Molecular Microbiology (2007) 64(1), 57–67

doi:10.1111/j.1365-2958.2007.05623.x

The bidirectional polar and unidirectional lateral
flagellar motors of Vibrio alginolyticus are controlled
by a single CheY species
Masaru Kojima,1 Rumi Kubo,1 Toshiharu Yakushi,1†Michio Homma1 and Ikuro Kawagishi1,2*
1
Division of Biological Science, Graduate School of
Science, and 2Institute for Advanced Research, Nagoya
University, Chikusa-ku, Nagoya 464-8602, Japan.
Summary
The bacterial flagellar motor is an elaborate molecularmachine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is
its swift switching of the rotational direction or speed
upon binding of the response regulator phosphoCheY, which causes the changes in swimming that
achieve chemotaxis. Vibrio alginolyticus has dual
flagellar systems: the Na+-driven polar flagellum (Pof)and the H+-driven lateral flagella (Laf), which are used
for swimming in liquid and swarming over surfaces
respectively. Here we show that both swimming andsurface-swarming of V. alginolyticus involve chemotaxis and are regulated by a single CheY species.
Some of the substitutions of CheY residues conserved in various bacteria have different effects on
the Pof and Laf motors, implying that CheY interacts
with the two motors differently. Furthermore, analyses of tethered cells revealed that their switchingmodes are different: the Laf motor rotates exclusively
counterclockwise and is slowed down by CheY,
whereas the Pof motor turns both counterclockwise
and clockwise, and CheY controls its rotational
direction.

Introduction
The bacterial flagellar motor is a molecular machine thatconverts ion-motive force into mechanical force into rotation of helical filament extending from the cell body (forreviews, see Macnab, 1996; Yorimitsu and Homma, 2001;
Kojima and Blair, 2004). Another remarkable feature of
the flagellar motor is that its direction of rotation switches
Accepted 13 November, 2006. *For correspondence. E-maili45406a@cc.nagoya-u.ac.jp; Tel. (+81) 52 789 2993; Fax (+81)
52 789 3001. †Present address: Faculty of Agriculture, Shinshu University, 8304 Minami Minowa, Nagano Prefecture 399-4598, Japan.

© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd

without changing the direction of ion flow (unlike F1Fo-ATP
synthase, in which reverse rotation produces reverse ionflow). This switching of the motor causes two alternate
modes of swimming, e.g. run/tumble (in peritrichously
flagellated bacteria) or forward/backward in polarly flagellated bacteria, and hence generates the random walk
behaviour of the cell. The switching frequency is modulated in response to external stimuli, thereby enabling acell to migrate towards an attractant or away from a repellent via biased random walk. Many bacterial species,
including Escherichia coli, switch the rotational sense of
the motor, i.e. between counterclockwise (CCW) andclockwise (CW) (Larsen et al., 1974; Macnab, 1996).
Other bacteria employ other modes of switching, such as
rotation/stop in Rhodobacter sphaeroides (Armitage andMacnab, 1987) and fast/slow in Sinorhizobium meliloti(Götz et al., 1982; Schmitt, 2002). However, the molecularmechanism underlying motor switching is largely
unknown.
Some marine Vibrio spp. (V. alginolyticus andV. parahaemolyticus) have two types of flagella, the polar
flagellum (Pof) and the lateral flagella (Laf) (Blake et al.,1980). The small, soluble protein CheY in its active phosphorylated form is the response regulator that binds tothe flagellar motor to promote switching. However, thegenome of V. parahaemolyticus (Makino et al., 2003; seebelow for detail) contains only one cheY gene. (Thegenome sequence of V. alginolyticus 12G01 is available
online, but the sequence has not been completed. The
che genes of V. alginolyticus are very similar to those of
V. parahaemolyticus in terms of sequence and gene orderon the chromosome. Considering this situation, we willrefer to the genome sequence of V. parahaemolyticus
throughout the text.) The two flagellar systems of marine
Vibrio play distinct roles in their life cycles: in liquid, arod-shaped swimmer cell is propelled by a single polar
flagellum, which is produced constitutively, whereas on asurface the cell elongates and generates numerous
lateral flagella as it differentiates into a swarmer cell thatcan migrate over the surface (McCarter and Silverman,
1990).
Consistent with their distinct physiological roles, the two
types of flagella have many distinct properties. (i) The
polar flagellum assembles at a cell pole, and its filament is

58 M. Kojima et al.
sheathed with an extension of the outer membrane (Allen
and Baumann, 1971), whereas the lateral flagella areperitrichous and are not sheathed. (ii) The two types offlagella are encoded by separate (non-overlapping) setsof genes (Makino et al., 2003), and no single mutation thatabolishes the Pof and Laf formation has been isolated(McCarter et al., 1988). (iii) The energy sources of the Pof
and Laf motors are the Na+- and H+-motive forces respectively (Atsumi et al., 1992; Kawagishi et al., 1995). (iv) Ina high-viscosity environment, the lateral flagella work
better than the polar flagellum, although swimming propelled by the polar flagellum is much faster in a lowviscosity environment (Atsumi et al., 1996). (v) The polar
flagellum serves as a sensor for the viscosity (via theflagellar rotation speed) to regulate laf gene expression(McCarter et al., 1988; Kawagishi et al., 1996).
Signal transduction in chemotaxis has been beststudied in E. coli (for reviews, see Sourjik, 2004;
Wadhams and Armitage, 2004; Parkinson et al., 2005;
Baker et al., 2006). Central to this pathway is a twocomponent regulatory system that consists of the histidine
kinase CheA and its substrate CheY. CheA is activatedwhen coupled to an unliganded chemoreceptor [alsoknown as methyl-accepting chemotaxis protein (MCP)].
CheA phosphorylates itself and transfers the phosphoryl
group to CheY. Binding of phospho-CheY to the switch
complex of the flagellar motor induces its CW rotation,
resulting in an abrupt change in its swimming direction
called a tumble.
Chemotaxis-defective (che) mutants of V. parahaemolyticus have been isolated. Detailed characterization of the mutants strongly argues that both Pof and Laf,which have their own motors, are controlled by a common
chemosensory signalling system (Sar et al., 1990; Kim
and McCarter, 2000; McCarter, 2004). Indeed, thegenome sequence of V. parahaemolyticus (Makino et al.,2003) has identified that it has one set of che genes andtwo sets of flagellar genes (McCarter, 2004). Thus, theflagellar and chemotaxis systems of V. parahaemolyticus
shows a sharp contrast to those of Vibrio cholerae: thelatter has a single flagellar system (Pof) but has three setsof che genes (Gosink et al., 2002), only one of which is
directly involved in chemotaxis (Gosink et al., 2002;
Hyakutake et al., 2005).
It is therefore likely that a single CheY species cancontrol two distinct flagellar motors in V. alginolyticus,
which is closely related to V. parahaemolyticus. However,the Laf and Pof homologues of the E. coli motor switch
component FliM, which binds phospho-CheY, are quitedissimilar in V. alginolyticus (see Discussion for details). If
a single CheY species indeed controls the Laf and Pof
systems, V. alginolyticus becomes a nice system for acomparative study of the two systems for control of flagellar rotation.

It should be noted, however, that swimming (Pof motility) and swarming (Laf motility) are quite different. In particular, surface motility of E. coli, which also differentiates
into an elongated and hyperflagellated swarmer cell on
surfaces (Harshey and Matsuyama, 1994), does not
require chemotaxis, although the chemotactic signallingsystem is essential (Burkart et al., 1998). Therefore, it
must be conclusively determined whether surfaceswarming of V. alginolyticus involves chemotaxis andwhether the lateral flagellar motors are under the control
of CheY when cells are swarming on surfaces.
In this study, we cloned and characterized the cheY
gene of V. alginolyticus and demonstrated that one CheY
protein regulates both Pof and Laf motility. We thenshowed unambiguously that surface-swarming of
V. alginolyticus involves chemotaxis. Furthermore, we
found that some of the mutations targeting CheY residues
conserved among various bacteria had different effects
on the Pof and Laf systems, implying that CheY interacts
with the two motors differently. We conclude that CheY,
presumably in its phosphorylated form, slows rotation
when it binds to the Laf motor, whereas it induces CW
rotation when it binds to the Pof motor.
Results
The cheY gene of V. alginolyticus
The V. alginolyticus cheY gene was cloned andsequenced. The deduced amino acid sequence of CheY
homologues from various species were aligned using
CLUSTAL W version 1.83 (Fig. 1). In Vibrio species, theCheY amino acid sequence shows high homology (morethan 94% identity and 96% similarity, in any combination).
The identity and similarity between the V. alginolyticus
and E. coli CheY proteins were 66% and 84%
respectively. The site of phosphorylation (Asp-57) and theother residues critical for phosphorylation (Asp-12,
Asp-13 and Lys-109) of E. coli CheY (Bourret et al., 1990)
were conserved in all of the Vibrio CheY proteins. Alignment with CheY of other species predicted that Asp-60 of
V. alginolyticus CheY is the site of phosphorylation. Other
key residues conserved among various response regulators (Volz, 1993) and FliM-interacting residues identified
by genetic and structural studies (Roman et al., 1992;
Shukla et al., 1998; Lee et al., 2001) were also generally
conserved (for details, see Fig. 1). The cloned cheY geneof V. alginolyticus also complemented the cheY defect ofE. coli (Fig. S1 in Supplementary material).
Deletion of cheY abolishes swimming andsurface-swarming by V. alginolyticus
We deleted the cheY gene in the wild-type strain 138-2(Pof+ Laf+) and characterized the motility of the resulting
strain (138-2DcheY). The 138-2DcheY cells did not spread© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

Control of dual flagellar motors 59
Fig. 1. Sequence alignment of the CheY
homologues of Vibrio species and E. coli. The
deduced amino acid sequence of CheY
homologues from various species werealigned using CLUSTAL W version 1.83.
Residues different from those of
V. alginolyticus CheY are shown in white.
Particularly important residues that have been
identified in CheY of E. coli are marked withsymbols: ‘ ’, the phosphorylation site; ‘^’,*other sites conserved among response
regulators; ‘+’, sites of interaction with FliM.
The residues changed in this study areshaded.

either in VPG-0.3% agar or on VPG-1.3% agar (Fig. 2),
indicating that the loss of the cheY gene disrupts bothPof-based and Laf-based motility (hereafter, spreading on
hard agar and in soft agar will be referred to as surfaceswarming and swimming respectively. Swimming in liquid
will be referred to as free-swimming). As uncontrolled
expression of CheY failed to complement the swarming
defects of the DcheY strain, cheY or its FLAG-tagged
derivative was cloned into pSU41 and the resulting plasmids were introduced into the DcheY strain with a lacIcontaining plasmid pMMB206. Cells expressing CheY or
CheY-FLAG spread both in VPG-0.3% agar and on VPG1.3% agar in the presence of proper concentrations of
IPTG (Fig. 2). These findings are consistent with thereported behaviour of V. parahaemolyticus cheY mutants(Sar et al., 1990; Kim and McCarter, 2000; McCarter,
2004) and directly demonstrate that a single CheY canregulate Pof and Laf.
Overexpression of CheY interferes with both Pof
and Laf
When CheY is overexpressed, E. coli or V. cholerae cells
tumble or reorient incessantly (Hyakutake et al., 2005).

Fig. 2. Complementation of the defect of the cheY-knockout
V. alginolyticus strain by the plasmid-borne cheY gene. Overnight
cultures of 138-2 (Pof+ Laf+) or 138-2DcheY cells carrying pSU41,
pVacheY or pVacheY-FLAG as well as pMMB206 were spotted
onto VPG-0.3% agar (for spreading by swimming) and VPG-1.3%
agar (for surface-swarming) supplemented with 2.5 mg ml-1
chloramphenicol, 100 mg ml-1 kanamycin and 10 mM IPTG. Plates
were incubated at 30°C for 8 (0.3% agar) or 25 (1.3% agar) h.

We examined whether overexpression of CheY has
similar effects in V. alginolyticus. Both swimming in VPG0.3% agar and surface-swarming on VPG-1.3% agar of138-2 (Pof+ Laf+) cells were inhibited by overexpression of
CheY from pVacheY in the absence of LacI (i.e. without
pMMB206) (Fig. 3A).
We then directly observed Pof-only or Laf-only cells
under a microscope. First, we observed free-swimmingof 138-2 cells that were grown in liquid media and hence
express the polar flagellum only. 138-2 cells harbouring
the vector plasmid (pSU41) showed longer runs andshorter ‘reverse’ runs, with the reorientation frequency ataround 1.5 s-1 (Fig. 3B, top left), whereas 138-2 cells
overexpressing CheY showed more frequent reorientation (~3.0 s-1) (Fig. 3B, top right). Similar results wereobtained for the Pof+ Laf– mutant strain YM4 (data notshown).
We used the Pof– Laf+ mutant strain YM19 as Laf-only
cells, as lateral flagella are constitutively expressed instrains defective in the formation of polar flagella(McCarter et al., 1988; Kawagishi et al., 1996). YM19
cells harbouring the vector plasmid swam smoothly(Fig. 3B, bottom left). When CheY was overexpressed,
the motile fraction became zero: no swimming or tumbling
cell was observed (Fig. 3B, bottom right). This was notdue to the lack of Laf, as immunoblotting to detect Laf
flagellin revealed that Laf-only cells carrying pVacheY
expressed the same level of Laf flagellin as do cells carrying the vector plasmid (data not shown). We also
observed surface-swarming of Laf-only cells under adigital microscope and found that cells overexpressing
CheY hardly move (data not shown). These results
suggest that the modes of CheY control on the Laf and
Pof motors are different.

Surface-swarming of V. alginolyticus requires
chemotaxis
Although the effects of CheY on surface-swarming andfree-swimming Laf-only cells are similar, the actual© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

60 M. Kojima et al.

Fig. 3. Inhibition of Pof-based swimming and Laf-based surface-swarming by overexpression of V. alginolyticus CheY.
A. Overnight cultures of 138-2 (Pof+ Laf+) cells carrying the vector (+) or the CheY-encoding plasmid (+++) were spotted onto VPG-0.3% agar
(for spreading by swimming) and VPG-1.3% agar (for surface-swarming). The agar was supplemented with 100 mg ml-1 kanamycin. Plates
were incubated at 30°C for 5 h (0.3% agar) or 15 h (1.3% agar).
B. Free-swimming behaviours in liquid medium of 138-2 and YM19 (Pof– Laf+) cells overexpressing CheY. Swimming of 138-2 or YM19 cells
carrying the vector (+) or the CheY-overexpressing plasmid (+++) was observed under a dark-field microscope. Motility is represented byintegrated traces of the recorded images for 3.3 s.

movements observed under a digital microscope arequite different. Capillary assays with V. parahaemolyticus and V. alginolyticus already revealed that Laf-only
cells are chemotactic in liquid (Sar et al., 1990; Homma
et al., 1996), but it has not been established whether
they are also chemotactic on surfaces. We therefore
examined the effects of saturating concentrations of theattractant L-serine on spreading on hard-agar surfaces
as well as in soft agar (Fig. 4). Very high concentrationsof serine should saturate the relevant chemoreceptor,
thereby inhibiting chemotaxis. As shown in Fig. 4B,
chemotactic rings in VPG-0.3% agar became smaller forYM4 (Pof+ Laf–), YM19 (Pof– Laf+) and wild-type (Pof+Laf+) strains with increasing concentrations (0–50 mM)
of L-serine. The addition of 1 mM L-serine enhanced
surface-swarming of the wild-type and Pof– Laf+ strains
on VPG-1.3% agar, but higher concentrations (10 mM or
higher) inhibited swarming (Fig. 4C). These higher concentrations of serine did not affect cell growth (data notshown). In contrast, L-aspartate failed to inhibit orenhance either chemotactic-ring formation in 0.3% agar
or surface-swarming on 1.3% agar (Fig. 4D and E).
When serine and aspartate were spotted on the opposite sides of a streak of YM19 cells on VPG-1.3% agar,
cells swarmed faster towards serine than towards aspartate (Fig. 4F). Similar results were obtained with synthetic medium agar. For the wild-type strain, the additionof 1 and 5 mM L-serine enhanced swimming andsurface-swarming, respectively, but higher concentrations of serine inhibited them (Fig. 4G and H). These
results indicate that Laf motility of V. alginolyticus can be
modulated by external stimuli and that surface-swarming
involves chemotaxis.

Substitution of Ala for Asp-60, affects both typesof swarming
We examined the role of the putative phosphorylation site,
Asp-60, in the control of the Pof and Laf motors by making
the D60A substitution. The D60A mutant cheY gene failed
to complement both swimming and surface-swarming
defects of the 138-2DcheY strain (Pof+ Laf+) (data notshown). The mutant protein (CheY-D60A) was overproduced in 138-2 (Pof+ Laf+) cells carrying pMMB206 (carrying the lacI gene) and swimming in VPG-0.3% agar andswarming on VPG-1.3% agar of the resulting strains were
examined (Fig. 5). In the presence of 100 mM or more
IPTG, cells expressing CheY-D60A swam and swarmed
faster than cells expressing wild-type CheY, indicating thatthe mutant protein interferes less with both the Pof and
Laf motors.
We also examined the role of CheZ, which facilitates
dephosphorylation of phospho-CheY. A cheZ-defective
mutant of 138-2 lost both Pof-based swimming and Lafbased surface-swarming (data not shown). This result
suggests that phosphorylation at the same site of CheY isinvolved in the control of both types of flagellar motors.

Replacement of conserved CheY residues can havedifferent effects on the Pof and Laf motors
We introduced the following substitutions, which are predicted to affect the interaction with FliM, into the cheY
gene: A93V, I98V, Y109L and V111M (Fig. 1). These correspond to the A90V, I95V, Y106L and V108M replacements in E. coli CheY. In E. coli, A90V and V108M
decrease the affinity of CheY to CheA and FliM (Shukla© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

Control of dual flagellar motors 61
Fig. 4. Both chemotactic-ring formation inswim agar and surface-swarming require
chemotaxis.
A–E. Effects of increasing concentrations ofserine and aspartate (0, 1, 5, 10 and 50 mM,
from left to right) on spreading by swimming(VPG-0.3% agar) (serine, B; aspartate, D)and surface-swarming (VPG-1.3% agar)(serine, C; aspartate, E) of V. alginolyticus
strains 138-2 (Pof+ Laf+), YM4 (Pof+ Laf–) andYM19 (Pof–Laf+) (all arranged as shown in A).
F. Surface-swarming of V. alginolyticus
towards serine. An overnight swarm culture of
YM19 cells was streaked on VPG-1.3% agar,
and aliquots (10 ml each) of 0.5 M serine andaspartate were spotted at the positions
indicated by a star and a circle respectively.
G and H. Effects of increasing concentrationsof L-serine (0, 0.01, 0.1, 1, 5, 10 and 100 mM,
from left to right) on spreading by swimming(synthetic medium-0.3% agar) (G) andsurface-swarming (synthetic medium-1.3%
agar) (H) of strain 138-2.
Plates were incubated at 30°C for 5 (B and
D), 9 (C), 18 (E), 20 (F), 12 (G) or 36 (H) h.

et al., 1998), whereas I95V increases the affinity to the
N-terminal residues of FliM (Schuster et al., 2000). Thesemutations therefore would cause CCW- and CW-biased
motor rotation respectively. Y106L confers a null phenotype without affecting phosphorylation or binding to FliM
(Zhu et al., 1996). We examined whether these mutant
cheY genes could complement the swimming and swarming defects of 138-2DcheY cells. Immunoblotting demonstrated that the expression levels of the mutant CheY
proteins were essentially the same as that of wild-type
CheY at any concentration of IPTG tested (Fig. 6A).
The colony diameters of 138-2DcheY cells expressing
wild-type or mutant CheY protein were measured in VPG0.3% agar and on VPG-1.3% agar and plotted against theconcentrations of IPTG (Fig. 6B). Cells expressing wildtype CheY swam and swarmed best when induced by
10 mM and 25–50 mM, respectively, IPTG. The V111M
protein required higher concentrations of IPTG for bothswimming and swarming than wild-type CheY, indicating

Fig. 5. Inhibition of chemotactic-ring formation in swim agar andsurface-swarming by the overexpression of CheY or its mutant
derivative CheY-D60A. Overnight cultures of 138-2 (Pof+ Laf+) cells
carrying the vector or the plasmid encoding CheY or CheY-D60A
were spotted onto VPG-0.3% agar (for swimming) and VPG-1.3%
agar (for surface-swarming) supplemented with 2.5 mg ml-1
chloramphenicol and 100 mg ml-1 kanamycin with 0–1000 mM IPTG
(as indicated). Plates were then incubated at 30°C for 5 h (0.3%
agar) or 15 h (1.3% agar).

© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

62 M. Kojima et al.
Fig. 6. Effects of mutant CheY proteins on
swimming and surface-swarming.
A. Expression levels of wild-type and mutant
CheY proteins at various concentrations of
IPTG. Whole-cell extracts of 138-2DcheY(Pof+ Laf+) cells carrying the LacI-encoding
plasmid pMMB206 and the vector pSU41 (v)or the plasmid encoding wild type (WT) or
each mutant CheY protein (93, A93V; 98,
I98V; 109, Y109L; 111, V111M) wereseparated by SDS-PAGE (13% acrylamidegel) followed by immunoblotting with
anti-FLAG antibody.
B. Swimming and surface-swarming of138-2DcheY cells expressing wild-type ormutant CheY proteins. Fresh overnight
cultures of 138-2DcheY cells carrying the
LacI-encoding plasmid pMMB206 and thevector plasmid pSU41 or the plasmid
encoding wild type or each mutant CheY
protein were inoculated onto VPG-0.3% and1.3% agar plates. Plates were incubated at
30°C for 7 (0.3% agar) or 24 (1.3% agar) h.
Mean diameters of the chemotactic rings, runin triplicate for each strain, are plotted against
the concentration of IPTG. The colour code is:blue, vector; magenta, wild-type; green, A93V;
sky blue, I98V; orange, Y109L; and red,
V111M.

that it has lower affinities for both Pof and Laf motors.
Cells expressing the A93V protein showed a similar swimming pattern than those expressing the V111M protein.
However, the IPTG concentration (25 mM) required for
maximal complementation of surface-swarming by the
A93V protein was essentially the same as that requiredwith wild-type CheY, although the swarm diameters werelarger than with wild-type CheY. The Y109L protein conferred swimming and swarming phenotypes similar to, but
stronger than, those associated with V111M; even higher
concentrations of IPTG were required for optimal chemotactic ring formation in VPG-0.3% agar, and the rings were
smaller. These complementation profiles are quite different from that shown by the corresponding Y106L CheY of
E. coli, which has a null phenotype (Zhu et al., 1996).
In contrast to the other three mutant proteins, the I98V
protein required lower concentrations of IPTG to complement the swimming defect, and higher levels of expression inhibited formation of chemotactic rings, a profile
consistent with the phenotype associated with the E. coli
I95V protein. However, I98V CheY behaved just like wildtype CheY for surface-swarming. Thus, some substitutions at conserved CheY residues had different effects on
the Pof and Laf motors, which suggests that CheY may
interact with the two motors in different ways.

The Laf motor rotates only CCW and may be slowed
down by phospho-CheY
We observed rotation of Laf motors by preparing tethered
YM19 (Pof– Laf+) cells. Although good tethered cells weredifficult to obtain, all of the stably rotating tethered YM19
cells rotated exclusively CCW, unlike tethered E. coli
cells, which rotate in both CCW and CW directions(Fig. 7). The CCW rotation of the Laf motor is supposed torender Laf to form a bundle considering our preliminary
microscopic observation based on the method by
Shimada et al. (1975) that Laf forms a left-handed helicalbundle in liquid (Y. Maekawa, I. Kawagishi and Y. Imae,
unpublished). When CheY was overexpressed, no rotating tethered cells were observed. Thus, control of the Laf
motor does not involve switching between rotational
senses.
To examine the role of CheY in the control of the Laf
motor, we deleted the cheY gene in the Laf-only mutant
strain (YM19). YM19DcheY cells showed a characteristic
free-swimming pattern (Table 1): both the motile fraction
and the mean swimming speed were higher than those of
the parental strain, raising the possibility that CheY
interferes with motility itself. 138-2DcheY (Pof+ Laf—) cells
grown in liquid medium swam without apparent reorienta-© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

Control of dual flagellar motors 63
overproduction of the wild-type CheY protein, but not thatof the D60A mutant protein, increases the reorientation
frequency of 138-2 cells grown in liquid medium (Fig. S2
in Supplementary material), these observations suggestthat phospho-CheY induces CW rotation of the Pof
motor.

Fig. 7. Analysis of rotational direction of the Laf motor in tetheredcells. Tethered cells were prepared as described in Experimental
procedures and observed under a microscope. For each strain, 50
tethered cells were recorded for 15 s and then classified into one ofthree categories: (i) rotating exclusively CW (open bar), (ii) rotatingexclusively CCW (hatched bar) and (iii) alternating CW and CCW
rotation (filled bar)., Va, Vibrio alginolyticus; Ec, Escherichia coli.

tion but their swimming speed is similar to those of 138-2
cells (data not shown). Rotation of tethered YM19 andYM19DcheY cells was recorded, and the video images
were analysed by using the software Image J (http://rsb.
info.nih.gov/ij/). Rotation angles of a tethered cell were
integrated with CCW and CW directions as positive andnegative values, respectively, and plotted against frame
numbers. As a control, we analysed E. coli RP437 cells(Che+). The time-course of the integrated rotation angle ofa tethered RP437 cell gave a zigzag line (not shown). So
did that of tethered V. alginolyticus YM4 (Pof+ Laf–) cells,
although we found very few tethered cells for this strain(not shown). The rotational speeds of RP437 and YM4cells showed both positive and negative values (Fig. 8A
and B), clearly demonstrating that the V. alginolyticus Pof
motor, like the E. coli flagellar motor, alternates between
CCW and CW rotation. In general, cells tethered withpolar flagella are not suited for detailed analyses, but
tethered Pof-only DcheY cells were found to rotate exclusively CCW (data not shown) as shown in optical nanometric measurements of the Pof rotation of polar-only
Che– cells (Sowa et al., 2003). Together with the fact that
Table 1. Motile fractions and mean swimming speeds of YM19 andYM19DcheY cells.

StrainMotile fraction(%)

Mean swimming
speed (mm s-1)

YM19
YM19DcheY

34.7
75.0

15.2 ⫾ 4.1
23.1 ⫾ 6.3

Swimming speed was measured as described previously ( Nishiyama
et al., 1997).

Fig. 8. Detailed analyses of tethered cells. Video images oftethered cells were analysed by Image J software. Rotational
speeds of tethered RP437 (A), YM4 (Pof+ Laf–) (B) and YM19
(Pof–Laf+) (C) cells are plotted against video frames (30frames = 1 s). For each strain, data of a representative cell are
shown. For strains YM19 (open symbols) and YM19DcheY (closed
symbols), integrated rotational angles of three representative cells(each labelled with different symbols) of each strain were fitted witha straight line, and deviations from the fitted lines are plotted in (D).
Arrowheads indicate slow episodes.

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64 M. Kojima et al.
In contrast, the integrated rotation angles of tethered
YM19 (Pof– Laf+) cells increased monotonously over time,with some intervals of more gentle slopes (not shown).
This feature is more apparent when rotational speeds
are plotted (Fig. 8C, indicated by arrow heads). For
YM19DcheY cells, however, no obviously slower intervals
were seen. When integrated rotational angles of three
representative cells of YM19 and YM19DcheY were fittedwith straight lines and deviations from the fitted lines wereplotted (Fig. 8D), YM19DcheY cells clearly had muchsmaller speed fluctuations than YM19 cells. These results
strongly suggest that the function of CheY relative to Laf isto slow down unidirectional CCW rotation of the motor.
Discussion
Vibrio alginolyticus possesses two distinct flagellar
systems: the polar flagellum (Pof), which is well suited forswimming in sea water, and the lateral flagella (Laf),which enable swarming over biotic and non-biotic surfaces (Blake et al., 1980; McCarter and Silverman, 1990).
The results presented here show that not only Pof-basedand Laf-based swimming but also Laf-based surfaceswarming involve chemotaxis. A single CheY protein controls both the Pof and Laf motors. The behaviour of cells
in which the putative phosphorylation site of CheY, Asp60, is replaced with Ala suggests that phosphorylation of
CheY is involved in the control of both of the motor
systems.
The I98V and Y109L substitutions in V. alginolyticus
CheY had differential effects on the Pof and Laf motors,
suggesting that the motor interaction surfaces of CheY
and/or the conformational changes of CheY required forswitching are different between the Pof and Laf motors.
Tethered cell analyses revealed that phospho-CheY
affects the Pof and Laf motors differently. The Pof motor
undergoes switching between the CCW and CW rotation,
whereas the Laf motor slows down in response tophospho-CheY. Although each of these responses isknown from different species, this is the first report that asingle CheY species can exert both types of control in thesame organism.
The genome sequences of V. alginolyticus andV. parahaemolyticus, which is closely related toV. alginolyticus, contain an additional gene annotated as
cheY, but its product seems unlikely to control flagellar
motility, as: (i) the gene maps outside of the che genecluster and is presumably co-transcribed with a neighbouring gene encoding a non-CheA histidine kinase, and(ii) the overexpression of the orthologue in the related
species V. cholerae does not affect flagellar rotation(Hyakutake et al., 2005). V. cholerae and other unrelated
bacteria, including R. sphaeroides and S. meliloti, havemultiple sets of che genes with a single flagellar system(Ferrandez et al., 2002). V. cholerae has four cheY homologues, including the putative cheY described above, but
only one of them (cheY3) directly controls flagellar rotation (Hyakutake et al., 2005). In R. sphaeroides, multiple
che genes are thought to cross-talk with each other, but
only one cheY gene directly regulates the stop-and-gocontrol of the flagellar motor (Porter and Armitage, 2002;
2004; Porter et al., 2002). S. meliloti has a single CheA
protein and two CheY proteins. Only one of them directly
controls the flagellar motor (slow-and-fast type). The otherserves as a phosphate sink (Sourjik and Schmitt, 1996;
1998; Schmitt, 2002).
In E. coli, phospho-CheY binds to the FliM protein, acomponent of the cytoplasmic ring (C-ring) of the flagellar
motor (Welch et al., 1993). The N-terminal residues of
FliM constitute a CheY-docking site (Bren and Eisenbach,1998; Lee et al., 2001). In V. alginolyticus, mutagenic
analyses of cheY were consistent with the notion that
phosphorylation at the same site of CheY is involved in
the control of both types of flagellar motors. Furthermore,
the substitution I98V affected only the Pof motor but not
the Laf motor. The corresponding substitution, I95V, in
E. coli, decreases binding to FliM and interferes withchemotaxis of swimming cells but does not affect surfaceswarming (Mariconda et al., 2006). We aligned the
deduced amino acid sequences of the fliM genes for Pof
and Laf that have been identified in the genome sequenceof V. parahaemolyticus (Makino et al., 2003; McCarter,
2004) and V. alginolyticus with those of the fliM from otherbacterial species (not shown). The polar flagellar FliM (Pof
FliM) protein has a well-conserved N-terminal CheYdocking site. In Rhodospirillum centenum, which also has
two flagellar systems (Ragatz et al., 1995), both the
Pof and Laf FliM proteins have putative CheY-docking
sites (McClain et al., 2002). In contrast, the Laf FliMs
of V. parahaemolyticus and V. alginolyticus lack thissequence, and the rest of the proteins show a relatively
low similarity to that of their Pof counterparts (not shown).
It is therefore likely that the CheY-binding site on the Laf
motor of marine Vibrio differs from that on the Pof motor:
it could reside in a different part of FliM, or other proteinsmight be involved in CheY binding.
When placed on a surface, a rod-shaped marine Vibrio
swimmer cell with a single polar flagellum undergoes asubstantial morphological change to become a highly
elongated cell with numerous (> 100) lateral flagella. The
polar flagellum, however, is retained. How can CheY copewith such a large increase in number of its targets (i.e. theflagellar motors)? The swimmer-to-swarmer differentiation might be accompanied with an increased intracellularlevel of CheY. Alternatively, CheY might have a higher
affinity for the Laf motor, or control of the Laf motor might
require binding of fewer CheY molecules. In any case,identification of the CheY-binding sites is a next obvious© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

Control of dual flagellar motors 65
step towards understanding control of the unidirectional
Laf motor and the bidirectional Pof motor.

Experimental procedures
Bacterial strains, plasmids, growth conditions and media
Bacterial strains and plasmids used in this study are listed inTable S1 in Supplementary material. V. alginolyticus strainsused are a polar flagellum-defective mutant, YM19 (Pof–Laf+), a lateral flagellum-defective mutant, YM4 (Pof+ Laf–),
and their parental strain 138-2 (Pof+ Laf+). A 138-2-derivative
strain, 138-2DcheY, in which the cheY gene is deleted, wasconstructed as described below. All of the E. coli strains arederivatives of K12. Strain RP437 is wild type for chemotaxis(Parkinson and Houts, 1982). Strain RP4979 (J.S. Parkinson,
pers. comm.) is a DcheY mutant.
Vibrio alginolyticus cells were cultured at 30°C in VC
medium (0.5% polypeptone, 0.5% yeast extract, 0.4%
K2HPO4, 3% NaCl, 0.2% glucose), VPG medium [1%polypeptone, 0.4% K2HPO4, 3% NaCl, 0.5% (w/v) glycerol] or
synthetic medium [0.3 M NaCl, 10 mM KCl, 2 mM K2HPO4,
0.01 mM FeSO4, 15 mM (NH4)2SO4, 5 mM MgSO4, 1% glycerol, 50 mM Tris-HCl (pH 7.5)]. E. coli cells were cultured at
37°C in LB or TG [1% tryptone, 0.5% NaCl, 0.5% (w/v)
glycerol] medium. When necessary, chloramphenicol andkanamycin were added to final concentrations of 2.5 and100 mg ml-1 for Vibrio, or 25 and 25 mg ml-1 for E. coli
respectively.

Deletion of the cheY gene
An in-frame deletion in the cheY gene was constructed byoverlap-extension PCR (Ho et al., 1989; Urban et al., 1997).
The 1.4 kb SphI–SacI fragment with the in-frame deletion incheY was constructed as described in the note of Table S2and subcloned into the suicide vector pKY704 (Xu et al.,1994). The resulting suicide plasmid (pKY704-DVacheY) was
first introduced into the E. coli train SM10lpir (Miller and
Mekalanos, 1988) which expresses p protein to support replication of the plasmid, and then transferred by conjugation
into V. alginolyticus strains. The suicide plasmid is expected
to integrate into chromosome by homologous recombination
at chromosomal sequences flanking cheY. Chlorampenicolresistant (Cmr) transconjugants were inoculated on VPG0.3% agar to screen for chemotaxis-defective mutants. Two
Che– clones were found among 100 Cmr transconjugants.
Next, these Cmr Che– cells were cultured in liquid brothwithout antibiotics, and single colonies were isolated on
chloramphenicol-free VC-3.0% agar. From 2000 colonies, wefound one Cms Che– clone. We confirmed the deletion by
examining the sizes of PCR-amplified fragments and thecomplementation of the strain with pVacheY. This mutant
strain was named 138-2DcheY.

Analyses of amino acid sequences
Amino acid sequences were aligned using the software of
CLUSTAL W version 1.83. Sequence homology and similarity
were calculated using the BLAST 2 sequence program(Tatusova and Madden, 1999).

Cloning and mutagenesis of the cheY gene
Primers used for cloning the cheY gene of V. alginolyticus arelisted in Table S2 in Supplementary material. The cheY gene
was amplified by PCR using chromosomal DNA of strain138-2 as the template, cloned into the vector plasmid pCR2.1(Invitrogen). The resulting plasmid was named pCR2.1VacheY. The 1.1 kb EcoRI fragment of pCR2.1-VacheY was
subcloned into the multicloning site of the vector plasmid
pSU41 to yield pVacheY. A KpnI site was introduced at the 3′
end of the coding region. The resulting DNA fragment wasdigested with EcoRI and KpnI and cloned into the vector
plasmid pFLAG-CTC (Sigma) to yield pFLAG-CTC-VacheY,
so that the FLAG tag is fused to the C-terminus of the CheY
protein. Plasmid pVacheY-FLAG was constructed by cloning
the 0.6 kb HindIII–SacI fragment of the PCR product into themulticloning site of pSU41. Mutant cheY genes were constructed by a two-step PCR method with the primers listed inTable S2 essentially as described previously (Umemura
et al., 1998).

Swim and swarm agar assays
VPG-0.3% and 1.3% agar were used for V. alginolyticus swim
and swarm assays respectively. TG-0.3% agar was used forE. coli swim assays. When necessary, chloramphenicol,
kanamycin, 10–500 mM IPTG, and/or 1–500 mM serine or
aspartate (pH 7.0) were added. An aliquot (1 ml) of overnight
culture was spotted onto a plate, which was then incubated at
30°C for 5–20 h.

Observation of swimming behaviour
Free-swimming cells were observed essentially as described
previously (Homma et al., 1996; Nishiyama et al., 1997).
V. alginolyticus cells were resuspended in VPG medium, andcells were observed under a dark-field microscope andrecorded on videotape. Swimming speed was measured as
described previously (Nishiyama et al., 1997).

Nucleotide sequencing
The nucleotide sequence of the cloned gene was determined by the dideoxy-chain termination method, using
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequence of the cheY gene of V. alginolyticus has been deposited to the DDBJ database (Accession
No. AB246040).

Observation of tethered cells
Tethered E. coli and V. alginolyticus cells (grown at 30°C)
were prepared according to the protocol of Silverman andSimon (1974), with minor modifications, and were observed
under a dark-field microscope at room temperature andrecorded on videotape.

© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67

66 M. Kojima et al.
Immunoblotting
Immunoblotting was carried out essentially as described
previously (Okumura et al., 1998). An overnight culture of
V. alginolyticus in VC medium was diluted 1:30 into fresh
VPG medium supplemented, if necessary, with IPTG. Cells
were grown with vigorous shaking, harvested at midlogarithmic phase and washed with Vibrio buffer [25 mM TrisHCl (pH 8.0), 300 mM NaCl, 10 mM MgCl2]. The samples
were subjected to SDS-PAGE (13% polyacrylamide) followed
by immunoblotting with anti-FLAG M2 antibody (Sigma).

Acknowledgements
We thank Drs Yoshiyuki Sowa (Oxford University) and Seiji
Kojima (Nagoya University) for discussion and encouragement and Dr Michael Manson (Texas A&M University)
for critically reading the manuscript. This work was supported in part by grants-in-aid for scientific research from theJapan Society for the Promotion of Science (to T.Y., M.H.
and I.K.).

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Supplementary material
The following supplementary material is available for thisarticle online:
Fig. S1. Complementation of the defect of the cheY knockout E. coli strain by the CheY homolog of V. alginolyticus.
Fig. S2. Reorientation frequencies of wild-type (138-2) cellwith or without overexpression of wild-type a mutant (D60A)
CheY protein.
Table S1. Bacterial strains and plasmids.
Table S2. Primers used in this study.
This material is available as part of the online article fromhttp://www.blackwell-synergy.com© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 64, 57–67