JOURNAL OF BACTERIOLOGY, June 2000, p. 3314–3318
0021-9193/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 182, No. 11

A Slow-Motility Phenotype Caused by Substitutions at Residue
Asp31 in the PomA Channel Component of a Sodium-Driven
Flagellar Motor
SEIJI KOJIMA,† TOMOKAZU SHOJI, YUKAKO ASAI, IKURO KAWAGISHI, AND MICHIO HOMMA*
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
Received 3 January 2000/Accepted 17 March 2000

PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of Vibrio
alginolyticus. We have found that some cysteine substitutions in the periplasmic region of PomA result in aslow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of highconcentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild typebut with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C
mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C
mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein
is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue
is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or
efficiency of the motor.
body, and PomB could be coprecipitated with PomA by antiPomA antibody (30). These results support the hypothesis that
PomA and PomB form a complex. Recently, it has been shown
directly that the complex consisting of PomA and PomB catalyzes sodium influx in reconstituted proteoliposomes (24).
Slow-motility mutants caused by mutations in MotA havebeen isolated in E. coli and Salmonella serovar Typhimurium(5, 29). It was reported that in most of the slow-motility mutants motor torque was normal at low speeds but reduced athigh speeds. These mutants generated approximately equal
torques in H2O and D2O, but their swimming speeds weresignificantly reduced in D2O compared with those in H2O (5).
These results suggested that motor rotation is kinetically limited in these mutants by a process that might involve protontransfer. In the case of the sodium-driven motor, rates of
sodium ion flux are easily altered by changing the NaCl concentration or by using sodium channel-specific inhibitors. Thisapproach allows a direct assessment of the relationship between slow rotation and ion flux through the motor. We havegenerated a series of cysteine-substituted mutations in the
periplasmic residues of PomA and found that some of thesemutants show a slow-motility phenotype (2). In this study, oneof these slow-motility mutants, which could not swim in a low
concentration of NaCl, was characterized.
The PomA mutation D31C causes slow polar flagellar motility. We first examined the relationship between swimming
speed and NaCl concentration in the slow-motility pomA mutants isolated previously. The plasmid pYA301 (pomA⫹ Kmr)
(15) containing wild-type or mutant pomA was introduced into
the V. alginolyticus strain NMB188 (Laf⫺ PomA⫺ Che⫺) (15)and cultured to late logarithmic phase at 30°C in VPG medium(1% polypeptone, 0.4% K2HPO4, 3% NaCl, 0.5% glycerol).
Swimming speeds were measured in TMN medium containing
50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM glucose, andvarious concentrations of NaCl and KCl to keep the sum of theconcentrations of NaCl and KCl at 300 mM. Most of theslow-motility mutants obtained by cysteine substitutions in the
periplasmic residues of PomA showed a dependence on sodium ion concentration similar to that of the wild-type strain,
but with swimming speeds significantly lower than for the wild-

Flagella are the filamentous organelles responsible for motility of most bacteria. Flagellar rotation is driven by a reversible rotary motor embedded in the cytoplasmic membrane at
the base of each flagellar filament. The energy for rotation of
the flagellar motors comes from the electrochemical gradientof specific ions across the cytoplasmic membrane (protons inEscherichia coli and Salmonella enterica serovar Typhimurium,or sodium ions in Vibrio spp. and alkalophilic Bacillus strains)(4, 11, 12, 17).
The essential component for energy conversion in the motor
is thought to be a force-generating unit that functions as an ion
channel and interacts with the coupling ion to produce mechanical force. In the proton-driven motor, the channel is composed of a complex of MotA and MotB (5, 9, 10, 25, 27, 28),
membrane proteins with four and one single transmembranesegments, respectively (6, 7, 26, 31). It is believed that thecytoplasmic domain of MotA interacts electrostatically with
the C-terminal domain of the rotor protein FliG (19, 32). Fourproteins, PomA, PomB, MotX, and MotY, are essential for
rotation of the sodium-driven polar-flagellar motors of themarine bacteria Vibrio alginolyticus (1, 8, 23) and Vibrio parahaemolyticus (13, 20, 21). PomA and PomB are homologs of
MotA and MotB and are thus thought to form a sodium channel in the motor. The functions of MotX and MotY are unknown, although MotX may also be a sodium channel component of the motor, because overproduction of MotX is lethal in
E. coli in proportion to the external sodium ion concentration,
and also because this lethality is suppressed by the addition of
amiloride, a specific inhibitor of the sodium-driven motor (20).
Mutations resistant to phenamil, an amiloride analog, wererecently identified in PomA and PomB, and these mutations
showed synergistic effects on motility (13, 15). In addition,
PomA could be coprecipitated with PomB by anti-PomB anti* 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.
† Present address: Department of Biology, University of Utah, Salt
Lake City, UT 84112.
3314

VOL. 182, 2000

FIG. 1. Sodium concentration dependence of slow-motility mutants. (A)NMB188 (PomA⫺ Che⫺) cells containing wild-type (filled circle) or mutant(I175C [triangle] or D31C [open circle]) pomA cells were harvested in latelogarithmic phase and resuspended in TMN medium (pH 7.5) containing various
concentrations of NaCl. Motility of the cells was observed at room temperature
under a dark-field microscope and recorded on videotape. Swimming speed wasdetermined as described previously (3). The average swimming speed was obtained by measuring more than 20 swimming tracks. (B) The predicted transmembrane regions of PomA. The residue Asp31 is marked by a filled circle andwhite letter. The topology of PomA was predicted from the hydropathy profile of
PomA and the topology of the MotA protein of E. coli (31).

type strain (Fig. 1A; PomA I175C is a typical example). Amongthe slow-motility mutants, only D31C showed a significantly
different dependence on sodium ion concentration (Fig. 1A).
This mutant did not swim at low concentrations of NaCl (0 to19 mM). It could swim in medium containing 38 mM NaCl,and its swimming speed increased gradually as the concentration of NaCl in the medium increased. It swam at about 20
␮m/s in 300 mM NaCl, a speed about one-fourth that of cells
expressing the wild-type PomA and comparable to the speed of
the PomA I175C mutant. As shown in Fig. 1B, Asp31 of PomA
is thought to be located in the periplasmic loop region. We
found that the swimming speed of the PomA D31C mutant was
further reduced by the sulfhydryl modifying reagents dithionitrobenzoic acid and N-ethylmaleimide, although motility of
the wild-type strain, which does not contain cysteine in PomA,
was not reduced. This result suggests that this residue is exposed to the outside (2).
Site-directed mutagenesis of PomA Asp31. Given the presumed location of Asp31 in the periplasmic space (Fig. 1B), thenegative charge of this residue might affect rates of ion influx

NOTES

3315

through the motor. We next carried out site-directed mutagenesis to replace Asp31 of PomA with various residues. Mutations were made in pomA carried in the plasmid pYA301 by
the two-step PCR method described previously (15). Plasmids
carrying the mutated pomA were transformed to the PomA⫺
Che⫺ strain NMB188, and swimming speeds were measured atvarious NaCl concentrations (Fig. 2). We generated 10 new
mutants, all of which, except the D31F mutant, exhibited motility at 300 mM NaCl. The mutants were classified into fourgroups according to their NaCl thresholds for motility andtheir maximal swimming speeds in 300 mM NaCl. The type I
mutant D31E showed nearly the same sodium dependence andswimming speed as the wild-type strain. Type II mutants wereslightly shifted in their threshold for motility, being motile in 13
to 19 mM NaCl, and they exhibited a reduced maximal swimming speed relative to the wild-type strain. Mutants carrying
substitutions with neutral side chains (D31N, D31Q, andD31S) fell into this group. The single type III mutant (D31G)
showed the same NaCl dependence and maximum speed as
D31C. Type IV mutants had a higher NaCl threshold for motility, requiring more than 75 mM NaCl for motility and showing reduced maximum swimming speeds relative to D31C. Substitution with small (D31A), large (D31Y), or positively
charged residues (D31K and D31R) produced this profile.
These results show that various mutations in Asp31 of PomA
affect the sodium ion concentration dependence of motility.
Charge-reversed mutations (D31K and D31R) greatly reduced
motility, but the charge-conserved mutation (D31E) did notaffect motility very much, suggesting that a negative charge at
this position might be important for optimal function of the
motor.
To investigate whether Asp31 is located near ion-bindingsites in the channel complex, we examined the inhibition of
motility of the D31C mutant by the sodium channel-specificinhibitors phenamil and amiloride. Motility of both the D31C
mutant and the wild-type strain was inhibited by 50 ␮M phenamil or 3 mM amiloride in 100 mM NaCl (data not shown).
Thus, Asp31 of PomA might not be directly involved in binding
sodium ions. We also performed immunoblotting of whole-cell
proteins of the mutants, using a polyclonal anti-PomA peptide
antibody. All of the mutant PomA proteins were detected at
levels sufficient to support motor function (data not shown).
Analysis of speed fluctuations in the PomA D31C mutant. It
has been shown, by adjusting the sodium ion concentration in
the medium, that rotation of the polar flagellar motor of V.
alginolyticus is stable as the motor speed is varied over a wide
range (from about 50 to about 1,000 revolutions per second[rps]) (22). Thus, if sodium flux is reduced in the D31C motor
but other aspects of function are unaffected, the rotation speedof the motor should not fluctuate. However, if the force-generating units of the mutant bind unstably to sites in the motor,
this effect should slow the motility and increase fluctuations inrotation speed. To test this idea, we measured flagellar rotation in NMB188 cells containing wild-type or D31C mutant
PomA protein by using laser dark-field microscopy (LDM), as
described previously (16). In this method, a rapidly rotating
flagellum is irradiated by a thin beam of laser light, and its
rotation rate is measured from the intensity change of scattered light (18).
NMB188 cells containing wild-type or D31C PomA protein
were harvested at late logarithmic phase, washed once with
TMN medium, and resuspended in the same medium. The cellsuspension was poured into the space between the slide andthe cover glass on which thin spacers were placed. After 10
min, cells not stuck to the cover glass were washed by a flow of
TMN medium. To allow speed fluctuations to be compared at

3316

NOTES

J. BACTERIOL.

FIG. 2. Sodium-dependent motility of the PomA Asp31 mutants. Mutants were classified into four groups based on the Na⫹ concentration threshold for motility.
NMB188 cells expressing each mutant PomA were harvested at late logarithmic phase and suspended in TMN medium containing various concentrations of NaCl.
Swimming speeds were measured as described in the legend to Fig. 1. WT, wild type.

the same average rotation speed, wild-type cells were studied
in 6 mM NaCl and mutant cells were studied in 300 mM NaCl.
Figure 3A shows the intensity of scattered light versus time forboth the wild type and the mutant. Both mutant and wild-typeflagella rotated steadily at about 200 rps (Fig. 3B). To evaluate
speed fluctuations of the motors, we made the following calculation for multiple samples: (standard deviation of the rotation rate)/(average rotation rate) ⫻ 100. This value was 10 and
12% for the wild type and the D31C mutant, respectively,
suggesting that force-generating units of the mutant D31C
motor are stably bound to the motor.
Possible role of negative charge of Asp31 for motor rotation.
In this study, we characterized slow-motility mutants thatmight have defects in essential steps of torque generation by
the flagellar motor. Two questions are raised by these slowmotility mutants. First, what slows the rotation of these mutant
motors? Second, what causes a threshold for motility? Two
factors are likely to be important: the rate of sodium influx
through the motors and the efficiency of energy conversion in
the motors. That is, ion flux through the mutant motors might
be decreased and/or the efficiency of energy coupling in the
mutant motors might be reduced by defects in the channel
complex. The simplest model for such a defect is that mutationof Asp31 of PomA makes ions pass more slowly through thechannel (for example, the pore size might become smaller).
Mutations might reduce the rate of flow so much that it results
in a threshold for motility. Similarly, reduction in the efficiencyof energy conversion in the motor might cause this phenotype.
Based on this study, we cannot conclude which model ismore likely to explain the effect on motor rotation imposed byreplacements of Asp31. Asp31 is located in the first periplasmic loop (loop1-2) of PomA, and it has been suggested thatloop1-2 may be in contact with other proteins, such as PomB,
MotX, or MotY, or that it may be embedded in the pore regionof the channel (2). In either case, the negative charge of thisresidue appears to be important. Introduction of a positive
charge (D31K or D31R), and also changes in the size of theside chain (D31A and D31Y), caused large increases in thethreshold NaCl concentration needed for motility. These mutations (type IV) probably do not cause unstable binding of theforce-generating units to the motor, because the motors of
D31C mutant rotated steadily. Thus, the mutant phenotype isprobably due to defects in the channel complex itself and not
in its installation into the motor.
The periplasmic segment of PomA contains two other negative charges, D170 and D171 (in loop3-4). These negative
charges, together with D31, may function to recruit sodium
ions into the channel complex. An analogous recruitment
mechanism for hydrogen ions has been suggested for bacteriorhodopsin (14). The D170C or D171C single mutant reduced
motility; however, both of them did not have NaCl thresholds
for motility as D31C did. Double mutations of these two residues might affect motility more severely. According to sequence alignment of MotA proteins, these three residues areconserved in some species, but not perfectly. Alignment also
shows that MotA proteins have several charged residues inperiplasmic domains, and in most cases, their net charges arenegative. This might imply that the negatively charged area inVOL. 182, 2000

NOTES

3317

FIG. 3. Flagellar rotation of wild-type and D31C mutant cells detected by LDM. Rotation of a single polar flagellum of a stuck cell of NMB188 expressing wild-typeor D31C PomA protein was measured by using LDM for about 6 s in the presence of 6 mM NaCl (wild type) or 300 mM NaCl (D31C), as described previously (16).
(A) Intensity changes of the scattered light from the rotating flagellum. The interval between peaks corresponds to the rotation period. (B) Fluctuations in rotation
rate of the wild-type and D31C mutant motors plotted as the reciprocal of the interpeak intervals from the data in panel A. Average rotation rates were 197 ⫾ 20.7
rps (wild type) and 187 ⫾ 16.9 rps (D31C).

MotA proteins can function similarly to recruit protons into
MotA/MotB channels.
Recently, our group succeeded in measuring sodium ion
flux, in response to a potassium diffusion potential, throughpurified PomAB channel complexes reconstituted in proteoliposomes (24). Such a direct measurement of the current
passed through the channel complex will be needed to clarify
the function of residue Asp31. We hope to determine thecause(s) of the Na⫹ concentration threshold and thus gain
insight into the mechanism of torque generation.
We thank David F. Blair and Ken Sato for critically reading themanuscript.
This work was supported in part by grants-in-aid for scientific researches (to I.K. and M.H.) from the Ministry of Education, Science
and Culture of Japan and the Japan Society for the Promotion of
Science (to S.K. and Y.A.).

3318

NOTES

J. BACTERIOL.
REFERENCES

1. Asai, Y., S. Kojima, H. Kato, N. Nishioka, I. Kawagishi, and M. Homma.
1997. Putative channel components for the fast-rotating sodium-driven
flagellar motor of a marine bacterium. J. Bacteriol. 179:5104–5110.
2. Asai, Y., T. Shoji, I. Kawagishi, and M. Homma. 2000. Cysteine-scanningmutagenesis of the periplasmic loop regions of PomA, a putative channel
component of the sodium-driven flagellar motor in Vibrio alginolyticus. J.
Bacteriol. 182:1001–1007.
3. Atsumi, T., Y. Maekawa, T. Yamada, I. Kawagishi, Y. Imae, and M. Homma.
1996. Effect of viscosity on swimming by the lateral and polar flagella ofVibrio alginolyticus. J. Bacteriol. 178:5024–5026.
4. Blair, D. F. 1995. How bacteria sense and swim. Annu. Rev. Microbiol.
49:489–522.
5. Blair, D. F., and H. C. Berg. 1990. The MotA protein of E. coli is a protonconducting component of the flagellar motor. Cell 60:439–449.
6. Chun, S. Y., and J. S. Parkinson. 1988. Bacterial motility: membrane topology of the Escherichia coli MotB protein. Science 239:276–278.
7. Dean, G. E., R. M. Macnab, J. Stader, P. Matsumura, and C. Burks. 1984.
Gene sequence and predicted amino acid sequence of the motA protein, amembrane-associated protein required for flagellar rotation in Escherichia
coli. J. Bacteriol. 159:991–999.
8. Furuno, M., N. Nishioka, I. Kawagishi, and M. Homma. 1999. Suppression
by the DNA fragment of the motX promoter region on long flagellar mutantsof Vibrio alginolyticus. Microbiol. Immunol. 43:39–43.
9. Garza, A. G., R. Biran, J. A. Wohlschlegel, and M. D. Manson. 1996. Mutations in motB suppressible by changes in stator or rotor components of thebacterial flagellar motor. J. Mol. Biol. 258:270–285.
10. Garza, A. G., P. A. Bronstein, P. A. Valdez, L. W. Harris-Haller, and M. D.
Manson. 1996. Extragenic suppression of motA missense mutations of Escherichia coli. J. Bacteriol. 178:6116–6122.
11. Hase, C. C., and J. J. Mekalanos. 1999. Effects of changes in membrane
sodium flux on virulence gene expression in Vibrio cholerae. Proc. Natl. Acad.
Sci. USA 96:3183–3187.
12. Imae, Y., and T. Atsumi. 1989. Na⫹-driven bacterial flagellar motors.
J. Bioenerg. Biomembr. 21:705–716.
13. Jaques, S., Y. K. Kim, and L. L. McCarter. 1999. Mutations conferring
resistance to phenamil and amiloride, inhibitors of sodium-driven motility ofVibrio parahaemolyticus. Proc. Natl. Acad. Sci. USA 96:5740–5745.
14. Kimura, Y., D. G. Vassylyev, A. Miyazawa, A. Kidera, M. Matsushima, K.
Mitsuoka, K. Murata, T. Hirai, and Y. Fujiyoshi. 1997. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389:
206–211.
15. Kojima, S., Y. Asai, T. Atsumi, I. Kawagishi, and M. Homma. 1999. Na⫹driven flagellar motor resistant to phenamil, an amiloride analog, caused bymutations in putative channel components. J. Mol. Biol. 285:1537–1547.
16. Kojima, S., T. Atsumi, K. Muramoto, S. Kudo, I. Kawagishi, and M.
Homma. 1997. Vibrio alginolyticus mutants resistant to phenamil, a specificinhibitor of the sodium-driven flagellar motor. J. Mol. Biol. 265:310–318.
17. Kojima, S., K. Yamamoto, I. Kawagishi, and M. Homma. 1999. The polar
flagellar motor of Vibrio cholerae is driven by an Na⫹ motive force. J.
Bacteriol. 181:1927–1930.
18. Kudo, S., Y. Magariyama, and S.-I. Aizawa. 1990. Abrupt changes in flagellar
rotation observed by laser dark-field microscopy. Nature 346:677–680.
19. Lloyd, S. A., F. G. Whitby, D. F. Blair, and C. P. Hill. 1999. Structure of the
C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor. Nature 400:472–475.
20. McCarter, L. L. 1994. MotX, the channel component of the sodium-typeflagellar motor. J. Bacteriol. 176:5988–5998.
21. McCarter, L. L. 1994. MotY, a component of the sodium-type flagellar
motor. J. Bacteriol. 176:4219–4225.
22. Muramoto, K., I. Kawagishi, S. Kudo, Y. Magariyama, Y. Imae, and M.
Homma. 1995. High-speed rotation and speed stability of the sodium-driven
flagellar motor in Vibrio alginolyticus. J. Mol. Biol. 251:50–58.
23. Okunishi, I., I. Kawagishi, and M. Homma. 1996. Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus. J. Bacteriol. 178:2409–2415.
24. Sato, K., and M. Homma. 2000. Functional reconstitution of the Na⫹-driven
polar flagellar motor component of Vibrio alginolyticus. J. Biol. Chem. 275:
5718–5722.
25. Sharp, L. L., J. Zhou, and D. F. Blair. 1995. Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in
Escherichia coli. Biochemistry 34:9166–9171.
26. Stader, J., P. Matsumura, D. Vacante, G. E. Dean, and R. M. Macnab. 1986.
Nucleotide sequence of the Escherichia coli motB gene and site-limited
incorporation of its product into the cytoplasmic membrane. J. Bacteriol.
166:244–252.
27. Stolz, B., and H. C. Berg. 1991. Evidence for interactions between MotA and
MotB, torque-generating elements of the flagellar motor of Escherichia coli.
J. Bacteriol. 173:7033–7037.
28. Tang, H., T. F. Braun, and D. F. Blair. 1996. Motility protein complexes inthe bacterial flagellar motor. J. Mol. Biol. 261:209–221.
29. Togashi, F., S. Yamaguchi, M. Kihara, S.-I. Aizawa, and R. M. Macnab.
1997. An extreme clockwise switch bias mutation in fliG of Salmonella
typhimurium and its suppression by slow-motile mutations in motA and motB.
J. Bacteriol. 179:2994–3003.
30. Yorimitsu, T., K. Sato, Y. Asai, I. Kawagishi, and M. Homma. 1999. Functional interaction between PomA and PomB, the Na⫹-driven flagellar motor
components of Vibrio alginolyticus. J. Bacteriol. 181:5103–5106.
31. Zhou, J., R. T. Fazzio, and D. F. Blair. 1995. Membrane topology of the
MotA protein of Escherichia coli. J. Mol. Biol. 251:237–242.
32. Zhou, J., S. A. Lloyd, and D. F. Blair. 1998. Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc. Natl. Acad. Sci.
USA 95:6436–6441.