J. Mol. Biol. (2007) 367, 692–701

doi:10.1016/j.jmb.2007.01.015

Visualization of Functional Rotor Proteins of theBacterial Flagellar Motor in the Cell Membrane
Hajime Fukuoka 1 , Yoshiyuki Sowa 2 , Seiji Kojima 1 , Akihiko Ishijima 2
and Michio Homma 1 ⁎1Division of Biological Science,
Graduate School of Biological
Science, Nagoya University,
Chikusa-ku, Nagoya, 464-8602,
Japan
2

Department of Applied Physics,
Graduate School of Engineering,
Nagoya University,
Chikusa-Ku, Nagoya, 464-8603,
Japan

The bacterial flagellar motor is a rotary motor driven by the electrochemical
potentials of specific ions across the cell membrane. Direct interactions
between the rotor protein FliG and the stator protein MotA are thought togenerate the rotational torque. Here, we used total internal reflection
fluorescent microscopy to observe the localization of green fluorescentprotein (GFP)-fused FliG in Escherichia coli cells. We identified three types offluorescent punctate signals: immobile dots, mobile dots that exhibited
simple diffusion, and mobile dots that exhibited restricted diffusion. When
GFP–FliG was expressed in a ΔfliG background, most of the cells were notmobile. When the cells were tethered to a glass side, however, rotating cells
were commonly observed and a single fluorescent dot was always observedat the rotational center of the tethered cell. These fluorescent dots werelikely positions at which functional GFP–FliG had been incorporated into a
flagellar motor. Our results suggest that flagellar basal bodies diffuse in thecytoplasmic membrane until the axial structure and/or other structuresassemble.
© 2007 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: bacterial flagella; FliG; stator; GFP

Introduction
Many bacteria sense external signals from theenvironment and can swim toward favorable conditions by rotating locomotive organelles calledflagella. The bacterial flagellum consists of a helicalfilament that acts as a propeller, a basal body thatacts as a rotary motor embedded in the cytoplasmic
membrane, and a hook that acts as a universal joint
connecting the filament to the basal body.1 The
rotary motor of a bacterial flagellum is driven by theflux of specific ions across the cell membrane;
Escherichia coli and Salmonella enterica serovar Typhimurium use H+, whereas marine Vibrio species andPresent addresses: Y. Sowa, Clarendon Laboratory,
University of Oxford, Parks Road, Oxford OX1 3PU, UK;
A. Ishijima, Institute of Multidisciplinary Research for
Advanced Materials, Tohoku University, Aoba-ku,
Sendai, 980-8577, Japan.
Abbreviations used: GFP, green fluorescent protein;
TIR, total internal reflection; CW, clockwise; CCW,
counterclockwise.
E-mail address of the corresponding author:
g44416a@cc.nagoya-u.ac.jp

alkalophilic Bacillus species use Na+.2–4 In Bacillus
subtilis, distinct stators for coupling either Na+ or H+
flux to force generation are thought to function in
the same bacterial cell.5
The rotor in the flagellar basal body consists of anaxial rod and two rings: the MS ring and the C ring.
The MS ring is embedded in the cytoplasmic
membrane, whereas the C ring is attached to thecytoplasmic surface of the MS ring. In Gramnegative bacteria, two additional rings, the L andP rings, surround the axial rod and are attached tothe outer membrane and the peptidoglycan layer,
respectively.6 These rings are thought to act as
bearings for the axial rod. During flagellar assembly,
FliF first multimerizes to form the MS ring in thecytoplasmic membrane.7 Next, FliG, FliM, and FliN
assemble to form the C ring on the cytoplasmic
surface of the MS ring.6 The axial proteins are thenexported to the outside of the cytoplasmic membrane by the export apparatus attached to the insideof the C ring to form the rod, hook, and flagellar
filament.8
The stator of the flagellar motor consists of PomA
and PomB in the Na+ -driven motors of Vibrio
alginolyticus, or MotA and MotB in the H+-driven
motors of E. coli.9–11 These stator complexes are

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

Visualization of Functional Rotor Proteinsthought to function as specific ion channels.12,13 The
A and B stator subunits are incorporated into themotor as a stator complex after completion of thehook-basal body; deletions in the C-terminal
domain of the B subunit have been shown to block
this incorporation process.14,15 FliG, a cytoplasmic
protein that is thought to be directly involved inforce generation by the flagellar motor, forms theswitch complex with FliM and FliN that changes the
direction of motor rotation between clockwise (CW)
and counterclockwise (CCW) in response to chemotactic signals.16–19 Mutational analyses of the H+driven flagellar motor have identified charged
residues in the C-terminal region of FliG and thecytoplasmic domain of MotA that are important forforce generation.20,21 In the crystal structure of the
middle and C-terminal domains of Thermotoga
maritima FliG, the corresponding charged residues
are aligned in a C-terminal ridge that is exposed on
the surface of the FliG molecule.22 It has been
predicted that the electrostatic interactions between
the charged residues of FliG and the cytoplasmic
regions of MotA cause motor rotation.23 The precise
mechanism underlying motor rotation, however, is
not fully understood.
FliG molecules of the flagellar rotor have beenvisualized at the base of the polar flagellum in V.
alginolyticus using fusion proteins carrying green
fluorescent protein (GFP).24 Moreover, FliG molecules tagged with yellow fluorescent protein weredetected as several fluorescent foci on the surface ofthe cell body of S. typhimurium, a bacterium that hasperitrichous flagella.25 In the present work, weconstructed functional fusion proteins of GFP and
the E. coli rotor protein FliG and observed therotating motor using total internal reflection (TIR)
fluorescent microscopy.

Results
Motility of cells producing GFP–FliG
The gfp gene was genetically fused to the 5′ end of
the fliG gene to form the gfp-fliG construct, theproduct of which was GFP–FliG. Wild-type fliG and
gfp-fliG were cloned and placed under the control ofarabinose-inducible promoters in the pYF2 andpYF1 plasmids, respectively. We investigated the
motility of the cells producing GFP–FliG or wildtype FliG as a rotor component in semi-solid agar
containing arabinose. In a medium containing
0.0005% (w/v) arabinose, ΔfliG cells (DFB225)
producing wild-type FliG from pYF2 showed thesame degree of swarming as RP437 cells, a strain
with wild-type motility and chemotaxis (Figure1(a)). ΔfliG cells producing wild-type FliG needed
at least 0.0002% arabinose in the medium to achieve
the same degree of motility as RP437 cells (data notshown), and the cells overproduced the FliG protein
when they were placed in the medium containing
0.0002% arabinose (Figure 1(a)). On the other hand,

693

Figure 1. (a) Swarming behavior of cells producing
GFP–FliG. pYF1 (encoding gfp-fliG)-carrying DFB225 cells,
pYF2 (encoding wild-type fliG)-carrying DFB225, pTY200(encoding gfp)-carrying DFB225 cells, and pBAD33 (vector)-carrying RP437 cells were grown in TG-0.27% (w/v)
soft agar medium containing 0.0005% (w/v) arabinose at
30 °C for 9 h. RP437, wild-type for chemotaxis; DFB225,
ΔfliG strain. (b) Immunoblots for GFP–FliG or wild-type
FliG in whole-cell extracts. Proteins were detected with
anti-FliG antibodies or anti-GFP antibodies. Molecularmass values (kDa) are shown on the left side of the panels.
The band between 47.5 kDa and 62 kDa is probably a nonspecific band detected by the anti-GFP antibodies because
the band was detected in all of the lanes. Lane 1, pBAD33carrying RP437 cells grown in TG medium containing noarabinose; lane 2, pYF2-carrying DFB225 cells grown in
TG-0.0002% arabinose medium; lanes 3 and 4, pTY200carrying and pYF1-carrying DFB225 cells grown in TG0.0005% arabinose medium, respectively; lane 5, pYF1carrying RP3098 cells (ΔflhDC) grown in TG-0.002%
arabinose medium. Cells were grown at 30 °C for 4 h.

the ΔfliG cells producing GFP–FliG from pYF1 did
not swarm in the semi-solid agar containing
0.0005% arabinose (Figure 1(a)). Under a light microscope, however, ΔfliG cells producing GFP–FliG
were observed to swim, although their swimming
ability was much lower than that of wild-type cells(Table 1). Similar amounts of plasmid-derived wildtype FliG and GFP–FliG proteins were detected by

Visualization of Functional Rotor Proteins

694
Table 1. Motility parameters of the cells
Strains

FliG variants

Swimming
fraction (%)aSwimming
speed (μm/s)b

DFB225DFB225
RP437

GFP–FliG
Wild-typeWild-typec

3±3
85 ± 696 ± 312 ± 326 ± 428 ± 6The cells were grown in media containing no arabinose (RP437),
0.0002% arabinose (DFB225 cells harboring the plasmid encodingfliG), or 0.0005% arabinose (DFB225 cells harboring the plasmid
encoding gfp-fliG).
a
The average value calculated from more than eight visual
fields.
b
The average value calculated from more than 40 cells.
c
Wild-type FliG encoded on the chromosome.

immunoblot analysis of whole-cell extracts fromthe ΔfliG cells (Figure 1(b)). On the other hand,the cells producing GFP–FliG showed a reduced
level of flagellin expression (Figure 2); the intensity of the band of flagellin derived from thesecells was approximately ten times less than theintensity of the band derived from the cells producing wild-type FliG. In addition, the number
and the length of the flagella observed with electron microscopy were greatly reduced in the cells
producing GFP–FliG (data not shown). Thus, thefusion of the GFP molecule to FliG probably
affected flagellar assembly.
Next, to investigate the function of individual
motors, the cells were tethered to the surface of acoverslip using the FliC sticky filament, which has
been shown to readily attach to glass surfaces.26,27
FliC sticky proteins were produced from a second
plasmid, pYS11. 27 The tethered cells robustly
rotated on the coverslip in spite of their weak
motility in liquid media. Immunoblot analysis ofwhole-cell extracts detected GFP–FliG at a position
corresponding the expected molecular mass of
64 kDa (Figure 1). Very weak bands were detectedjust under the GFP–FliG bands (Figure 1(b), open
arrowhead); these bands may have resulted from aslight degradation or mistranslation of GFP–FliG.
Degradation products with a molecular mass
corresponding to that of wild-type FliG, however,were not detected, indicating that GFP–FliG
was functional in individual flagellar motors. The

rotational velocities of the tethered cells producing
wild-type FliG and GFP–FliG were ca 6 and ca 3
revolutions/s, respectively (Table 2). For the wildtype FliG motor, the switching frequency and thepercentage of time spent rotating CCW were 0.9
reversal/s and 65%, respectively. On the other
hand, the switching frequency and the percentageof time the motor rotated CCW for the GFP–FliG
motor were 0.4 reversal/s and more than 92%,
respectively. Flagellar motors that incorporated
GFP–FliG preferentially rotated CCW. Although
the activity of the GFP–FliG motors was lower
than that of the wild-type motors, the GFP-FliG
motors were sufficiently functional for further
analysis.
Subcellular localization of GFP–FliG
We observed the localization of GFP–FliG in the
ΔfliG and ΔflhDC strains. Because the flhDC genes
are master genes that control the expression of allof the flagellar genes, the ΔflhDC strain produces
no flagellar components. Similar amounts of GFP–FliG protein were detected by immunoblot analysisof whole-cell extracts from the two cell lines(Figure 1(b), lanes 4 and 5). When cells at midlog phase were immobilized on the surface of apolylysine-coated coverslip and observed using
TIR fluorescent microscopy, we detected 5 ± 1(mean ± SD) fluorescent dots near the cell surface
in each of the ΔfliG cells producing GFP–FliG(Figure 3; ΔfliG, TIR). On the other hand, in the
ΔflhDC cells producing GFP–FliG, no fluorescentdots were detected and a diffuse fluorescent signal
was observed throughout the cell body (Figure 3;
ΔflhDC, TIR). Using axial (brightfield) epi-illumination (epi-illumination), GFP–FliG was also
detected throughout the cell bodies of the ΔflhDC
cells (Figure 3; ΔflhDC, Epi). Therefore, theclustering of the GFP–FliG molecules was dependent on flagellar structures or one or more of theflagellar proteins. Under epi-illumination, we
detected 11 ± 3 fluorescent dots in each ΔfliG cellthough the background fluorescent signal waslarger than that observed using TIR illumination(Figure 3; ΔfliG, Epi and TIR). The number offluorescent dots in each cell was more than the
Table 2. Rotational parameters of the cells

Figure 2. Detection of flagellin. Proteins were separated and immunoblotting was performed using antiflagellin antibodies. Lane 1, pBAD33-carrying RP437 cells
grown in TG medium containing no arabinose; lane 2,
pYF2-carrying DFB225 cells grown in TG-0.0002%
arabinose medium; lane 3, pYF1-carrying DFB225 cells
grown in TG-0.0005% arabinose medium; lanes 4 and 5,
pBAD33-carrying DFB225 cells and pTY200-carrying
RP3098 cells grown in TG medium containing no arabinose, respectively.

Strains

FliG variants

DFB225DFB225

GFP-FliG
Wild type

Rotational
Switchingvelocitya Rotational biasb frequencyc
3±2
6±2

92 ± 1565 ± 190.4 ± 0.4
0.9 ± 0.8

More than 40 tethered cells were investigated. Images were
captured at 60 frames/s and 600 frames (10 s) were analyzed. The
cells were grown in media containing 0.0002% arabinose (DFB225
cells harboring the plasmid encoding fliG), or 0.0005% arabinose(DFB225 cells harboring the plasmid encoding gfp-fliG).
a
Rotation velocity is shown in revolutions/s.
b
Rotational bias is shown as the percentage of time spentrotating CCW.
c
Switching frequency is shown as the number of reversals/s.

Visualization of Functional Rotor Proteins695
reported number of flagella for an E. coli cell.28,30
This was probably due to flagellar basal bodies
that lacked flagellar filaments.
Movement of GFP–FliG on the cytoplasmic
membrane

Figure 3. Sub-cellular localization of GFP–FliG. Cells
producing GFP–FliG were grown at 30 °C for 4 h inmedium containing 0.0005% arabinose (ΔfliG) or 0.002%
arabinose (ΔflhDC), and were observed with a fluorescence microscope. TIR indicates the images obtained
under TIR illumination, whereas Epi indicates images
obtained under axial (brightfield) epi-illumination. BF
indicates bright field images. The adjacent fluorescent andbright field images are images of the same cells. The bar
represents 1.6 μm.

In the ΔfliG cells producing GFP–FliG, we
tracked the movement of the fluorescent dots inthe cell membrane using TIR fluorescent microscopy. After the cells were immobilized on apolylysine-coated coverslip, we detected immobile
and mobile fluorescent dots on the cell membrane(Figure 4). In each cell, approximately two immobile and four mobile fluorescent dots weredetected. The positional distributions of the centersof the immobile dots on the cell membrane wererestricted to a very narrow area (Figure 4(d)). On
the other hand, as shown in Figure 4(a) and (b), one
fluorescent dot (dot 1) moved in the direction of thecell pole, whereas the movement of anotherfluorescent dot (dot 2) was restricted within alimited region for more than 12 s. We analyzed the
movement of these mobile dots by plotting themean squared displacement versus the time interval(MSD-Δt) and examining the relative deviation.
The MSD-Δt plot for dot 1 appeared to fit a linear
function, which is indicative particle undergoing
simple diffusion (Figure 4(c)). On the other hand,

Figure 4. Movement of thefluorescent dots. (a) A sequence ofimages (TIR illumination) of fluorescent dots formed by GFP–FliG.
The Figure shows a 2 s image
sequence, and every tenth frame isshown. Images were captured at a
normal video rate (30 frames/s).
The bar represents 1.6 μm. (b)Movement trajectories of fluorescent dot 1 and fluorescent dot 2(arrowheads 1 and 2 in (a), respectively). Trajectories of the centers of
the fluorescent dots over 12 s areshown. Each trajectory color represents the movement of the dot
during a period of 1 s, and the
movements of the dots in firstsecond through the sixth second
are denoted in pink, blue, green,orange, turquoise, and red, respectively; this order of the colors isthen repeated for the movements ofthe dots in the seventh second
through the 12th second. Start andfinish indicate the position of the
dot in the first video frame and thelast video frame, respectively. (c)MSD-Δt plots for the trajectories ofthe fluorescent dots. Blue and red
circles indicate the MSD-Δt plots
for dot 1 and dot 2, respectively. The blue continuous line and the red broken line are the linear-fitted lines at 2Δt, 3Δt, and
4Δt for the MSD values (Δt is the video frame time: 33 ms). RD (N, n) was calculated as described in Materials andMethods. (d) Trajectory of an immobile fluorescent dot in another cell.

696
the MSD-Δt plot of dot 2 is characteristic of aparticle undergoing restricted diffusion (Figure4(c)). The relative deviation, RD (300, 90), of dot 1
and dot 2 were calculated to be 1.1 and 0.2,
respectively. From a comparison with the theoretical RD (300, 90) reported by Kusumi et al.,29 thetrajectory of dot 1 was determined to be a result ofsimple diffusion, whereas the trajectory of dot 2
was determined to be a result of restricteddiffusion. We analyzed the trajectories of 15 otherfluorescent dots using RD (300, 90) or RD (200, 90).
Three of these trajectories were determined toresult from simple diffusion and the average RD
value for these dots was 0.9. Eight of the trajectories
were determined to result from restricted diffusion
and the average RD value for these dots was 0.2.
The diffusion modes of the four remaining dots
were not determined, because there were notenough data points for analysis. Estimating the
diffusion coefficients (D) of dot 1 and dot 2 using
the slope of each of the MSD-Δt plots revealed that

Visualization of Functional Rotor Proteins

the diffusion coefficients of dot 1 and dot 2 were
5.6 × 10− 11 cm2/s and 2.5 × 10− 11 cm2/s, respectively. The average diffusion coefficient for all 17 of
the mobile dots was 4.9 × 10− 11 (± 3.1 × 10− 11 )cm2/s.
Observation of GFP–FliG in tethered cells
If the GFP–FliG protein retains its function andattaches to basal bodies as a component of the rotor,
the rotational center of each tethered cell should be
fluorescently labeled (Figure 5(a), central dot). To
determine the position of the rotational center, wechose slowly rotating cells (approximately 1 revolution/s). For each of the GFP–FliG-producing cells, a
single immobile fluorescent dot was observed at the
midpoint of the cell body, which was the rotational
center of the tethered cell (Figure 5(b), arrowhead
C). In order to precisely determine the position of
the central dot and the rotational center of thetethered cell, we determined the centers of thefluorescent signals from a central dot (arrowhead C)

Figure 5. Sub-cellular localization of GFP–FliG in a tethered celland determination of the rotational
center. (a) Schematic diagrams toillustrate the concept. A flagellar
filament (sticky filament) extending
from the cell body is immobilizedon the coverslip and the cell bodyrotates around this point. Central
and satellite fluorescent dots are
localized at the rotational centerand the cell pole, respectively.
These two dots were used to define
the rotational center of the tetheredcell. (b) A sequence of fluorescentimages of a tethered cell. Images
were captured at the normal video
rate (30 frames/s) and every framein the sequence is shown. C denotes
the central dot and S marks thesatellite dot. The bar represents
1.6 μm. (c) The distributions of thepositions of a satellite dot and acentral dot in a tethered cell.
Shaded squares indicate the distribution of the positions of the central
dot (150 points). The small open
squares indicate the centers of theellipses fitted from each rotation of
the satellite dot (we analyzed fiverotations). The positions of thecenters of the fitted ellipses were
calculated using the least-squares
method from the center of thesatellite dot. In this Figure, theposition of the rotational center(large open square) was defined as
the average position of the five small open squares. The origin of this graph represents the center of the ellipse fitted basedon the rotation of the satellite dot. Histograms indicate the distributions of the central dot along the X axis and Y axis. (d)Histograms of positional data obtained for central dots from 11 tethered cells. For each tethered cell, the positions of thecentral dot were obtained from 100 frames, and the range of the distribution of the central dots was normalized to thecenter of the fitted ellipse.

Visualization of Functional Rotor Proteins

and a satellite dot (arrowhead S) (Figure 5(a) and(b)) from more than 100 frames (images were
captured at 30 frames/s). As shown in the inset in
Figure 5(c), the center of the satellite dot followed acircular path with a radius of approximately 1.2 μm(shaded squares). The rotational center of thetethered cell was defined as the center of the ellipse
calculated by least-squares fitting of the trajectory of
the center of the satellite dot (Figure 5(c), large open
square). In this cell, the standard deviations for thecenter of the fitted ellipse were ± 12.8 nm along the X
axis and ± 19.2 nm along the Y axis. The positions of
the center of the central dot were distributed withina range of approximately 80 nm along both the X
and Y axes (Figure 5(c), shaded squares). The
rotational center calculated from the path of thesatellite dot was located within the area defined by
the distribution of the positions of the central dot(Figure 5(c)). Figure 5(d) shows the distributions of
the positions of the central dots from 11 tetheredcells. For each of these cells, the rotational center
calculated from the movement of a satellite dot was
located within the range defined by the distributionof the positions of the central dot. These results
indicate that the GFP–FliG molecules localized at therotational centers of the tethered cells.

Discussion
The bacterial flagellar motor is a rotary motor
driven by the electrochemical potentials of ions. Theinteraction between the rotor and the stators isthought to generate torque, thereby converting theenergy of ion flux into mechanical power. In theflagellar motor of E. coli, FliG is a rotor component
that is thought to interact directly with the stator
complex formed by MotA and MotB.23 Here, weused the functional fusion protein GFP–FliG and
TIR fluorescent microscopy to observe rotating
motors and to characterize the motion of basal
bodies in the cell membrane of E. coli.
To construct a flagellum, FliF first assembles toform the MS ring in the cytoplasmic membrane. FliG
then attaches to the cytoplasmic surface of the MS
ring to form the C ring with FliM and FliN.6,7 In the
ΔflhDC deletion strain, the MS ring is not formed
because none of the flagellar proteins, including FliF,
are produced.1 Accordingly, no fluorescent dots
were observed in GFP–FliG-producing ΔflhDC cells;
a diffuse fluorescent signal was instead detected
throughout the ΔflhDC cells (Figure 3). In contrast,
fluorescent dots were detected in GFP–FliG-producing ΔfliG cells, indicating that the formation of thefluorescent dots was dependent on the flagellar
proteins. It can be concluded that the fluorescentdots corresponded to locations at which GFP–FliG
bound to MS rings, and in some cases, assembled toform the rotor in the basal body of a flagellar motor.
Although we may not have detected all of thefluorescent dots due to the high background
fluorescence, at least 11 fluorescent dots wereobserved in each cell. Interestingly, this number of697
dots is more than the reported number of flagella for
an individual cell.28,30 The larger than expected
number of fluorescent puncta was probably not due
to the overproduction of GFP–FliG. In ΔfliG cells,
the number of MS rings is probably the same as in
RP437 cells because FliF is produced from thechromosome. This means that the number offluorescent dots was limited by the number of MS
rings. Thus, the maximum number of fluorescentdots would not exceed the number of MS rings even
if GFP–FliG was overproduced. Therefore, E. coli
cells must have complete flagellar structures andpartial flagella that lack filaments, such as basal
bodies that are not completely assembled and/or
basal bodies that have lost the flagellar filaments. It
has been reported that there are an average of 3.4
flagella in each E. coli cell.30 This suggests that there
are twice as many unfinished basal body structures
as there are fully assembled basal bodies with intact
flagella.
We observed mobile and immobile fluorescentdots in cells producing GFP–FliG. After the construction of the MS ring and the C ring, the export
apparatus attached to the inside of the C ringtransports the axial proteins outside of the cytoplasmic membrane, where they assemble to form therod, hook, and filament.8 Structures consisting ofan MS ring and a C ring without the axial components can probably diffuse in the cell membrane,
whereas basal bodies with axial structures thatpenetrate the peptidoglycan layer and/or the outermembrane are likely fixed in the cell membrane. The
diffusion coefficient of the mobile fluorescent dotsroughly estimated from the MSD-Δt plots was
4.9 × 10− 11(± 3.1 × 10− 11) cm2/s. If each mobile fluorescent dot corresponded to a single flagellar basal
body, the molecular size of the fluorescent dots wasat least 3200 kDa (26 molecules each of FliF, FliG,
and GFP). In the cell membrane of E. coli, thediffusion coefficient (D) of a TatA–GFP fusion
protein (9.6 kDa) was estimated to be 1.3 × 10− 9
cm2 /s using fluorescence recovery after photobleaching.31 On the other hand, the D value of the
MotA4–MotB2 complex, which is the stator of thebacterial flagellar motor, was estimated to be
8.8 × 10− 11 cm2/s using single particle tracking.32
Because the D value is dependent on the Stokes
radius (r) of the transmembrane domain in the lipid
bilayer, it is not surprising that the D values of thecomplexes composed of GFP–FliG and MotA4–
MotB2 are of the same order of magnitude (× 10− 11
cm2/s). On the other hand, the observation that the
D value of the GFP–FliG complex is smaller thanthat reported for the TatA fusion protein is reasonable, because TatA was predicted to have one
transmembrane segment by the SOSUI program.
Some of the mobile fluorescent dots diffused
within a restricted area (Figure 4(b) and (c), dot 2).
Structures in the peptidoglycan layer and/or
the outer membrane, such as L rings and P rings,
might capture flagellar basal bodies that are diffusing in the cytoplasmic membrane. On the other
hand, in eukaryotic cells, several elements have beenVisualization of Functional Rotor Proteins698
proposed to restrict the movement of membraneproteins, including a membrane skeleton/cytoskeleton fence or transient confinement zones(TCZs). 33–35 In bacteria, MreB forms actin-likefilaments that run along the inner surface of thecytoplasmic membrane,36 whereas FtsZ, a tubulin
homolog that is required for cell division, forms aring structure at the site of division.37 Membrane
domains enclosed by cytoskeletal-like structures or
TCZ-like membrane domains might restrict the
diffusion of flagellar basal bodies. This restriction
might be involved in determining positions at which
flagella form.
Only 3% of cells producing GFP–FliG wereobserved to swim, and the swimming speed ofthese cells was half of the speed observed for cells
producing wild-type FliG from a plasmid. Thisphenotype may be attributable to a reduction in thenumber of flagella. The percentage of GFP–FliGproducing cells with flagella was low and theamount of flagellin in these cells was approximately
ten times less than the amount of flagellin in the cells
producing wild-type FliG. The fliC gene, which
encodes flagellin, is a class III gene in the expression
hierarchy of the flagellar genes. The expression ofclass III genes is repressed by the anti-sigma factor
FlgM, and this repression is removed following theexcretion of FlgM through the flagellar export
apparatus.1 The low expression level of flagellinsuggests that anti-sigma factor FlgM was notefficiently pumped out of the GFP–FliG-producing
cells; this would prevent the cells from downregulating the expression of the class II flagellar
genes and increasing the expression of the class III
genes. Our observations suggest that the activity ofthe export apparatus was reduced or the assemblyof the export apparatus was inhibited by the GFP
molecule fused to FliG.
When the GFP–FliG-producing cells were tethered
to glass coverslips, they were able to rotate although
the rotation speed was about half of that observed
for cells producing wild-type FliG. A puncta of
GFP–FliG was localized at the rotational center of
each tethered cell (Figure 5). This indicates that (i)GFP–FliG was located at the site of the motor, (ii)
GFP–FliG assembled on an MS ring or in a flagellar
basal body, and (iii) the fusion protein functioned as
a component of the rotor in the flagellar motor.
Compared to cells with wild-type motors, therotation of the motors was biased to the CCW
direction and the switching frequency was low in
the GFP–FliG-expressing cells. GFP is a large protein(27 kDa) with a β-barrel structure.38 Fusion of thisprotein to the N terminus of FliG may have affected
the structure of FliG, the interaction between FliG
and MotA, and/or the interaction between FliM andphosphorylated CheY, which switches the rotationof the motor from CCW to CW. Alternatively, thelow level of expression of the class III genes or thelate genes (the che and mot genes) might haveaffected the rotation of the motor.1
Here, we have successfully observed fluorescently
labeled, rotating flagellar motors and the diffusionof flagellar basal bodies in the cytoplasmic membrane. We do not know the precise number of FliG
molecules that are in an individual rotating motor.
According to a rough estimate, the fluorescentintensity of one of the fluorescent dots appeared to
be about ten times stronger than that of a single
molecule of GFP (data not shown), suggesting thatat least ten molecules of GFP–FliG were present ineach fluorescent dot. As shown in a recent report,32
we may be able to determine the precise number of
FliG molecules that are incorporated into a functional motor by measuring single-step photobleaching of GFP–FliG in a rotating cell. Additionally,
development of this technique may allow us to
examine the interaction between the flagellar motor
components, such as the rotor and the stator, using
fluorescence resonance energy transfer.

Materials and Methods
Bacterial strains, growth conditions, and media
The bacterial strains used in this study are listed inTable 3. E. coli cells were cultured in LB broth (1% (w/v)
tryptone peptone, 0.5% (w/v) yeast extract, and 0.5%(w/v) NaCl) or TG broth (1% (w/v) tryptone peptone,
0.5% (w/v) NaCl, and 0.5% (w/v) glycerol) at 30 °C.
Chloramphenicol was added to a final concentration of
25 μg/ml and ampicillin was added to a final concentration of 50 μg/ml.
Plasmids
The plasmids used in this study are listed in Table 3. The
plasmids carrying the fliG gene under the control of thearaBAD promoter were pBAD33-based plasmids. The
SacI/HindIII fragment from pSL27 was inserted into the
SacI/HindIII sites of pTY102, resulting in the pYF2
plasmid. A fragment encoding the fliG gene, which was
amplified by PCR using pYF2 as a template, the sense
primer BsrGI–fliG, and an antisense primer that annealed
to a region downstream of the multiple cloning site wasTable 3. Bacterial strains and plasmids
Description
A. Strains
RP437

Source orreference

DFB225
RP3098F−thi thr leu his met eda rpsL
(wild-type for motility)
RP437 ΔfliG
Δ(flhD–flhA)442
43

B. Plasmids
pBAD33
pBR322
pTY200pTY102
pSL27
pYF2
pYF1
pYS11

Cmr, PBAD
Apr, Tcr
his6-gfp in pBAD33
fliG of V. alginolyticus in pBAD33
fliG of E. coli in pAlter-1
fliG of E. coli in pBAD33
his6-gfp-fliG of E. coli in pBAD33
fliC sticky in pBR322

44
45
24
24
41
This work
This work
27

41

Cmr, chloramphenicol resistant; Apr, ampicillin resistant; Tcr,
tetracycline resistant; PBAD, araBAD promoter; his6, hexahistidine tag.

Visualization of Functional Rotor Proteinsinserted into the BsrGI/HindIII sites of pTY200; thisplasmid was named pYF1.
Preparation of cells for observation with fluorescencemicroscopy
Overnight cultures of cells harboring the plasmids were
inoculated into TG medium (1/100) in the presence ofarabinose and cultured at 30 °C for 4 h. The cells were
harvested by centrifugation and suspended in motility
medium: 10 mM potassium phosphate (pH 7.0), 0.1 mM
EDTA, and 85 mM NaCl. Sixty μl of the cell suspension
was loaded into the space between a slide glass and acoverslip with a spacer. The slide was then inverted andincubated for 2 min. Another 60 μl of motility medium
was loaded into the space between the slide and thecoverslip to remove the remaining unattached cells.
Microscopic system
We used an Olympus IX-71 microscope with a modified
mirror turret to allow side entry of the excitation light into
the microscope. A laser beam (λ = 488 nm) for excitation
was focused at the back focal plane of the objective lens(Olympus PLAPON 60XOTIRFM, NA = 1.45). For objective TIR illumination39 the angle of the incident laser beam
was adjusted with a mirror set in front of the focusing lens
( f = 180) so that the laser beam would pass near the edge ofthe objective lens. In our experiments, the incidence angle
was 71.4°, and thus the evanescent light reached a depth ofapproximately 70 nm (the value when the sample on theglass surface was water). The image was projected to an
EM-CCD camera (DV860-BV, Andor Technology, South
Windsor, CT) through a projection lens (5×); as a result, 1
pixel corresponded to 80 nm in the specimen plane. All
images were captured at the normal video rate (30frames/s). We confirmed the positional resolution of thismicroscope system was 5 nm in an experiment using a
100 nm fluorescent bead.
Determination of the rotational center of a tetheredcell
We used the free ImageJ software† for analysis of all of theimages. The position of the rotational center was defined as
the center of a fitted ellipse calculated by the least-squares
method from the center of a fluorescent satellite dot. For thedefinition of the rotational center of a tethered cell, the positions of the center of the fluorescent satellite dot obtained
from more than 100 video frames were used. The distribution of positions of the central dots was calculated from thecenters of their fluorescent signals. To calculate the center of
each dot, we analyzed video frames that contained morethan 16 pixels that were brighter than the threshold intensity. We selected tethered cells rotating CCW for analysis.

699
video frame. For the trajectory of each fluorescent dot, themean square displacement (MSD) was calculated according to the following formula:29MSDðnytÞ ¼

N1n
X nh
1
xð jyt þ nytÞ  xð jytÞ2
N  1  n j¼1
hi2
þ yð jyt þ nytÞ  yð jytÞ ;g

where δt is the video frame time (33 ms) and (x( jδt +nδt) − x( jδt), y( jδt + nδt) − y( jδt)) describes the positionof the dot following a time interval Δtn = nδt after
starting at position (x( jδt), y( jδt)); N is the total numberof frames in the recording sequence, n and j are positive
integers, and n determines the time increment. The
diffusion coefficients (D2–4) of the mobile dots were
estimated by fitting the MSD values at 2Δt, 3Δt, and
4Δt using a straight line.29 Mobile dots that weretracked for more than 200 frames were analyzed for the
calculation of the diffusion coefficient. The relative
deviation RD (N, n) was used for the determination of
the diffusion mode of a mobile fluorescent dot.29 The
relative deviation is defined as
RDðN; nÞ ¼

MSDðN; nÞ
4D24nytwhere MSD(N, n) represents the MSD at the timeinterval nδt from a sequence of N video frames. 4D2–4 isthe expected average MSD value for fluorescent dots
undergoing simple diffusion with a diffusion coefficientof D2–4. The value of RD (300, 90) or RD (200, 90) wasused for the determination of the diffusion mode ofeach mobile fluorescent dot.29
Detection of GFP–FliG and flagellin in E. coli cells
E. coli cells harboring a plasmid encoding wild-type
FliG or GFP–FliG were incubated at 30 °C for 4 h in TG
medium containing arabinose. The cells were harvested
by centrifugation and suspended in motility medium to an
A660 of 10. The same volume of SDS loading buffer (033M
Tris-HCl, pH 6.8, 42% glycerol, 5% SDS, 0.017% bromophenol blue, 17% 2-mercaptoethanol) was added to thecell suspensions and the samples were boiled at 100 °C for
5 min. The proteins were separated by SDS–PAGE andimmunoblotting was performed using anti-FliG (a giftfrom D. Blair) and anti-GFP antibodies (GFP monoclonal
antibodies, Clontech).
To detect flagellin, a 1/10 volume of trichloroacetic acidwas added to the cell culture (A660 = 1) and the cells were
harvested by centrifugation. The pellet was washed withacetone and dried. The pellet was suspended in a 1/10
volume of the twofold diluted SDS loading buffer to theoriginal culture volume. The proteins were separated by
SDS–PAGE and immunoblotting was performed using
anti-flagellin antibodies (laboratory stock).

Tracking the movement of fluorescent dots
To track the movement of the mobile fluorescent dots,
ΔfliG cells producing GFP–FliG were attached to polylysine-coatedcoverslips and observed with TIR fluorescent
microscopy. Images were captured at 30 frames/s and thecenter of the fluorescent mobile dot was calculated in each
† http://rsb.info.nih.gov/ij/

Measurement of the swimming fraction and swimming
speed
Cells were grown at 30 °C for 4 h in TG medium
containing arabinose. The cell suspensions were diluted
50-fold in fresh TG medium containing 20 mM serine,
which was used to suppress CW rotation. Cell motility
was observed under a dark-field microscope and recorded
on videotape. Swimming speed was determined as

700
described.40 The average swimming speed was obtainedby measuring at least 40 swimming tracks. The swimming
fraction was measured from the same videotape.

Acknowledgements
We are grateful to Dr Richard Berry for criticallyreading the manuscript and thank Dr Toshiharu
Yakushi for helpful discussions. This work was
supported in part by grants-in-aid for scientificresearch from the Japan Society for the Promotion of
Science (to M. H. and S. K.), from the Ministry of
Education, Culture, Sports, Science and Technologyof Japan (to A. I.), from JSPS Research Fellowships
for Young Scientists (to Y. S.), and from the Japan
Science and Technology Agency (to M. H., S. K. and
A. I.).

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Edited by J. Karn
(Received 24 August 2006; received in revised form 29 December 2006; accepted 4 January 2007)
Available online 12 January 2007