JOURNAL OF BACTERIOLOGY, Aug. 1999, p. 5103–5106
0021-9193/99/$04.00⫹0Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 16

Functional Interaction between PomA and PomB, the Na⫹-Driven
Flagellar Motor Components of Vibrio alginolyticus
TOMOHIRO YORIMITSU, KEN SATO, YUKAKO ASAI, IKURO KAWAGISHI, AND MICHIO HOMMA*Division of Biological Science, Graduate School of Science, Nagoya University,Chikusa-Ku, Nagoya 464-8602, Japan
Received 1 March 1999/Accepted 21 May 1999

Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodiumdriven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to forma sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB.
As a result, we could demonstrate that PomA and PomB functionally interact with each other.
dicted from the DNA sequence of each gene. Those for antibody PomA91 were raised against mixtures of fragment 1(K126 to P151), fragment 2 (T185 to T212), and fragment 3(Q226 to K246). Those for antibody PomA1312 were raised
against fragment 4 (P231 to E253). Those for anti-PomB antibody were raised against mixtures of fragments 5 (T104 toG128), 6 (K219 to S243), and 7 (S266 to R290).
The reactivities of antibodies PomA91 and PomB93 against
cellular proteins were assessed by immunoblotting (Fig. 1). An
overnight culture of NMB191 cells in VC medium (3) wasdiluted 1:50 into VPG medium. At mid-log phase, cells wereharvested by centrifugation and suspended at an optical density at 660 nm of 10 in sodium dodecyl sulfate (SDS) loading
buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 1% SDS,0.1% bromophenol blue) containing ␤-mercaptoethanol.
The suspension was boiled for 5 min, and then proteins inthe samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to a
polyvinylidene difluoride membrane (Millipore Japan, Tokyo)
by using a semidry blotting apparatus (Biocraft, Tokyo, Japan).
PomA91 or PomB93 antisera were used for the primary antibody, and an alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was used as the secondary antibody. Detection
was performed as described previously (19). The plasmids wereintroduced into cells by electroporation as described previously(13).
With antibody PomA91, a protein with a molecular mass of
25 kDa was specifically detected in cells expressing the pomA
gene from a plasmid. Antibody PomB93 specifically recognizeda protein of 37 kDa in cells harboring a plasmid carrying pomB.
The molecular masses of 25 kDa and 37 kDa correspond to thepredicted molecular masses of PomA (27,224 Da) and PomB
(35,461 Da), respectively. The appearance of those bands wascorrelated to the presence of the pomA or pomB gene on theplasmid. Both proteins were detected in a membrane fraction(data not shown).
In this detection system, neither PomA nor PomB was detected in cells expressing at wild-type levels. However, twoproteins with masses of 25 and 37 kDa could be immunoprecipitated from lysates of 35S-labeled wild-type cells (Fig. 2). For
the immunoprecipitation assay, cells of strains VIO5 (wild-typepolar flagella and NMB155 multipolar flagella) were cultured
overnight in VC medium and inoculated 1:50 in synthetic medium (25). At mid-log phase, Tran35S-label (ICN Biomedicals
Inc., Costa Mesa, Calif.) was added to 100 ␮Ci/ml; then theMany bacteria swim by rotating flagellar filaments, which arepowered by rotary motors. The flagellar filament is attached
via a flexible coupling to the basal body. Multiple torquegenerating units exist in the cytoplasmic membrane surrounding the basal body and function independently (1). Energy todrive the motor comes from the transmembrane electrochemical potential of specific ions. For some bacteria, such as Escherichia coli and Bacillus subtilis, the flagellar motor is driven by
the proton motive force, whereas alkaliphilic Bacillus and marine Vibrio species are driven by the sodium motive force (11,16).
E. coli has two motor proteins, MotA and MotB, which areessential for rotating the proton-driven flagellar motor. MotAand MotB are localized in the cytoplasmic membrane and havefour and one transmembrane segment(s), respectively (8, 22).
From genetic and biochemical studies, it has been suggested
that MotA and MotB form a complex via transmembraneregions and function as a proton channel (6, 21, 23, 24). The C
terminus of MotB, which has a peptidoglycan binding motif, isthought to bind to peptidoglycan to make MotA–MotB complexes function as the stator (7, 9). Vibrio alginolyticus has two
types of flagellar motor in the same cell: sodium driven (polarflagella) and proton driven (lateral flagella) (4, 12). Fourgenes, pomA, pomB, motX, and motY, have been shown to be
essential for rotation of the sodium-driven polar flagella (3, 17,18, 20). PomA and PomB exhibit about 20 to 30% similarity tothe proton-driven motor proteins, MotA and MotB, respectively. Thus, it is plausible that PomA and PomB have roles
similar to those of MotA and MotB (3). In this study, we
prepared antipeptide antibodies against PomA and PomB andinvestigated the functional interaction between expressed
PomA and PomB proteins.
Strains and plasmids. Strains and plasmids used in thisstudy are listed in Table 1.
Detection of PomA and PomB by antipeptide antibody. Antipeptide antibodies against PomA or PomB, which were called
PomA91 or PomB93, respectively, were produced by Biologica(Nagoya, Japan). Another antipeptide antibody against PomA,called PomA1312, was produced by Sawady Technology (Tokyo, Japan) and was affinity purified. Peptide fragments weresynthesized to correspond to the amino acid sequence pre* 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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J. BACTERIOL.

TABLE 1. Bacterial strains and plasmids
Strain orplasmid

Genotype or descriptiona

Source orreference

V. alginolyticus
strains
VIO5
NMB155
NMB191blaf (Pof⫹ Laf⫺)
laf (Pofm Laf⫺)⌬pomAB mutant of VIO520
S. Kojima
This work

Plasmids
pSU41
pYA301
pYA303
pSK603kan (Kmr) Plac lacZ␣
pSU41 Plac pomA
pSU41 Plac pomA pomBpSU41 Plac pomB

5141414aKmr, kanamycin resistant; Plac, lac promoter; Pof, polar flagella; Laf, lateral
flagella; Pofm, multipolar flagella.
bThe pomAB deletion mutant was constructed by a procedure similar to that
described elsewhere (2). pYA303 was digested with DraI (inside the pomA gene)and HpaI (inside the pomB gene) and ligated. The resulting plasmid (pYA303⌬pomAB) contains codons 1 to 68 of pomA fused to codons 126 to 301 of pomB.
An XbaI-SacI fragment of pYA303 ⌬pomAB was moved to a suicide vector,pKY704. The deletion was introduced into strain VIO5.

mixture was incubated at 30°C for 30 min. The radioactively
labeled cells were harvested by centrifugation and lysed at4°C for 30 min with 1 ml of TNET buffer (50 mM Tris-HCl[pH 7.8], 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100);
then the lysate was centrifuged at 10,000 ⫻ g for 30 min. The
labeled proteins were immunoprecipitated with either antibody PomA1312 or antibody PomB93 by a method described
previously (10). The resulting precipitates were subjected toSDS-PAGE followed by fluorography.
In both VIO5 and NMB155 cells, the 25-kDa PomA protein
or the 37-kDa PomB protein was immunoprecipitated. The 25and 37-kDa bands were not detected in a ⌬pomAB mutantstrain. These results indicate that PomA and PomB can be
specifically detected by the antibodies prepared in this study.
Other bands in addition to the PomA and PomB bands werealso detected by the antibodies. A 45-kDa band was always
present in cells overexpressing PomA. This band might represent the different conformations of PomA or an SDS-resistantcomplex of PomA with itself or another protein.

FIG. 1. Detection of proteins by immunoblotting with antipeptide antibodies
against PomA (A) or PomB (B). Lanes 1, pSU41; lanes 2, pYA301; lanes 3,pYA303; lanes 4, pSK603. Molecular mass markers are indicated.

FIG. 2. Immunoprecipitation assays with PomA1312 (A) and PomB93 (B).
Lanes 1, NMB191 transformed with pSU41; lanes 2, VIO5; lanes 3, NMB155;
lanes 4, NMB191 transformed with pYA303.

Stabilities of PomA and PomB proteins in vivo. MotA andMotB proteins of E. coli are thought to be associated in acomplex (21, 23, 24). It has been shown that MotB is unstable
unless excess MotA is expressed together in the same cell(26). The PomA and PomB proteins might show features similar to those of MotA and MotB. In fact, the amount of PomB
was much smaller when it was expressed alone than when
PomA and PomB were expressed simultaneously (Fig. 2, lanes
3 and 4).
To examine the stabilities of PomA and PomB, NMB191
cells harboring the pomA gene and/or the pomB gene werecultured for 3, 6, 12, or 24 h and the amount of expressed
PomA or PomB in the cells was analyzed by immunoblotting(Fig. 3). The intensity of the PomA band did not change during
the entire period of the experiment, whether PomB waspresent or not (Fig. 3A). In contrast, PomB was stable when
coexpressed with PomA but decreased in amount, and disappeared completely after 12 h, when expressed by itself (Fig.
3B). Pulse-chase analysis (Fig. 4) supported the PomA-dependent stabilization of PomB, and the half-time for disappearance of PomB was calculated to be about 4.5 h. These results
suggest that PomA and PomB functionally interact with eachother. They show that PomA is quite stable whether expressedby itself or together with PomB. PomB is slowly degraded in
the absence of PomA, however, which indicates that simultaneous synthesis of PomA may facilitate the overproduction ofPomB. This result is similar to that observed with the MotA
and MotB proteins of E. coli (26). These facts support the idea

FIG. 3. Stabilities of PomA and PomB. Overnight cultures of NMB191 cellswith the pomA and/or pomB gene on a plasmid were inoculated at 1:50, and cells
were harvested after 3, 6, 12, and 24 h. PomA or PomB was detected byimmunoblotting with antibody PomA91 (A) or antibody PomB93 (B), respectively, as described in the text. Numbers below the lanes correspond to the times
when cells were harvested.

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FIG. 4. Pulse-chase analysis of PomB. NMB191 cells harboring plasmids
carrying pomA plus pomB (pYA303) (A) or pomB alone (pSK603) (B) werecultured in synthetic medium. At mid-log phase, Tran35S-label was added to 100
␮Ci/ml, and the mixture was incubated at 30°C for 30 min (pulse). Excess
unlabeled methionine and cysteine were added to stop the labeling (chase at timezero). At the times indicated above the lanes, the cells were harvested by centrifugation, suspended in 1 ml of TNET buffer, and then immunoprecipitatedwith antibody PomB93, followed by SDS-PAGE and fluorography, as describedin the text.

that PomA and PomB may interact with each other in thesodium-driven motor, as MotA and MotB do in the protondriven motor.
Interaction of PomA and PomB. To test for physical interaction between PomA and PomB, we carried out immunoprecipitation assays (Fig. 5). Radiolabeled whole-cell lysate wasincubated with antibody PomA1312, and PomA was immunoprecipitated; then the protein associated with PomA was analyzed by SDS-PAGE followed by fluorography. In addition tothe 25-kDa band due to PomA, a 37-kDa band correspondingto PomB was also observed. The 37-kDa band was seen only incells harboring pomA and pomB genes (Fig. 5A), which suggests that PomA forms a complex with PomB. Similar results
were obtained with antibody PomB93. A 25-kDa protein wasspecifically coprecipitated along with PomB when the pomA
gene was present (Fig. 5B). These results indicate that PomA
and PomB physically interact with each other.
Amiloride and its analogs, which are inhibitors of Na⫹ channels, Na⫹/H⫹ exchangers, and Na⫹/Ca⫹ exchangers, specifically inhibit the Na⫹-driven flagellar motor. Phenamil is themost potent inhibitor of the Na⫹ motor. Previously, we isolated mutants whose motility is resistant to phenamil (Mpar)
and mapped the Mpar mutations to specific sites in pomA andpomB (14, 15). Further mutational analysis suggested that acertain structural change around these sites affects the sensitivity of the motor to phenamil. When mutations were present
in both PomA and PomB, motility was less sensitive to phenamil than with either mutation alone. These findings also support the proposal that PomA and PomB interact with eachother.
MotA and MotB are required for the rotation of proton-FIG. 5. Coprecipitation of PomA and PomB. NMB191 cells transformedwith the plasmid were labeled with Tran35S-label for 30 min and lysed, and thesamples were immunoprecipitated either with antibody PomA1312 (A) or withantibody PomB93 (B) as described in the text. Samples were analyzed by SDSPAGE followed by fluorography. The transformed plasmids were as follows:
lanes 1, pSU41; lanes 2, pYA301; lanes 3, pSK603; lanes 4, pYA303.

NOTES

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driven flagella, while sodium-driven flagella require four components: PomA, PomB, MotX, and MotY (3, 17, 18, 20). The
motX and motY genes, whose homologs are not found in theE. coli genome, are located apart from each other on the Vibrio
chromosome. On the other hand, pomA and pomB are in thesame operon. It has been proposed that MotX is a sodium
channel component and MotY functions to connect the stator
to peptidoglycan (17, 18). Thus, we might speculate that thesodium-driven motor contains a complex containing four proteins rather than only two as in the proton-driven motor. Further analysis will be required to determine the functions ofMotX and MotY.
We thank R. Elizabeth Sockett and David F. Blair for criticallyreading the manuscript and S. Kojima of our laboratory for providing
us with the Pofm strain.
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 from the Japan Society for the Promotion ofScience (to Y.A.).
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