JOURNAL OF BACTERIOLOGY, Aug. 1996, p. 5024–5026
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology

Vol. 178, No. 16

Effect of Viscosity on Swimming by the Lateral and Polar
Flagella of Vibrio alginolyticus†
TATSUO ATSUMI,1 YUKIYO MAEKAWA,2 TAKU YAMADA,2 IKURO KAWAGISHI,2YASUO IMAE,2 AND MICHIO HOMMA2*Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01,2and Department of Physics, Suzuka College of Technology, Suzuka, Mie 510-02,1 Japan
Received 15 April 1996/Accepted 6 June 1996

By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof 1 Laf 2) or only lateral flagella (Pof 2Laf 1), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof 1 Laf 2 cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof 2 Laf 1cells swam at 20 mm/s. The Pof 2 Laf 1 cells swam at about 20 mm/s at normal viscosity (1 cP) without theviscous agent, and the speed increased to 40 mm/s at about 5 cP and then decreased gradually as the viscosity
was increased further. These results show the functional difference between polar and lateral flagella in viscousenvironments.

Many bacteria use flagella to move. The helical flagellar
filaments extending from the cell body are rotated by a motor
located in the proximal portion of the flagellar structure andembedded in the cell membrane (14, 19). The rotation of thehelical filaments against the surrounding medium generates
the driving force for cell swimming. Swimming speed is dependent on motor torque, cell shape, and the shape, length, number, and location (polar or peritrichous) of the filaments.
Those parameters vary among bacterial species.
Vibrio alginolyticus has two types of flagella, polar (Pof) andlateral (Laf), on one cell (5). When grown in a liquid environment, it expresses mainly a single polar flagellum at the cellpole and swims rapidly using it. When transferred to the surface of a solid medium, it expresses lateral flagella in addition
to polar flagella and the cells elongate and become able tomove and spread on the surface (3, 23, 31, 32). By using lateral
flagellum-defective mutants (Pof1 Laf2) and polar flagellumdefective mutants (Pof2 Laf1), it has been shown that lateral
flagella, but not polar flagella, are essential for movement on asurface such as solid agar (16, 30). It has been proposed thatpolar flagella act as dynamometers to sense features of the outer environment, such as viscosity, and regulate lateral flagellar
expression (15, 22, 23). It has been observed that lateral flagella work better for swimming in a high-viscosity environment
than the polar flagellum (27).
Polar flagella are sheathed with a membrane structure whichis contiguous to the outer membrane (1). Their diameter is 24
to 30 nm, and the diameter of the central filament is 14 to 16
nm (1, 7). Lateral flagella are not sheathed and have a diameter of 14 to 15 nm (1). The helical wavelengths of polar andlateral flagella are about 1.5 and 0.9 mm, respectively. Their
constituent proteins, flagellins, have different molecular weights
and antigenicities (22, 29). Moreover, the energy sources ofpolar and lateral flagellar motors are different, Na1 and H1motive forces, respectively (2, 16). Thus, polar and lateral
flagella are distinct in many regards. The rotational frequencyof polar flagella has been observed at over 1,000 Hz in liquid

media (21, 24), whereas the rotational frequency of peritrichous flagella of Salmonella typhimurium or Escherichia coli ismuch lower, up to 200 or 300 Hz (17, 18). The torque property
may be different among the motors.
In this study, we examined the propulsive ability of polar andlateral flagella to clarify the functional difference between thetwo flagellar systems.
Effect of viscosity on swimming speed. The flagellum-defective mutants, YM4 (Pof1 Laf2) and YM19 (Pof2 Laf1), which
had been isolated from a parental strain, 138-2 (Pof1 Laf1),
were incubated in VC medium or VPG medium (26), and theculture at the late log phase was directly diluted into HEPES(N-2-hydroxyethylpiperazine-N9-ethanesulfonic acid) buffer(16) or Tris motility buffer (50 mM Tris-HCl [pH 7.5], 300 mM
NaCl, 5 mM MgCl2, 5 mM glucose) with 1 mM serine, which
is an attractant and prevents the directional change of swimming cells (13, 27). Within 1 min after dilution, the cells wereobserved under a dark-field microscope at 258C (maintained byair conditioning) and recorded on videotape. The video images
were processed by an image-analyzing system (AVIO ImageS-II and EXCEL) to generate the motility tracks of cells. Theswimming speed of cells was measured from the tracks (Fig. 1).
The viscosity of motility buffer containing 300 mM NaCl wasvaried by addition of polyvinylpyrrolidone K-90 (PVP; WakoPure Chemical Industries) and was measured by an Ubbelohde
viscometer in a water bath at 258C. YM4 (Pof1 Laf2) swam atabout 60 mm/s in the buffer without PVP (viscosity, 0.99 cP). Its
swimming speed decreased rapidly as the buffer viscosity wasincreased. At 200 cP (12% PVP), YM4 hardly moved. YM19(Pof2 Laf1) swam at 20 mm/s in the buffer without PVP. In
contrast with YM4, the swimming speed of YM19 increased
with viscosity to about 5 cP, at which YM19 swam maximally at
40 mm/s. Beyond 5 cP, the swimming speed decreased gradually, but even at 200 cP YM19 swam well at about 20 mm/s.
Swimming speed of wild-type cells. Viscous agents induce
production of lateral flagella, while polar flagella are constitutively expressed (2, 22, 23). So, wild-type cells (138-2) cultured
without PVP and with 7% PVP should be Pof1 Laf2 and Pof1
Laf1, respectively. Swimming profiles against viscosity for wildtype cells, which were grown in the absence and the presenceof PVP, were similar to those of the Pof1 Laf2 mutant and thePof2 Laf1 mutant, respectively (Fig. 2). However, Pof1 Laf2

* Corresponding author. Phone: 052-789-2992. Fax: 052-789-3001.
Electronic mail address: g44416a@nucc.cc.nagoya-u.ac.jp.
† Professor Yasuo Imae died suddenly of a cerebral hemorrhage on
2 July 1993. This article is dedicated to him.
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FIG. 1. Functional difference between Laf and Pof with regard to viscosity.
YM4 (Pof1 Laf2) (F), YM19 (Pof2 Laf1) (E), and E. coli AS-1 (Ç) cells weregrown in VC medium (YM4 and YM19) or TG medium (AS-1). The swimming
speed was measured in the HEPES motility buffer (pH 7.0) containing 300 mM
NaCl for YM4 and YM19 or in 10 mM phosphate buffer (pH 7.0) containing 0.1
mM EDTA and 0.5% glycerol for E. coli. The viscosities of the buffers werechanged by the addition of PVP.

cells of the wild-type strain swam more slowly at low viscosity
than the Pof1 Laf2 mutant cells, while at high viscosity theyswam faster than the mutant cells. These results may be explained by the small amount of lateral flagella expressed evenin the absence of PVP. We actually have detected lateral
flagellin in extracellular space of low-viscosity medium (25).
Pof1 Laf1 cells of the wild-type strain swam at 30 mm/s in thebuffer without PVP, which is slower than Pof1 Laf2 mutant
cells but faster than Pof2 Laf1 mutant cells. The overall relation of swimming speed and buffer viscosity for Pof1 Laf1 cells
was more similar to the Pof2 Laf1 mutant YM19 (Fig. 1).
From these results, the polar flagellar function seems to be

FIG. 2. Effect of viscosity on wild-type cells. Strain 138-2 cells were grown in
VC medium without PVP (F) or with 7% PVP (E). The swimming speed wasmeasured in HEPES motility buffer (pH 7.0) containing 300 mM NaCl andsupplemented with various concentrations of PVP.

NOTES

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FIG. 3. Effects of viscosity at various sodium concentrations. YM4 (Pof1Laf2) cells were grown in VPG medium, and their swimming speed was measured in Tris motility buffer (pH 7.5) containing 50 (E), 10 (F), 5 (h), or 2.5 (å)
mM NaCl and supplemented with various concentrations of PVP. The total salt
concentration was adjusted to 300 mM by addition of KCl.

restricted in the presence of lateral flagella. However, at a lowviscosity lateral-flagellum synthesis seems to be effectively
down-regulated (22, 30), so the polar flagellar function is notseverely disturbed.
Effect of viscosity on E. coli flagella. In order to further
examine lateral flagellar function, we measured the swimmingof E. coli, a well-studied peritrichous bacterium, as a functionof viscosity (Fig. 1). E. coli cells grown in TG medium (1%Bacto Tryptone, 0.5% NaCl, 0.5% glycerol) were harvested
and resuspended in 10 mM phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 0.5% glycerol. E. coli cells showedmaximal speed at around 2 cP, their swimming speed rapidly
decreased with increasing viscosity, and they could not move at
200 cP. This profile is more similar to the V. alginolyticus polar
flagella than their lateral flagella except for the lower speed at
low viscosity and the slight increase in speed with initial increase in viscosity. In spite of the same peritrichous state ofV. alginolyticus and E. coli, the abilities to propel the cell athigh viscosity are very different in the two species.
Effect of viscosity on various initial swimming speeds produced by Na1-driven polar flagella. The effects of viscosity on
the swimming speed of the Pof1 Laf2 cells were examined atvarious initial swimming speeds (Fig. 3). The initial swimming
speed, which has a roughly linear relation with the flagellar rotation rate (20, 24), was controlled by the external Na1 concentrations. At 50 mM NaCl, swimming speed was ca. 70 mm/s
without PVP and decreased with increasing viscosity. On theother hand, at 2.5 mM NaCl, the initial swimming speed wasca. 20 mm/s, increased with increasing viscosity until around 5cP, and then decreased. Though the initial swimming speeds
were very different at 10, 5, and 2.5 mM NaCl, the swimmingspeeds at around 10 cP were almost the same. These swimming
profiles were also observed when the initial swimming speed
was reduced by amiloride, which is a specific inhibitor of theNa1-driven flagellar motor, or by the protonophor carbonyl
cyanide m-chlorophenylhydrazone, which disrupts the H1 motive force-dependent Na1 pumping system (data not shown).
The swimming speeds of YM19 and E. coli also showed peaks
at around 5 and 2 cP, respectively (Fig. 1). Thus, at lower
viscosity, the hydrodynamic efficiency of flagellar rotation

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NOTES

J. BACTERIOL.

seems to increase with viscosity. A peak in swimming speed asa function of various viscosities has been found for many bacteria, both peritrichous and monotrichous (4, 6, 8, 9, 28). It
may be speculated that the shape change of rotating flagellaoccurs at various viscosities and the degree of the shape change
dependent on viscosity differs with initial swimming speed.
Swimming ability of lateral flagella in a high-viscosity environment. There might be three causes for the swimming
ability at high viscosity: peritrichous state, number of filaments,and motor torque itself. First, the comparison of the result forYM19 with E. coli suggests that peritrichous state is not asufficient condition for driving the cell body at high viscosity.
Second, in E. coli flagella or Vibrio lateral flagella, the filaments make bundles to swim. We could assume that the torqueof the flagella in a bundle is the summation of the individual
torques. It would be possible that many flagella generate alarge enhancement of torque, especially in the region of low
rotation frequency. Consequently, the swimming speed would
be increased with increasing number of flagella especially inthe region of high viscosity relative to liquid (no PVP). Other
bacteria which can swarm on the surface, e.g., Proteus mirabilis
and Serratia marcescens, have 6 to 10 flagella in liquid. On thesurface, they elongate the cell body and increase the number offlagella to hundreds or thousands (10, 12). Recently, it has
been reported that E. coli and S. typhimurium can differentiate
into filamentous and hyperflagellated cells that navigate thesurface of solid media (10, 11). However, in the high-viscosity
medium used, V. alginolyticus cells were not hyperflagellated(data not shown) and the number of flagella does not seemsufficient to explain the swimming ability at high viscosity. The
third possibility is that different motors have different torquecharacteristics. Polar motors could apparently generate torqueat higher frequencies than the motors of E. coli and lateral
flagella. Lateral motors apparently generate high torque at low
rotational frequency. At this stage, we speculate that both thenumber of flagella and the torque property of the motor may
be responsible for the distinct characters of polar and lateral
flagella. To clarify the swimming ability of flagella, their torqueproperties need to be compared.
We thank Seishi Kudo for invaluable discussions. We especiallythank Robert M. Macnab for critically reading the manuscript.
This work was supported in part by grants-in-aid for scientific research (to T.A., I.K., and M.H.) from the Ministry of Education,
Science and Culture of Japan.
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