) or VPG medium (1% Polypepton, 0.4% K2HPO4, 3% NaCl, 0.5%
glycerol). When necessary, chloramphenicol and kanamycin were added to final

concentrations of 2.5 and 100 ␮g/ml, respectively. E. coli cells were cultured at
37°C in LB medium (1% Bacto Tryptone, 0.5% yeast extract, 0.5% NaCl). For E.
coli, chloramphenicol and kanamycin were added to final concentrations of 25
and 50 ␮g/ml, respectively. The swimming speed was measured in Tris motility
buffer TMN300 (300 mM NaCl, 50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM
glucose), TMK300 (300 mM KCl, 50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM
glucose) or mixtures of TMN300 and TMK300 to vary the sodium concentration.
DNA manipulations. Routine DNA manipulations were carried out by standard procedures (39). Restriction endonucleases and other enzymes for DNA
manipulations were purchased from Takara Shuzo (Siga, Japan) and New England Biolabs (Beverly, Mass.).
Plasmid construction and site-directed mutagenesis. The inserted SphI-SalI
fragment of pRED1 (41) contains the whole motAB operon including the putative ␴54-dependent promoter region. At first, we inserted the HindIII-EcoRI
fragment of pRED1 (including that SphI-SalI fragment and parts of the multiplecloning site) into vector pSU41, which can be maintained in Vibrio. The resulting
plasmid was named pYA603 (see Fig. 1b). Next, we digested pYA603 with SmaIand ligated it to delete the latter half of motB. The multiple-cloning site ofpSU41 does not have a SmaI site, but the transferred parts of the multiplecloning site from pRED1 have such a site. This deletion derivative was named
pYA6031. We constructed a plasmid, pYA701, including the PCR-amplified
motA fragment from 40 bp upstream of the start codon to 3 bp downstream ofthe stop codon. The PCR amplification was done as described previously (25). In
the reaction, we used the end primers RsmotA.B1 and RsmotA.E2, as well as
pYA6031, as templates. RsmotA.B1 has a BamHI site and is homologous to thesense strand of the upstream RsmotA gene, 5⬘-GCGGGATCCTGCCGCTCCG
GACCTGGATGA-3⬘. RsmotA.E2 including an EcoRI site is complementary to
the antisense downstream of the gene, 5⬘-CTCGAATTCGCCTCACGCCGCC
TTGCGCT-3⬘. This PCR-amplified fragment was inserted into pSU41 between
the BamHI site and the EcoRI site. The motA gene of pYA701 is under the lacpromoter directly and does not contain the native promoter.
To introduce mutations into pYA701, we used the two-step PCR method (25).
We synthesized pairs of mutant primers, RsmotAT186C.1, designed for the sense
strand (5⬘-GCTGCTCACGTGCCTCTACGGCG-3⬘), and RsmotAT186C.2, designed for the antisense strand (5⬘-CGCCGTAGAGGCACGTGAGCAGC-3⬘).
In addition to these primers, we used both of the end primers and amplified thefull-length R. sphaeroides motA gene. This mutated motA fragment was cloned
into pSU41 (6), and the sequence was checked.
Transformation of the Vibrio cells. Transformation of Vibrio cells was carriedout by electroporation as described previously (24). The cells were subjected toosmotic shock and washed thoroughly with 20 mM MgSO4. Electroporation was
carried out with the Gene Pulser electroporation apparatus (Japan Bio-Rad
Laboratories, Tokyo, Japan) at an electric field strength between 5.0 and 7.5
kV/cm.
Construction of a pomA null mutant mutated on the chromosome. The 0.4-kb
HindIII fragment of a mutant pomA gene (allele 2 in reference 26) from

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J. BACTERIOL.

pMK201-M2, which has a 211-bp deletion in the gene, was replaced with the
0.6-kb HindIII fragment of pomA in pYA303, and the resultant plasmid wasnamed pYA303-M2. The XbaI-SacI fragment in pYA303-M2 was moved to asuicide vector, pKY704 (46). The resultant suicide plasmid (pYA801) was transformed into E. coli SM10␭pir (34), and conjugal transfer was done to introduce
pYA801 into V. alginolyticus VIO5. The suicide plasmids are expected to integrate into the chromosomal pomA gene. Transconjugants which showed chloramphenicol resistance were inoculated on a semisolid agar VPG plate to selectmotility mutants. Three Pom⫺ colonies were found in 100 chloramphenicolresistant transconjugants. Next, these Cmr Pom⫺ cells were cultured in liquid
broth without antibiotics and inoculated into new broth every day, and single
colonies were isolated on a chloramphenicol-free VC hard agar plate. On day 4,
we found one Cms Pom⫺ colony. We confirmed the deletion mutation in thechromosomal pomA gene by the amplified fragment size of PCR and the complementation test with pYA301 and pYA303. This mutant was named
NMB190(⌬pomA).
Detection of R. sphaeroides MotA protein by antipeptide antibody. Preparationof cells and immunoblotting were done as described previously (48). For the firstantibody of immunoblotting, we used antipeptide antibody against R. sphaeroides
MotA (RsMotA) or V. alginolyticus PomA (VaPomA). The production andaffinity purification of anti-RsMotA antibody were carried out by Sawady Technology (Tokyo, Japan). The selected peptide fragment was synthesized artificially, with the sequence NH2-CIEDQMVCALDRKQQMKRKAA-COOH,
which is located at the C terminus of MotA. A cysteine was added to the N
terminus, and the synthesized fragment was conjugated to keyhole limpet hemocyanin by it. Rabbits were immunized by the conjugated keyhole limpet
hemocyanin. PomA91 antibody was used as an anti-VaPomA antibody, generated against three synthetic peptides designed for the different regions of PomA
(48). For the second antibody, we used an alkaline phosphatase-conjugated goatanti-rabbit immunoglobulin G antibody.
Measurement of swimming speed. An overnight culture in VC medium was
inoculated into fresh VPG medium at a 100-fold dilution and grown at 30°C toexponential phase. Cells were harvested by centrifugation and suspended in
TMN50 (50 mM NaCl, 250 mM KCl, 50 mM Tris-HCl [pH 7.5], 5 mM MgCl2,
5 mM glucose). The cell suspension was diluted about 100-fold into Tris motility
buffers containing various concentrations of NaCl and 20 mM serine (to suppress
the directional change of swimming). Motility of the cells was observed under adark-field microscope and recorded on videotape. The swimming speed wasdetermined as described previously (3).
Behavioral assay. Tumbling frequency was determined as described previously(16). Cell suspension as prepared for measurement of swimming speed was
diluted about 100-fold into TMN50, and individual chemoeffectors were added.
Within 1 min of addition, cell motility was observed under a dark-field microscope and recorded on videotape. Each frame (30 frames in 1 s) on the videotapes was played back on a monitor and the number of times the cell changed
direction in 1 s was noted. At least 20 cells were measured for any given
condition.

RESULTS
Introduction of R. sphaeroides motA and motB into the pom
mutants of V. alginolyticus. The proton-type MotA of R.
sphaeroides is very similar to the sodium-type PomA of V.
alginolyticus (2). Both are 253-amino-acid proteins, and they
have ca. 40% identity over the entire region (Fig. 1a). Moreover, the transmembrane regions of MotB and PomB have alsohigh similarity (2, 40).
As shown in Fig. 1b, in pYA301, pYA303, and pYA701, thepomA, pomAB, and Rhodobacter motA genes were controlled
under the lac promoter directly. pYA603 and pYA6031 include Rhodobacter motAB and motA genes under the native
promoter. These plasmids were introduced into the pomA mutant, VIO586, and the pomB mutant, NMB161. Figure 2ashows the motility of the transformants. The transformed
VIO586 was restored to motility by pYA603, pYA6031 (datanot shown), and pYA701, which contain the motA gene of R.
sphaeroides, although the swarm size was smaller than that ofcells transformed by pYA301, which contain the pomA gene.
On the other hand, the transformed pomB mutants, NMB161
and NMB152, could not have motility restored.
We thought that the defective PomA of VIO586 might besuppressed by MotA, and we identified the pomA mutation siteof VIO586 by amplifying and sequencing the PCR fragment of
the whole pomAB operon. Only the G154R mutation was
found (Fig. 1a). If nonfunctional PomA proteins were ex-

FIG. 1. (a) Amino acid alignments of V. alginolyticus PomA and R.
sphaeroides MotA. White letters in black boxes, diamond, and star show identicalresidues, the mutation site of the PomA mutant VIO586 (G154R), and thethreonine residue that is highly conserved among all species of MotA, respectively. Dotted bars indicate putative transmembrane regions. (b) Restriction mapof plasmids. White boxes and the direction of the arrowheads indicate the vector
part of pSU41 and the direction of translation from the lac promoter, respectively. Insertional fragments are indicated by solid lines. The bold lines in theinserted fragments indicate the region of the native promoter. The open arrows
show the coding regions of PomA, PomB, MotA, and MotB. Abbreviations: B,
BamHI; H, HindIII; E, EcoRI; Sp, SphI; X, XhoI; Sm, SmaI; Sl, SalI.

pressed in VIO586, it was also possible that R. sphaeroides
MotA could “suppress,” not complement, this PomA mutation. Thus, we constructed a pomA null mutant to examine thepossibility.
The pomA deletion allele has been isolated by Kojima et al.
(26). Using this mutant pomA and suicide vector pKY704, weconstructed a strain (NMB190) carrying the chromosomal deletion mutation of pomA. Even in this strain, motA of pYA701
could restore motility to the same extent as in VIO586 (Fig.
2b).
The threonine residue in the fourth transmembrane region(Fig. 1a) is conserved among sodium-type PomA and all of thereported proton-type MotAs. This residue corresponds to position 202 in E. coli, and 186 in V. alginolyticus and R.
sphaeroides. It was proposed that a mutation such as T202W in
E. coli and T186C or I in V. alginolyticus would be nonfunctional (26, 42). We introduced the T186C mutation into
pYA701 and examined if the mutated R. sphaeroides MotA

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6335

FIG. 3. Detection of the R. sphaeroides MotA protein by the antipeptide
antibody. NMB190 cells harboring each plasmid (lanes 1 and 5, pSU41; lanes 2
and 6, pYA301; lanes 3 and 7, pYA701; lane 4, pYA701-T186I) were cultured
until the mid-log phase, harvested, and suspended in distilled-deionized water.
Immunoblottings were done with anti-RsMotA antibody (a) and anti-PomA
antibody (b).

FIG. 2. Swarming abilities of transformants. Fresh colonies were inoculated
in 0.3% agar VPG plates containing 100 ␮g of kanamycin per ml and incubatedat 30°C for 5 h. (a) Motility of the pomA mutant (VIO586) and the pomB mutant(NMB161) as the host strains were transformed with plasmids pYA301, pYA603,
pYA701, pSU41, and pYA603. The introduced plasmids and the host strains arenoted on the left and right, respectively. (b) Effect of the T186C mutation of R.
sphaeroides MotA. Two pomA mutants, VIO586 and NMB190, were transformed
with plasmids pYA701 and pYA701-T186C.

could restore motility (Fig. 2b). Wild-type MotA (pYA701)
restored motility to both pomA mutants. In contrast, mutant
MotA (pYA701-T186C) did not. Other nonfunctional aminoacid substitutions at highly conserved residues, T186I andP199L (P199 corresponds to P222 in E. coli) (9, 26, 51), also
did not restore motility (data not shown). These results suggested that R. sphaeroides MotA, which is a proton component,
could work as a replacement for PomA in V. alginolyticus.
Detection of R. sphaeroides MotA. We tried to detect the R.
sphaeroides proteins by immunoblotting with anti-RsMotA
synthetic peptide antibody (Fig. 3a). A band with a molecularmass of 25 kDa was detected specifically from NMB190 harboring pYA701 (lane 3). The molecular mass (25 kDa) isalmost equal to the predicted size of R. sphaeroides MotA (27kDa). On the other hand, in the case of anti-VaPomA antibody(Fig. 3b), no band appeared in NMB190 harboring pYA701(lane 7); however, the V. alginolyticus PomA band could be
detected in NMB190 harboring pYA301 (lane 6). From theseresults, we assessed that this anti-RsMotA antibody could detect specifically R. sphaeroides MotA. None of the nonfunctional mutant R. sphaeroides MotA proteins could be detectedby our system (for example, T186I in lane 4). The mutant
proteins might be degraded more rapidly than the wild type ormight be changed in this antigenicity.

Characterization of the hybrid motor between the Hⴙ andNaⴙ types. NMB190 with pYA701 should have hybrid motors
composed of H⫹-type MotA and Na⫹-type PomB, MotX, andMotY. Which ions couple to the flagellar rotation in the motors? We examined this question by performing several experiments. In the first, we asked whether cells can swim in theNa⫹-free buffer TMK300 and whether the swimming speed of
the cells is dependent on the Na⫹ concentration. As the results(Fig. 4a) show, the hybrid motor (pYA701/NMB190) could not
rotate in Na⫹-free buffer as the Na⫹-type motor (pYA301/
NMB190). Moreover, the speed of swimming generated by thehybrid motor increased with the Na⫹ concentration, although
the maximum speed was low and saturated at lower Na⫹ concentration than that by the Na⫹-type motor of pYA301/
NMB190. Second, we asked whether the free swimming is
inhibited by phenamil, an Na⫹-driven motor-specific inhibitor.
The swimming speeds were measured in various concentrations of phenamil (Fig. 4b). We found that pYA701/NMB190
cells were inhibited in motility strongly with the increase of thephenamil concentration, and stopped almost completely with
50 ␮M phenamil. pYA301/NMB190 cells showed similar results. They were also inhibited in motility by amiloride (Fig.
4c). The motility inhibition of pYA701/NMB190 cells by amiloride was competitive with sodium ions such as that of pYA301/
NMB190 cells, although their initial swimming speeds weredifferent (Table 2). Finally, we tested the effects of a protonophore, CCCP (Table 3). In alkaline Tris motility buffer,
TMN300 (pH 8.5), YM19 (Pof⫺ Laf⫹) cells could swim in theabsence of 20 ␮M CCCP and could stop completely in thepresence of CCCP, because lateral flagella have proton-driven
motors. On the other hand, pYA301/NMB190 and pYA701/
NMB190 cells did not stop in the presence of 20 ␮M CCCP,
but they both stopped completely after the further addition of
20 ␮M 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a specific inhibitor of the Na⫹ pump (45). We concluded from theseresults that the hybrid motor of pYA701/NMB190 rotates theflagella by using Na⫹-motive force.

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TABLE 3. Effect of pH and CCCP on swimming speedNa⫹ concn
(mM)

pH

300

7.5

CCCP concn
(␮M)

8.5
0

7.5

Swimming speeda (␮m 䡠 s⫺1) for:pYA301/NMB190pYA701/NMB190

YM19

0
20
0
20

63
10
64
64 (0)

28
8
26
23 (0)

27
0
20
0

0

0

0

22

a

The swimming speed after HQNO (20 ␮M final) addition is shown in parentheses.

DISCUSSION

FIG. 4. Swimming speed of NMB190 cells harboring pYA301 or pYA701. (a)The cells suspended in Tris motility buffer were diluted 100-fold into buffer
containing various concentration of sodium ions, and the swimming speed wasmeasured. (a⬘) Enlargement of the low-Na⫹-concentration part of panel a. (band c) The swimming speed was measured at various concentrations of thespecific inhibitor of the sodium motor, phenamil (b) or amiloride (c). The sodium
concentration was 50 mM in all measurements.

We tested the chemotaxis of pYA701/NMB190, because
pYA701/NMB190 formed a very small swarm on a semisolid
agar plate even though its swimming speed in liquid medium
was not so low. The tumble frequency of pYA301/NMB190
was increased from the basal level after 2 mM phenol wasadded as a repellent (Table 4). After the further addition of 20
mM serine as an attractant, the tumble frequency decreased to0.40 s⫺1 from 3.10 s⫺1. In contrast, pYA701/NMB190 showedsmooth-biased swimming as a basal condition and little response to either repellent or attractant.

TABLE 2. Effect of Na⫹ concentration on the inhibition of motility
by amiloride
Swimming speed (␮m 䡠 s⫺1) for:Na⫹ concn(mM)

Amiloride concn(mM)

pYA301/NMB190

pYA701/NMB190

10

0
0.8
0
0.8
0
0.8

33
6
54
18
56
27

23
2
28
6
28
12

50
300

Recently, two sodium-driven motor components, PomA and
PomB, were identified in addition to MotX and MotY (2).
They are similar to the proton motor components MotA and
MotB and different from MotX and MotY. Thus, we thoughtthat PomA and PomB might function as a sodium channel andhoped that it might be possible to study the ion specificity byvarious chimeric proteins between PomA and MotA. However,surprisingly, we found that the whole R. sphaeroides motA gene
could partially complement a pomA mutant of V. alginolyticus.
This worked despite the different codon usages of Rhodobacter
and Vibrio genes (which may account for the less than 100%
complementation that was achieved). We had previously tested
for motility restoration of pomA mutants by E. coli MotA, but
it did not work (data not shown). This is probably because E.
coli MotA has lower similarity to Vibrio PomA than does R.
sphaeroides and could not precisely interact with PomB. Thus,
the lateral motor components LafT and LafU of Vibrio seemnot to be interchangeable with PomA and PomB, because they
are highly similar to E. coli MotA and MotB but not PomA and
PomB (32). As shown in Fig. 1a, the similarity between the
PomA and R. sphaeroides MotA transmembrane regions is veryhigh. The large cytoplasmic loop regions have poor similarity,
although some important residues for torque generation wereconserved, for example, two proline, arginine, and aspartate
residues proposed by Zhou et al. in E. coli MotA (51). It has
been also proposed that the cytoplasmic loop of E. coli MotA
interacts with the rotor part directly (52). Therefore, we speculate that if the transmembrane regions could form a functional channel, the conformational change coupling with theion flux could be transmitted to the rotor since some important
residues were at least conserved. If this is true, a chimeric
protein, which consists of the transmembrane regions of R.
sphaeroides MotA and the cytoplasmic loop of V. alginolyticus
PomA, might propel the cell at the high speed generated by thenative motor. This is currently being studied.
Which ion flux, proton or sodium, was coupled to the force
generation in this hybrid motor? We conclude that sodium ionswere involved in the torque generation of the hybrid motor for
the following reasons. V. alginolyticus has two types of flagella,
TABLE 4. Chemotaxis of NMB190 cells having pYA301 or pYA701
Tumble frequency (s⫺1) for:

Phenol concn(mM)

Serine concn(mM)

pYA301/NMB190

pYA701/NMB190

0
2
2

0
0
20

1.81
3.10
0.40

0.20
0.25
0.05

VOL. 181, 1999

HYBRID MOTOR WITH Na⫹- AND H⫹-DRIVEN COMPONENTS

polar and lateral; the latter is a proton type, and it is known
that the mutants defective in polar flagella can swim in sodiumfree buffer by using the proton-driven Laf (4, 23) (Table 3).
However, the hybrid motor could not rotate in sodium-free
motility buffer at pH 7.5, when V. alginolyticus can make asufficient proton gradient. Phenamil and its analogs, which arepotent inhibitors of the sodium channels of various organisms(5), inhibit polar flagellar motors but not lateral ones. In particular, phenamil is a more specific inhibitor for the motor
function because target sites of phenamil are found in thesodium motor proteins PomA and PomB (17, 25). Both thehybrid and native motors were inhibited, being severely dependent on the concentration of phenamil (Fig. 4b) and amiloride(Fig. 4c). The motility inhibition of the hybrid motors by amiloride was also competitive with sodium ions (Table 2). Thisshows that the effect of amiloride is specific in the hybrid
motor. Moreover, it has been shown that the sodium-driven
polar flagella can rotate even though the proton motive force
is abolished by CCCP, because Vibrio has respiration-coupledNa⫹ pumps that are active only in an alkaline pH range (4, 23).
The hybrid motor in pYA701/NMB190 and the sodium motor
in pYA301/NMB190 did not stop in the presence of 20 ␮M
CCCP under the alkaline condition. These results suggest thatthe hybrid motor is not dependent on the proton motive force.
This raises the question of which components decide ion
selectivity. The most potent candidate seems to be PomB. Its
N-terminal transmembrane region is thought to form a sodium
channel with PomA, and it includes the important Asp that
seems to be involved in proton transfer. The similarity between
MotB and PomB in the N-terminal transmembrane region isvery high. In this study, we showed that R. sphaeroides MotB
could not suppress the pomB mutants (Fig. 2a). Thus, it is notclear whether PomB decides ion selectivity. The other candidates are MotX and MotY, sodium-type specific components(31, 32). Actually, it was suggested that MotX was a channel
component of a sodium motor from the evidence that overproduction of MotX was lethal to E. coli at some sodium
concentrations and that this effect was suppressed by the addition of amiloride (31). From the present information, we
speculate that PomA, PomB, MotX, and MotY act together toform a sodium channel complex in Vibrio motor; moreover,multiple ion binding sites are located in these motor complexes
and may recognize sodium ions.
It has been shown that chemotactic signals are transduced to
the C ring of the flagellar rotor by phospho-CheY and that thebinding of phospho-CheY changes the rotor conformation,although little is known about how the direction is determined.
As shown in Table 4, pYA701/NMB190 cells rarely change
direction, compared with pYA301/NMB190 cells. To explain
this phenotype, we suggest that the MotA cytoplasmic loop
interacts poorly with the C ring in hybrid motors because it haspoor similarity to the PomA cytoplasmic loop and that thisimperfect rotor-stator interaction is enough to generate torquebut not to change the direction of flagellar rotation properly. It
was reported that a single mutation in the E. coli MotA
periplasmic loop resulted in a CW-biased phenotype (14), andthe mutation in periplasmic space is supposed to affect thestructure of the cytoplasmic loop and change the interaction
with the switch complex of the rotor.
This work has shown that a component of a proton-driven
flagellar motor can substitute for a component of a sodiumdriven motor. It has opened the way for further studies on
chimeric motor proteins, which should determine how different ions can be used to power flagellar rotation and should leadto a greater understanding of the whole flagellar rotation andswitching processes themselves.

6337

ACKNOWLEDGMENTS
We thank K. Yamamoto for plasmids and R. Macnab for criticallyreading the manuscript.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan(to I.K. and M.H.) and the Japan Society for the Promotion of Science(to Y.A.) and by grants from the Royal Society (to R.E.S.).
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