Journal of Bacteriology, August 1999, p. 5103-5106, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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
 |
ABSTRACT |
Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in
force generation of the sodium-driven polar flagella of Vibrio
alginolyticus. Among these, PomA and PomB seem to be associated and to form a 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.
| |
TEXT |
Many bacteria swim by rotating
flagellar filaments, which are powered by rotary motors. The flagellar
filament is attached via a flexible coupling to the basal body.
Multiple torque-generating units exist in the cytoplasmic membrane
surrounding the basal body and function independently (1).
Energy to drive 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 are
essential for rotating the proton-driven flagellar motor. MotA and MotB
are localized in the cytoplasmic membrane and have four 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 transmembrane regions and function as a proton channel
(6, 21, 23, 24). The C terminus of MotB, which has a
peptidoglycan binding motif, is thought 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 (polar flagella) and proton driven (lateral
flagella) (4, 12). Four genes, 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 to the 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 and investigated
the functional interaction between expressed PomA and PomB proteins.
Strains and plasmids.
Strains and plasmids used in this study
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 were synthesized to correspond to the amino acid
sequence predicted 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 to G128), 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) was diluted 1:50 into VPG medium. At
mid-log phase, cells were harvested 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 in the 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 were introduced into cells by electroporation as described previously (13).
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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.
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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 recognized a protein of 37 kDa
in cells harboring a plasmid carrying pomB. The molecular masses of 25 kDa and 37 kDa correspond to the predicted molecular masses of PomA (27,224 Da) and PomB (35,461 Da), respectively. The
appearance of those bands was correlated to the presence of the
pomA or pomB gene on the plasmid. 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, two proteins 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-type polar 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 µCi/ml; then the mixture was incubated at 30°C for
30 min. The radioactively labeled cells were harvested by
centrifugation and lysed at 4°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
to SDS-PAGE followed by fluorography.
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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.
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|
In both VIO5 and NMB155 cells, the 25-kDa PomA protein or the 37-kDa
PomB protein was immunoprecipitated. The 25- and 37-kDa bands were not
detected in a 
pomAB mutant strain. 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 were also 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-resistant complex of PomA
with itself or another protein.
Stabilities of PomA and PomB proteins in vivo.
MotA and MotB
proteins of E. coli are thought to be associated in a
complex (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 were cultured 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 was
present 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 each other. They show that PomA is
quite stable whether expressed by 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 of
PomB. This result is similar to that observed with the MotA and MotB
proteins of E. coli (26). These facts support the
idea that PomA and PomB may interact with each other in the
sodium-driven motor, as MotA and MotB do in the proton-driven motor.
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FIG. 3.
Stabilities of PomA and PomB. Overnight cultures of
NMB191 cells with 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 by immunoblotting 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) were cultured in synthetic medium.
At mid-log phase, Tran35S-label was added to 100 µCi/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 time zero). At the times indicated above the lanes, the cells
were harvested by centrifugation, suspended in 1 ml of TNET buffer, and
then immunoprecipitated with antibody PomB93, followed by SDS-PAGE and
fluorography, as described in the text.
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|
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 was
incubated with antibody PomA1312, and PomA was immunoprecipitated; then
the protein associated with PomA was analyzed by SDS-PAGE followed by
fluorography. In addition to the 25-kDa band due to PomA, a 37-kDa band
corresponding to PomB was also observed. The 37-kDa band was seen only
in cells 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 was specifically
coprecipitated along with PomB when the pomA gene was
present (Fig. 5B). These results indicate that PomA and PomB physically
interact with each other.
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FIG. 5.
Coprecipitation of PomA and PomB. NMB191 cells
transformed with the plasmid were labeled with
Tran35S-label for 30 min and lysed, and the samples were
immunoprecipitated either with antibody PomA1312 (A) or with antibody
PomB93 (B) as described in the text. Samples were analyzed by SDS-PAGE
followed by fluorography. The transformed plasmids were as follows:
lanes 1, pSU41; lanes 2, pYA301; lanes 3, pSK603; lanes 4, pYA303.
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|
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 the most 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 and
pomB (14, 15). Further mutational analysis
suggested that a certain 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 each other.
MotA and MotB are required for the rotation of proton-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 the
E. coli genome, are located apart from each other on
the Vibrio chromosome. On the other hand, pomA
and pomB are in the same 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 the sodium-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 of MotX and MotY.
| |
ACKNOWLEDGMENTS |
We thank R. Elizabeth Sockett and David F. Blair for critically
reading 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 of
Science (to Y.A.).
| |
FOOTNOTES |
*
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{at}nucc.cc.nagoya-u.ac.jp.
| |
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Journal of Bacteriology, August 1999, p. 5103-5106, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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