doi:10.1016/j.jmb.2003.09.052

J. Mol. Biol. (2003) 334, 567–583

The Conserved Charged Residues of the C-terminal
Region of FliG, a Rotor Component of the Na1-driven
Flagellar Motor
Professor Robert M. Macnab, who was professor of Yale University in the Department of Molecular
Biophysics and Biochemistry, died on 7 September 2003 at 63 years old. This article is dedicated to him,a kind, gentle, fair, and unforgettable person

Tomohiro Yorimitsu, Atsushi Mimaki, Toshiharu Yakushi andMichio Homma*
Division of Biological Science
Graduate School of Science
Nagoya University, Chikusa-ku
Nagoya 464-8602, Japan

FliG is an essential component of the flagellar motor and functions inflagellar assembly, torque generation and regulation of the direction offlagellar rotation. The five charged residues important for the rotation of
the flagellar motor were identified in Escherichia coli FliG (FliGE). These
residues are clustered in the C terminus and are all conserved in FliGV of
the Naþ-driven motor of Vibrio alginolyticus (Lys284, Arg301, Asp308,
Asp309 and Arg317). To investigate the roles of these charged residues in
the Naþ-driven motor, we cloned the Vibrio fliG gene and introduced
single or multiple substitutions into the corresponding positions in FliGV.
FliGV with double Ala replacements in all possible combinations at these
five conserved positions still retained significant motile ability, althoughsome of the mutations completely eliminated the function of FliGE. All of
the triple mutants constructed in this study also remained motile. These
results suggest that the important charged residues may be located indifferent places and the conserved charged residues are not so important
for the Naþ-driven flagellar motor of Vibrio. The chimeric FliG protein(FliGVE), composed of the N-terminal domain from V. alginolyticus andthe C-terminal domain from E. coli, functions in Vibrio cells. The mutationsof the charge residues of the C-terminal region in FliGVE affected swarming ability as in E. coli. Both the FliGV and the FliGVE proteins with the triple mutation were more susceptible to proteolysis than proteins without
the mutation, suggesting that their conformations were altered.
q 2003 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: electrostatic interaction; flagellar motor; energy transduction;
sodium type; Vibrio

Introduction
Present address: T. Yorimitsu, Department of
Molecular, Cellular and Developmental Biology,
University of Michigan, Ann Arbor, MI 48109, USA.
Abbreviations used: CCW, counter-clockwise; CW,
clockwise; FliGE, E. coli FliG; FliGV, V. alginolyticus FliG;
FliGEV, E. coli– V. alginolyticus chimeric FliG; FliGVE,
V. alginolyticus – E. coli chimeric FliG; AAQQQ mutant,
PomA mutant introduced R88A/K89A/E96Q/E97Q/
E99Q substitutions; GFP, green fluorescent protein.
E-mail address of the corresponding author:
g44416a@cc.nagoya-u.ac.jp

Bacterial flagella, which are filamentous organelles extending from the cell body, rotate reversibly by using the energy from the electrochemical
potential of a specific ion, either Hþ or Naþ, acrossthe cell membrane. The energy is harnessed by a
motor complex that is located at the base of the flagellum. From studies of Salmonella typhimurium
and Escherichia coli, which have Hþ-driven flagellar
motors, about 50 kinds of proteins are involved inflagellar assembly and function. Among them,
only three proteins, MotA, MotB, and FliG, are0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

568

Charged Residues in the C Terminus of FliG

Figure 1. The stator and rotor components of the bacterial flagellar motor. (a) Schematic diagram of the flagellar
motor. In the bacterial flagellar motor, the stator, which is composed of the PomA – PomB complex in the Naþ-driven
flagellar motor (MotA– MotB complex in the Hþ-driven flagellar motor), generates torque by the specific ion flux andtransfers it to FliG of the rotor component. It is proposed that four PomA (MotA) and two PomB (MotB) form one
unit of the complex.34,37 (b) Alignment of the sequences of the rotor components, FliG of V. alginolyticus (VaFliG) andCharged Residues in the C Terminus of FliG

thought to be directly involved in the energy conversion to generate the rotation from the ion
flux.1 – 3
MotA and MotB, which are membrane-spanning
proteins, form a Hþ-channel complex. MotA has
four transmembrane segments and a single large
cytoplasmic loop, and MotB has a single transmembrane segment.4,5 The C-terminal domain of
MotB is believed to bind with the peptidoglycan
layer6,7 and fix the MotA – MotB complex as a stator
around the basal body.8 The MotA –MotB complex
converts the Hþ flux into mechanical power for flagellar rotation.9,10 It is thought that the ion flow
induces the conformational changes of the complex
and then changes the interactions between thecytoplasmic regions of MotA and FliG, which is asoluble protein, to drive the motor (Figure 1(a)).
FliG, FliM and FliN proteins are components ofthe switch complex, which has been geneticallyassigned, and together they form the C-ring structure identified by electron microscopy.11,12 These
three proteins are needed for flagellar assembly,
torque generation, and regulation of the directionof flagellar rotation, counter-clockwise (CCW) or
clockwise (CW).13 – 15 Several lines of evidence indicate that FliG forms a ring structure 300 Å in diameter, which is composed of 25 –40 copies, and isperipherally attached to the cytoplasmic side of
the inner membrane by assembling with FliF,
which forms the MS ring of the basal body16 – 18
(Figure 1(a)). Among the motor components, thestructure of FliG from the thermophile Thermotoga
maritima, which possesses a flagellar motor whose
ion specificity has not been determined, wasresolved at atomic resolution and two crystal structures have been reported. One is the C-terminal
domain (FliG-C) at 2.3 Å resolution19 and the otheris the middle and C-terminal domains (FliG-MC)
at 2.8 Å resolution.20
It is known that some Vibrio species, V. alginolyticus, V. parahaemolyticus, and V. cholerae, and alkalophilic Bacillus species have Naþ-driven flagella.21 – 25
In Vibrio species, PomA and PomB, homologs of
MotA and MotB, respectively, are essential for therotation of the flagellar motor. Like MotA and
MotB, PomA and PomB have been shown to befunctional partners. In the MotB or PomB component, a single negatively charged residue is conserved in the putative transmembrane segment
and is suggested to be an ion-binding site.26 – 28 The
additional components MotX and MotY, which are

569

specific for the Vibrio motor, are needed for torquegeneration.29 – 32 They are localized to the outermembrane, although their functions are still
unknown.33 The PomA – PomB complex, whenreconstituted into proteoliposomes, exhibited Naþ
uptake activity without MotX and MotY, but
PomA alone did not. Because the molecular massof the purified PomA –PomB complex was estimated to be 175 kDa by gel permeation chromatography, PomA and PomB are suggested to form acomplex as oligomers, which might consist of four
PomA and two PomB molecules34 (Figure 1(a)).
From the following lines of evidence, PomA is
inferred to function as at least a dimer in the
PomA –PomB complex: (i) PomA alone forms astable dimmer;34 (ii) a genetically fused tandem
PomA dimer functions in the Naþ-type motor;35
and (iii) when either one or both of the PomA subunits in the homodimer is mutated, the resulting
complex is completely defective in motor
function.35 This inference is supported by crosslinking experiments using Cys-substituted mutantsof PomA.36 Recent systematic cross-linking analysis
against the transmembrane segment of MotB alsosuggested that MotA and MotB are together oligomerized to work in the Hþ-driven flagellar motor.37
Although the molecular mechanism of the flagellar motor is not yet clearly understood, several
studies suggest that both the Hþ-driven motor andthe Naþ-driven motor use a common mechanism
for torque generation. MotA of the Hþ-type motorof Rhodobacter sphaeroides functions in V. alginolyticus cells as the Naþ-driven motor component.38 In
addition, MomB, a chimera between the N terminus of MotB from R. sphaeroides and the C terminusof PomB can functionally substitute for PomB
in V. alginolyticus cells as a Naþ-driven motor.39
A recent study also demonstrated that PotB, a chimera between the N terminus of PomB and the C
terminus of MotB from E. coli constitutes the Naþdriven motor together with PomA not only in
V. alginolyticus but also in E. coli cells even in theabsence of MotX or/and MotY.40 Furthermore,
Sowa et al.41 analyzed the relationships between
torque and speed in detail and concluded thatcharacteristics are shared between the Hþ-driven
and Naþ-driven flagellar motors.
In the Hþ-driven flagellar motor, a mechanicalmodel for the interactions between MotA and FliG
during motor rotation has been presented.42 The
residues in the cytoplasmic loop of MotA and theof E. coli (EcFliG). White letters in black boxes indicate conserved residues between FliG of V. alginolyticus and of E. coli.
The conserved charged residues mutagenized in this work are indicated by filled arrowheads (positively charged) andopen arrowheads (negatively charged). The numbers over the arrowheads represent the positions of the amino acidresidues in V. alginolyticus FliG. The arrow indicates the position of the junction in the chimeric FliG proteins between
V. alginolyticus and E. coli. (c) Charged residues of the cytoplasmic loop of the stator part (PomA or MotA) and the
C-terminal domain of the rotor part (FliG). Arg88 and Glu96 of PomA correspond to Arg90 and Glu98 of MotA,
respectively. Charged residues, mutagenized here and previously, are suggested to be located at the interface between
the stator and rotor parts.

570

C-terminal region of FliG were analyzed by mutagenesis, and the charged residues important for
torque generation were identified to be Arg90 and
Glu98 in MotA and Lys264, Arg281, Glu288,
Glu289 and Arg297 in FliG43,44 (Figure 1(c)). In the
crystal structure of the T. maritima FliG, it is predicted that these charged residues are localized tothe edges toward the outside of the FliG ring, andthe electrostatic interactions that are exerted
between the charged residues of MotA and FliG inthe cytoplasmic regions cause motor rotation.19,20
PomA shares two conserved charged residues in
the cytoplasmic loop with MotA (Arg90 and
Glu98, which correspond to Arg88 and Glu96 in
PomA of V. alginolyticus), and has other charged
residues, namely Lys89, Glu97 and Glu99, locatednear the two conserved residues (Figure 1(c)).
When the charged residues of MotA are substituted with neutral residues, MotA completely
eliminates motor rotation. On the other hand, substitutions of the charged residues, including thenon-conserved residues of PomA do not affect thetorque generation of the Naþ-driven flagellar
motor.45 This discrepancy has suggested thatelectrostatic interactions are not required for therotation of the flagellar motor or that the othercharged residues are involved in the electrostaticinteractions of the Naþ-driven flagellar motor.
FliG in the Hþ-driven motor of E. coli and theNaþ-driven motor of V. alginolyticus are more similar to each other than MotA and PomA. The fivecharged residues (Lys284, Arg308, Asp308, Asp309
and Arg317) identified to be important for E. coli
flagellar rotation are all conserved in the V. alginolyticus FliG (Figure 1(b) and (c)). In the present
work, we cloned and characterized the fliG geneof the polar flagellum of V. alginolyticus, and examined whether the conserved charged residues are
involved in driving the Naþ-driven flagellar motor.

Charged Residues in the C Terminus of FliG

Polar flagellar FliG of V. alginolyticus

Figure 2. Expression of fliG. (a) Swarms of YM4 ( fliG þ)or NMB107 ( fliG 2) cells harboring a vector plasmid(pBAD33) or a plasmid encoding FliG (pTY101) under
the control of the arabinose promoter on a 0.25% VPG
plate in the absence (upper panel) or presence (lower
panel) of 1 mM arabinose. (b) Detection of FliG proteins.
YM4 or NMB107 cells harboring a vector plasmid or aplasmid encoding FliG were grown in the absence orpresence of 1 mM arabinose, collected at mid-log phaseand subjected to SDS-PAGE followed by immunoblottingwith anti-FliG antibody. The asterisk indicates a nonspecific band.

To clone the V. alginolyticus fliG gene, we amplified the fragment containing fliG by PCR as
described in Materials and Methods. The cloned
fliG sequence of V. alginolyticus encodes 351 aminoacid residues, and the predicted FliG protein has
40% amino acid sequence identity with the E. coli
FliG protein (Figure 1(b)) and 96% and 86% identity with FliG from the related Vibrio species
V. parahaemolyticus and V. cholerae, respectively.
To examine whether the cloned fliG gene functions for the polar flagellum, we tried to identify afliG mutant from the laboratory stocks of Fla2
strains, which are derived from YM4,46 a strain
that posses the polar flagellum but has the defectof lateral flagella. One strain, NMB107, retained aswarm in the presence of arabinose only whentransformed with the plasmid encoding the fliG
gene under an arabinose-inducible promoter(Figure 2(a)). Although NMB107 cells have no flagella, after expression of fliG in the strain, thepolar flagellum was easily observed by dark-fieldmicroscopy (data not shown). The site of themutation in NMB107 was located at the codon for
Arg135 (CGA) of fliG, which was altered to theopal codon (TGA).
To detect FliG protein by immunoblotting, weraised antibody against the synthetic peptide
deduced from the amino acid sequence of FliG.
Cells grown in the presence or absence of 1 mM
arabinose were collected during log-phase andproteins were subjected to SDS-PAGE followed
by immunoblotting with anti-FliG antibody(Figure 2(b)). In the presence of arabinose, theapproximately 40 kDa FliG band (as estimated by

Results

Charged Residues in the C Terminus of FliG

molecular markers) was observed with strain
NMB107 harboring the plasmid carrying the fliG
gene, and also independently with strain YM4grown on arabinose. The molecular mass of FliG
deduced from the amino acid sequence is38,587 Da. FliG protein was not detected with
NMB107 cells containing a vector control or theplasmid-borne fliG gene in the absence of arabinose, indicating that, as expected, NMB107 does
not produce FliG protein due to the fliG mutation
determined by sequence analysis.

571

Subcellular localization of GFP – FliG protein
To characterize the polar flagellar FliG protein of
V. alginolyticus, we constructed green fluorescentprotein (GFP)-fused FliG and expressed it under
the control of an arabinose promoter in NMB107
cells. GFP was tagged to the N and C termini of
FliG, and the proteins were designated GFP – FliG
and FliG – GFP, respectively (Figure 3(a)). To
examine whether these GFP-tagged FliG proteinsare functional, we carried out swarm assays. Cells

Figure 3. Fusion of FliG with
GFP. (a) Diagram of the GFP-fused
FliG proteins. Hatched boxes indicate the GFP sequence and open
boxes indicate the FliG sequence.
GFP was fused with the N and C
termini of FliG, termed GFP–FliG and GFP –FliG, respectively.
(b) Swarms of NMB107 cells harboring a vector plasmid (pBAD33)
or a plasmid encoding FliG
(pTY101), GFP (pTY200), GFP –FliG(pTY201) or FliG –GFP (pTY202)
under the control of the arabinose
promoter on 0.25% VPG plates in
the absence (left panel) or presence(right panel) of 1 mM arabinose.
(c) Detection of the GFP-fused FliG
proteins. NMB107 cells harboringa vector plasmid or a plasmid
encoding FliG, GFP or GFP – FliG
were grown in the absence orpresence of 1 mM arabinose,
collected at mid-log phase andsubjected to SDS-PAGE followed
by immunoblotting with anti-FliG
antibody (upper panel) or anti-GFP
antibody (lower panel).

572

carrying both GFP –FliG and FliG – GFP were functional, but the swarm sizes were smaller than thatmade by cells carrying wild-type FliG. In addition,
cells carrying GFP – FliG swarmed better thanthose carrying FliG – GFP (Figure 3(b)). By darkfield microscopy, the polar flagella were observedin cells with GFP – FliG or FliG – GFP as well as
those with untagged FliG. Therefore, we used
GFP – FliG for subsequent experiments.
To detect the GFP –FliG protein in cells, we carried out immunoblotting (Figure 3(c)). Immunoblot
analysis with anti-FliG antibody or anti-GFP antibody showed that GFP –FliG was produced stably
in NMB107 cells and no cleavage of the GFP
moiety region was observed. These results suggestthat GFP – FliG is functionally incorporated into
the motor structure.
Next, we examined the subcellular localizationof GFP –FliG in living cells (Figure 4). Cells harvested at mid-log phase were observed in a fluorescent microscope as described in Materials andMethods. A fluorescent dot was observed at thepole of NMB107 cells producing GFP – FliG, while
no dot was observed in cells producing GFP(Figure 4(a); upper panels). Strain YM14 was previously isolated as an rpoN mutant and completely
lacks the flagellar structure including the basal
body.47 There was no fluorescent dot at the pole of
YM14 cells with GFP – FliG, as with GFP, and only
dispersed fluorescence was seen (Figure 4(a);lower panels). To observe whether the fluorescent
dot is co-located with the flagellar structure, weused a primary antibody against the polar flagellum and a second antibody coupled with rhodamine. As shown in Figure 4(b), a green dot of
GFP – FliG co-localized with the red-staining flagellum at the same pole. Because the polar flagellum
is covered with a lipid sheath connected to theouter membrane,48 the cell body also seems to be
stained. These results indicate that GFP – FliG is
incorporated into the polar flagellar motor andfunctions in it.
Site-directed mutagenesis of individual
charged residues
It has been reported that the five conserved
charged residues located in the C-terminal regionof FliG are functionally important for flagellar
rotation of the Hþ-driven flagellar motor of E. coli
by electrostatically interacting with the charged
residues of MotA42 – 44 (Figure 1(c)). All five charged
residues are conserved in the Naþ-driven motor of
V. alginolyticus (Figure 1(b)). To study the role ofthe conserved charged residues of V. alginolyticus
FliG, we replaced each of the charged residues
with neutral amino acid residues. When substitutions with Ala were carried out, all of the mutant
proteins were stably expressed in NMB107 cells(Figure 5(a)). The motility of cells expressing
mutant FliG was examined by the swarm assay ina soft agar plate and the swimming speed in liquidcontaining 50 mM NaCl (Figure 5(a)). As shown

Charged Residues in the C Terminus of FliG

for E. coli FliG mutants with single Ala substitutions, none of the single mutations with Alaseverely affected swarm formation or swimming
speed. The swarm size of the R301A and R317A
mutants decreased to 73% and 61% of that of cellswith wild-type FliG, respectively, but the swimming speeds were not affected.
A previous study of E. coli FliG showed that
motility is more severely impaired by reversing
the charges of some residues than by introducing
neutral residues.43 Therefore, we substituted residues with opposite charges, replacing the Lys284
residue with Glu, the Arg301 and Arg317 residues
with Asp, or the Asp308 and Asp309 residues
with Lys. FliG charge-reversed mutant proteins
were produced as well as wild-type FliG (Figure5(b)). The D289K mutation of E. coli FliG, whichsignificantly impaired motility in E. coli, corresponded to the D309K mutation of V. alginolyticus
FliG. However, cells expressing either D308K or
D309K mutant FliG were capable of swarming
and swimming as well as those expressing wildtype FliG (Figure 5(b)). The R301D mutant also
swarmed, but the R281D mutant of E. coli FliG
was non-motile. On the other hand, the K284E
and R317D mutations caused more severe swarming
defects in V. alginolyticus than the corresponding
K264E and R297D FliG mutations in E. coli, respectively. In spite of the poor swarms made by the
K284E and R301D mutants, these strains were able
to swim as fast as the strain carrying wild-type
FliG. Cells expressing the R317D mutant were
never motile. The polar flagellum assembly in all
the mutants was observed by dark-fieldmicroscopy and was similar to that of wild-type(results not shown).
Double substitutions of charged residues
It was reported previously that introduction ofcertain double mutations at positions of charged
residues in E. coli FliG synergistically impairs the
motor function.43 To test this in V. alginolyticus
FliG, we changed two of the five charged residues
to Ala to generate ten double Ala-substituted
mutants and also changed Asp308 and Asp309
into Asn residues (Figure 6). All of the double
mutant proteins were stably produced in Vibrio
cells (Figure 6, upper panel), and a normal polar
flagellum was detected by microscopic observationin all of the mutant strains (results not shown). The
swarming ability and the swimming speed of cells
expressing FliG mutants with double substitutions
were examined (Figure 6, lower panel). All of the
double mutants have the ability to swim at a
speed nearly comparable to that of the wild-type
FliG. A slightly more severe swarming impairment
was observed with cells expressing the mutants
K284A/R301A, K284A/R317A, R301A/R317A or
D309A/R317A than with those expressing eachof singly Ala-substituted mutants. Similar to thephenotypes of the corresponding E. coli FliG
mutants, a significant swarming ability wasCharged Residues in the C Terminus of FliG

573

Figure 4. Subcellular localizationof GFP – FliG. (a) NMB107 (upper
panels) or YM14 (rpoN) (lower
panels) cells harboring a plasmid
encoding GFP (pTY200) (left) or
GFP – FliG (pTY201) (right) weregrown to mid-log phase in the presence of 1 mM arabinose andobserved under a fluorescencemicroscope. (b) NMB107 cells
expressing GFP – FliG grown to
mid-log phase in the presence of
1 mM arabinose were treated with
anti-polar flagella antibody followed by rhodamine-conjugatedanti-rabbit IgG antibody andobserved under a fluorescencemicroscope. Images of four independent cells are shown.

observed in cells with the K284A/D308A or K284A/
D309A mutations in V. alginolyticus FliG. Five
double mutants (R301A/D308A, R301A/D309A,
D308A/D309A, D308A/R317A and D308N/
D309N) were wild-type in their ability to swarm
and swim, although the corresponding E. coli FliG
double mutants were severely impaired in motility.
Triple substitutions of charged residues
The crystal structure of the C-terminal region of

the T. maritima FliG reveals that the conserved
charged residues are aligned in two individual
lines, Lys266-Arg283-Asp290 and Arg283-Glu291Arg299, suggesting that each combination has
some role in the motor function.19,20 To examine
this in V. alginolyticus FliG, we constructed triple
Ala mutants, K284A/R301A/D308A and R301A/
D309A/R317A corresponding to the residues ofT. maritima FliG. The protein production of thetwo triple mutants was similar to that of the wildtype FliG as detected by immunoblotting (Figure 7,

574

Charged Residues in the C Terminus of FliG

Three other charged residues present in E. coli
FliG (Arg281, Asp288 and Asp289) have been
identified as a functionally important combination
for flagellar rotation and are hypothesized to beminimally
involved
intherotor–stator
interaction.42,43 We examined whether the equivalent triple Ala substitution (R301A/D308A/
D309A) in the V. alginolyticus FliG affects motility(Figure 7). Although all possible combinations ofdouble mutations of these three residues did notprevent motility, as shown in Figure 7, cells carrying the R301A/D308A/D309A triple mutant (produced at wild-type levels) swarmed poorly (20%of wild-type) but swam at the same speed as cells
carrying the wild-type FliG. A triple mutant
R301A/D308N/D309N was also constructed tominimize the effects of the substitutions of the
Asp residues. The D308N/D309N double mutant
exhibited completely normal swarming ability andswimming speed. The R301A/D308N/D309N
mutation conferred a better swarming ability thanthe R301A/D308A/D309A mutation. Whenobserved in the microscope, the flagellar formationof all of the triple mutants was similar to that of thewild-type strain.
Chimeras between FliG proteins derived fromV. alginolyticus and from E. coli
Figure 5. Effect of single substitutions of charged residues with an Ala residue on swarm formation andswimming speed (a) and of reversing charged residues(b). Detection of FliG mutant proteins (upper panel).
NMB107 cells harboring a vector plasmid (pBAD33) ora plasmid encoding wild-type FliG or indicated FliG
mutant (pTY101) were grown in the absence or presenceof 1 mM arabinose, collected at mid-log phase and subjected to SDS-PAGE followed by immunoblotting with
anti-FliG antibody. Swarm sizes and swimming speedsof the mutants with single substitution of charged residues (lower panel). Swarm diameters (open bars) andswimming speeds (filled bars) of NMB107 cells containing the various FliG mutants are shown in the histogram
as a fraction of the swarm diameters and the swimming
speeds of cells containing wild-type FliG. Swimming
speeds in 50 mM NaCl were measured as described inMaterials and Methods.

upper panel). Although both of the triple mutantsremained motile, the swarm sizes of the K284A/
R301A/D308A
and
R301A/D309A/R317A
mutants were decreased to 28% and 5%, respectively, of that of a strain carrying wild-type FliG
(Figure 7, lower panel). In liquid medium, themotile fraction of cells with the K284A/R301A/
D308A mutation was similar to that of cells withwild-type FliG, while the number of motile cells in
the R301A/D309A/R317A mutant population wasvery low (a small percentage of wild-type). We
speculate that the motor rotation may be hard to
be started by the mutant FliG. However, both of
the triple mutants exhibited swimming speeds
comparable to that of the wild-type strain.

It has been reported that chimeric proteins
between V. cholerae and E. coli FliG have been
constructed.49 A chimera consisting of the N-terminal region of V. cholerae FliG fused to the C-terminal domain of E. coli FliG is able to function in the
V. cholerae fliG null strain, but not the E. coli strain.
The opposite V. cholerae –E. coli chimeric FliG alsofunctioned in E. coli but not in V. cholerae fliGdeleted cells. We generated chimeric proteins
between the V. alginolyticus and E. coli FliG proteins. A fusion FliG composed of the N-terminal
portion of V. alginolyticus FliG and the C-terminal
region of E. coli FliG was named FliGVE and theinverted fusion was designated FliGEV (Figure8(a)). Swarm assays were carried out in the fliGmutant strains V. alginolyticus NMB107 and E. coli
DFB225 (Figure 8(b)). NMB107 cells complemented
by the FliGVE chimera swarmed as well as by wildtype V. alginolyticus FliG, but FliGEV did not complement. In contrast, E. coli FliG and the FliGEV chimera conferred the swarming ability to E. coli
DFB225 cells, but FliGVE did not.
To further analyze the C-terminal region of FliG,
we introduced mutations at the charged residues
in the FliGVE chimera (Figure 8(c)). Such substitutions resulted in a loss of function in E. coli FliG
but not in V. alginolyticus FliG. When the two residues Asp308 and Asp309 of FliGVE were replacedwith the Asn residues, swarming ability was significantly decreased to 11% of the wild-typeswarm size. The number of motile cells decreased
significantly in the D308N/D309N mutant of
FliGVE, but had swimming speeds comparable tocells carrying FliGV. Although the strain with

Charged Residues in the C Terminus of FliG

575

Figure 6. Effect of double substitutions of charged residues on
swarm formation and swimming
speed. Detection of FliG mutant
proteins (upper panel). FliG mutant
proteins were detected as describedin the legend to Figure 5. Swarm
sizes and swimming speeds of themutants with the double substitutions of charged residues (lower
panel).

FliGV carrying the R301A/D308N/D309N triple
mutation showed a 40% swarm ability comparedto the wild-type, the introduction of the triple
mutation into FliGVE resulted in completely nonmotile cells. The chimeric FliGVE mutant proteins
were produced in Vibrio cells at the same level as
the intact FliG mutant proteins as determined byimmunoblotting (Figure 8(d)), and the flagellation
patterns observed by microscopy appeared to benormal (data not shown).
Effect of substitutions of charged residues on
the proteolytic susceptibility of membranelocated FliG protein

Figure 7. Effect of triple substitutions of charged residues on swarm formation and swimming speed. Detection of FliG mutant proteins (upper panel). FliG mutant
proteins were detected as described in the legend to
Figure 5. Swarm sizes and swimming speeds of themutants with the triple substitutions of charged residues(lower panel).

In the flagellar motor, FliG is peripherally
embedded in the cytoplasmic side of the membrane by assembling with FliF, which is a component of the basal body.50 To confirm this, wecloned the fliF gene of V. alginolyticus and co-produced the V. alginolyticus FliG and FliF in E. coli
cells. In the absence of FliF co-production, the FliG
protein was detected only in the supernatant fraction after high-speed centrifugation. In contrast,
when expressed with FliF, FliG was predominantly
pelleted in the membrane fraction (Figure 9(a)). To
examine whether co-expression of PomA and
PomB affects the protease susceptibility of FliG, we
prepared membrane vesicles from cells expressing
FliF and FliG with or without PomA and PomB
and treated them with the proteases, proteinase K
and trypsin. As shown in Figure 9(b), without coexpression of PomA and PomB, FliG proteins
were degraded by both proteases more significantly than those with PomA and PomB, indicatingthat PomA and PomB affect the protease sensitivityof membrane-localized FliG.
We next examined whether introduction of
mutations into the charged residues of FliGV and576

Charged Residues in the C Terminus of FliG

Figure 8. Characterization of chimeric FliG. (a) Schematic diagramsof chimeric FliG proteins. Chimeric
proteins FliGVE and FliGEV wereconstructedasdescribed
inMaterials and Methods. White
boxes indicate the V. alginolyticus
FliG-derived sequence and hatchedboxes indicate the E. coli FliG
sequence. Numbers correspond to
the amino acid residues. (b) Complementation by chimeric FliG
proteins. DFB225 or NMB107 cells
expressing the FliG proteins were
cultured overnight and spotted on
a TB or VPG 0.25% agar plate containing chloramphenicol in thepresence or absence of 1 mM
arabinose. FliGE and FliGV indicate
FliG derived from E. coli FliG andfrom V. alginolyticus, respectively.
(c) Swarm profiles of the chimeric
FliGVE mutants. Swarm diametersof NMB107 cells containing FliGVor FliGVE with D308N/D309N or
R301A/D308N/D309N mutations
are shown in the histogram as afraction of the swarm diameters ofcells with the wild-type FliG.
(d) Detection of chimeric FliGVE
mutant proteins. FliG and FliGVE
mutant proteins were detected byimmunoblotting as described in thelegend to Figure 5.

FliGVE affects the protease susceptibility of these
proteins (Figure 9(c)). FliGV proteins with the
D308N/D309N substitution, which does not affectthe motility as shown above, exhibited a susceptibility equivalent to the wild-type FliGV. However,the R301A/D308N/D309N triple mutant, whichcaused reduced swarming to less than 40% of
wild-type, showed an increased susceptibility toproteolysis. The susceptibility to proteases was
also examined with the chimeric FliGVE proteins.
The FliGVE protein without substitutions was
slightly more susceptible to proteolysis than FliGV
protein. As shown with the FliG proteins, theD308N/D309N-mutagenized FliGVE protein wasdigested to the same level as the FliGVE protein
without substitutions and the R301A/D308N/
D309N-mutagenized FliGVE protein was slightly
more digested than FliGVE protein.

Discussion
FliG has multiple roles in the operation of theflagellar motor, the flagellar architecture, torquegeneration and the clockwise (CW) to counterclockwise (CCW) rotational switch.13,15,51 In thiswork we first cloned the fliG gene for the polar flagellum of V. alginolyticus and carried out a basic
characterization. Based on previous biochemicaland structural evidence, it is known that FliG
forms a switch complex with FliM and FliN.11,12,52
Kubori et al.50 have reported that only FliF isneeded for FliG localization, but FliM and FliN
are not. Sourjik & Berg53 observed the assembly of
FliM – YFP (FliM fused with yellow fluorescent protein which is a variant of GFP) on the switch complex in E. coli cells and found that FliM – YFP
localization was dependent on FliG but independent

Charged Residues in the C Terminus of FliG

577

Figure 9. Localization and protease sensitivity of FliG proteins.
(a) Localization was examined
during co-expression of FliG with
FliF. Cell suspensions of JM109
cells harboring plasmids carrying
FliG alone (pTY101) or FliG with
FliF (pTY601) were passed througha French press, and cytoplasm(S100) and membrane (P100) fractions were prepared from thelysates (Total) by centrifugation as
described
inMaterials
andMethods. Immunoblotting was carried out with anti-FliG antibody.
(b) Effect of co-expression of PomA
and PomB on the protease susceptibility of membrane-associated FliG.
Membrane vesicles of JM109 cells
harboring a plasmid carrying
V. alginolyticus FliF and FliGV(pTY601) with or without plasmids
carrying
PomA
and
PomB
(pJN131) were treated with trypsin
or proteinase K at the indicatedconcentrations, or without protease(2). (c) Effect of mutations ofcharged residues on protease susceptibility of FliG or the FliGVE chimera. Membrane vesicles of JM109
cells harboring plasmids carrying
fliF and fliG V (pTY601) (left panel)or a FliGVE chimera (pTY602) (right panel), with or without the indicated mutations shown on the left side, together
with the plasmid carrying PomA and PomB (pJN131) were treated with trypsin at the indicated concentrations. None
indicates the wild-type. Immunoblotting was carried out with anti-FliG antibody.

of FliN. It is known that the direction of flagellar
rotation is regulated by phosphorylation or dephosphorylation of CheY and that phospho-CheY
binds to FliM of the switch complex to change thedirection of rotation from CCW to CW.54 – 57
Here, we were able to observe the assembly of
GFP-fused FliG (GFP –FliG) in the polar flagellar
structure in living Vibrio cells. The present work
showed that FliG localization is not observed inan rpoN strain, which does not synthesize flagella.47
We have cloned the fliF gene of the polar flagellumof V. alginolyticus, which functionally complemented the DfliF strain, NMB196, generated from thelateral-flagella-defective strain VIO5. The localization of GFP – FliG in the membrane of E. coli cells
was observed only when expressed together with
V. alginolyticus FliF (data not shown), and the interaction between FliF and FliG was shown by ourbiochemical analysis. These results are in agreement with the previous results for E. coli andS. typhimurium as described above, suggesting thatthe mechanism for the assembly of the components
in the switch complex is basically shared between
peritrichous and polar-flagellated cells. The functional GFP –FliG should be a useful tool for studying the mechanism of flagellar assembly.
For the flagellar motor rotation, the stator portion (the PomA –PomB complex for the Naþ-type
and the MotA –MotB complex for Hþ-type) con-verts the ion flux generated by the electrochemical
potential into mechanical energy and transfers it
to the rotor portion (FliG) (Figure 1(a)). In the Hþdriven flagellar motor, it has been proposed thatthe charged residues in the C-terminal domain of
the rotor protein FliG and in the cytoplasmic loopof the stator protein MotA have a role in the electrostatic interaction, which is a functionally important event for the motor rotation42 (Figure 1(c)).
Those residues in the Hþ-driven components areconserved in the Naþ-driven components (Figure1(c)). However, we have demonstrated that thesecharged residues are not essential for motor
rotation, suggesting that the stator component(PomA) of the Naþ-driven motor of V. alginolyticus
has extra or additional essential charged residues,or alternatively, that the specific charge interaction
is not as important in the Naþ-driven motor.45 Several studies suggest that the mechanism of torquegeneration in the Naþ and Hþ-type motors isshared. Rhodobacter sphaeroides MotA, which is theHþ-driven motor component, functions in pomAdefective Vibrio cells as a component of the Naþtype motor.38 The V. cholerae – E. coli FliG fusion,49
the chimera FliGVE, is functional in V. cholerae anda similar result was obtained in this study using
V. alginolyticus. It has been shown that PomA and
PotB (a chimera of V. alginolyticus PomB and E. coli
MotB) can function with E. coli FliG in E. coli cells

578

and the E. coli flagellar motor can rotate using theNaþ flux in the absence of MotX or/and MotY.40
When amino acid sequences of V. alginolyticus
FliG and E. coli FliG are aligned, their C-terminal
regions have much higher homology than the cytoplasmic loops of PomA and MotA. We focused on
the five conserved charged residues Lys284,
Arg301, Glu308, Glu309 and Arg317 of V. alginolyticus FliG that correspond to the E. coli FliG residues
predicted to be crucial for function.43 When theE. coli FliG was mutagenized, some conserved
charged residues affected the motor rotation synergistically. In V. alginolyticus FliG, not only single,
but also multiple substitutions with neutral residues did not affect torque generation, although
the swarming ability of the strains was somewhat
decreased. These results suggest that these conserved residues of V. alginolyticus FliG are notessential for rotating the Naþ-driven motor but are
involved in controlling the direction of the rotationof the flagellar motor. From the crystal structure of
the T. maritima FliG, that can function in E. coli, a
subset of three of the five key charged residues
can be linearized in two individual arrangements
and each set of arrangements is speculated tomake the torque for one direction, CCW or CW.19,20
We triply mutagenized the residues corresponding
to each of the combinations of T. maritima FliG.
Cells with either of the triple mutants were poor
swarmers, but they were able to swim at speeds
comparable to cells with wild-type FliG. Conformational changes in FliG might induce the change
between CCW and CW state of the flagellar
rotation or might affect the force generation of the
motor in response to increasing load.
When the three residues Arg301, Asp308 andAsp309 of V. alginolyticus FliG were neutralized(the R301A/D308N/D309N mutant), the triple
mutants still retained motility. Triple mutations of
the equivalent three residues, Arg281, Asp288 andAsp289 in E. coli FliG resulted in a complete lossof function and were suggested to be primarily
important for electrostatic interactions in E. coli.43
To exclude the possibility that misreading of theopal mutation in the fliG gene in strain NMB107(which was used as the fliG mutant in this work)
results in the production of some functional protein, we generated a fliG deletion mutant,
NMB198. When the mutant FliG proteins wereexpressed in NMB198, we obtained the same
results as with NMB107 (data not shown). Basedon these lines of evidence in FliG and PomA of
V. alginolyticus, we may infer that electrostaticinteractions are not required, or at least are notimportant, for the rotation of the Naþ-driven
motor. However, it is highly possible that othersubsets of charged residues that are not sharedwith E. coli FliG, are located in the C-terminal
domain of V. alginolyticus FliG and are involved in
the Naþ-driven motor. The candidates may be thecharged residues K300 and E311, which are predicted to locate on the same surface of the conserved residues. To clarify the roles of charged

Charged Residues in the C Terminus of FliG

residues in V. alginolyticus, the interaction of thecharged residues between PomA and FliG should
be investigated.
Proteolysis analysis showed that co-expressionof PomA and PomB affects the protease sensitivityof FliG. We speculate that the PomA – PomB complex directly blocks the proteolysis of FliG protein
or alters the FliG conformation because of their
physical interaction. Either FliGV or FliGVE (the chimera between the N-terminal region of V. alginolyticus FliG and the C-terminal region of E. coli FliG)
with the R301A/D308N/D309N substitution isless resistant to proteolysis than the wild-type proteins or the D308N/D309N mutant proteins. This
protease susceptibility of the mutant proteins has
also been observed in the E. coli FliG mutants.43
We speculate that the charged residues are
required for sustaining the functional structure,and the conformational changes caused by themultiple mutations are enough to impair FliG function or the functional (electrostatic) interaction
with the stator.
In the present mutational study of the V. alginolyticus FliG, the R317D mutant was the only nonfunctional FliG. Double and triple mutants combined with the Ala mutation of the Arg317 residue
decreased the function more severely than theother mutations. According to these results, theArg317 residue could be one of the candidates fora functionally important charged residue in theNaþ-driven flagellar motor. The corresponding
residue of E. coli FliG, Arg297, which is locatedbetween helix 5 and helix 6 as deduced from thecrystal structure of T. maritima FliG,19,20 is found to
be moderately important for function.43 There arealso other possible candidates in the C-terminal
segment of V. alginolyticus FliG, which contains
three basic and four acidic residues. Further
studies are needed to identify the essential residues for the Naþ-driven flagellar motor if thoseresidues do exist. The flagellar motor is likely tocontain 25– 40 copies of FliG, which are assembled
in a ring-shaped structure and attach to the MSring directly.16 – 18 The predicted stoichiometry of
PomA and PomB or MotA and MotB in the complex is 4:2, so the PomA –PomB (MotA – MotB)
complex has four cytoplasmic loops, of which the
charged residues are predicted to interact with the
charged residues of the C-terminal FliG (Figure1(a)). At this time, it is difficult to predict the functional contacts exerted between the cytoplasmic
regions of FliG and PomA.
It has been reported that the CW – CCW switching of Vibrio cells is abnormal in the Naþ-driven
motor composed of the R. sphaeroides wild-type
MotA or of the quadruple-mutant form of PomA
(AAQQQ mutant).38,45 Some of the FliG mutants
described here also showed a discrepancy between
the swarm and swimming speed, which is often
seen in chemotaxis mutants. It is hypothesized thatthe rotor–stator interface is first aligned correctly by
the electrostatic interaction, and this alignment is a
trigger for the following torque-generating events.

579

Charged Residues in the C Terminus of FliG

It is easy to speculate that the stator – rotor arrangement affects CW – CCW switching. Such a systemof regulating the rotation by electrostatic interactions has been proposed for the F1-ATPase,
which is also known to be a rotary motor.58 The b
subunit of F1-ATPase has a conserved acidic
motif, the DELSEED motif, which was assumed tocontact the g subunit and to be involved in torquegeneration. However, Ala-scanning mutagenesisof the DELSEED motif in the thermophilic Bacillus
PS3 F1-ATPase showed that all of examinedmutants rotate with as much torque as wildtype.59 However, the 1 subunit of F1-ATPase, an
intrinsic inhibitor of the enzyme, is unable to inhibit the ATPase activity in the DELSEED mutants,60
indicating that the DELSEED motif does not havea direct role in torque generation, but that the electrostatic interactions with the 1 subunit function toexert the inhibitory activity. Further, it has beensuggested that the 1 subunit of the F1Fo-ATP
synthase regulates the switch between the ATPhydrolysis mode and the ATP synthase mode largely by changing conformation, which might regulate the direction of the rotation of the enzyme.61
In the Hþ-driven flagellar motor, one model forthe motor rotation coupling with Hþ flow has
been presented as follows.62 (i) In the stator, MotB
has an Asp32 residue in an ion-binding site in atransmembrane segment. However, a channel ofthe MotA –MotB complex is not opened and Hþ is
not accessible to the binding site. (ii) A conformational change of the stator involving the Pro173
residue causes a gate to open in the channel,
which is induced by an electrostatic interaction
between the rotor and the stator. (iii) Hþ can enter
the channel from the periplasm and bind to
Asp32. (iv) The binding of Hþ triggers a further
conformational change to drive the rotor movement and then Hþ is released into the cytoplasm.
(v) The rotor moves farther and the stator returns
to the first conformation. This model could beapplied basically to the Naþ-driven motor, because
the two Pro residues, Pro173 and Pro222, in theE. coli model are conserved in PomA (Pro153 andPro199, respectively). The Asp32 residue of MotB
is conserved in PomB. Asp24 is the only charged
residue in the transmembrane segment of PomBand substitution of that residue causes cells to
become non-motile. However, a mutant PomA
with a substitution at Pro199 was unable to associate with PomB, suggesting that unlike Pro222 of
MotA, Pro199 may play a role in forming or stabilizing the PomA – PomB complex.35 It seems thatelectrostatic interactions are not important for therotation of the Naþ-driven motor, so the conformational changes that cause the opening of thegate might be unnecessary for the Naþ-driven
motor. Ions might pass more effectively throughthe channel of the Naþ-driven flagellar motor,
which could confer a much higher rotational
speed on the Naþ-driven flagellar motor than theHþ-driven motor (1700 rps versus 300 rps,
respectively).63,64

Materials and Methods
Bacterial strains, plasmids, growth conditions
and media
The bacterial strains and plasmids used in this study
are listed in Table 1. V. alginolyticus cells were cultured
at 30 8C 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 VPG medium (1% (w/v)
polypeptone, 0.4% (w/v) K2HPO4, 3% (w/v) NaCl, 0.5%(w/v) glycerol). For the swarm assay, overnight culturesof cells were spotted onto 0.25% (w/v) agar-VPG plates
containing 2.5 mg/ml of chloramphenicol and incubatedat 30 8C. E. coli cells were cultured 30 8C in TB medium(1% (w/v) tryptone, 0.3% (w/v) NaCl) and overnight
cultures of cells were spotted onto 0.25% agar-TB plates
containing 25 mg/ml of chloramphenicol and incubatedat 30 8C. If necessary, L -arabinose was added to a final
concentration of 1 mM.
Routine DNA manipulations were carried out according to standard procedures.65 Plasmid pSU41 carries thelac promoter.66 Plasmid pBAD33 carries the araBAD promoter and the araC gene encoding the positive and negative regulator.67 Plasmid pJY19 is a derivative of plasmid
pTTQ18 (Amersham), and carries the tac promoter, thelacI q gene and the different multiple cloning site ofTable 1. Bacterial strains and plasmids used in this study
Strain orplasmid

Description

Source orreference

A. V. alginolyticus strains
VIO5
Pofþ, Laf2
YM4Pofþ, Laf2
YM14
Pof2, Laf2, rpoN
NMB107
Pof2, Laf2, fliG
NMB196
Pof2, Laf2, DfliF
NMB198Pof2, Laf2, DfliG

31,47
46
47
This work
This work
This work

B. E. coli strains
JM109
DFB225
DfliG

74
15

C. Plasmids
pSU41
pBAD33
pJY19
pEGFP
pSL27
pJN131
pAM101
pAM305
pTY101
pTY102
pTY200pTY201
pTY202
pTY301
pTY401
pTY402
pTY501

66
67
This work
Clontech
15
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work

pTY601
pTY602

Kmr, Plac lacZa
Cmr, PBAD
Apr, Ptac lacZa, lacI q
GFP expression vector
E. coli FliG in pAlter-1
PomA and PomB in pJY19
V. alginolyticus FliG in pCR2.1
V. alginolyticus FliG in pSU41
V. alginolyticus FliG in pBAD33
V. alginolyticus FliG in pBAD33
GFP in pBAD33
GFP–FliG in pBAD33
FliG–GFP in pBAD33
E. coli FliG in pBAD33
FliGVE chimera in pBAD33
FliGEV chimera in pBAD33
V. alginolyticus FliF in pGEM-T
Easy
V. alginolyticus FliF and FliG in
pBAD33V. alginolyticus FliF and FliGVEchimera in pBAD33

This work
This work

Kmr, kanamycin resistant; Cmr, chloramphenicol resistant;Apr, ampicillin resistant; Plac, lac promoter; Ptac, tac promoter;
PBAD, araBAD promoter.

580

pTTQ18. The pAlter1 (Promega)-based plasmid, pSL27,
carries the E. coli fliG gene, which was kindly provided
by D.F. Blair.68 Plasmid pEGFP encodes the enhanced
green fluorescent protein (EGFP) (Clontech). For the production of FliG (i.e. FliG, GFP –FliG and FliG – GFP proteins) and GFP proteins in this study, the coding regions
were placed downstream of the araBAD promoter on
the vector pBAD33. Plasmid pTY401 carrying the FliG
chimera was prepared by replacement of a SalI-HindIII
fragment of pTY102 containing the C-terminal region of
the V. alginolyticus fliG gene with a SalI-HindIII fragment
of pTY301 containing the C-terminal region of the E. coli
fliG gene. Plasmid pTY402 was prepared by replacementof a SalI-HindIII fragment of pTY301, the C-terminal
region of the E. coli fliG gene, with a SalI-HindIII
fragment of pTY102, the C-terminal region of theV. alginolyticus fliG gene.
Cloning fliG and fliF genes
The primers (50 -GCTCAAGTAGTGAAGAACTGGATGCA-30 and 50 -TCCCGCACCGTAATCCGGNAGNCCCCA-30 ) were synthesized from the genome sequenceof V. cholerae and the fliG gene and surrounding
sequences were amplified by PCR using V. alginolyticus
VIO5 chromosomal DNA as the template. The 1.2 kb
amplified fragment was cloned into pCR2.1 (Invitrogen),resulting pAM101, and the 1.2 kb SphI-BamHI fragment
was subcloned into pSU41, resulting in plasmid
pAM305.
For the fliF gene cloning, the primers (50 -GCCACTGTTCAAATCCGCAATAAGC-30 and 50 -GCTCAACTGGCATTCCCGCG-30 ) were synthesized based on sequencesfrom V. parahaemolyticus and V. alginolyticus, respectively,
and the fliF gene and surrounding sequences were
amplified by PCR using V. alginolyticus VIO5 chromosomal DNA as the template. The 1.9 kb amplified fragment
was cloned into pGEM-T Easy vector (Promega), resulting in pTY501. The cloned fliG and fliF genes were confirmed by DNA sequencing.

Charged Residues in the C Terminus of FliG

Detection of FliG proteins
For immunoblotting, anti-FliG antibody, called aFliG2,
was raised against a synthetic peptide (NH2-PVELDVSTITGEEKA-COOH) corresponding to a partial sequence(Pro16 to Ala30) of V. alginolyticus FliG and affinity-purified as described.71 Vibrio cells harboring a vector or plasmid encoding wild-type FliG or various FliG variants
were cultured in VPG medium in the presence orabsence of 1 mM L -arabinose and collected by centrifugation at mid-log phase. Collected cells were solubilizedwith SDS-sample buffer and subjected to SDS-12%
PAGE followed by immunoblotting with antibody
against GFP (Molecular Probe) or the FliG peptide.
Observation of the subcellular localization of
GFP –FliGNMB107 or YM14 cells carrying the pBAD33-based
plasmids encoding GFP (pTY200), GFP –FliG (pTY201)
or FliG– GFP (pTY202) were grown in VPG medium
with 1 mM arabinose at 30 8C. At late exponential
phase, a small aliquot of the cell suspension was spotted
onto glass slides coated with 0.5% agarose and observed
under a fluorescence microscope (Olympus, BX50), as
described.72 The images were recorded and processedby using a digital camera (Hamamatsu Photonics,
C4742-95) and imaging software (Scanalytics, IP Lab
ver. 3.2). For observation of GFP – FliG with the flagellum, NMB107 cells carrying plasmid encoding GFP –FliG (pTY201) were grown as described above. The cellsuspension on the glass slide covered by a cover-glasswith a spacer was washed twice with V-buffer (25 mM
Tris – HCl (pH7.5), 10 mM MgCl2, 300 mM NaCl) andreacted with V-buffer containing anti-polar flagellum(Pof) antibody. After washing with V-buffer, anti-rabbit
IgG conjugated with rhodamine was applied and thenwashed with V-buffer. Cells on the cover-glass wereobserved in the microscope as described above.
Measurement of swimming speed

Construction of fliF and fliG deletion mutants
The fliG deletion mutant (NMB198) and the fliF deletion mutant (NMB196) were generated by homologous
recombination using a suicide vector as described.69 An
in-frame deletion of fliG was constructed by using thethree internal AvaII sites, resulting in the deletion ofamino acid residues 55 – 175. An in-frame deletion of fliF
was constructed by using the internal HindIII (filled
in by T4 polymerase) and EcoRV sites, resulting in thedeletion of amino acid residues 106– 294. The deleted
alleles were introduced into VIO5. Motility-defective
mutants with resistance to chloramphenicol were selected first on semi-solid agar plates, and chloramphenicolsensitive mutants were identified.
Site-directed mutagenesis
To introduce amino acid substitutions or a Sal I-digestion site into fliG, we used a previously described PCR
method.70 We synthesized pairs of mutant primers homologous to either the sense or antisense strand of thefliG gene, with a 1 – 3 base mismatch at the mutationsite. We amplified the full plasmid sequence containing
the fliG gene. The presence of the fliG mutations was confirmed by DNA sequencing.

Cells were harvested at late logarithmic phase andsuspended in TMN buffer (50 mM Tris – HCl (pH 7.5),5 mM MgCl2, 50 mM NaCl, 250 mM KCl). The cell suspension was diluted 100-fold in TMN buffer, and cells
were observed at room temperature under a dark-fieldmicroscope and recorded on videotape. Their swimming
speed was determined as described.73
Membrane preparation and protease treatment
The procedure for membrane preparation was basically as described by Sato & Homma.34 JM109 cells harboring plasmids were grown in TB medium in thepresence of arabinose until late-log phase. If necessary,
IPTG was added to a final concentration of 1 mM. Harvested cells were suspended in lysis buffer (20 mM potassium phosphate (pH 8.0), 150 mM NaCl, 10 mM
MgSO4, 10% (w/v) glycerol) containing 30 mg/ml of
DNase I and the suspension was passed twice througha French press at 4000 lb/in.2 Unbroken cells were
removed by low-speed centrifugation, and the membrane pellet (P100) and the cytoplasm supernatant(S100) were recovered after centrifugation at 100,000g
for one hour. The P100 fraction (membrane) was resuspended in lysis buffer, and the membrane proteins(0.5 mg/ml) were digested with trypsin or proteinase K

Charged Residues in the C Terminus of FliG

at the indicated concentrations on ice for 30 minutes. The
samples were precipitated with trichloroacetic acid,washed with acetone, and then analyzed by SDS-PAGE,
followed by immunoblotting with anti-FliG antibody.

Acknowledgements
We thank Dr D. F. Blair for providing plasmids
and bacterial strains. This work was supported, inpart, by grants-in-aid for scientific research from
the Ministry of Education, Science and Culture ofJapan (to M.H. and to T.Y.) and from the Japan
Society for the Promotion of Science (to T.Y.).

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Edited by J. Karn
(Received 14 July 2003; received in revised form 23 September 2003; accepted 25 September 2003)