Article No. jmbi.1998.2377 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 285, 1537±1547

Na-driven Flagellar Motor Resistant to Phenamil, an
Amiloride Analog, Caused by Mutations in Putative
Channel Components
Seiji Kojima1, Yukako Asai1, Tatsuo Atsumi2, Ikuro Kawagishi1
and Michio Homma1*
1

Division of Biological Science
Graduate School of Science
Nagoya University, Chikusa-ku
Nagoya 464-8602, Japan2

Department of Physics
Suzuka National College ofTechnology Shiroko-cho, Suzuka
Mie 510-0244, JapanThe rotation of the Na-driven ¯agellar motor is speci®cally and strongly
inhibited by phenamil, an amiloride analog. Here, we provide the ®rst
evidence that phenamil interacts directly with the Na-channel components (PomA and PomB) of the motor. The alterations in Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or
pomB genes. We cloned and sequenced pomA and pomB from two Mparstrains, NMB205 and NMB201, and found a substitution in pomA
(Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Bothresidues are predicted to be near the cytoplasmic ends of the putativetransmembrane segments. Mutational analyses at PomA-Asp148 and
PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Co-expression of
the PomA D148Y and PomB P16S proteins resulted in an Mpar phenotype which seemed to be less sensitive to phenamil than either of thesingle mutants, although motility was more severely impaired in theabsence of inhibitors. These results support the idea that PomA and
PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-af®nity phenamil-binding site at the inner face of the PomA/PomB channel complex.
# 1999 Academic Press*Corresponding author

Keywords: ¯agella; phenamil; amiloride; motor; sodium channel

Introduction
Bacterial ¯agellar motors are very tiny molecularengines driven by electrochemical gradients of a
speci®c ion, either H or Na, across the cytoplasmic membrane (Blair, 1995; Imae & Atsumi,1989). These motors propel the cells by rotating
helical ¯agellar ®laments, which function as propellers. A motor has several functionally independent force-generating units which are directly
responsible for the mechanochemical coupling in
the motor (Block & Berg, 1984; Blair & Berg, 1988;
Muramoto et al., 1994). In the H-driven motor,
two integral membrane proteins, MotA and MotB,
Present address: T. Atsumi, Protonic Nanomachine
Project, ERATO Japan Science and Technology
Corporation, 1-7 Hikaridai, Seika, Kyoto, 619-0237,
Japan.
E-mail address of the corresponding author:
g44416a@nucc.cc.nagoya-u.ac.jp0022-2836/99/041537±11 $30.00/0

are thought to be the components of the force-generating units (Dean et al., 1984; Stader et al., 1986).
MotA and MotB have four and one transmembrane segments, respectively (Zhou et al., 1995;
Chun & Parkinson, 1988), and it is inferred thatthey together form a proton-conducting channel(Blair & Berg, 1990; Stolz & Berg, 1991; Garza et al.,
1995; Sharp et al., 1995b). MotB has a long extracellar domain that is believed to attach to the peptidoglycan layer (Chun & Parkinson, 1988; Blairet al., 1991; De Mot & Vanderleyden, 1994). It has
therefore been suggested that the MotA/MotB
channel complex is the stator, anchored to the cellwall by the peptidoglycan-binding domain of
MotB.
Some critical residues of MotA and MotB
involved in torque generation have been reported.
Random mutagenesis of motA and motB revealed
that many mutations in the membrane domains of
MotA or MotB abolished their functions (Blair &Berg, 1991; Blair et al., 1991). Especially, Asp32 of# 1999 Academic Press

1538
MotB, a unique acidic residue in the membranesegment of MotB, is speculated to function in conveying protons. This Asp residue is well conserved
among species. Recently 15 different substitutions
were studied at this position; only the conservative
replacement D32E mutant retained any function(Zhou et al., 1998b). On the other hand, cytoplasmic domains of MotA might interact with therotor, especially with the switch proteins (FliG,
FliM, FliN) that function to control the direction of
the rotation of the motor, and which are all locatedon the cytoplasmic side of the membrane and constitute the C-ring (Irikura et al., 1993; Sockett et al.,
1992; Francis et al., 1994). Among these switch proteins, it has been shown that FliG is most directly
involved in torque generation, and might be associated with MotA (Tang et al., 1996; Lloyd et al.,1996). A recent report suggested that three charged
residues of FliG, Arg281, Asp288, Asp289, predicted to be on one surface, play important roles intorque generation (Lloyd & Blair, 1997). Moreover,it was suggested that two cytoplasmic charged
residues of MotA, Arg90 and Glu98, are involved
in torque generation (Zhou & Blair, 1997). It is
speculated that these charged residues of FliG and
MotA might engage in electrostatic interactions
with each other and that these interactions areimportant for motor function (Zhou et al., 1998a).
The Na-driven motor has some characteristics
distinct from the H-driven type. The Na-driven
polar ¯agellar motor of Vibrio alginolyticus rotates
stably and remarkably fast, up to 1700 r.p.s.
(Magariyama et al., 1994; Muramoto et al., 1995), as
measured by laser dark-®eld microscopy (LDM)(Kudo et al., 1990). Four transmembrane proteins
essential for torque generation, PomA, PomB,
MotX, and MotY (Figure 1), were recently identi®ed in the Na-driven polar ¯agellar motor of
V. alginolyticus (Okunishi et al., 1996; Asai et al.,1997). MotX and MotY, which were ®rst identi®ed
in V. parahaemolyticus (McCarter, 1994a,b), eachhave a single putative transmembrane segment,
and PomA and PomB have four and one transmembrane segments, respectively. Because of
sequence similarity to MotA and MotB, PomA and
PomB are thought to be the Na channel com-

Phenamil Resistant Mutations

ponents. MotX is also inferred to be a Na channel
component of the motor because overproduction of
MotX is lethal in Escherichia coli in proportion to
the external Na concentration, and because thislethality is suppressed by the addition of amiloride, which is known to be a potent inhibitor of Nachannels (Cragoe et al., 1992) and of Na-driven¯agellar motors (Sugiyama et al., 1988). Now,
residues in these components that might play
important roles in torque generation are being
intensively investigated.
Amiloride speci®cally inhibits Na-driven ¯agellar motors by competing with Na in the medium,suggesting that it interacts with the sodium binding sites located at the outer side of the motors(Sugiyama et al., 1988). Among amiloride analogs,
phenamil is the most potent inhibitor for theNa-motor. In contrast to amiloride, inhibition byphenamil is non-competitive with respect to Na
in the medium (Atsumi et al., 1990), suggestingthat a major interaction site of phenamil on the
motor is not identical to the sodium binding siteon the motor. Interestingly, motor rotation rate
¯uctuates greatly in the presence of phenamil, but
not amiloride, a phenomenon that can be explainedin terms of a relatively slow dissociation rate ofphenamil from the force-generating units ascompared with amiloride (Muramoto et al., 1996).
Phenamil might interact more strongly with its siteand bind more stably than amiloride to the Nachannel of the motor. In addition, because ofstructural similarity, phenamil might also be able
to associate with the amiloride-binding site by low
af®nity. Previously, we isolated motility mutants
resistant to phenamil (Mpar), whose motors rotate
more stably than wild-type in the presence ofphenamil (Kojima et al., 1997). This steadierrotation of the Mpar motors can be explained by
the rapid dissociation of phenamil from the highaf®nity binding site in the Na channel of Mpar
motor as compared with the wild-type. Therefore,
these Mpar mutations presumably affect genes
encoding components of the force-generating units,
such as pomA, pomB, motX and motY. Here, wehave mapped the Mpar mutations to speci®c sites
in PomA and PomB, the putative Na channelforming proteins. The notable importance of thesesites in torque generation is discussed.

Results
The Mpar mutation of strain NMB201 is in
pomA and/or pomB

Figure 1. Bacterial ¯agellar motors of the sodiumdriven and proton-driven type.

On the basis of evidence presented in theprevious study of Mpar strains (Kojima et al., 1997),
we expected that the Mpar mutations would befound in the genes coding the channel componentsof the Na-driven motor, such as the pom (polar¯agellar motility) genes (Okunishi et al., 1996;
Figure 1). We therefore sought to analyze the pom
genes of the Mpar strains NMB201 and NMB205
by using the pom mutants with paralyzed polarPhenamil Resistant Mutations

1539¯agella derived from them. Such a Pomÿ Mpar
double mutant was isolated ®rstly from NMB201,
and named NMB161. Motility was restored to thisstrain when the plasmid pHK2, harboring wildtype pomA and pomB genes, was introduced. Next,
sensitivity to phenamil was examined in the transformed cells (NMB161/pHK2). As shown in
Figure 2, motility of the NMB161/pHK2 cells wascompletely inhibited by 50 mM phenamil, as isobserved in the PomAÿ strain VIO586 whichharbored pHK2 (wild-type control), suggestingthat the Mpar phenotype of NMB161 was suppressed by the wild-type pomA and/or pomB gene.
This suggests that the Mpar mutation of NMB201(and NMB161) is in the pomA or pomB gene.
The PomA mutation D148Y confers
phenamil resistance
Since NMB201 and NMB205 show very similar
Mpar phenotypes (Kojima et al., 1997), we tried toclone fragments containing the pomA and pomB
genes from NMB201 and NMB205 by shot-gun
cloning. Fortunately, a 2.9 kb SacI fragment containing pomA and pomB derived from NMB205 was®rstly isolated by the complementation of thepomA mutant VIO586, so we advanced the analysis
of Mpar mutation of NMB205. Then, from this SacI
fragment we subcloned the pomA and pomB genes
into the plasmids pSK502 and pSK602, respectively(Figure 3(a)). We measured swimming speeds invarious concentrations of phenamil of the VIO586(PomAÿ) or NMB104 (PomBÿ) cells containing
pSK502 or pSK602, respectively (Figure 3(b)). Motility of the wild-type control strains, VIO586/
pSK1(pomA) and NMB104/pHK4(pomB), werecompletely inhibited by 50 mM phenamil. On theother hand, cells of the VIO586/pSK502 strain,
which harbored pomA derived from the mutant
NMB205, showed phenamil-resistant motility,

Figure 2. Inhibition of motility of NMB161/pHK2
cells by phenamil. Cells were harvested at late logarithmic phase and suspended in TMN medium (pH 7.5,
50 mM NaCl). The cell suspension was diluted about
100-fold into TMN medium containing various concentrations of phenamil. Swimming speed was measured as
described in Materials and Methods.

Figure 3. Single amino acid substitution in thecytoplasmic face of PomA causes phenamil resistance.
(a) Restriction map of plasmids containing genes of awild-type strain (pomA and pomB; pHK2, pomA; pSK1,
pomB; pHK4) or Mpar strain NMB205 (pomA and pomB;
pSK401, pomA; pSK502, pomB; pSK602). Genes from
NMB205 were isolated by shot-gun cloning, using the
VIO586 (PomAÿ) strain as a recipient, and thensubcloned. (b) Cells containing pomA derived from the
Mpar strain NMB205 show phenamil-resistant motility.
Swimming speed of cells containing either the pomA
gene (left panel) or the pomB gene (right panel) derived
from the Mpar strain NMB205 (*), or from a wild-typestrain (*), was measured in TMN medium (NaCl
50 mM) containing various concentrations of phenamil,
as described in Materials and Methods. For the hoststrain, VIO586 (PomAÿ) and NMB104 (PomBÿ) wereused, in the left and right panels, respectively. (c) The
predicted transmembrane regions of PomA. The Mpar
mutation of PomA D148Y is located near thecytoplasmic end of a membrane segment of PomA. The
topology of PomA was predicted from the hydropathy
pro®le of PomA, and the topology of the MotA proteinof E. coli (Zhou et al., 1995).

indicating that the Mpar mutation in NMB205 wasin the pomA gene.
Nucleotide sequencing of the fragments in
pSK502 and pSK602 was carried out to ®nd themutations. A single-base substitution (G442 to T)
was detected in pomA, which is predicted to cause
the substitution D148Y. No nucleotide changes
were detected in pomB. Based on the membrane1540
topology of E. coli MotA (Zhou et al., 1995), theresidue Asp148 of PomA should be located near
the cytoplasmic end of the third transmembranesegment (Figure 3(c)). Asp148 is conserved in
MotA of E. coli and Rhodobacter sphaeroides (Asaiet al., 1997), suggesting that this residue is not
speci®c to Na-motors. When pSK502 wasintroduced into the Mpas (wild-type) strain, thetransformants showed an Mpar phenotype.
Mutational analysis of PomA Asp148
To probe the requirements for function and forphenamil resistance at position 148, we replaced
residue Asp148 of PomA by Tyr (the Mpar
mutation of NMB205), Phe, Cys, Asn and Arg. To
measure swimming speed more easily, we isolated
the strain NMB188 (PomAÿ Cheÿ; see Materials
and Methods), and used it for host. NMB188 cells
harbored the plasmids containing pomA whichswim smoothly and do not change the swimming
direction at all (che phenotype). The swimming
speeds of NMB188 (PomAÿ Cheÿ) cells containing
the mutant pomA genes were measured in the presence of various concentrations of phenamil(Figure 4). In the absence of phenamil, NMB188
cells containing wild-type PomA swam at about 53

Figure 4. Phenamil resistance of PomA Asp148
mutants. Swimming speeds were measured in TMN
medium containing various concentrations of phenamilas indicated in Materials and Methods, for the strain
NMB188 (PomAÿ Cheÿ) expressing the mutant PomA
proteins D148F, D148C, D148N, D148R, and D148Y.
Filled (*) and open (*) symbols indicate speeds of themutants and wild-type, respectively.

Phenamil Resistant Mutations

mm/second. The PomA mutations D148Y, D148F,
D148C, and D148N reduced swimming speeds toabout 50-65 % of this value. The D148R mutation
impaired motility more severely. Motility of thewild-type strain was completely inhibited at 50 mM
phenamil, but the D148Y, D148F and D148C
mutants could be motile in that condition. The
mutation D148N gave an Mpas phenotype comparable to wild-type. The concentrations of phenamilrequired for 50 % inhibition of motility (IC50) wereobtained from the graph; 1.3 mM for wild-type,
8.7 mM for D148Y, 8.7 mM for D148F, 3.3 mM for
D148C, 1.3 mM for D148N and 8.7 mM for D148R.
D148R mutant cells could not swim at 50 mM phenamil, but the IC50 value is comparable with thephenamil-resistant mutants. So D148R mutation
might confer the resistance to phenamil. These
results suggest that a negative charge at residue
148 is not essential for sensitivity to phenamil, andthat both large (Y and F) and small (C) side-chainsat this position can weaken the interaction withphenamil.
We also examined the sensitivity of the Asp148
mutants to amiloride (Figure 5). Mutants that weresigni®cantly resistant to phenamil (Y, F and C)
were also slightly resistant to amiloride, as compared with the wild-type. The IC50 values for amiloride were about 0.5 mM for wild-type, 1.5 mM
for D148Y, 1.2 mM for D148F, 1.0 mM for D148C,
0.5 mM for D148N and 0.7 mM for D148R.

Figure 5. Sensitivity to amiloride of PomA Asp148
mutants. The experimental conditions and symbols are
as indicated for Figure 4, except the TMN medium contained the indicated concentrations of amiloride.

1541

Phenamil Resistant Mutations

Another Mpar mutation is in the pomB geneAs described above, we assumed that the Mpar
mutation in the strain NMB201 was likely to be in
pomA or pomB, so we cloned these genes from
NMB201 by using PCR, making plasmids pSK5021(pomA) and pSK6021 (pomB), respectively. These
plasmids were introduced into strain VIO586(PomAÿ) or strain NMB104 (PomBÿ), and swimming speeds in various concentrations of phenamilwere measured. NMB104/pSK6021 cells, carrying
the pomB gene derived from NMB201, showedmotility resistant to phenamil (Figure 6(a)).
VIO586/pSK5021 cells, carrying the pomA genederived from NMB201, showed the Mpas pheno-

type (data not shown). The Mpar mutation in
NMB201 is therefore in pomB. DNA sequence
analysis revealed two base changes in the pomB
gene from NMB201. One caused the substitution
P16S (C46 to T), whereas the other was silent (T834
to A; T278T). This silent mutation might haveoccurred during the PCR cloning. We removed thissilent mutation from pSK6021 by replacing an
EcoRI fragment from nucleotides at position 317 to
the end of the pomB with that of wild-type pomB,
derived from plasmid pHK4. NMB104 cells harboring the resulting plasmid, pSK603-P16S, also
showed the Mpar phenotype. We also introduced
the P16S mutation into wild-type pomB by sitedirected mutagenesis, and con®rmed Mpar phenotype. Hence, we concluded that the P16S mutation
caused the phenamil resistance. Based on the membrane topology of E. coli MotB (Chun & Parkinson,1988), residue Pro16 of PomB should be located inor near the cytoplasmic end of the transmembranesegment (Figure 6(b)). When pSK6021 was introduced into the Mpas strain VIO5, the transformed
cells showed an Mpar phenotype.
Mutational analysis of PomB Pro16

Figure 6. The mutation of another Mpar strain,
NMB201, maps to the pomB gene. (a) Cells containing
pomB derived from the Mpar strain NMB201 showedphenamil-resistant motility. The pomB gene of this strain,cloned by using PCR, was introduced into the pomB
mutant (NMB104). Swimming speeds of these cells were
measured as described in Materials and Methods.
(b) The predicted transmembrane region of PomB. The
Mpar mutation of PomB P16S is located near the cytoplasmic end of the membrane segment of PomB. The
topology of PomB was predicted by the hydropathy
pro®le of PomB and the topology of the MotB protein of
E. coli (Chun & Parkinson, 1988).

To prove the requirements for function and phenamil resistance at position 16 of PomB, we carriedout four replacements, with Phe, Tyr, Cys, andAla. Plasmids harboring the P16C, P16F or P16Y
mutations proved dif®cult to introduce into strain
NMB104 (PomBÿ). Because this might be causedby the presence of large amounts of mutant PomB
proteins, which might affect cell growth (mutant
pomB fragments were directly connected to the lacpromoter), we co-transformed cells with the
mutant plasmids and pMMB206, which contains
the lacIq gene. By this procedure, we succeeded in
introducing wild-type as well as mutant pomB
genes into the cells. Expression of the pomB genes
could be then induced by the addition of 1 mM
IPTG.
Swimming speeds of the pomB mutants were
measured in various concentrations of phenamil(Figure 7). All of the mutations that introduced
small side-chains (S, C and A) conferred resistanceto phenamil. In the absence of phenamil, the swimming speeds of the P16S and P16A mutants weresimilar to that of wild-type cells, whereas the P16C
mutation decreased swimming speed to 60 % of
wild-type. By contrast, the motilities of the large
side-chain mutants (P16F, P16Y) were severely
impaired even in the absence of phenamil, and the
P16Y but not the P16F mutant, could swim at
50 mM phenamil. The IC50 values for phenamilwere obtained from the graph: 1.3 mM in the wildtype, 8.3 mM in the P16S, 25 mM in the P16C,
8.7 mM in the P16A, 50 mM in the P16Y and 18 mM
in the P16F. P16F cells could not swim at 50 mM
phenamil, but the IC50 value is comparable withthe phenamil-resistant mutants. So P16F mutation
might confer the resistance to phenamil. These
results suggest that replacement of Pro16 with

1542

Phenamil Resistant Mutations

Figure 8. Motility inhibition of the PomA D148Y/
PomB P16S double mutant by phenamil and amiloride.
The plasmid containing both PomA D148Y and PomB
P16S mutations (pYA303-D148Y/P16S) was introduced
into the strain NMB188 (PomAÿ Cheÿ). Swimming
speeds were measured as indicated in Materials andMethods.

Figure 7. Phenamil resistance of PomB Pro16 mutants.
Plasmids coding the PomB mutants P16S, P16A, P16C,
P16F and P16Y were co-introduced into the strain
NMB104 along with pMMB206, which contains the lacIq
gene. Mutant pomB genes were induced by 1 mM IPTG,
as described in Materials and Methods, and swimming
speeds were measured in TMN medium containing
various concentrations of phenamil, as indicated inMaterials and Methods. Filled (*) and open (*)
symbols show the speed of mutants and wild-type,respectively.

both large and small residues can cause structuralchanges that result in phenamil-resistant motility,
and that the large side-chains at this position cangreatly impair motor function.
We also examined sensitivity to amiloride in
the PomB Pro16 mutants. As was observed in the
PomA Asp148 mutants, the Pro16 mutants thatshowed signi®cant resistance to phenamil (S, C,
A, Y and F) also exhibited some resistance toamiloride (data not shown). The IC50 for amiloride was about 0.5 mM in the wild-type, 1.4 mM
in the P16S, 1.2 mM in the P16C, 1.2 mM in the
P16A, 1.2 mM in the P16Y and 1.0 mM in the
P16F.
PomA D148Y/PomB P16S double mutant
To examine the combined effect of the two
independently identi®ed Mpar mutations in pomAand pomB, we constructed a plasmid containing
both PomA D148Y and PomB P16S mutations(pYA303-D148Y/P16S), and introduced it into thePomAÿ Cheÿ strain NMB188. In the absence ofphenamil, motility of the double mutant was
severely impaired. As the concentration of phenamil in the medium increased, however, the motility was inhibited very little. At 50 mM phenamil,
the double mutant swam at 11 mm/second, a ratecomparable with the D148Y and P16S single
mutants (Figure 8). The double mutant exhibited
resistance to amiloride similar to that of the P16Y
mutant. When the double mutant D148Y/P16S
was expressed in NMB104 (PomBÿ), similar
results were observed. These results suggest thatthe double mutant is less sensitive to phenamilthan either of the single mutants; although swimming is poorer in the absence of phenamil, phenamil seems to have practically no effect on this.

Discussion
Since phenamil speci®cally inhibits the rotationof the Na-driven ¯agellar motor, the site of interaction with phenamil might be involved, directly
or indirectly, in force-generation in the motor.
Direct evidence that phenamil interacts with theforce-generating units in the motor has not beenreported. Previously, we isolated motility mutants
resistant to phenamil (Mpar mutants), and speculated that mutations occurred in a high-af®nity
phenamil-binding site (Kojima et al., 1997). Here,
we showed that two Mpar mutations are located in
pomA and pomB, whose products are putative
channel components in the motor. This is the ®rst
molecular evidence that phenamil directly interacts
with the Na channel components essential for
rotation of the motor.
Two Mpar mutations were identi®ed in pomA
(D148Y) and pomB (P16S), respectively. This result
provides evidence that both PomA and PomB
contribute to phenamil sensitivity, essential forforce-generation in the Na motor. Moreover,both of the mutated residues are predicted to belocated near the cytoplasmic ends of transmembrane segments. This suggests a novel possibility

Phenamil Resistant Mutations

that the high-af®nity phenamil-binding site might
be located on the cytoplasmic face of the motor.
In order to gain more insight into the phenamilinteraction site, we carried out site-directed mutagenesis of residues Asp148 in PomA and Pro16
in PomB. In the case of PomA, the negative
charge of Asp148 did not prove of primary
importance for the resistant phenotype, whereaschanges in the size of the side-chain, either to larger (Tyr and Phe) or smaller (Cys), gave signi®cant phenamil resistance. Mutational analysis ofresidues Pro16 in PomB showed similar results,
in that substitution with either small side-chains(Ser, Cys and Ala) or large side-chains (Tyr andPhe) gave signi®cant phenamil resistance. These
Mpar mutations in PomA and PomB also
conferred some amiloride resistance. In addition,
the competition of amiloride with Na in themedium was signi®cantly decreased in these
Mpar mutants (data not shown). This may indicate that the amiloride-binding site is affected by
the Mpar mutations, consistent with previous
results (Kojima et al., 1997). The motility of somemutants, especially the PomA D148R, PomB P16F
and P16Y mutants, was impaired, suggesting thatthese mutant residues may affect the structure of
the force-generating unit or may prevent ion ¯ux.
Interestingly, the pomB genes with Mpar mutation
which showed the signi®cant impaired motility(P16C, P16F and P16Y) were dif®cult to introduce
into cells unless the pomB expression on the plasmids was regulated. We think that the large
amounts of the mutant PomB proteins by them-

Figure 9. Working model for the interaction of phenamil with the PomA/PomB Na channel complex. If phenamil interacts directly with PomA Asp148 or PomB
Pro16, it has to go through the channel pore ( 1 ) or
permeate the lipid bilayer ( 2 ) to reach the cytoplasmicside of the transmembrane segments.

1543
selves, or the ion leakage by the mutant proteinsmight give a toxic effect.
In the case of the H-motor, it was suggested
that MotA and MotB form a complex thatfunctions as a H channel (Stolz & Berg, 1991;
Garza et al., 1995, 1996a,b; Sharp et al., 1995a,b).
We assumed that PomA and PomB, which arehomologous to MotA and MotB, respectively, form
the Na channel complex in the motor (Figure 9).
Based on this assumption, it is conceivable that
residues Asp148 of PomA and Pro16 of PomB
might be located near each other and may form ahigh-af®nity binding site for phenamil at the innerface of the PomA/PomB channel complex. If so,
the observation that at 50 mM phenamil the
D148Y/P16S double mutant exhibited an Mpar
phenotype comparable to each single mutationsuggests the possibility that multiple residues,
including Asp148 of PomA and Pro16 of PomB,
constitute a high-af®nity phenamil-binding site. On
the other hand, the D148Y/P16S double mutation
showed a synergistic effect, impairing motor function, much more severely than the individual
mutations, even in the absence of inhibitors. This isconsistent with the proposal that PomA and PomB
function together to form a channel complex.
Muramoto et al. (1996) suggested that binding ofphenamil to the motor might induce additionaldrag on the motor, in addition to blocking Na
in¯ux. Strong direct interaction of phenamil withan internal binding site on the PomA/PomB channel complex might induce such function in the
motor. From the present results, however, we cannot conclude that phenamil directly interacts withthese residues.
The amiloride-binding site might also be locatedat the inner side of the channel complex, because
Mpar mutations in PomA and PomB conferredsome amiloride resistance and an internal Nabinding site was suggested by Yoshida et al. (1990).
In such a model, in order to reach the binding site,
phenamil and amiloride would have to go throughthe channel pore, or cross the lipid bilayer to reachthe cytoplasmic side (Figure 9). To discuss the precise mechanism for amiloride interaction, we havebeen trying to isolate motility mutants resistant toamiloride.
Amiloride-binding sites have been studied ineukaryotic sodium channels and transporters. In
the eukaryotic epithelial sodium channel (ENaC),
amiloride is effective only from the external sideof the lipid bilayer (Ismailov et al., 1995). Amiloride-resistant mutations were identi®ed at externalor transmembrane region of ENaC, and also in
the Na/H exchanger. Mutations that cause alarge decrease in Ki for amiloride were found tooccur in the second transmembrane segment of
the a subunit of rat ENaC (Waldmann et al.,1995), and in residues of a hydrophobic region
termed the pre-M2 domain that immediately precedes the second membrane domains of several
different ENaC proteins (Schild et al., 1997; Fuller
et al., 1997). Ismailov et al. (1997) showed that

1544

Phenamil Resistant Mutations

deletion of a putative amiloride binding domain``WYRFHY'' within the extracellar domain ofarENaC, whose existence was suggested byexperiments using an anti-amiloride monoclonal
antibody (Lin et al., 1994), caused a large
decrease in amiloride sensitivity. In addition, theysuggested that the His residue in this tract mightinteract primarily with the Cl atom on the pyrazine ring moiety of amiloride, to stabilize thebinding. In the case of the eukaryotic Na/H
exchanger and prokaryotic Na/H antiporter,
residues within the putative second (Kuroda et al.,1997), fourth (Counillon et al., 1993, 1997) andninth transmembrane domains (Orlowski &Kandasamy, 1996) are involved in amiloride
binding. This putative amiloride-binding site in
the fourth transmembrane domain contains asequence, VFF, which is also found in PomA at
the cytoplasmic end of the putative fourth transmembrane domain. In the case of the transporter,
replacing these F residues with Y decreased thesensitivity to amiloride. However, PomA F197Y
or PomA F198Y mutants showed an Mpas phenotype comparable with the wild-type strain (datanot shown), so the VFF residues in PomA seemnot to be involved in sensitivity to amiloride orphenamil.
The Na motor of V. alginolyticus has other putative channel components, MotX and MotY
(McCarter, 1994a,b; Okunishi et al., 1996). MotX in
particular has been suggested to be a target of ami-

loride. We speculate that the native Na channel
complex in the motor would consist of PomA,
PomB, MotX and MotY, and function only whenthese four proteins assemble properly. Identifying
more mutations resistant to phenamil will help inunderstanding the Na channel complex essential
for torque generation.

Materials and Methods
Bacterial strains, plasmids, growth conditions,and media
The strains and plasmids used are described inTable 1. 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). For swimming speed analysis, cells
were cultured to late logarithmic phase at 30  C in VPG
medium (1 % polypeptone, 0.4 % K2HPO4, 3 % NaCl,
0.5 % (w/v) glycerol), and the swimming speed wasmeasured in the TMN medium containing 50 mM TrisHCl (pH 7.5), 5 mM MgCl2, 5 mM glucose, 50 mM
NaCl, 250 mM KCl. For the induction of the pom genes
in plasmids, cells were grown in VC medium containing
1 mM IPTG (isopropyl-1-thio-b-D-garactoside). E. coli
cells were cultured at 37  C in LB medium (1 % tryptone,
0.5 % yeast extract, 0.5 % NaCl). When necessary, chloramphenicol and kanamycin were added to ®nal concentrations of 2.5 mg/ml and 100 mg/ml for Vibrio cells, or
25 mg/ml and 50 mg/ml for E. coli cells, respectively.

Table 1. Bacterial strains and plasmids
Strains or plasmids
V. alginolyticus strains
VIO586
NMB104
NMB201
NMB205
NMB161NMB188

Genotype or descriptiona

References or sources

Rifr Pof Lafÿ PomAÿ
Pof Lafÿ PomBÿ
Rifr Pof Lafÿ Mpar
Rifr Pof Lafÿ Mpar
Rifr Pof Lafÿ Mpar PomBÿ
Rifr Pof Lafÿ PomAÿ Cheÿ(Asai et al., 1997)(Asai et al., 1997)(Kojima et al., 1997)(Kojima et al., 1997)(Asai et al., 1997)
This work

XL1-BlueFÿ lÿ recA1 hsdR17 endA1 supE44 thi-1 relA1 gyrA96 (argF-lacZYA)U169
80dlacZM15
recA1 hsdR17 supE44 (lac-proAB){F0 ::Tn10 proAB lacIqZM15}(Grant et al., 1990)
Stratagene

Plasmids
pSU21
pSU41
pYA301
pYA303
pHK2
pHK4b
pSK1
pSK1-28
pSK401
pSK502
pSK602bpSK5021
pSK6021
pSK603
pMMB206cat(Cmr) PlaclacZakan(Kmr) PlaclacZa (MCS same as that in pSU21)
0.8 kb BamHI fragment (Plac-pomA) in pSU41
1.9 kb BamHI-SacI fragment (Plac-pomAB) in pSU41
2.9 kb SacI fragment (pomAB) in pSU21
1.2 kb HindIII-SacI fragment (Plac-pomB) in pSU41
1.5 kb XbaI-EcoRI fragment (pomA) in pSU41
329 bp deleted from XbaI site of pSK1 (lack of the native promoter of pomA)
2.9 kb SacI fragment (pomAB) from NMB205 in pSU21
1.5 kb XbaI-EcoRI fragment (pomA) from NMB205 in pSU41
1.2 kb HindIII-SacI fragment (Plac-pomB) from NMB205 in pSU41
0.8 kb BamHI-EcoRI fragment (Plac-pomA) from NMB201 in pSU41
1.1 kb BamHI-SacI fragment (Plac-pomB) from NMB201 in pSU41
1.1 kb BamHI-SacI fragment (Plac-pomB) in pSU41
cat(Cmr) IncQ lacIq bla Ptac-lac lacZa(BartolomeÂ et al., 1991)(BartolomeÂ et al., 1991)
This work
This work(Asai et al., 1997)
This work(Asai et al., 1997)(Asai et al., 1997)
This work
This work
This work
This work
This work
This work(Morales et al., 1991)

E. coli strains
DH5aaCmr, chloramphenicol-resistant; Kmr, kanamycin-resistant; MCS, multicloning site; Plac, lac promoter; Ptac-lac, tandemly located tac
and lac promoters; Rifr, rifampicin-resistant.
b
The 1.2 kb HindIII-SacI fragments contain 30 -terminal 155 bp of pomA and full-length pomB.

1545

Phenamil Resistant Mutations
Chemicals
Amiloride was purchased from Sigma Chemical Co.
Phenamil was synthesized by the method of Cragoe et al.
(1967).
DNA manipulations and sequencing
Routine DNA manipulations were carried out according to standard procedures (Sambrook et al., 1989).
Restriction endonuclease and other enzymes for DNA
manipulation were purchased from Takara Shuzo(Kyoto) and New England Biolabs (Beverly, MA). The
nucleotide sequence was determined by the dideoxy
chain termination method using the ABI PRISMT2 Dye
Terminator Cycle Sequencing Ready Reaction Kit andthe ABI PRISM2 377 DNA sequencer (Perkin-Elmer
Corporation).
pom gene cloning
Plasmid pSU21 (BartolomeÂ et al., 1991) and chromosomal DNA from V. alginolyticus Mpar strain NMB205(Kojima et al., 1997) were digested with SacI and ligated.
These DNA libraries were introduced into cells of thepomA mutant (VIO586) by electroporation and they wereincubated at 30  C on 0.25 % agar VC plates containing
chloramphenicol. To isolate the pom genes from the Mparstrain NMB201 (Kojima et al., 1997), PCR ampli®cation
was carried out with the Takara Ex Taq kit (Takara
shuzo) and Thermal Cycler (Perkin-Elmer Japan). PCRprimers complementary to each end of pomA were synthesized. The sense-primer (designated pomA-B1) was 50 dCTCGGATCCGCCCAATGGCAGTATTTGA-30 ,andgenerated a BamHI site. The antisense-primer (designated pomA-E2) was 50 -dCTCGAATTCAAGTTACTCGTCAATCTCA-30 , and generated an EcoRI site. PCRprimers complementary to each end of pomB were alsosynthesized: The sense-primer (designated pomB-A) was
50 -dATTGGATCCCGTGCCCTTGAGATTGAC-30 ,andgenerated a BamHI site. The antisense-primer (designated pomB-Z) was 50 -dTAGGATCCGAGCTCACTTTTAAGGCG-30 , and contained both a SacI site and a
BamHI site.
Site-directed mutagenesis
A two-step PCR method was used to introduce
mutations (White, 1993). We synthesized pairs of mutant
primers that were homologous to either the sense orantisense strands of pomA or pomB, with the exception ofa one to three base mismatch at the site of mutation. In
the ®rst step, two PCR reactions were carried out separately, one using an antisense-mutant primer and wildtype sense-primer, and the other, a mutant-sense primer
and wild-type antisense-primer. Each of the ampli®ed
fragments was separated from the remaining primers bypolyacrylamide gel electrophoresis. In the second step,these fragments were mixed together, then the full target
genes were ampli®ed by using the appropriate end primers. PCR ampli®cation was carried out by using the
Takara Ex Taq kit (Takara shuzo) and Thermal Cycler(Perkin-Elmer Japan). PCR reactions were repeated for
25 cycles of 30 seconds at 94  C, 60 seconds at 60  C, and60 seconds at 72  C. Ampli®ed mutant fragments containing the full-length genes were digested with BamHI
and EcoRI for pomA, or with BamHI and SacI for pomB,then ligated into the kanamycin-resistance vector pSU41(BartolomeÂ et al., 1991). The resultant plasmids carrying
mutant pomA or pomB genes have the same length offragments as pYA301 (pomA) and pSK603 (pomB),
respectively. For the construction of the PomA-D148Y/
PomB-P16S double mutant, 1.2 kb HindIII-SacI fragment
containing the 30 -terminal 155 bp of pomA and full-length
pomB which has the PomB-P16S mutation was ampli®ed,digested with HindIII and SacI, and ligated into pSU41(named pHK4-P16S). Then the HindIII fragment from
pYA301-D148Y was inserted into the HindIII site of
pHK4-P16S to make full-length pomA and pomB containing two mutations. The resultant plasmid has the samelength of fragment as pYA303 (pomAB), so it wasnamed pYA303-D148Y/P16S.
Isolation of PomAÿ Cheÿ double mutant
In order to analyze the swimming speeds of pomA
mutants easily, we isolated a pomA che double mutant.
The plasmid pSK1-28 (Asai et al., 1997) was introduced
into strain VIO586 (PomAÿ), and EMS mutagenesis was
carried out on the transformants (Asai et al., 1997). A che
mutant was selected as described (Homma et al., 1996).
The plasmid pSK1-28 was removed from this chemutant by growing in VC medium without kanamycin,
and a kanamycin-sensitive clone was selected. The isolated pomA che mutant, named NMB188, could swim
with a Cheÿ phenotype and the same swimming speed
as the wild-type strain when a plasmid containing pomA
was introduced.
Electroporation
Transformation of Vibrio cells by electroporation was
carried out as described (Kawagishi et al., 1994) with theGene Pulser (Japan Bio-Rad Laboratories, Tokyo) at anelectric ®eld strength between 5.0 and 7.5 kV/cm.
Measurement of swimming speed
Cells were harvested at late logarithmic phase, andresuspended in TMN medium. Then a small amount of
the cell suspension was diluted about 100-fold into TMN
medium with or without the inhibitors, and motility of
the cells was observed at room temperature under adark-®eld microscope and recorded on video tape.
Swimming speed was determined as described (Atsumi
et al., 1996). The average swimming speed was obtainedby measuring more than 20 swimming tracks.

Acknowledgments
We thank Dr Seishi Kudo for invaluable discussions.
We especially thank Dr David F. Blair for critically reading the manuscript. This work was supported in part by
grants-in-aid for scienti®c researches (to S.K., Y.A., T.A.,
I.K. and M.H.) from the Ministry of Education, Science
and Culture of Japan.

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Edited by J. Karn
(Received 17 August 1998; received in revised form 3 November 1998; accepted 4 November 1998)