Molecular Microbiology (2006)

doi:10.1111/j.1365-2958.2006.05435.x

The Vibrio motor proteins, MotX and MotY, areassociated with the basal body of Na+-driven flagellaand required for stator formation
Hiroyuki Terashima, Hajime Fukuoka,
Toshiharu Yakushi,† Seiji Kojima andMichio Homma*
Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-Ku, Nagoya
464-8602, Japan.
Summary
The four motor proteins PomA, PomB, MotX and
MotY, which are believed to be stator proteins, areessential for motility by the Na+-driven flagella ofVibrio alginolyticus. When we purified the flagellar
basal bodies, MotX and MotY were detected in thebasal body, which is the supramolecular complex
comprised of the rotor and the bushing, but PomA
and PomB were not. By antibody labelling, MotX andMotY were detected around the LP ring. These results
indicate that MotX and MotY associate with the basal
body. The basal body had a new ring structure
beneath the LP ring, which was named the T ring. This
structure was changed or lost in the basal body froma DmotX or DmotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or
MotY, we demonstrated that PomA and PomB were
not localized to a cell pole. From the above results, wesuggest that MotX and MotY of the T ring are involvedin the incorporation and/or stabilization of the PomA/
PomB complex in the motor.

Introduction
Many bacteria can swim using flagella, which function asa rotary machine like a corkscrew. The flagellar motor
uses the electrochemical potential of specific ions (H+ or
Na+) across the cytoplasmic membrane as the energy
source (Yorimitsu and Homma, 2001; Blair, 2003). The
flagellum consists of the filament, the hook and the basal
body. In Gram-negative bacteria, the basal body, which is
Accepted 11 September, 2006. *For correspondence. E-mailg44416a@cc.nagoya-u.ac.jp; Tel. (+81) 52 789 2991; Fax (+81)
52 789 3001. †Present address: Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, Japan.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd

the supramolecular complex consisting of the rotor andthe bushing, consists of the rod and the four ring structures, L, P, MS and C rings. The L, P and MS rings arethought to associate with the outer membrane, the peptidoglycan layer and the inner (cytoplasmic) membrane
respectively. The C ring is located on the cytoplasmic faceof the MS ring. The MS and C rings and the rod arethought to act as the rotor, and the L and P rings arethought to act as the molecular bushing for the rod(Macnab, 1996; Derosier, 1998).
mot mutants, which have paralysed phenotypes formotility (Mot–), can produce flagella but cannot rotate
them. Five genes, motA, motB, fliG, fliM and fliN, areresponsible for the Mot– phenotype in Escherichia coliand Salmonella. motA and motB are involved only inmotor function. On the other hand, fliG, fliM and fliN are
also involved in flagellar formation and switching of rotational direction because null mutations give a flagelladefective phenotype (Fla–), and some mutations cause
the motor to rotate in only one direction (Che–). Genetic
and physiological evidence has suggested that MotA
and MotB, which are cytoplasmic membrane proteins,
together comprise the stator complex of the motor andfunction as a proton channel complex to generate torque(Blair, 2003). MotA has four transmembrane segments
and a large cytoplasmic loop and MotB has a single
transmembrane domain and a peptidoglycan-binding
motif through which the motor is anchored to the cellwall (De Mot and Vanderleyden, 1994). MotB has an
Asp residue in the putative transmembrane region,
which is speculated to be protonated during torque generation (Zhou et al., 1998a). PomA and PomB are orthologues of MotA and MotB, respectively, in the Na+-driven
polar flagellum of Vibrio alginolyticus (Asai et al., 1997).
The purified PomA/PomB complex was shown to havesodium-conducting activity in vitro (Sato and Homma,
2000a) and is predicted to be in a 4A:2B stoichiometrycomplex (Sato and Homma, 2000b; Yorimitsu et al.,2004). The PomA/PomB complex is thought to function
as the Na+ channel and is referred to as a stator similar
to the MotA/MotB complex (Asai et al., 1997). The cytoplasmic domains of MotA and PomA are thought to interact with FliG, one of the rotor components, to generate
torque for the flagellar motor in the H+- and Na+-driven

2 H. Terashima et al.
motors (Zhou et al., 1998b; Yakushi et al., 2006).
However, such an interaction has not yet been biochemically observed (Driks and DeRosier, 1990). The
stator proteins must be installed into the flagellar structure as a stator complex that surrounds the rotor.
Recently, it has been shown that the flagellar structure
and C-terminal domain of PomB are required for the
localization of the PomA/PomB complex at the cell pole
where the flagellum is generated (Fukuoka et al., 2005;
Yakushi et al., 2005).
Two additional genes, motX and motY, are also
required for motility in the Na+-driven Vibrio flagella, andare thought to be stator proteins because null mutants
have a Mot– phenotype (Gosink and Häse, 2000;
Yagasaki et al., 2006). MotY has a putative peptidoglycanbinding motif in its C-terminal region as do MotB and
PomB. The functions of MotX and MotY have not yet beenclarified.
Recently, MotX and MotY were shown to localize to theouter membrane and to interact with each other (Okabe
et al., 2002; 2005). MotX affects the membrane localization of PomB when MotY is absent, suggesting that MotX
interacts with PomB (Okabe et al., 2005). In this study, weshow that MotX and MotY are associated with the flagellar
basal body of V. alginolyticus and form a T ring. We demonstrated the interaction of the stator proteins with thebasal body in the flagellar motor. We propose that the
T ring formed by the MotX and MotY proteins is requiredfor incorporation and stabilization of the PomA/PomB
complex in the motor complex.

A new ring structure in the basal body of V. alginolyticus
Purified hook-basal bodies from V. alginolyticus wereobserved by electron microscopy, and their overall structure was similar to those of other Gram-negative bacteria(Fig. 1B). The structure of the hook-basal body of Gramnegative bacteria is composed of three rings and an axial
rod connected to the hook. Interestingly, in the
V. alginolyticus basal body, we found an additional new
ring structure that appeared as an appendage on the
periplasmic side of the P ring, and it was named the T ring.
The diameter and axial dimension of the T ring were
31 ! 2 (SD) nm and 8 ! 1 (SD) nm (n = 31) (Fig. 1C).
Such T rings have not been previously observed in thebasal bodies of other Gram-negative bacteria. When thebasal body structure was compared with that from Salmonella, the LP ring from V. alginolyticus looked like two thinrings. The diameter of the LP ring was 33 ! 3 (SD) nm(n = 31). The LP ring of the V. alginolyticus polar flagellumis larger than that of Salmonella flagella, which is approximately 27 nm (Sosinsky et al., 1992). When the hookbasal body preparation from V. alginolyticus and that from
E. coli were compared directly, the diameter of the LP
rings of the V. alginolyticus polar flagella was clearlylarger than that of the E. coli flagella (Fig. 1D). When the
structure of the purified hook-basal bodies of strain
KK148, which is the flhG mutant with multiple flagella,
were compared with those of the parent strain VIO5 byelectron microscopy, we did not see any differences
between the two basal body structures (data not shown).
Detection of MotX and MotY in the hook-basal bodyResults
Purification of the hook-basal body of V. alginolyticus
Vibrio alginolyticus cells have a single polar flagellum andmultiple lateral flagella. To obtain a large amount of thehook-basal bodies of the sodium-driven polar flagella, weused the flhG mutant (KK148) which forms multiple polar
flagella and was isolated from the lateral flagella defective
mutant, VIO5 (Kusumoto et al., 2006). The fractions ofpurified hook-basal bodies were analysed by SDSpolyacrylamide gel electrophoresis (PAGE) and stainedwith Coomassie brilliant blue to visualize proteins (Fig. 1A).
We reproducibly detected 11 bands with molecular massesof 62, 50, 47, 46, 40, 33, 32, 31, 28, 26 and 25 kDa. The
banding pattern on the SDS-PAGE gel was similar to that ofthe purified hook-basal bodies from Salmonella typhimurium (Aizawa et al., 1985). We chose the major 50 kDa
band and determined its N-terminal amino acid sequenceby Edman degradation. The obtained sequence wasSYVSL, which corresponds to the hook protein, FlgE, ofVibrio parahaemolyticus, which is a closely related
species.

We analysed components of the purified hook-basal
bodies by immunoblotting using the anti-PomA, antiPomB, anti-MotX and anti-MotY antibodies. MotX andMotY were detected in the purified hook-basal body,whereas PomA and PomB were not (Fig. 2A). These
results suggest that MotX and MotY are associated with thebasal body. Recently, it was shown that the overexpressed
MotX and MotY tend to oligomerize by themselves (Okabe
et al., 2005). We examined the hook-basal body fraction
further by using sucrose density gradient centrifugation to
see whether MotX and MotY co-fractionate with the basal
body proteins. The P ring component, FlgI, was used as amarker protein for the basal body. The fractionation profilesof both MotX and MotY corresponded well to that of the FlgI
protein (Fig. 2B). We propose that MotX and MotY arecomponents of the flagellar basal body.
Localization of MotX and MotY on the basal body
In order to directly observe the localization of MotX andMotY in the basal body, we performed immunoelectron
microscopy using anti-MotX or anti-MotY antibodies andgold-labelled anti-rabbit IgG as the secondary antibody© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

Na+-driven Vibrio flagella motor 3

Fig. 1. A. Purification of the hook-basal bodies of V. alginolyticus (KK148). The proteins of the purified hook-basal body were separated by
SDS-PAGE. The 50 kDa band and the other 10 bands with molecular masses of, 62, 47, 46, 40, 33, 32, 31, 28, 26 and 25 kDa are shownwith an arrow and arrowheads respectively. The N-terminal amino acid sequence, SYVSL, of the 50 kDa protein was determined by Edman
degradation, identifying the hook protein FlgE.
B. Electron microscopy image of the hook-basal bodies from V. alginolyticus strain KK148. The new structure named the T ring is indicated by
the arrow. The hook-basal bodies were negatively stained with 2% phosphotungstic acid (pH 7.4). Bar, 50 nm.
C. Cartoon of the flagellar basal body of V. alginolyticus. The diameter of the LP ring is 33 ! 3 (SD) nm (n = 31). The diameter and axial
dimension of the T ring of the flagellar basal body are 31 ! 2 (SD) nm and 8 ! 1 (SD) nm (n = 31).
D. Comparison of the basal bodies purified from V. alginolyticus (left: KK148) and E. coli (right: RP437). Bar, 25 nm.

(Fig. 3). When the basal bodies from the parental strain
KK148 were treated with anti-MotX or anti-MotY antibodies, the fuzzy structure of the antibody was observed
around the LP ring or the T ring and gold particles mainly
attached around the basal body or tended to be present
around the LP ring or the T ring. On the other hand, when
the same treatment was carried out with the basal bodies
isolated from the DmotX DmotY strain (TH3), which weconstructed from strain KK148, gold particles were not
localized around the basal body or the LP ring. These
results strongly suggest that MotX and MotY are associ-ated with the basal body. Thus, we hypothesized that the
T ring detected as appendages around the P ring is composed of MotX and MotY.
The basal body structure from DmotX or DmotY cells
If the T ring is composed of MotX and MotY, the basal
bodies from DmotX and/or DmotY cells might have analtered appearance. Therefore, we constructed DmotX(TH1) and DmotY (TH2) strains from KK148 andobserved their basal bodies by electron microscopy. The© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

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H. Terashima et al.

Fig. 2. Co-purification of MotX and MotY with the basal body.
A. Whole cell (W) and the purified hook-basal bodies (HBB) of V. alginolyticus strain KK148 were subjected to SDS-PAGE, followed byimmunoblotting using anti-MotX, anti-MotY, anti-PomA, anti-PomB and anti-FlgI antibodies.
B. The purified hook-basal bodies were separated by sucrose density gradient centrifugation. The gradient was divided into 18 fractions fromthe bottom. MotX, MotY and FlgI in each fraction were subjected to SDS-PAGE and detected by immunoblotting using anti-MotX, anti-MotY
and anti-FlgI antibodies.

basal body of strain TH1 still had an appendage, but thestructure seemed to be smaller than the T ring observedin the parental strain (Fig. 4A). Therefore, the appendage may be derived from the MotY protein. We detected
the MotY protein in the purified hook-basal body of strain
TH1 by immunoblotting (Fig. 4B). The basal body ofstrain TH2 did not have a T ring (Fig. 4A), and MotX
protein was also not seen in whole cell extracts or the
purified hook-basal body preparation by immunoblotting(Fig. 4B). It has been suggested that MotX is rapidly
degraded in the absence of MotY (Okabe et al., 2001;
Yagasaki et al., 2006). The T ring was not observed in
the basal bodies purified from strain TH3 or strain TH2,
in which no MotX protein was detected (Fig. 4A). These

results demonstrate that MotX and MotY are responsible
for the T ring structure.
Polar localization of PomA/PomB complex requires
MotX and MotY
Recently, it has been suggested that MotX interacts with
the PomA/PomB complex as well as MotY (Okabe et al.,2005). In the present study, we showed that MotX andMotY were associated with the basal body or the LP ring,
therefore they might be rotor or bushing components.
Although we know that MotX and MotY are essential forthe function of the sodium-driven motor, their roles are notclear. Now, we speculate that MotX and MotY are involved

Fig. 3. Immunogold labelling of the basal bodies using anti-MotX and anti-MotY antibodies. The basal bodies from KK148 (left) or TH3 (DmotX
DmotY) (right) were treated with anti-MotX (upper) or anti-MotY (lower) antibodies, which were prepared as described previously (Yagasaki
et al., 2006), and 5 nm colloidal gold-conjugated anti-rabbit IgG antibody. The samples were negatively stained with 2% phosphotungstic acid(pH 7.4). Bar, 50 nm.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

Na+-driven Vibrio flagella motor 5

Fig. 4. The basal bodies from V. alginolyticus motX and/or motY deletion mutants.
A. Electron micrographs of the basal bodies from V. alginolyticus strains TH1 (DmotX), TH2 (DmotY) and TH3 (DmotX DmotY) (upper andmiddle) and enlarged images of the middle panel corresponding to the dotted frame (lower). The hook-basal bodies were negatively stainedwith 2% phosphotungstic acid (pH 7.4). Bar, 25 nm.
B. Whole cell (W) and the purified hook-basal bodies (HBB) of V. alginolyticus strains TH1 (DmotX), TH2 (DmotY) and TH3 (DmotX DmotY)
were subjected to SDS-PAGE, followed by immunoblotting using anti-MotX, anti-MotY and anti-FlgI antibodies.

© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

6

H. Terashima et al.

Fig. 5. Cellular localization of GFP-fused PomA or PomB in the motX and motY deletion strain. The cells harbouring plasmids encoding
GFP-fused PomA and PomB (pHFGAB and pHFGBA2) were grown at 30°C for 4 h in VPG500 medium containing 0.02% (w/v) arabinose andwere observed under a fluorescence microscope as described in Experimental procedures.

in the stabilization or incorporation of the PomA/PomB
complex into the flagellar motor to connect to the rotor. To
address this possibility, we examined the in vivo behaviour of GFP-fused PomA and PomB proteins, which havebeen shown to localize at a cell pole in a flagellumdependent, partner protein-dependent and C-terminal
region of PomB-dependent manner (Fukuoka et al.,2005). In the DpomAB strain, NMB191, the polar localization of GFP-PomA or GFP-PomB proteins was observedin the presence of the partner protein, PomB or PomA, as
described previously (Fig. 5, left). On the other hand, thefluorescent GFP signals were diffused in most of the
DmotX (TH4) or DmotY (TH5) cells, which were constructed from NMB191 (Fig. 5, middle and right). Although
GFP-PomA produced in the TH4 or TH5 cells was diffuse,some fluorescent clusters were observed on themembrane. GFP-PomB produced in TH4 or TH5 cells was
uniformly diffuse. Such intracellular localizations werealso observed in cells deficient in the flagellum, thepartner proteins or the C-terminal region of PomB as
described in the previous report (Fukuoka et al., 2005).
These results suggest that MotX and MotY are required
for the polar localization of the PomA/PomB complex, atthe base of the flagellum.
Discussion
PomA, PomB, MotX and MotY are essential for motor
function to rotate the Na+-driven flagellar motor in Vibrio.
In this study, we clarified that MotX and MotY were associated with the basal body at a position beneath the LP

ring and formed a T ring structure based on the following
results: (i) the basal body from V. alginolyticus had a T
ring; (ii) the MotX and MotY proteins were detected in thebasal body by immunoblotting; (iii) the periphery of the LP
ring or the T ring was decorated with anti-MotX or antiMotY antibody; and (iv) the basal bodies from DmotY and
DmotX DmotY strains lost the T ring. We think that the Tring is associated with the LP ring of the bushing rather
than the rod of the rotor. It seems most likely that MotX
and MotY associate with the LP ring and do not rotate
because they function as stator proteins. The MotX andMotY proteins are translocated across the inner membrane in a signal sequence-dependent manner using the
Sec machinery as are the L and P ring proteins, FlgH and
FlgI, and they probably assemble onto the LP ring (Okabe
et al., 2002). The unused MotX and MotY proteins for the
T ring assembly can be isolated as outer membraneproteins.
We demonstrated that the GFP-PomA/PomB and
PomA/GFP-PomB complexes were rarely localized at thecell pole in the absence of MotX or MotY. These results
show that MotX and MotY are required for the polar localization of PomA and PomB and suggest that MotX andMotY have a role in incorporating the PomA/PomB
complex into the flagellar motor. What roles do MotX andMotY have for incorporating the PomA/PomB complex?
The basal body from the DmotX strain has a partial T ring
structure, which is likely to be composed of only the MotY
proteins. In the DmotX strain, the PomA/PomB complex
appeared not to be incorporated into the flagellar motor
because GFP-PomA and GFP-PomB did not localize to© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

Na+-driven Vibrio flagella motor 7
the cell pole. MotX is likely to interact with PomB andrecruit the PomA/PomB complex to the motor (Okabe
et al., 2005). MotY may be required to anchor MotX to thebasal body. We propose the incorporation process of
PomA and PomB into the flagellar motor as the following
scheme. A peptidoglycan-binding motif initially anchors
PomB to the cell wall rather than to the motor. Because
MotX seems to interact with PomB and MotY, the T ringcomposed of MotX and MotY appears to assist in thisbinding process. The T ring may link the PomA/PomB
complex with the LP ring. MotX and MotY form a ring
structure and may fix the PomA/PomB complex in theVibrio polar flagellar motor. Subsequently, stator formation
may be completed and may activate the PomA/PomB
complex. The T ring composed of MotX and MotY might
be important for generating high-speed rotation, which in
the Vibrio motor reaches a maximum of 1700 Hz and an
average of 1100 Hz (Magariyama et al., 1994). The rotation speeds of E. coli and Salmonella flagella are 300 Hz
and 200 Hz respectively (Lowe et al., 1987; Kudo et al.,1990), and they do not have MotX and MotY paralogues.
A chimeric stator of PomA and PotB, whose periplasmic
region was replaced by the corresponding region of MotB
from E. coli, functioned in Vibrio cells without MotX or
MotY, although the swarming ability was very poor (Asaiet al., 2003). The periplasmic region, which contains the
peptidoglycan-binding motif, may determine the requirement for MotX and MotY. It has been shown that MotY of
Pseudomonas aeruginosa is important for flagellar rotation in that organism (Doyle et al., 2004). Moreover it has
been shown that the MotYL of the H+-driven lateral flagellar motor of V. parahaemolyticus is also important forlateral flagellar motility (Stewart and McCarter, 2003). As
the whole genomes of many species of bacteria havebeen decoded, both putative motX and motY genes havebeen found in several species of bacteria, such as Idiomarina loihiensis (Hou et al., 2004) and Photobacterium profundum (Vezzi et al., 2005). In contrast, only putative
motY genes have been found in Pseudomonas syringae(Buell et al., 2003) and Legionella pneumophila (Cazalet
et al., 2004). It will be interesting to determine whether theflagellar basal body has an additional structure composedof the MotY protein in Pseudomonas or Legionella sp. As
the motX gene has not been found in these species, theMotY protein of these species may have a different function from the Vibrio protein.
It has been suggested that the periplasmic protein
MotC of Sinorhizobium meliloti interacts with MotB andregulates the rotation of the flagellar motor (Platzer et al.,1997). MotE, a periplasmic chaperone specific for MotC,
affects the amount of MotC and also regulates motor
rotation (Eggenhofer et al., 2004). These proteins do nothave significant primary sequence homology with MotX
and MotY, although their functions may be similar.

Because MotX interacts with PomB, MotX might regulate
some activity of the PomA/PomB complex: for example,
opening and closing of the Na+-channel of the PomA/
PomB complex or the interaction between PomA and FliG
through PomB, which is thought to be important for torquegeneration.
It will be necessary to analyse the stoichiometry of
MotX and MotY in the basal body, and generate fineimages of the basal body. The stoichiometry of MotX andMotY proteins with the basal body and the detailed shapeof the T ring may help determine the number and/or
arrangement of the PomA/PomB complexes with the
motor. The PomA/PomB complex should be arranged
around the basal body to carry out the interaction between
PomA and FliG, which is thought to be essential for therotation of the motor. However, as the PomA/PomB
complex does not co-purify with the basal body, it has notbeen directly demonstrated that the PomA/PomB
complex is arranged around the basal body. Although
PomB has been suggested to interact with MotX, thePomA/PomB complex did not co-purify with the basal
body of the V. alginolyticus polar flagellum. The purification method used in this study seemed likely to result in
disruption of the interaction between MotX and PomB. If
MotX could be bound to PomB in some way, for example,
in a fusion or by chemical cross-linking, we would be able
to co-purify the rotor with the stator and reconstitute theflagellar motor in vitro.

Experimental procedures
Bacterial strains, growth conditions and media
The strains and plasmids used are shown in Table 1.
V. alginolyticus was cultured at 30°C in VC medium [0.5%(w/v) polypeptone, 0.5% (w/v) yeast extract, 0.4% (w/v)
K2HPO4, 3% (w/v) NaCl, 0.2% (w/v) glucose] or in VPG500
medium [1% (w/v) polypeptone, 0.4% (w/v) K2HPO4, 500 mM
NaCl, 0.5% (w/v) glycerol]. E. coli was cultured in LB broth[1% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v)
NaCl] or TG medium [1% (w/v) bactotryptone, 0.5% (w/v)
NaCl, 0.5% (w/v) glycerol]. In the second selection to selectmutants, V. alginolyticus was cultured at 30°C in sucrosecontaining plates [1% (w/v) polypeptone, 30 mM NaCl,
55 mM KCl, 10% (w/v) sucrose, 1.25% (w/v) agar]. Chloramphenicol was added to a final concentration of 2.5 mg ml-1
for V. alginolyticus and 25 mg ml-1 for E. coli. Kanamycin
was added to a final concentration of 100 mg ml-1 for
V. alginolyticus.

Isolation of the flagellar basal bodies
The isolation of the flagellar basal bodies was carried out asdescribed previously with several modifications (Aizawa
et al., 1985; Kobayashi et al., 2003). An overnight culture(grown on VC medium) was inoculated at a 100-fold dilution© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

8

H. Terashima et al.

Table 1. Bacterial strains and plasmids.
Strain or plasmid
V. alginolyticus strains
VIO5
KK148
TH1
TH2
TH3
NMB191
TH4
TH5
E. coli strains
SM10lpir
RP437
Plasmids
pSU41
pMO401
pIO6
pKJ601
pKY704
pHFS401
pMO200pJN559pTH101
pJN507
pJN506
pTH102
pHFGAB
pHFGBA2

Genotype or description

Reference

VIK4 laf (Rifr Pof+ Laf–)
VIO5 flhG (Rifr multi-Pof+)
KK148 DmotX (Rifr multi-Pof+ Pom–)
KK148 DmotY (Rifr multi-Pof+ Pom–)
KK148 DmotX DmotY (Rifr multi-Pof+ Pom–)
VIO5 DpomAB (Rifr Pof+ Pom–)
NMB191 DmotX (Rifr Pof+ Pom–)
NMB191 DmotY (Rifr Pof+ Pom–)Okunishi et al. (1996)
Kusumoto et al. (2006)
This study
This study
This study
Yorimitsu et al. (1999)
This study
This study

thi thr leu tonA lacy supE recA::RP4-2-Tc::Mu Km lpirRK6
F–thi thr leu his met eda rpsL

Miller et al. (1988)
Parkinson and Houts (1982)

kan (Kmr) Plac lacZa
pSU41, 0.7 kb ClaI-BamHI fragment (motX+)
pSU38, 1.0 kb HindIII-XbaI fragment (motY+)
pKJ502 (motY+), 0.7 kb XbaI-BamHI fragment (motX+)
Suicide vector (Cmr)
pSU41, 2.0 kb EcoRV-XbaI fragment (sacB+)
pSU21, 1.0 kb EcoRI-EcoRI fragment (motX+)
pMO200, 385 bp deletion in motX
pKY704, 0.6 kb EcoRI-EcoRI fragment (motX) from pJN559,
2.0 kb XbaI-XbaI fragment (sacB+) from pHFS401
pUC18, 1.6 kb XbaI-HaeIII fragment (motY+)
pJN507, 871 bp deletion in motY
pKY704, 0.8 kb EcoRI-EcoRI fragment (motY) from pJN506
2.0 kb XbaI-XbaI fragment (sacB+) from pHFS401
his6-gfp-pomA and pomB in pBAD33
his6-gfp-pomB and pomA in pBAD33

Bartolome et al. (1991)
Okabe et al. (2001)
Okunishi et al. (1996)
Okabe et al. (2002)
Xu et al. (1994)
This study
Okabe et al. (2001)
This study
This study
This study
This study
This study
Fukuoka et al. (2005)
Fukuoka et al. (2005)

Rifr, rifampicin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Plac, lac promoter; Pof+, normal polar flagellar formation; Laf–,defective in lateral flagellar formation; multi-Pof+, multiple polar flagellar formation.

into VPG500 medium (500 ml) and cultured at 30°C. After asubsequent 3.5 h cultivation, the cells were harvested in a
sucrose solution (0.5 M sucrose, 50 mM Tris-HCl at pH 8.0)
and converted into spheroplasts by adding lysozyme and
EDTA to final concentrations of 0.1 mg ml-1 and 2 mM
respectively. The spheroplasts were lysed by adding Triton
X-100 to a final concentration of 1% (w/v). To reduce theviscosity of the lysed samples, MgSO4 and DNase I wereadded to final concentrations of 5 mM and 0.1 mg ml-1
respectively. After the viscosity of the solution decreased,
EDTA was added to a final concentration of 5 mM. Unlysed
cells and cellular debris were removed by centrifugation at
17 000 g for 20 min. Polyethylene glycol 6000 and NaCl wereadded to the lysate to final concentrations of 2% and 100 mM
respectively. The samples were then incubated at 4°C for
60 min. The suspension was centrifuged at 27 000 g for
30 min at 4°C and the pellet was resuspended in 6 ml of TET
buffer [10 mM Tris-HCl at pH 8.0, 5 mM EDTA, 0.1% (w/v)
Triton X-100]. To remove cellular debris, the suspension wascentrifuged at 1000 g for 15 min at 4°C. The supernatant wascentrifuged at 100 000 g for 30 min and the pellet was resuspended in 200 ml of TET buffer. To dissociate the flagellar
filaments into monomeric flagellin, the suspension was
diluted 30-fold in 50 mM glycine-HCl (pH 3.5) containing
0.1% (w/v) Triton X-100 and shaken for 60 min at room
temperature. After the solution was centrifuged at 1000 g for
15 min at 4°C, the supernatant was centrifuged at 150 000 g
for 40 min at 4°C and the pellet was resuspended in 100 ml of
TET buffer. To isolate the basal bodies from E. coli RP437, an

overnight culture was grown on LB medium and then asecond culture was grown on TG medium for 6 h at 30°C. The
basal bodies were isolated using the same procedure as
described above, except for using 50 mM glycine-HCl (pH 2.
75) containing 0.1% (w/v) Triton X-100 at the filament dissociation step.

Electron microscopy
The isolated flagellar structures were negatively stained with
2% phosphotungstic acid (pH 7.4) and observed with a JEM1200EX electron microscope (JEOL, Japan).

Determination of N-terminal amino acid sequence
The hook-basal bodies were prepared as described above.
The proteins were separated by SDS-PAGE and transferred
electrophoretically to a polyvinylidene difluoride membrane(Millipore) and stained with Coomassie blue R250. The bandcorresponding to approximately 50 kDa was excised. The
N-terminal amino acid sequence was determined by Aproscience (Tokushima, Japan), based on the Edman degradation method.

Detection of proteins in cells
Vibrio cells were cultured at 30°C for 3 h in VPG500 medium.
The cells collected by centrifugation were suspended in SDS© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

Na+-driven Vibrio flagella motor 9
loading buffer at a concentration corresponding to an A660 of100. The 10 ml preparations of the purified basal body weremixed with 10 ml of SDS loading buffer. The samples wereboiled at 90°C for 10 min. SDS-PAGE and immunoblotting
were performed as described previously (Yorimitsu et al.,1999). The antibodies against MotX (MotXB0080), MotY
(MotYB0079), PomA (PomA1312), PomB (PomB93) and FlgI
were used as previously described (Nambu et al., 1999;
Yorimitsu et al., 1999; Yagasaki et al., 2006).

(10 mM Tris, 5 mM EDTA, 150 mM NaCl at pH 8.0). The
mixture was placed on a carbon-coated copper-mesh grid
and was left at room temperature for 1–2 h. The grid waswashed with buffer-B, and 5 nm colloidal gold-conjugatedanti-rabbit IgG antibody diluted twofold with 2 ml of buffer-C(20 mM Tris, 10 mM EDTA, 300 mM NaCl at pH 8.0) was puton the grid, and this sample was left at room temperature for
1–2 h. Then, the grid was washed with buffer-B, negatively
stained with 2% phosphotungstic acid (pH 7.4) and observedwith an electron microscope.

Sucrose density gradient centrifugation
The preparation of basal bodies was applied to a 20–60%(w/w) stepwise sucrose gradient in TET. After centrifugation
at 80 000 g for 13 h at 4°C, the gradient was divided into 18
fractions from the bottom to the top.

Gene disruption
The motX and motY deletion mutants were generated byhomologous recombination using a suicide vector as
described (Asai et al., 1999; Yagasaki et al., 2006). To makethe DmotX mutant, the sequences upstream (-227 to +72)
and downstream (+457 to +756) of the motX coding region of
pMO200 were amplified by PCR with an artificial StuI
sequence (AGGCCT) and ligated with the StuI site, and theresultant plasmid was named pJN559. The 0.6 kb EcoRI
fragment of the deleted motX gene was excised from pJN559
and introduced into the suicide vector pKY704 (Cmr). To
make the DmotY mutant, the sequences upstream (-440 to-2) and downstream (+871 to +1237) of the motY coding
region of pKJ507 were amplified by PCR with an artificial StuI
sequence (AGGCCT) and ligated with the StuI site, and theresultant plasmid was named pJN506. The 0.8 kb XbaI-SacI
fragment of the deleted motY gene was excised from pJN506
and introduced into the suicide vector pKY704 (Cmr). The
sacB gene (sucroses), which was obtained from pHFS401
using XbaI, was also introduced into pKY704 as a selection
marker for the second recombination. The suicide plasmids,
pTH101 (containing the DmotX fragment) and pTH102 (containing the DmotY fragment), were used to transform E. coli
SM10lpir. The deletion alleles were introduced into KK148
by a conjugation-based method. Chloramphenicol-resistantmutants were selected first on VC plates, and thenchloramphenicol-sensitive sucrose-resistant mutants wereidentified on sucrose-containing plates. We confirmed thedeletion mutations in the chromosomal motX and motY
genes based on the sizes of the amplified PCR fragments
and the complementation tests with pMO401, pIO6 andpKJ601. These mutants were named TH1 (DmotX), TH2(DmotY) and TH3 (DmotX DmotY). We also generated genedisruptions in NMB191 by the same methods; these mutants
were named TH4 (DpomAB DmotX) and TH5 (DpomAB
DmotY).

Immunogold labelling
The 3 ml basal body sample was mixed with 1 ml of buffer-A(10 mM Tris, 5 mM EDTA, 600 mM NaCl at pH 8.0) and with
1 ml of the antibody solution diluted 20-fold with buffer-B

Observation of GFP-PomA and GFP-PomB
The cells harbouring plasmids encoding GFP-fused PomA
and PomB (pHFGAB and pHFGBA2) were grown at 30°C for
4 h in VPG500 medium containing 0.02% arabinose. Cultured cells (1 ml) were harvested by centrifugation and suspended in 200 ml of V buffer (50 mM Tris, 5 mM MgCl2,
300 mM NaCl, at pH 7.5). A small aliquot of the cell suspension was spotted onto a glass slide, and cells were fixed on
the slide with an equal volume of 0.5% agarose dissolved in
V buffer. Cells were observed by fluorescence microscopy(Olympus BX50). The fluorescent images of GFP-PomA and
GFP-PomB were taken with a 1 s exposure. The images
were recorded and processed using a digital camera(Hamamatsu Photonics, C4742-80-12AG) and imaging softwares (BD Biosciences, IPLab, Ver. 3.9.5 r2 and Adobe,
Photoshop, Ver. 7).

Acknowledgements
We thank Shin-Ichi Aizawa and Masaomi Kanbe for technicalassistance to purify the basal bodies and for helpful
discussions. This work was supported in part by grants-in-aid
for scientific research from the Ministry of Education,
Science, and Culture of Japan; the Japan Science and Technology Corporation (to M.H. and to T.Y.); from the Soft NanoMachine Project of the Japan Science and Technology
Agency (to M.H.); and from Strategic International Cooperative Program of JST (to H.F. and M.H.).

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© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology