JOURNAL OF BACTERIOLOGY, Sept. 1995, p. 5158–5160
0021-9193/95/$04.0010
Copyright q 1995, American Society for Microbiology

Vol. 177, No. 17

Isolation of the Polar and Lateral Flagellum-Defective Mutantsin Vibrio alginolyticus and Identification of Their
Flagellar Driving Energy Sources
IKURO KAWAGISHI,1 YUKIYO MAEKAWA,1 TATSUO ATSUMI,2MICHIO HOMMA,1* AND YASUO IMAE1†
Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01,1and Department of Physics, Suzuka College of Technology, Suzuka 510-02,2 Japan
Received 22 August 1994/Accepted 21 June 1995

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only apolar flagellum (Pof1 Laf2) or only lateral flagella (Pof2 Laf1). Using these mutants, we demonstrated thatthe energy sources of the lateral and polar flagellar motors in V. alginolyticus are H1 and Na1 motive forces,respectively, as in the related species V. parahaemolyticus.
Vibrio alginolyticus and V. parahaemolyticus, which areclosely related, have similar flagellar systems. They have twotypes of flagella, named polar flagella (Pof) and lateral flagella(Laf), in one cell (1, 4). When they are grown in a liquid
environment, they express mainly a single polar flagellum attheir cell poles and swim by it rapidly. When they are transferred to a solid agar medium, they express lateral flagellaperitrichously in addition to polar flagella, and the cells elongate and begin to move and spread on the surface (3, 9, 12, 13).
Recently, by using those flagellar mutants with only polar flagella and with only lateral flagella, it has been shown that in V.
parahaemolyticus, the energy sources of polar and lateral
flagellar motors are Na1 and H1 motive forces, respectively(2). Results of experiments using wild-type cells have suggested
that the polar and lateral flagella of V. alginolyticus are also
powered by different ion motive forces (2, 5). Although some
flagellum-defective mutants have been isolated from V. alginolyticus (12), the organism has not yet been characterized in thisregard. In this study, we isolated mutants with only polar orwith only lateral flagella from V. alginolyticus, which is lesspathogenic than V. parahaemolyticus (4) and is well characterized in terms of respiration-coupled primary sodium pumps(10, 11). Furthermore, an energetic analysis of the polar andlateral motors was carried out.
Isolation of lateral or polar flagellum-defective mutants.
The parental strain V. alginolyticus 138-2 (Pof1 Laf1) (11) wasexposed to UV light at the 0.01% survival level. The cells werespotted on the center of 1.5% agar HI plates (1.5% agar in HI
broth, which contained [per liter of distilled water] 25 g ofheart infusion broth [Difco] and 20 g of NaCl) and incubatedat 308C overnight. To enrich nonswarming cells on 1.5% agar,
the cells from the original spot location were grown in HI brothto stationary phase. After the enrichment procedure was repeated, the diluted cultures from the center were spread on
2.5% agar HI plates (2.5% agar in HI broth). As a result, we
isolated the three independent mutants which could not swarm
on the 1.5% agar plate. The lateral flagellum-defective mutants(Pof1 Laf2) were named YM4, YM5, and YM6. Strains YM4
and YM6 did not give rise to Laf1 revertants when the cells

were inoculated on 1.5% agar plates and incubated at 308C for
3 days, but YM5 did.
Next, we tried to isolate polar flagellum-defective mutants(Pof2 Laf1), which were expected to swarm on either 1.5 or
0.3% agar plates by using lateral flagella. Therefore, first we
isolated Pof2 Laf2 mutants from the Pof1 Laf2 mutants by aprocedure similar to that used for the Pof2 Laf1 isolation butusing 0.3% agar HI plates (0.3% agar [Difco] in HI broth).
After enrichment procedures, the swarming ability of colonies
was tested in 0.3% agar plates. We obtained the following Pof2
Laf2 mutants, which could be regarded as independent mutants: YM14 from YM4; YM17, YM18, and YM32 from YM5;and YM44 from YM6. Next, the Pof2 Laf2 mutants wereinoculated in lines on HI 1.5% agar plates and incubated at308C. After 2 or 3 days, we observed in YM17 and YM32
several spontaneous swarming revertants which recovered thesame swarming ability by lateral flagella as wild-type cells. ThePof2 Laf1 mutants from YM17 and YM32 were named YM19and YM38, respectively.
In V. parahaemolyticus, polar and lateral flagellar genes havebeen characterized by using transposon mutagenesis and conjugation (8). The mutants isolated in this study were not genetically characterized because of the lack of a good genetic
system for V. alginolyticus. However, a reliable transformation
procedure for V. alginolyticus has been established recently byusing a P15A replicon-based plasmid vector system and electroporation (7). This development promises to improve thegenetic analysis of the mutants.
Characterization of isolated mutants. We determined thatthe polar and lateral flagella of the isolated mutants had thesame appearances as those of the wild type by electron microscopy. Motility profiles of the flagellum-defective mutants on
1.5 and 0.3% agar plates are shown in Fig. 1. The swarm ringsof the wild type (Pof1 Laf1) and YM4 (Pof1 Laf2) weresimilar in size on the 0.3% agar plate (Fig. 1A). However, therings of YM19 (Pof2 Laf1) on the 0.3% agar plate were
smaller than those of the wild type and YM4. From these linesof evidence, it is inferred that wild-type cells swim mainly byusing polar flagella on the 0.3% agar plate. Swarming ringprofiles of the wild type (Pof1 Laf1) and YM19 (Pof2 Laf1)
on the 1.5% agar plate were identical (Fig. 1B), which suggeststhat polar flagella do not contribute to swarming on the 1.5%
agar plate. The Pof2 Laf1 mutants of V. parahaemolyticus havebeen isolated directly from wild-type cells by screening for* Corresponding author. Phone: 81-52-789-2992. Fax: 81-52-7893001. Electronic mail address: g44416a@nucc.cc.nagoya-u.ac.jp.
† This article is dedicated to Yasuo Imae, who died suddenly of acerebral hemorrhage on 2 July 1993.
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FIG. 1. Motility profiles of the flagellar mutants on 0.3% (A) and 1.5% (B)agar HI plates. Strains: wild type (WT), 138-2; Pof1 Laf2, YM4; Pof2 Laf2,YM18; Pof2 Laf1, YM19. Three microliters of an overnight culture of eachstrain was spotted on each plate and incubated at 308C for several hours. Because
swarming rings on 1.5% agar are made of thin layers of the cells and are hardly
visible, cells were stained for about 3 min with 0.1% Coomassie brilliant blue inmethanol-acetic acid-water (5:1:4) and destained with methanol-acetic acid-water.

motility-defective mutants in minimal medium solidified with
0.3% agar (9). Though we did not do such a selection to obtainPof2 Laf1 mutants, their swarm size in the semisolid agar was
smaller than those of the wild-type strain and the Pof1 Laf2mutant (Fig. 1). This may be because the movement in semisolid agar by lateral flagella is slower than that by polar flagellain V. alginolyticus and V. parahaemolyticus, as observed forfree-swimming cells.
It has been proposed that the polar flagellum is a sensor ofenvironmental conditions and controls lateral flagellar expression in V. parahaemolyticus (9). The Pof2 Laf1 cells (YM19and YM38) expressed lateral flagella constitutively in liquid
media without polyvinylpyrrolidone (PVP) and were not elongated even in viscous medium. A similar observation has beenreported for the polar flagellum-defective mutants of V. para-FIG. 2. Na1 dependency and amiloride sensitivity of swimming speeds ofYM19 (Pof2 Laf1) and YM4 (Pof1 Laf2) cells. Swimming speed was measuredin N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES) motility
buffer, which contained 50 mM HEPES-KOH (pH 7.0), 5 mM glucose, 10 mM
MgCl2, and various concentrations of NaCl in the presence of 3.5% PVP (A) andin HEPES motility buffer (pH 7.0) containing 50 mM NaCl and 3.5% PVP
without MgCl2 and supplemented with various amounts of amiloride (B).

NOTES5159

FIG. 3. CCCP sensitivity of swimming speeds of YM4 (Pof1 Laf2) andYM19 (Pof2 Laf1) cells. The swimming speed was measured in HEPES motility
buffer containing 300 mM NaCl at pH 7.0 or pH 8.7 and supplemented withvarious amounts of CCCP.

haemolyticus (8), which suggests that the two species have thesame sensor system by polar flagella.
Energy source of flagellar motors. We confirmed the energy
source of flagellar motors in V. alginolyticus by using the mutants which possessed only polar or lateral flagella. The culture
at the late log phase was directly diluted 100- or 500-fold intoa motility medium, and the cell motility was observed under adark-field microscope. Within 1 min after dilution, the swimming speed was measured at 258C (maintained by air conditioning) by a photographic method (6). The motility medium
was supplemented with 10 mM L-serine as an attractant. Afterthe addition, the cells swim smoothly without directionalchange for at least 1 min (14). To improve the swimming speed
by lateral flagella, PVP was added to the medium in someexperiments, but the addition of PVP reduces the swimming
speed by polar flagella. The motility of YM4 (Pof1 Laf2) cells
was dependent on the Na1 concentration and inhibited byamiloride, which is a specific inhibitor of Na1-driven flagellar
motors (Fig. 2). In contrast, YM19 (Pof2 Laf1) showed good
motility even without Na1 in the motility medium, and its
motility was not affected by amiloride. V. alginolyticus has respiration-coupled H1 and Na1 pumps, and the Na1 pump isonly active in an alkaline pH range (10, 11). In the neutral toslightly acidic pH range, the Na1 motive force is secondarily
generated by the Na1/H1 antiporter from the H1 motiveforce. Upon addition of the protonophore carbonyl cyanide
m-chlorophenylhydrazone (CCCP) at pH 7.0, the H1 motiveforce and hence the Na1 motive force are lost. Under thiscondition, swimming speeds of both YM19 and YM4 werereduced drastically (Fig. 3). On the other hand, at the alkaline
pH in the presence of CCCP, the H1 motive force is lost butthe Na1 motive force is generated by the primary Na1 pump.
As shown in Fig. 3, at pH 8.7, the motility of YM19 was lost at
high CCCP concentrations but YM4 remained motile in thepresence of CCCP. These results showed that the polar flagellar motor is Na1 driven and the lateral flagellar motor is H1
driven in V. alginolyticus, as in the related species V. parahaemolyticus (2).
We thank Miho Imagawa for assisting with electron micrographs
and Seishi Kudo for invaluable discussion. We especially thank Linda
McCarter for critically reading the manuscript.
This work was supported in part by grants-in-aid for scientific re-5160

NOTES

J. BACTERIOL.

search to I.K. from the Ishida Foundation and to I.K. and M.H. fromthe Ministry of Education, Science and Culture of Japan.
REFERENCES
1. Allen, R. D., and P. Baumann. 1971. Structure and arrangement of flagellain species of the Beneckea and Photobacterium fischeri. J. Bacteriol. 107:295–302.
2. Atsumi, T., L. McCarter, and Y. Imae. 1992. Polar and lateral flagellar
motors of marine Vibrio are driven by different ion-motive forces. Nature(London) 355:182–184.
3. Belas, M., and R. R. Colwell. 1982. Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus. J. Bacteriol.
150:956–959.
4. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Diseases of humans (otherthan cholera) caused by vibrios. Annu. Rev. Microbiol. 34:341–367.
5. Dibrov, P. A., V. A. Kostyrko, R. L. Lazarova, V. P. Skulachev, and I. A.
Smirnova. 1986. The sodium cycle. I. Na1-dependent motility and modes ofmembrane energization in the marine alkalotolerant Vibrio alginolyticus.
Biochim. Biophys. Acta 850:449–457.
6. Imae, Y., H. Matsukura, and S. Kobayasi. 1986. Sodium-driven flagellar

motors of alkalophilic Bacillus. Methods Enzymol. 125:582–592.
7. Kawagishi, I., I. Okunishi, M. Homma, and Y. Imae. 1994. Removal of theperiplasmic DNase before electroporation enhances efficiency of transformation in the marine bacterium Vibrio alginolyticus. Microbiology 140:2355–2361.
8. McCarter, L., M. Hilmen, and M. Silverman. 1988. Flagellar dynamometercontrols swarmer cell differentiation of V. parahaemolyticus. Cell 54:345–351.
9. McCarter, L., and M. Silverman. 1990. Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus. Mol. Microbiol. 4:1057–1062.
10. Skulachev, V. P. 1989. Bacterial Na1 energetics. FEBS Lett. 250:106–114.
11. Tokuda, H., M. Asano, Y. Shimamura, T. Unemoto, S. Sugiyama, and Y.
Imae. 1988. Roles of the respiratory Na1 pump in bioenergetics of Vibrio
alginolyticus. J. Biochem. 103:650–655.
12. Ulitzur, S. 1975. The mechanism of swarming of Vibrio alginolyticus. Arch.
Microbiol. 104:67–71.
13. Ulitzur, S. 1974. Induction of swarming in Vibrio parahaemolyticus. Arch.
Microbiol. 101:357–363.
14. Yoshida, S., S. Sugiyama, Y. Hojo, H. Tokuda, and Y. Imae. 1990. Intracellular Na1 kinetically interferes with the rotation of the Na1-driven flagellar
motors of Vibrio alginolyticus. J. Biol. Chem. 256:20346–20350.