JOURNAL OF BACTERIOLOGY, Aug. 2004, p. 5281–5291
0021-9193/04/$08.00⫹0 DOI: 10.1128/JB.186.16.5281–5291.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 186, No. 16

Interaction of PomB with the Third Transmembrane Segment of
PomA in the Na⫹-Driven Polar Flagellum of Vibrio alginolyticus
Toshiharu Yakushi,* Shingo Maki, and Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan
Received 20 January 2004/Accepted 5 May 2004

The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY,
responsible for its Naⴙ-driven flagellar rotation. PomA and PomB are integral inner membrane proteins
having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel
complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB,
and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys
substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34,
Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C
and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (fromMet-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double
mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it.
Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it wasrecovered by reducing reagent. The strain with this combination showed a lower affinity for Naⴙ than thewild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of
PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that
TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between
these two TMs is required for coupling of Naⴙ conduction to flagellar rotation.
inhibitor of Na⫹ channels, specifically inhibits Na⫹-driven flagella but not H⫹-driven flagella (7, 23, 28).
Four genes that are required for the function of the Na⫹driven motor of V. alginolyticus have been identified: pomA,
pomB, motX, and motY (2, 35, 37, 39). PomA and PomB are
orthologs of MotA and MotB, respectively, which are the motor proteins of H⫹-driven flagella in Escherichia coli, Salmonella spp., and other species (33). PomA (MotA) and PomB
(MotB) are integral cytoplasmic membrane proteins having
four and one transmembrane segments (TMs), respectively.
Figure 1 shows a model of the predicted membrane topology of
PomA and PomB (5, 14, 19, 44, 45, 58). A multimeric complex
composed of four PomA and two PomB proteins participatesin the ion influx to drive the flagellar rotation (15, 30, 42, 43,
55). PomB (MotB) has a putative peptidoglycan-binding motif
to attach the motor complex to the peptidoglycan layer in the
periplasmic space (2, 17). MotX and MotY are exclusively
found in Vibrio species, and they have been shown to bepresent in the outer membrane (38). It is not known how theouter membrane proteins function in the motor.
The large (ca. 100-amino-acid) cytoplasmic region of PomA
connecting the second TM (TM2) and the third TM (TM3) isthought to interact with the rotor protein FliG, which is associated with the MS ring (52). The 32nd Asp residue in MotB,
which corresponds to Asp-24 of PomB, is located near thecytoplasmic end of its TM and is thought to bind ions (H⫹ or
Na⫹) and play a critical role in ion flux and the energy conversion (57). It is thought that successive conformationalchanges in MotA, associated with the protonation and deprotonation of the MotB TM, drive the rotation of the FliG-MS
ring complex (29). It is hypothesized that the cytoplasmic region of MotA interacts with the rotor component FliG, where

The chemotactic behavior of bacteria is a consequence of
two swimming states, smooth swimming and tumbling (11, 33),
and the alternative, which can be backward swimming (22),turns (40), or stopping (1). The flagellar filament is the propeller that drives the cell body, and the bacterium utilizes it toswim. It is driven by the rotation of the motor embedded in thecytoplasmic membrane, which is connected to the filament via
the rod and hook structures. The basal body of the flagellum ingram-negative bacteria consists of the rod and several ring
structures: the L ring, P ring, MS ring, and C ring, which residein the outer membrane, peptidoglycan layer, cytoplasmic membrane, and cytoplasm, respectively (18). The MS and C rings
are referred to as the rotor, and the motor protein complexes(see below) that surround the rotor are referred to as the stator(27).
The flagellar motor is energized by an electrochemical potential using either H⫹ or Na⫹ as the coupling ion (21, 34). The
marine bacterium Vibrio alginolyticus and the pathogenic bacterium Vibrio parahaemolyticus have two different types of flagella, H⫹-driven lateral flagella and Na⫹-driven polar flagella(8, 26). The lateral flagella are useful for movement underviscous conditions, even on solid surfaces, but this motility isrelatively slow. The study of the rotation mechanism of Na⫹driven flagellar motors has two clear advantages: (i) it is easy tomanipulate the ion-motive force by changing the Na⫹ concentration of the medium, and (ii) phenamil, which is a known* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku,
Nagoya 464-8602, Japan. Phone: 81-52-789-2992. Fax: 81-52-789-3001.
E-mail: 4juji@cc.nagoya-u.ac.jp.
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FIG. 1. Putative membrane topology of PomA and PomB and sites of mutations used in this study. (A) The predicted structure of PomA (left)
is taken from Asai et al. (5). PomA has four TMs, TM1 to TM4, and no Cys residues. PomA has a ca. 100-amino-acid cytoplasmic region (dashes)that plays a critical role in interactions with the rotor protein FliG. Wild-type PomB (right) has three Cys residues, Cys-8, Cys-10, and Cys-31 (whiteletters in filled circles). The predicted topology of PomB is from Asai et al. (2) and Braun and Blair (14) but modified as described in Discussion.
PomB has an essential 24th Asp residue in its TM (bold circle) and a large (length, ⬃270 amino acids) C-terminal periplasmic domain (not shown),
which contains the putative peptidoglycan-binding region. (B) Alignments of the PomA TM3 and the PomB TM regions with the TM regions of
MotA and MotB. Va, V. alginolyticus; Rs, Rhodobacter sphaeroides; Ec, E. coli. Residues that are identical to those of V. alginolyticus are indicatedby white letters in black boxes. Arrows indicate cross-linked residues between PomA TM3 and PomB TM. The closed circle indicates the essential
Asp residue, which is thought to bind protons or sodium ions.

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TABLE 1. V. alginolyticus strains and plasmids
Genotype or descriptiona

Strain or plasmid

V. alginolyticus strain
VIO5
NMB136
NMB192
NMB191NMB195
PlasmidpSU41
pHK4
pYA303
pYA803
pYA802aReference or source

Rifr Laf⫺
Rifr Laf⫺ Che⫺
Rifr Laf⫺ ⌬pomB
Rifr Laf⫺ ⌬pomAB
Rifr Laf⫺ Che⫺ ⌬pomAB

39
This study
This study
50
This studykan (Kmr) Plac lacZ␣
pSU41, 1.0-kb HindIII-SacI fragment (pomB⫹)
pSU41, 1.9-kb BamHI-SacI fragment (pomAB⫹)
pKY704, 1.2-kb XbaI-HpaI and 500-base SnaBI-SacI fragments (pomA-⌬pomB)
pKY704, 250-base BamHI-DraI and 700-base HpaI-SacI fragments (⌬pomAB)

10
28
28
Y. Asai
50

Rifr, rifampin resistant; Kmr, kanamycin resistant; Laf⫺, defective in lateral flagellar formation; Che⫺, defective in chemotaxis; Plac, lac promoter-operator.

electrostatic interactions between conserved charges on FliGof the rotor and on MotA are important for the rotation of theH⫹-driven motor (59). According to these hypotheses, it canbe speculated that a conformational change in the MotB TM(PomB TM), which is evoked by binding of ions, is successivelytransferred to MotA (PomA), to its cytoplasmic region, andfinally to the rotor protein FliG to drive the motor. Thus, theinteractions between PomA and PomB are critical to understand the molecular mechanism of flagellar rotation. Several
chimeric motors composed of both H⫹ and Na⫹ types of components are functional (3, 4, 6, 20), suggesting that a similar
mechanism governs both types of motors. However, there aresome inconsistencies because the conserved charges of PomA
and FliG in the Na⫹-driven motor of V. alginolyticus are notparticularly important (53, 54).
Cys-scanning mutagenesis in PomA, especially in the
periplasmic loop regions, suggests that the environments
around loop1-2, which connects TM1 and TM2, and loop3-4,
which connects TM3 and TM4, are very different from eachother (5). Many Cys substitutions in loop3-4 impair motility,
implying that this loop region or the connecting TM3 or TM4
have important roles. The 5,5⬘-dithiobis(2-nitrobenzoic acid)(DTNB) modification of PomA-D170C at the periplasmic
boundary of TM3 was affected by NaCl concentrations, suggesting that Na⫹ competes with DTNB and that this region
may be involved in ion conductance in the PomA/PomB complex (5). The mutations conferring phenamil resistance forNa⫹-driven flagellar rotation have been mapped near the cytoplasmic boundaries of PomA TM3 and PomB TM (23, 28).
The charged residue Asp-24 of PomB TM, the hypotheticalNa⫹-binding site, is fairly close to the position of the phenamilresistant mutations. In the H⫹-driven motor, Trp scanning of
MotA showed that several different amino acid substitutions in
TM3 impaired motility (44). Furthermore, Pro-173 (TM3) of
MotA is suggested to be close to Asp-32 of MotB (16).
In order to understand the molecular mechanisms that connect the ion conductance and the subsequent conformationalchange of PomA, it is important to examine the interactions
between PomA, which interacts with FliG, and PomB, which
has a putative Na⫹-binding site. In this study, we assessed thepossibility of functional and physical interactions between
PomA TM3 and PomB TM, because they appear to be important TMs in the PomA/PomB complex. Through biochemicaland physiological analyses using combinations of Cys replace-ments of PomA and PomB, two major conclusions are described: (i) the PomA TM3 and PomB TM are associated atboth the periplasmic and cytoplasmic faces of the membrane,and (ii) at the periplasmic side, PomA TM3 and PomB TM
may comprise part of an entrance for Na⫹ into the PomA/
PomB complex.
MATERIALS AND METHODS
Bacterial strains, plasmids, media and growth conditions. E. coli strain JM109[recA1 endA1 gyrA96 thi hsdR17 relA1 supE44 ␭⫺ ⌬(lac-proAB) F⬘ traD36 proAB
lacIq⌬M15) was used for DNA manipulations (49). The strains of V. alginolyticus
used in this study are listed in Table 1. V. alginolyticus strain NMB136, which is
defective in chemotaxis (Che⫺), was isolated after ethyl methanesulfonate treatment (22) from the lateral-flagellar-defective strain VIO5. V. alginolyticus strain
NMB195, which has a pomAB deletion and Che⫺ phenotype, was constructedfrom NMB136 by using the suicide plasmid pYA802 carrying ⌬pomAB as described previously (50). V. alginolyticus cells were cultured at 30°C on VC medium (0.5% tryptone, 0.5% yeast extract, 0.4% K2HPO4, 3% NaCl, 0.2% glucose) or VPG500 medium (1% tryptone, 0.4% K2HPO4, 500 mM NaCl, 0.5%
glycerol). When necessary, kanamycin was added to a final concentration of 100
␮g/ml (for V. alginolyticus) or 50 ␮g/ml (for E. coli). The plasmids used are listed
in Table 1. The motor genes on the plasmids constructed in this study are
expressed from the lac promoter-operator in pSU41 (10). Transformation ofVibrio cells by electroporation was carried out as described previously (25). DNA
manipulations were carried out according to standard procedures (41). Aminoacid substitution was carried out using a site-directed mutagenesis kit (Stratagene).
Swarming assay. An aliquot (1 ␮l) of overnight culture in VC medium wasspotted onto VPG500 plates containing 0.25% agar and 100 ␮g of kanamycin/ml
and incubated at 30°C. Dithiothreitol (DTT) was added to final concentrations as
specified. DTNB was dissolved in dimethyl sulfoxide at 100 mM prior to use.
Measurement of swimming speed and data processing. An overnight culture
in VC medium was inoculated into VPG500 medium at a 50-fold dilution andgrown at 30°C to exponential growth phase. Cells were centrifuged at 3,500 ⫻ g
for 3 min, and the sedimented cells were resuspended in TMN50 (50 mM
Tris-HCl [pH 7.5], 5 mM MgSO4, 5 mM glucose, 50 mM NaCl, 450 mM KCl)
supplemented with 1 mM DTT. Cell suspensions were diluted 20-fold into TMN
containing 1 mM DTT and various concentrations of NaCl, and the motility of
the cells was observed immediately under a dark-field microscope. The KClconcentration was also changed to hold the total ion concentration at a fixed level(500 mM). Swimming speeds were determined from at least 20 individual cells as
described previously (9). The Na⫹ influx through the motor was estimated by the
kinetic treatment as described previously (47). Na⫹ influx corresponds to theequation v2/(ε ⫻ Na⫹-motive force), where v is the swimming speed and ε is theefficiency. For this estimation, the intracellular Na⫹ concentration and membrane potential were assumed to be 30 mM and ⫺150 mV, respectively. Apparent Km values for Na⫹ in the estimated Na⫹ influx were calculated from doublereciprocal plots of the kinetic treatment. The maximum velocity of Na⫹ influx
through the motor cannot be determined, because the efficiency ε cannot be
determined, whereas it is assumed to be constant.

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FIG. 2. Effect of the sulfhydryl reagent DTNB on swarming of cells with Cys-substituted variants of PomB. Vibrio strain NMB192 (⌬pomB)
harboring derivatives of pHK4 (pomB⫹) with Cys-substituted variants of pomB were spotted on semisolid plates containing no DTNB, 0.1 mM
DTNB, and 0.5 mM DTNB and photographed after 4 h of incubation at 30°C. Relative swarm sizes were normalized to that of the wild type under
the same conditions. The 31st residue is Cys in the wild-type PomB.

Detection of PomA, PomB, and the cross-linked products. Cells were cultured
in VPG500 medium at the mid-log phase of growth (optical density at 660 nm,⬃1.0). An equal volume (100 ␮l) of 20% (wt/vol) trichloroacetic acid (TCA) was
added to the culture. Alternatively, for a combination of PomA-D148C/PomBclP16C, cells were collected by centrifugation, disrupted by sonication, and incubated for 30 min at room temperature prior to TCA precipitation. TCA-insoluble materials were collected by centrifugation, washed with acetone anddiethylether, and dried. The dried materials were dissolved in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer containing 2 mM N-ethylmaleimide or 5% (vol/vol) 2-mercaptoethanol and subjected to
SDS-PAGE. Immunoblotting was performed using anti-PomA and -PomB antisera as described previously (50). The band densities were quantified by using theNIH Image program to view the captured gel images through a charge-coupleddevice camera.

RESULTS
Cys substitutions in the TM of PomB. PomB has a single
TM whose precise boundary has not yet been determined.
Each amino acid in the single TM of PomB was systematicallyreplaced with Cys (Asp-24 to Glu-41) to assess the effects ofeach substitution per se and, thereafter, the effects of treatment with the water-soluble sulfhydryl reagent DTNB.
The Cys derivatives were expressed in a ⌬pomB strain, andthe motor function was assayed by monitoring swarming behavior. The swarming ability of all mutants except two, D24C
and F33C, was comparable to that of the wild-type strain (Fig.
2). When cells were observed under the microscope, D24C wascompletely nonmotile and F33C showed very slow swimming(data not shown). The swarming diameter is assumed to reflect
the flagellar function under these conditions. Since all of the
PomB mutant proteins were detected at almost the same levelby immunoblotting (data not shown), any loss or decrease offunction must reflect the effects of the amino acid substitution
rather than protein stability or expression levels. Asp-24 of
PomB is an essential negatively charged residue, as has been
demonstrated in MotB of the H⫹-type motor, where it corre-

sponds to Asp-32 (57). Among the Cys substitutions, PomBS38C was the most susceptible to DTNB treatment: in thepresence of 0.1 mM DTNB, the swarm size of the PomB-S38C
mutant was reduced to ⬃5% of that in the control experiment.
Swimming cells were almost nonmotile in the presence of 0.1
mM DTNB, but motility could be restored by addition of the
reducing reagent DTT (data not shown). Around Ser-38 of
PomB, Cys substitutions at positions 35 to 39 were sensitive to
DTNB. This suggests that the environment around Ser-38 is
exposed to the solvent, i.e., to the periplasmic space.
Combinations of Cys substitutions in the periplasmic
boundaries of TMs. We tried to acquire topological information about the periplasmic regions of the PomA TMs and
PomB TM. Cys substitutions were performed at Gly-23, Ser34, Asp-170, and Ala-178, which correspond to the putative
periplasmic boundaries of TM1, TM2, TM3, and TM4 in
PomA, respectively (5). These mutants were combined with
PomB-S38C, which is the most susceptible to DTNB treatment. The swimming and swarming assays were done without
addition of oxidants. The periplasm is generally an oxidativeenvironment, so a disulfide bond is formed spontaneously if
two Cys residues are close to each other.
The swarming ability of the combined mutants was investigated in the presence or absence of the reductant DTT (Fig. 3).
An S34C mutation in PomA significantly decreased the swarming ability, both with the wild type and with PomB-S38C.
Swarming by the PomA-A178C or -G23C mutants was affectedby the Na⫹ concentration, especially when combined with
PomB-S38C (particularly PomA-G23C) (data not shown). No
significant restoration of swarming was observed upon additionof DTT to the A178C and G23C mutants. On the other hand,addition of DTT restored the swarming ability in the combination mutant carrying PomA-D170C and PomB-S38C.
Cross-link formation between PomA and PomB was as-

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FIG. 3. Combinations between Cys mutations in PomA TMs and PomB TM. Vibrio strain NMB191 (⌬pomAB) harboring derivatives of pYA303(pomAB⫹) with Cys-substituted variants of pomA and pomB were spotted on semisolid plates containing 500 mM NaCl and 0 mM (A) or 2 mM(B) DTT and photographed after 4 h of incubation at 30°C. wt, wild type.

sessed with the double Cys mutants. PomB has three Cys
residues, Cys-8 and Cys-10, which are predicted to be in thecytoplasm, and Cys-31, which is in the TM (Fig. 1) (2). PomB
forms a cross-linked dimer (PomB2) in the absence of reducing
agent but not in its presence (55). It has been shown that either
Cys-8 or Cys-10 is responsible for dimer formation, but Cys-31
is not involved. Thus, the cross-link-less PomB (PomB-C8A/
C10A [PomBxl]) and the Cys-less PomB (PomB-C8A/C10A/
C31A [PomBcl]) were used to eliminate the complications insome experiments. Cross-linked products of PomA and PomB
were not detected in the G23C, S34C, and A178C mutant
forms of PomA with PomB-S38C but were found with the
PomA D170C mutant protein (Fig. 4). In the combination of
PomA-D170C/PomB-S38C, three cross-linked products could
be detected without oxidant: a ca. 180-kDa product (X180) anda ca. 60-kDa product (X60), which were recognized by bothanti-PomA and anti-PomB antisera, and a ca.120-kDa product(X120), which was recognized by anti-PomB antiserum. The
X180 and X60 products could not be detected when a reducing
agent was added or when either PomA or PomB was wild type
(data not shown). The apparent molecular size of X60 is consistent with the sum of those of PomA (25 kDa) and PomB (37kDa). X180 can be assignable to linear cross-linkings of PomAPomB-PomB-PomA. On the other hand, the X120 product is
not readily known, because X120 is rarely detected by antiPomA antiserum. We suggest that the X120 product is assignable to an entangled complex of PomA-PomA and PomBPomB disulfides due to the following observations: (i) X120
was not detected in the absence of the D170C mutation (Fig.
4B, lane 3), and (ii) the band equivalent to X120 was detectedwith the combination of PomA-D170C and wild-type PomB,

although the amount was small (data not shown). Presumably
because PomB has two endogenous Cys residues, Cys-8 andCys-10, forming the cross-linked homodimer, several kinds ofcross-linked products of PomA and PomB would be formed.
We constructed the combination of PomA-D170C/PomBxlS38C to interpret the results of cross-linking between PomA
and PomB readily. Both X120 and X180 products were eliminated in this combination (Fig. 4A and B, lane 8). Among theCys mutants of PomA tested in Fig. 4, a PomA dimer (PomA2)
was only observed with D170C, which is consistent with resultsof a previous study (51). A PomA tetramer (PomA4), theca.100-kDa band, was also detected with PomA-D170C (Fig.
4A), because a small proportion of the wild-type PomA runs as
an SDS-resistant dimer, presumably because of the strong hydrophobic property (31). The PomA4 band otherwise may be
an intertwining of cross-linked PomA dimers.
Decrease in cross-linking of PomA TM3 and PomB TM by
the C31A mutation in PomB. The cells with PomB-C31A show
a lesser swarm size (55) and swim more slowly (T. Yakushi and
M. Homma, unpublished data) than those with wild-type
PomB. Furthermore, wild-type PomA seems to be dissociated more readily from PomB with the C31A mutation than
the wild-type protein under detergent-solubilized conditions(Yakushi and Homma, unpublished). When the C31A mutation was further introduced into PomBxl-S38C to construct
PomBcl-S38C, the amount of X60 significantly decreased (Fig.
5). Cross-linking between Cys-170 in PomA-D170C and Cys-31
in PomBxl was not observed (Fig. 5A and B, lane 3). These
results suggest that the formation of cross-linked PomA and
PomB requires interactions between PomA and PomB.

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FIG. 4. Cross-linking between PomA TM3 and PomB TM. Anti-PomA (A) and anti-PomB (B) immunoblots of whole-cell proteins of Vibrio
strain NMB191 (⌬pomAB) harboring plasmids producing various combinations of Cys mutations of PomA and PomB as indicated above each lane.
The whole-cell proteins were prepared as described in Materials and Methods. The details of X180, X120, and X60 are described in the text. wt,
wild type.

Effects of double Cys mutations at the periplasmic boundaries of PomA TM3 and PomB TM. To further investigate theperiplasmic boundaries of the PomA TM3 and PomB TM
interface, Cys substitutions around the putative periplasmic

FIG. 5. Cross-linking between PomA TM3 and PomB requires
their interaction. Whole-cell proteins of V. alginolyticus NMB191(⌬pomAB) harboring plasmids producing various combinations of Cys
mutations of PomA and PomB as indicated above each lane wereseparated by SDS-PAGE and immunoblotted with anti-PomA antibody (A) and anti-PomB antibody (B). wt, wild type.

boundary of PomA TM3 (M169-D171) and PomB TM (L37S40) were prepared, and the combinations substituted in thesesites were constructed in this study (Fig. 6).
For example, the swarming ability of the combined mutants
PomA-D170C and PomB-S38C was shown in the presence orabsence of the reductant DTT and the sulfhydryl reagent
DTNB (Fig. 6A). The swarming was reduced to about 20 ⫾ 6%(n ⫽ 4) of that of the wild type in the absence of DTT, but inthe presence of DTT, it recovered to 65 ⫾ 11% (n ⫽ 4) of thewild type level. The sulfhydryl reagent DTNB completely abolished the motility of a PomA-D170C/PomB-S38C mutant. On
the other hand, the combinations of PomA-D170C/PomB wild
type and PomA wild type/PomB-S38C are resistant to DTNB
treatment. The other combinations are shown in Fig. 6B. Most
combinations impaired the swarming ability, although single
mutations in PomA or PomB did not significantly affect theswarming ability. The swarming ability was improved by theaddition of DTT in D170C/F39C, D171C/S37C, and D171C/
S38C as well as D170C/S38C. PomA-D171C/PomB-F39C abolished the swarming ability completely with or without DTT.
The swarming ability of all combined mutants other than
combinations with PomB-S40C was impaired (⬎90% decrease) in the presence of 0.1 mM DTNB, although all combinations containing either wild-type PomA or PomB wereresistant to this treatment (data not shown).
A cross-linked product comprised of PomA and PomB was
detected as the X180 band (Fig. 6C). Asp-171 of PomA andSer-40 of PomB are presumably very close and in easy access ofeach other because the highest amount of X180 was observed.
However, the swarming ability was not lost in the presence ofthe combined mutations nor was it significantly affected by

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FIG. 6. Functional analysis and cross-link formation by the combinations of the PomA TM3 and PomB TM mutants in which Cys wasintroduced in the periplasmic boundaries. (A) Swarming ability of Vibrio strain NMB191 (⌬pomAB) harboring plasmids producing the PomA/
PomB combinations as indicated in the top square of the photographs: 2, PomA and PomB; 3, PomA-D170C and PomB; 4, PomA and
PomB-S38C; 5, PomA-D170C and PomB-S38C; or 1, vector plasmid. Semisolid agar plates contained no additions (top), 1 mM DTT (middle), or
0.1 mM DTNB (bottom). (B) Summary of relative swarm sizes of cells producing various PomA/PomB combinations. Each square represents a
combination of different versions of PomA and PomB and is split into two triangles, according to whether DTT was absent or present at 2 mM.
The cells are shaded according to the scale shown below. (C) Summary of cross-linking as represented by formation of X180 in each PomA/PomB
combination (see the text). Amounts of X180 in each combination were measured, and the relative amounts were normalized to that of
D171C/S40C as 100%. Each cell shows the results in the format from panel B and is shaded according to the scale shown below. wt, wild type.

DTT (Fig. 6B). On the other hand, the cells with the combination of PomA-D171C and PomB-F39C, which showed arelatively high amount of X180, had severely decreased motility even in the presence of DTT, although each single mutation
did not significantly affect motility.
Alteration of the Naⴙ requirement for the motility of PomAD170C/PomB-S38C. The swarming ability of the combination
mutant PomA-D170C/PomB-S38C was examined at two different NaCl concentrations, 50 and 500 mM in the presence ofDTT (Fig. 7A and B). As described above, the impaired
swarming ability of the PomA-D170C/PomB-S38C mutant issignificantly relieved by DTT (Fig. 6). The corresponding single mutants had almost the same swarming ability under thetwo salt conditions. However, swarming of the PomA-D170C/
PomB-S38C mutant was severely affected at the lower NaCl
concentration even in the presence of DTT. Swimming speeds
were measured as a function of NaCl concentration in thepresence of DTT for the PomA-D170C/PomB-S38C mutant

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TABLE 2. Swimming speeds and apparent Km values for estimatedNa⫹ influx through the motor in the swimming cells
PomA/PomBa

Swimming speedsb (␮m/sec)Kmc (mM)

wt/wt
D170C/wt
wt/S38C
D170C/S38C

77 ⫾ 6.2
69 ⫾ 6.3
66 ⫾ 8.0
47 ⫾ 5.5

4.1
9.7
10.4
20.7

a

wt, wild type.
Swimming speeds were measured at an NaCl concentration of 500 mM.
Swimming speeds of Vibrio strain NMB195 (Che⫺ ⌬pomAB) producing
PomA and PomB. PomA-D170C and PomB, PomA and PomB-S38C, and
PomA-D170C and PomB-S38C were measured under different NaCl concentrations. Apparent Km values for Na⫹ in the estimated Na⫹ influx were calculated
from the double-reciprocal plots (Fig. 7D) as described in Materials and Methods.
bcand the corresponding single mutants (Fig. 7C). If DTT wasomitted, cells having the PomA-D170C/PomB-S38C mutation
could swim slightly (data not shown). Table 2 shows kinetic
parameters of the single and the double mutants, as well as theswimming speeds at an NaCl concentration of 500 mM. The
Na⫹ influx through the motor can be estimated as described
previously (47). Apparent Km values for Na⫹ in the estimatedNa⫹ influx were calculated for each PomA and PomB mutation (Fig. 7D; Table 2). Each single mutation showed a lower
affinity for Na⫹ (⬃twofold-higher Km value) than wild-type
PomA/PomB. The affinity for Na⫹ in the combination mutant
D170C/S38C was affected further (⬃fivefold-higher Km value).
The cells required significantly higher Na⫹ concentrations toachieve their highest swimming speeds and therefore presumably to achieve maximal rates of Na⫹ influx. Such swimming
behaviors seem to correlate with the swarming results. These
results may suggest that Asp-170 of PomA and Ser-38 of PomB
functionally interact with each other and the interface is involved in the Na⫹ flux.
Cross-linking at the cytoplasmic boundaries of PomA TM3
and PomB TM. Phenamil-resistant mutations have been
mapped at both Asp-148 of PomA and Pro-16 of PomB, suggesting that they form a phenamil high-affinity site and are inclose proximity (28). The double mutant PomA-D148C/PomBclP16C was thus constructed to assess the physical interactions
between the two proteins. A 60-kDa cross-linked product (X60),
whose size corresponds to the sum of those of PomA and PomB,
could be detected by both anti-PomA and anti-PomB antisera

FIG. 7. Effect of NaCl concentration on the motility of the mutant
cells containing the combination of PomA-D170C and PomB-S38C.
Swarming behavior in the presence of 50 mM NaCl (A) or 500 mM
NaCl (B) of Vibrio strain NMB191 (⌬pomAB) harboring plasmids producing combinations of the following: 2, PomA and PomB; 3, PomA
and PomBxl; 4, PomA-D170C and PomBxl; 5, PomA and PomBxlS38C; 6, PomA-D170C and PomBxl-S38C; or 1, vector plasmid. Cells
were incubated on semisolid agar plates for 5 h at 30°C. Both plates
contained 2 mM DTT. (C) Swimming speed of Vibrio strain NMB195(Che⫺ ⌬pomAB) harboring PomA and PomB, PomA-D170C and
PomB, PomA and PomB-S38C, and PomA-D170C and PomB-S38C asa function of NaCl concentration. Symbols and error bars indicate theaverage values and standard deviations, respectively. (D) The doublereciprocal plots of the data shown in panel C after the kinetic treatment as described in Materials and Methods. Symbols and error bars
are as described for panel C.

VOL. 186, 2004

TRANSMEMBRANE SEGMENTS OF PomA AND PomB

FIG. 8. Cross-linking between PomA-D148C and PomBcl-P16C. A
P16C mutation was introduced into Cys-less PomB (PomBcl). AntiPomA (A) and anti-PomB (B) immunoblots of whole-cell proteins of
V. alginolyticus NMB191 (⌬pomAB) harboring plasmids producing various combinations of wild-type PomA or PomA-D148C and wild-type
PomB or PomBcl-P16C, as indicated above each lane. The whole-cell
proteins were derived from the cell lysates prepared by disruption
using sonicator prior to TCA precipitation as described in Materials
and Methods. wt, wild type.

(Fig. 8A and B, lane 6). X60 was not detected in the singly
substituted mutants (Fig. 8) and was lost upon addition of reducing reagents such as 2-mercaptoethanol (data not shown). Cells
expressing either PomA-D148C or PomBcl-P16C were motile but
less so than the wild type, as reported previously (28). Cells having
both mutations were not motile even in the presence of DTT(data not shown). This is probably a result of a synergistic effectbetween the two mutations. These results directly support the
proposal that Asp-148 of PomA and Pro-16 of PomB are close toeach other.
DISCUSSION
The flagellar motor proteins participate in the energy transduction from the electrochemical potential to a mechanicaloutput, the rotation of the flagellum. The PomA/PomB complex and MotA/MotB complex function as ion conductors in
the Na⫹-driven and H⫹-driven flagella, respectively (13, 43).
The interactions between PomA and PomB and the Na⫹ conductance are likely to be critical factors for the flagellar rotation. Cys-scanning mutagenesis is useful for studying the structure and function of membrane proteins for which thestereostructure has not been solved (24). In order to understand the interaction between PomA and PomB, site-directed
Cys mutagenesis was performed on the PomB TM, and the Cys
mutants of PomA TM3 and PomB TM were combined. Our
proposals are consistent with those for the H⫹-driven motor,
where it has been proposed that the essential Asp-32 of MotB
(corresponding to Asp-24 of PomB) faces MotA to make theH⫹-conducting channel because Asp-32 was presumed to point
away from the interface of the MotB dimer. Recently, a more5289

detailed hypothetical arrangement of the TMs of MotA/MotB
complex was proposed (12, 15).
Systematic substitutions in the TM of PomB showed that theputative Na⫹-binding site Asp-24 is essential for PomB function, consistent with data on Asp-32 of E. coli MotB (57). The
F33C mutation also significantly impaired motility. This phenylalanine, which has a bulky side chain, might play an important role in maintaining an interaction among the TMs. In E.
coli, MotB-F40C and MotB-W45C resulted in more severe
effects on motility than the other Cys mutations of the TM
region (14). Treatment with DTNB had the greatest effect on
the Cys mutant, PomB-S38C, and also had an effect on residues from L35 to F39, suggesting that this region may havehigh solvent accessibility. The TM of PomB has been tentatively defined by hydrophobicity analysis from Gly-20 to Phe-39(2); the precise periplasmic and cytoplasmic boundaries have
not yet been determined. According to the previous prediction,
Ser-38 is buried under the membrane, which is inconsistent
with the assumption in this study. Therefore, assignment of the
TM of PomB may be shifted from that of the previous study togive a tentative model as shown in Fig. 1. Plasmid-borne
PomB-S38C expressed with the chromosomal PomA was sensitive to DTNB (Fig. 2). However, the DTNB sensitivity of
PomB-S38C was repressed by coexpression of wild-type PomA
(Fig. 6A). The PomB protein should be in excess of the PomA
protein in the cells used in Fig. 2, whereas both PomA and
PomB proteins are overproduced from a plasmid in the cells
used in Fig. 6. An increase in the amount of PomA probably
facilitates the formation of the PomA/PomB complex. If the
PomA/PomB-S38C complex was influenced by the DTNB
treatment within the flagellar motor, we expect that the motility would be inhibited irrespective of coexpression of PomA. It
is likely that PomA affects the structure around the TM regionof PomB, resulting in an alteration of the sensitivity to DTNB
treatment. Therefore, we speculate that DTNB would affectthe PomB-S38C protein that does not associate with PomA but
it would no longer affect the PomB-S38C protein once the
PomA/PomB-S38C complex is formed.
In this study, cross-link formation between PomA TM3 and
PomB TM was detected. We suggest that the cross-linking
between PomA TM3 and PomB TM reflects the association in
the native PomA/PomB complex rather than aggregation due
to overproduction because of the following observations: (i)cross-links were not detected in cells with single Cys mutations
in either PomA or PomB and were specifically formed to theresidues where Cys was introduced, (ii) the C31A mutation in
PomB decreased the cross-linking between PomA-D170C and
PomB-S38C, and moreover, (iii) the cross-linked products corresponding to the X180, X120, and X60 were detected in thesame fraction where the wild-type PomA/PomB complex
eluted, when detergent-solubilized cross-linked and un-crosslinked PomA/PomB complexes were separated by gel filtration(D. Yoshimura, T. Yakushi, and M. Homma, unpublished results). Treatment with DTNB did little to enhance the crosslink formation (data not shown), whereas it significantly enhanced the inhibition of swarming ability (Fig. 6A). Asp-171 of
PomA and Ser-40 of PomB are presumed to be the closest
residues of the combinations tested in this study because thehighest amounts of cross-linked product were observed (Fig.
6C).

5290

YAKUSHI ET AL.

By using the wild-type PomB protein that has three endogenous Cys residues, the periplasmic substitutions resulted in
the formation of X60 and X180, which reacted with both antiPomA and anti-PomB antisera. PomA-D170C has a single Cys
residue, so the sulfhydryl group of D170C can react with either
that of another PomA-D170C or PomB-S38C. Two of thethree endogenous Cys residues of PomB, Cys-8 and Cys-10, areresponsible for the homotypic cross-link formation (55). Thus,
it is likely that X180 is a heterotetramer, PomA-PomB-PomBPomA: the cross-linked products of PomA-D170C and PomBS38C are cross-linked through the cytoplasmic Cys residues of
PomB to form the linear cross-linked product. The heterotetramer X180 seems to reflect a part of the complex of four
PomA and two PomB proteins, (PomA2/PomB)2 (43, 55). On
the other hand, X120 was clearly detected by anti-PomB antiserum, but just a faint band can be detected by anti-PomA
antiserum (Fig. 4). The D170C mutation in PomA and three
endogenous Cys of PomB are sufficient for the X120 formation(Fig. 4; data not shown). Because X120 are readily detectedwith anti-PomA antiserum through purification of the crosslinked products (Yakushi and Homma, unpublished), it islikely that X120 consists of PomA and PomB but there is somesteric hindrance from the antibody recognition. Therefore,
from the mobility of the X120 band on SDS-PAGE, we suggestthat X120 is an entangled complex of the homotypic crosslinked products of PomA-PomA and PomB-PomB, being
masked against anti-PomA antibody. The nature of PomA
forming an SDS-resistant dimer would not be involved in theformation of the two different types of the larger cross-linked
products, X120 and X180, because the combination of PomAD170C and PomBxl-S38C showed a single cross-linked product, X60 (Fig. 4 and 5).
As PomA-D170C/PomB-S38C, the combinations of a seriesof the Cys substitutions in PomA TM3 and PomB TM synergistically affected the swarming ability and formed cross-linked
products of PomA and PomB (Fig. 6). Furthermore, it has
been proposed that Asp-148 in TM3 of PomA and Pro-16 of
PomB may be close to each other and form a phenamil highaffinity site (28), results consistent with this study showing that
substituted Cys residues at these positions are actually crosslinked (Fig. 8). Since PomA TM3 and PomB TM are close toeach other on both faces of the membrane, it is reasonable toinfer that they remain in intimate contact throughout the entire TM region from the periplasmic to the cytoplasmic side.
It has been shown that the sensitivity of PomA-D170C to
DTNB treatment is affected by the NaCl concentration (5).
Asp-170 is predicted to face the pore of the channel and tointeract with Na⫹. The present study supports this possibility
because of the higher apparent Km value for Na⫹. The swarming ability of cells with PomA-D170C/PomB-S38C was significantly decreased at low NaCl concentrations even in the presence of DTT (Fig. 7), although the swarming ability of thesingle mutants was only slightly affected by the NaCl concentration. The kinetic analysis of swimming confirms that the
double mutant requires a higher concentration of Na⫹. Taken
together, it is suggested that an interface around Asp-170 of
PomA and Ser-38 of PomB participates in Na⫹ conductance todrive the motor. A working hypothesis has been proposed suchthat the motor has two ion-binding sites, extracellular andintracellular, for the ion flux (36, 46, 56). It is plausible that

J. BACTERIOL.

Asp-170 of PomA and Ser-38 of PomB may participate as an
entrance for extracellular Na⫹. In this work, we found that the
mutation of Gly-23 in PomA TM1 affects the Na⫹ requirement
for motility and shows synergism with the PomB-S38C mutation (Fig. 3; data not shown). It has been shown that the strains
with PomA substitutions at Asp-31 in periplasmic loop1-2 hada slow-motility phenotype and required higher Na⫹ concentrations to start swimming than those with wild-type PomA (32).
This may suggest that the periplasmic loop1-2 or the boundary
also contribute to an entrance for the Na⫹ translocation.
The cytoplasmic region of PomA, which connects TM2 andTM3, is involved in flagellar rotation and interacts with a rotor
protein, FliG (52). As PomB TM has a central role in Na⫹ flux,
we speculate that the Na⫹ flux in the interface between PomA
TM3 and PomB TM evoke conformational changes of thecytoplasmic region of PomA to drive the flagellar rotation. In
the H⫹-type motor of E. coli, it was shown that the protonationof Asp-32 in the MotB TM induces a conformational change inthe cytoplasmic region between TM2 and TM3 (29). The stator
complex is apparently composed of four PomA (MotA) and
two PomB (MotB) molecules or a larger complex than the 4:2
complex (48). At least 18 TMs of the complex are arranged in
the cytoplasmic membrane to form an ion channel or channels.
Based on the recent cross-linking data, a model for the arrangements of 10 TMs in the MotA/MotB complex of E. coli
has been proposed (15). The model is consistent with our
results demonstrating that the PomB TM and the PomA TM3
regions are in closest association to each other in the TMs. To
understand the mechanism of force generation by ion flux, thearrangement and structure of the TM regions must be clarified.
ACKNOWLEDGMENTS
We thank Noriko Ui and Emi Mitsuyama for technical assistanceand Ikuro Kawagishi, Yoshiyuki Sowa, and Tomohiro Yorimitsu for
stimulating discussions. We also thank Yukako Asai and Seiji Kojima
for construction of bacterial strains. We are grateful to the late Robert
M. Macnab for invaluable discussions and critically reading 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;
the Japan Science and Technology Corporation (to M.H. and T.Y.);and from the Soft Nano-Machine Project of Japan Science and Technology Agency (to T.Y. and M.H.).
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