C H A P T E R

T W O

Flagellar Motility in Bacteria:
Structure and Function ofFlagellar Motor
Hiroyuki Terashima,* Seiji Kojima,* and Michio Homma*Contents
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1. Introduction
1.1. Molecular architecture of flagella1.2. Gene regulation
1.3. Flagellar assembly
1.4. Regulation of rotation
2. Basal Structure of Flagella as Motor
2.1. Basal body
2.2. Export apparatus
2.3. Switch complex
2.4. Motor complex
3. Torque Generation
3.1. Interaction between stator and rotor
3.2. Ion-binding site
3.3. Ion specificity
3.4. Assembly of functional motor
4. Molecular Physiology of Motor
4.1. Torque–speed relationship4.2. Steps in rotation of motor
4.3. Fluorescent imaging of motor
5. Conclusion
References

Abstract
Bacterial flagella are filamentous organelles that drive cell locomotion. They
thrust cells in liquids (swimming) or on surfaces (swarming) so that cells canmove toward favorable environments. At the base of each flagellum, a reversible rotary motor, which is powered by the proton- or the sodium-motive force,* Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan

International Review of Cell and Molecular Biology, Volume 270
ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01402-0# 2008 Elsevier Inc.

All rights reserved.

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Hiroyuki Terashima et al.

is embedded in the cell envelope. The motor consists of two parts: the rotating
part, or rotor, that is connected to the hook and the filament, and the nonrotating part, or stator, that conducts coupling ion and is responsible for energy
conversion. Intensive genetic and biochemical studies of the flagellum havebeen conducted in Salmonella typhimurium and Escherichia coli, and more than
50 gene products are known to be involved in flagellar assembly and function.
The energy-coupling mechanism, however, is still not known. In this chapter, wesurvey our current knowledge of the flagellar system, based mostly on studies
from Salmonella, E. coli, and marine species Vibrio alginolyticus, supplemented
with distinct aspects of other bacterial species revealed by recent studies.
Key Words: Proton motive force, Sodium motive force, Energy transduction,
Vibrio alginolyticus, Salmonella typhimurium, Escherichia coli. ß 2008 Elsevier Inc.

1. Introduction
1.1. Molecular architecture of flagella
The flagellum consists of three parts: the filament (helical propeller), thehook (universal joint), and the basal structure (rotary motor) (Fig. 2.1). The
largest part of the flagellum is the filament, a helical structure whose shape
can vary among different helical forms, a phenomenon termed polymorphism (Asakura, 1970). This polymorphic alteration of flagellar shape isassociated with phase variation (Iino, 1969). When the cell swims, theflagellar filament serves as a screw propeller to convert rotary motion of
the motor into thrust (Berg and Anderson, 1973). In Salmonella, it grows toa length of around 15 mm and is composed of as many as 30,000 copies of asingle protein named flagellin (Minamino and Namba, 2004). Some bacteria, for example Vibrio, have several closely related flagellins that form thefilament (McCarter, 2001). The flagellin subunits (FliC in Escherichia coli and
Salmonella) are self-assembled to form a hollow concentric double-tubular
structure (inner and outer tubes) consisting of 11 protofilaments, which arearranged approximately parallel to the filament axis (Mimori et al., 1995;
Morgan et al., 1995). Formation of a helical structure is achieved by a
mixture of the protofilaments of two distinct conformations, the R- andL-type, distinguished by their helical handedness right or left (Asakura,
1970; Calladine, 1978). Each protofilament switches between these two
conformations by responding to a variety of factors including pH, ionicstrength, mechanical stress, and mutations (Kamiya and Asakura, 1976;
Macnab and Ornston, 1977). Later, X-ray fiber diffraction studies revealed
slightly different subunit packing between the R- and L-type, whose repeat
distances are 51.9 and 52.7 Å, respectively (Yamashita et al., 1998). To

41

Structure and Function of Flagellar Motor

A

Smooth swimming

Tumbling

Smooth swimming
C

Cap

B
Filament

Joint
Hook

OM
PG
IM

Mot complex

L ringP ring
Basal
body
Rod
MS ring
Switch complex
ExportmachinaryFigure 2.1 (A) Behavior of bacterial cells. (B) Electron micrograph of flagella isolated
from Salmonella typhimurium. (C) Schematic diagram of flagellar structure of Gramnegative bacteria.

understand the polymorphic switching mechanism, the crystal structure offragment containing most of the flagellin protein (F41 fragment of FliC) was
solved at 2 Å (Samatey et al., 2001). In the crystal, F41 fragments of the
R-type conformation form protofilaments arranged in an antiparallel

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Hiroyuki Terashima et al.

manner, and simulation using atomic model revealed that a small, distinct
conformational changes in the b-hairpin in D1, the domain that contributes
to outer tube structure, is responsible for the conformational switching
between L- and R-type protofilament. The crystal structure of F41 fragment lacks the D0 domain that forms inner tube structure. Later, electron
cryomicroscopy and helical image analysis made it possible to build complete atomic model of the R-type filament structure including D0 domainof flagellin, revealing that intersubunit hydrophobic interactions in the
inner tube (domain D0) make the filament structure mechanically stable,
and the diameter of central channel is only 2 nm (Yonekura et al., 2003).
This central channel serves as a transport pathway of flagellins that willpolymerize at the tip of the growing filament (Fig. 2.2).
The base of the filament is connected to the short tubular structure called
the hook, which is thought to function as a universal joint to smoothlytransmit the torque produced by the motor to the filament. The hookstructure of Salmonella is composed of about 120 copies of a single protein
FlgE and its length is controlled at 55 (6) nm (Hirano et al., 1994). The
core structure of the hook protein was solved by X-ray crystallography, andits atomic model was docked onto the density map of the hook obtained byelectron cryomicroscopy (Samatey et al., 2004).
The junction between hook and filament consists of the two proteins,
FlgK (HAP1) and FlgL (HAP3) (Homma and Iino, 1985; Ikeda et al., 1985).
About 13 molecules of each protein are present in each flagellum (Ikeda
et al., 1987). Mutational studies suggested that the junction acts as a buffering structure connecting two filamentous structures (hook and filament)with distinct mechanical properties (Fahrner et al., 1994).
The proximal end of the hook is connected to the basal body structure,
consisting of the rod and three coaxially mounted rings, termed as MS, P,
and L ring. The MS ring is embedded in the cytoplasmic membrane andmade of a single protein FliF (Ueno et al., 1992), the P and L rings areassociated with the peptidoglycan layer and the outer membrane, respectively, and are composed of FlgI and FlgH (Homma et al., 1987; Schoenhals
and Macnab, 1996). The rod structure is composed of three proximal rod
proteins FlgB, FlgC, FlgF, and a distal rod protein FlgG, and fully traverses
the periplasmic space. The L and P rings together form a quite rigid
assembly resistant to stringent chemical treatments, and the LP-ring complex is believed to act as a molecular bushing for the flagellar axial structure(Akiba et al., 1991). The basal body of Gram-positive bacteria is composedof only the MS ring and rod, and the LP ring is not present (Kobayashi et al.,2003), probably because Gram-positive bacteria do not have the outermembrane but have a thick peptidoglycan layer. When the basal body isisolated with more gentle treatment, a drum-shaped structure, called C ring,
was found on the MS ring facing the cytoplasm (Francis et al., 1994). It is
composed mostly of FliM and FliN proteins (Thomas et al., 2006; Zhao

43

Structure and Function of Flagellar Motor

A

B

C

V283

N315
K177
N100
N406

A191
A298
T346
A392
D140
N132
E153

D3

K160

D1

D2

D2a
D2b

N56

R450

Figure 2.2 (A) Polymorphic structure of the flagellar filament. The filaments of
L-type straight, normal, curly, and R-type straight are shown by the left to right in thisorder. (B) The flagellar filament structure revealed by electron cryomicroscopy.
The end-on view of the cross section (left) and the side view of long segment (right).
(C) The backbone trace of the flagellin (FliC) molecule of Salmonella typhimurium.
The figures were kindly supplied by K. Namba.

et al., 1996a,b). These proteins, together with FliG which is located beneaththe MS ring, have been known to form a complex, referred to as the switch
complex. Mutations in each of these three proteins cause defects in switching the rotational direction of the motor (Yamaguchi et al., 1986a). They
are also important for rotation, and mutational studies revealed that FliG
most closely participates in torque generation (Lloyd et al., 1996). Careful
preparation of the basal structure further revealed a central protrusion
within the C ring, that is probably the export apparatus essential for
assembly of flagellum (Katayama et al., 1996).

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Hiroyuki Terashima et al.

1.2. Gene regulation
More than 50 genes are required for flagellar formation and function(Macnab, 2003). Because the flagellum is such a big organelle, a large
amount of energy is consumed during the assembly process. Bacteria deal
with this problem by developing highly organized and regulated systems forflagellar assembly. Its characteristic feature is that the flagellar gene regulation temporally and tightly couples to the assembly process. Here we survey
this regulatory mechanism in Salmonella, which has been most extensively
studied. For further details of flagellar gene regulations in Salmonella enterica
serovar typhimurium and E. coli as well as the other bacteria, such as Caulobacter cresentus and Vibrio spp., see the reviews cited in Chilcott and Hughes(2000), McCarter (2004), Wolfe and Visick (2008), and Wu and Newton(1997).
In Salmonella, the flagellar/motility/chemotaxis genes constitute a regulon, and they are arranged in hierarchical order into three classes, early,
middle, and late (Fig. 2.3) (Kutsukake et al., 1990). At the top of thehierarchy is a single operon (class 1 master operon) containing the flhDC
genes (Liu and Matsumura, 1994). The FlhC and FlhD proteins form aheterotetrametic complex FlhC2FlhD2 that direct s70-RNA polymerase
complex to activate transcription from class 2 promoters upstream of theClass1

Class2

Class33

Stimuli

σ70
1
flhDC

FliT

FlhDC
σ70
FliZ

2
flgBCDEFGHIJ flgKL
2
fliFGHIJK
2
fliLMNOPQR
2flhBAE
3
2
motAB cheAW
fliE
3
23
tar cheRBYZ
3fliDST
23
fliC
FliA(σ28)
3fliAZY
3fliB
2
3flgA flgMN
hin fljBA
FlgM(anti-σ28)

Figure 2.3 Regulation of transcription of the flagellar regulon.The flagellar operons
are indicated by arrows. The numbers on the arrows show the class of transcriptional
hierarchy in the flagellar regulon. Descriptions of the transcriptional regulation andthe function of the gene products are described in text.

Structure and Function of Flagellar Motor45

middle gene operons. The flhDC operon is tightly regulated under thecontrol of a number of global regulatory signals such as cAMP-CRP, heatshock, DNA supercoiling, growth phase, surface-liquid transition, ClpXP
protease, and c-di-GMP (reviewed in Chilcott and Hughes, 2000; Wolfeand Visick, 2008). Expression of flhDC is also linked to the cell cycle,
showing that flagellation and cell cycle are interdependent processes (Prussand Matsumura, 1996, 1997).
More than 30 middle gene products from class 2 operons are primarily
required for the structure and assembly of the hook and basal body, andinclude regulatory proteins that control transcription of the genes from class
3 operons. Class 3 operons encode for proteins required late in the assembly
process, including flagellin, hook-associated proteins (HAPs), stator components, and chemosensory systems. Expression of class 3 operons is positively controlled by FliA and negatively by FlgM (Gillen and Hughes, 1991;
Kutsukake and Iino, 1994). FliA is the flagellum-specific transcription factors28 (Ohnishi et al., 1990), and it leads s28-RNA polymerase complex totranscribe class 3 late gene operons, whereas FlgM is an anti-s28 factor
specific for FliA (Ohnishi et al., 1992). Since filament formation requires
large resources of the cells, the stage to initiate expression of class 3 operons
is a critical checkpoint for flagellar gene regulation. Actually, what happensin vivo is that the late genes are not transcribed until the assembly of hookbasal body structures has been completed. Mutations in any one of thehook-basal body genes prevent the transcription of class 3 operons to avoid
unnecessary late gene expression that would cause an energy drain in thecell. This remarkable coupling between structure and gene expression isachieved by the balance of FliA and FlgM. FlgM inhibits FliA for the
expression of class 3 operons until hook-basal body completion, at whichtime FlgM is secreted out from the cells through hook-basal body structure
and released FliA turns on transcription of class 3 operons to achieve
filament formation and motor assembly (Hughes et al., 1993; Kutsukake,1994). Such a controlled expression is coupled to the ordered secretion ofeach gene products by the flagellum-specific export apparatus, putatively
located inside of the MS ring, to complete the self-assembly of flagella.
As described above, sensing hook-basal body completion followed by
FlgM secretion is such a critical checkpoint; Salmonella utilizes anotherfactor involved in negative regulation in FlgM secretion. This gene, flk(Karlinsey et al., 1997), also called as rflH (Kutsukake, 1997), was identified
as the factor that allows expression of class 3 operons only when mutated instrains defective in LP ring assembly. Flk is a cytoplasmic-facing protein
anchored to the inner membrane by a single, C-terminal transmembrane
domain, and it was revealed that turnover of FlgM was increased in flk
background due to FlgM secretion into periplasm where it is degraded,suggesting that Flk prevents premature secretion of the FlgM into periplasm, thus acting as a braking systems for the flagellar export system

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Hiroyuki Terashima et al.

(Aldridge et al., 2006). Loss of only Flk does not show any phenotype
related to motility, and flk gene is not located in the flagellar regulon, so
the role of Flk in the wild-type cell is still unclear (Karlinsey et al., 1997;
Kutsukake, 1997).

1.3. Flagellar assembly
As described in Section 1.2, flagellar assembly is tightly coupled to the gene
expression, and the monitoring system at the stage of hook completion
allows cells to achieve efficient and economical filament formation. The
sequence of events in flagellar assembly has been extensively studied in
Salmonella and E. coli by studying partial structures of flagella from mutants
defective in a certain flagellar gene (Kubori et al., 1992; Suzuki and Komeda,
1981; Suzuki et al., 1978). In general, assembly starts at the inner structure of
the basal body then proceeds to the outer ones (Fig. 2.4). The first built
structure is the MS ring and proximal rod, which is formed by a single
protein FliF (Ueno et al., 1992). The MS ring is the core structure of therotor and is embedded in the cytoplasmic membrane. The C ring attaches on

Flagellin
FlgD

HAP2

HAP3
HAP1
FlgE

FlgD

FlgH

Flgl

HAP2(FliD)

Cap

Flagellin(FliC)

Filament

HAP3(FlgL)
HAP1(FlgK)

Joint

FlgE

Hook

L ring(FlgH)
P ring(Flgl)
Rod(FlgBCFG)
MS ring(FliF)
C ring(FliGMN)

Basal body

FlhAB,
FliOPQR

Exportmachinary

Switch complex

FlgBCFG
FliF

FliGMN
FlhAB,
FliOPQR

Figure 2.4 Morphological assembly of bacterial flagellum. The model of pathway iscreated based on the assumption that the structure is built from a simple to complicatedform. The flagellar morphogenesis is thought to begin with the formation of MS ring
structure followed by the assembly of proteins for axial structures, each of which issecreted by the flagellum-specific export apparatus (except FlgA, FlgI, and FlgH).

Structure and Function of Flagellar Motor47

the cytoplasmic face of MS ring (Francis et al., 1994). The C ring contains
mostly two switch proteins FliM and FliN (Zhao et al., 1996a,b), and isassociated with MS ring via another switch/motor protein FliG, which
probably contributes to a part of the face of MS ring (Thomas et al., 2006).
Assembly of these three proteins on the basal body requires the MS-ring
platform, and mutations give rise to the nonflagellate phenotype. Inside theMS ring, there is the flagellum-specific export apparatus, visualized in freezefracture images as a protrusion inside the C ring (Katayama et al., 1996).
When the export apparatus is established in the flagellar base is still unclear.
Details of the export apparatus are described in Section 2.2.
After the export apparatus is constructed, structural proteins for the basal
body, expressed from class 2 operons, are secreted through the export
apparatus in the order described below. First, the proximal rod, composedof FlgB, FlgC, and FlgF, is added on the MS ring, probably in this order(Homma et al., 1990). FliE is needed for this assembly, joining FliF, andproximal rod as an adaptor (Minamino et al., 2000b). Then the distal rod,made of FlgG, is assembled on the proximal rod. Formation of the rod
requires FlgJ, which is exported to the periplasmic space via export apparatus and acts as a cap on the growing rod to facilitate the polymerization at
the tip (Kubori et al., 1992). FlgJ also has a muramidase activity at its
C-terminal half, hydrolyzing the peptidoglycan adjacent to the MS ring toallow the rod to penetrate the peptidoglycan layer (Nambu et al., 1999).
Then the P ring (made of FlgI) is formed around the distal rod, followed by
L-ring (FlgH) formation (Chevance et al., 2007). FlgI and FlgH are notsecreted through the export apparatus, but through the Sec pathway using asignal sequence at their N-termini (Homma et al., 1987). P-ring formation
requires the Dsb system, which is involved in intramolecular disulfide bondformation in the periplasm (Dailey and Berg, 1993). FlgI protein contains
two cysteine residues important for protein stability (Hizukuri et al., 2006).
P-ring formation also requires the FlgA protein that acts as a periplasmic
chaperone, assisting a polymerization reaction of FlgI into the P ring
through FlgI–FlgI interaction (Nambu and Kutsukake, 2000). The hookassembles next, from about 120 copies of FlgE proteins at the distal end of
the growing hook, with the aid of hook-capping protein FlgD. Hookelongation proceeds to the well-controlled length of 55  6 nm by asophisticated export switching mechanism (Hirano et al., 1994). After
hook reached to the defined length, FlgD dissociates from the tip of thehook, then replaced by the three HAPs, FlgK, FlgL, and FliD in this order(Homma and Iino, 1985). Addition of FlgK and FlgL is facilitated by thechaperone FlgN (Fraser et al., 1999), whereas that of FliD is facilitated byanother chaperone FliT (Bennett et al., 2001). Finally, the FliC filament
subunits (flagellin) are inserted at the distal end (Iino, 1969). FliD acts as acap to facilitate the filament elongation by inserting each FliC subunit
between the FlgL and FliD zones, with rotary cap mechanism revealed by

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Hiroyuki Terashima et al.

electron cryomicroscopic observation (Yonekura et al., 2000). Mutants thatlack FliD cannot polymerize the filament and excrete flagellin monomers
into the culture (Homma et al., 1984). However, when purified FliD isadded to this mutant, flagellins stop leaking out and start polymerizing on
the hook (Homma et al., 1986). Using this system, growth rate of thefilament was observed and revealed that initial growth rate is about
30 nm/min, which corresponds to one flagellin incorporated per second,suggesting that to reach the 10 mm long of the filament of wild-type cells, ittakes several generations (Ikeda et al., 1993).
Vibrio alginolyticus has a single polar flagellum, so components requiredfor polar flagellum are localized to a single cell pole. The mechanism fordirecting the MS ring (or to initiate MS-ring assembly) at the pole hasremained unknown, but recent studies revealed that two proteins, FlhF and
FlhG, are somehow involved in the process (Kusumoto et al., 2006). Almost
all of the cells of an flhF null strain do not have a polar flagellum, whereas an
flhG strain has multiple flagella at a pole. Overproduction of FlhF in thewild-type strain increased the number of polar flagella, whereas excess FlhG
reduced it, indicating that these two proteins function in opposing ways.
Although the flhFG double null strain also showed almost no flagella, a verysmall but significant fraction of the cells possesses several flagella at the lateralposition (Kusumoto et al., 2008). These results suggest that FlhF functions inpolar location of the flagellum. This idea is supported by a study in Pseudomonas putida, possessing polar flagella, which showed that an flhF mutant
exhibits a peritrichously flagellated phenotype (Pandza et al., 2000).
FlhF and FlhG show similarity to FtsY and MinD, respectively. FtsY is acomponent of the prokaryotic SRP receptor (Luirink and Sinning, 2004)
and MinD is a cell division inhibitor (Shapiro et al., 2002). MinD shows astructural similarity to Ffh (Cordell and Lowe, 2001), which is a prokaryotic
SRP that forms a complex with FtsY to function in a signal recognition
targeting pathway for protein secretion at the membrane (Focia et al., 2004).
Therefore, FlhF may function together with FlhG in the same manner with
FtsY/Ffh system to locate flagellar components, possibly the MS-ringprotein FliF, to the cell pole. The direct interaction between FlhF and
FlhG has been suggested (Kusumoto et al., 2008). A recently reported crystal
structure of FlhF from Bacillus subtilis revealed a dimer formation of FlhF
and significant structural similarity of FlhF to FtsY/Ffh, supporting the idea
described above (Bange et al., 2007). However, many features of the polar
localization mechanism remain unknown.

1.4. Regulation of rotation
Most flagellar motors are reversible rotary machines, able to rotate bothclockwise (CW) and counterclockwise (CCW) (Silverman and Simon,
1974). Rotational switching completes very quickly, within only 1 ms

49

Structure and Function of Flagellar Motor(Kudo et al., 1990). Rotational direction is controlled by environmental
stimuli, such as pH, temperature, and chemicals like sugars and amino acids.
Methyl-accepting chemotaxis proteins (MCPs) sense these stimuli andtransmit signals to the motor through a two-component phosphorelay
signaling cascade (Fig. 2.5). When a repellent signal is sensed by the
MCP, autophosphorylation activity of the CheA protein, associated with
MCP on the cytoplasmic side, is activated and a histidine residue of CheA isphosphorylated. Then this phospho group is transferred to the Asp residueof the response regulator CheY. Phosphorylated CheY protein (CheY-P)
then associated with the motor to trigger CW rotation. On the other hand,when an attractant signal is sensed by the MCP, autophosphorylation
activity of CheA is repressed, so that the level of CheY-P decreases andthe motor rotates in its default direction, CCW. The molecular mechanismsof MCP function and two-component signaling are reviewed elsewhere(Armitage, 1999; Parkinson et al., 2005).
The target of CheY-P in the motor is the switch complex, composed of
FliG, FliM, and FliN. As described above, FliG/FliM/FliN complex is also
called ‘‘the switch complex’’ because mutations in these proteins cause
defects in switching the CCW/CW rotation in response to tactic stimuli.
FliM functions most directly in regulation of the switching frequency bybinding to CheY-P (Welch et al., 1993). This binding of CheY-P to FliM
probably changes the FliG–FliM interaction, and causes movement of the
C-terminal domain of FliG (FliGC) that interacts with the stator protein
MotA, thereby altering the rotor–stator interface to switch the direction ofrotary motion.

Receptor

ATP

CheA

CheY-ppp
CheA-p

CheY

FliG
FliM
FliN

CheW
Signal
CH3
ADP

CheZ
pi

Flagellar motor

Figure 2.5 Schematic diagram of signal transduction of Escherichia coli chemotaxis.
The chemoreceptors are embedded in the cytoplasmic membrane and localized at apole. Chemotaxis substances or ligands bind to the receptor and the signals are transmitted into a cell and are transferred through the two-component phosphorelay system via
the Che proteins. The phosphorylated CheYcan bind to FliM and the direction of the
motor rotation is changed.

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Hiroyuki Terashima et al.

Studies of intracellular level of CheY-P in a single cell that causes switching from CCW to CW revealed that switching is a highly cooperative event,showing a Hill coefficient of about 10, suggesting that chemotactic signal isamplified within the switch (Cluzel et al., 2000). Fluorescence resonanceenergy transfer-based observation of CheY interaction with FliM by using
CFP-FliM and CheY-YFP showed that binding of CheY-P to FliM is muchless cooperative than motor switching (Hill coefficient of 1.7  0.3) (Sourjik
and Berg, 2002). This result was further supported by in vitro biochemical
studies showing that CheY-P binding to the isolated intact switch complex
was not cooperative (Hill coefficient is around 1) (Sagi et al., 2003). These
results indicate that the chemotactic signal is amplified within the switch, but
subsequent to the CheY-P binding to FliM.
Some bacteria respond to tactic stimuli using modes, other than directional switching. Rhodobacter sphaeroides has a unidirectional flagellar motorthat alternates between CW rotation and brief stops, during which thebacterium is reoriented by Brownian motion and changes in flagellar filament morphology (Armitage and Macnab, 1987). In the case of Sinorhizobium meliloti, the motor also rotates unidirectionally in the CW direction andswimming cells respond to tactic stimuli by modulating the flagellar rotary
speed (Schmitt, 2002). The marine bacterium V. alginolyticus has dual flagellar systems, Naþ-driven polar flagellum (Pof ) and Hþ-driven lateral flagella(Laf ), and their switching modes are different: the Laf motor rotates unidirectionally in CCW and responds to tactic signals by slowing down, whereas
the Pof motor turns in both directions (Kojima et al., 2007). In each casedescribed, tactic signals are transmitted through the two-component signaling pathway, and CheY-P association to the motor modulates rotation.

2. Basal Structure of Flagella as Motor
2.1. Basal body
Structural features and the components of the basal body are described in
Section 1.1. The supramolecular complex of the basal body is constructedby 7 kinds of proteins and 130 molecules in total are assembled (Macnab,
2003). Aizawa et al. (1985) established a method to isolate the basal body of
Salmonella with high yield and purity. Using this method, but greatly modified
to allow C-ring isolation, the structure of the flagellar basal body and C ring ofSalmonella had been investigated by the single particle analysis using negatively
stained- or cryo-electron microscopy (Francis et al., 1994). A resolution was
obtained at 20 Å that is used for building the three-dimensional reconstitution

Structure and Function of Flagellar Motor51

of images (Suzuki et al., 2004; Thomas et al., 2001, 2006). The flagellar basal
body has a rotational symmetry with its axis in the center of the rod. The
center of the rod is a hollow tube and the flagellar components required foraxial structure are exported through this tube. The detailed single particle
analysis of the MS ring revealed an interface between the MS ring and rod(Suzuki et al., 2004). The MS ring looks like a cylinder mounted on two disks.
Suzuki et al. (2004) reported that the MS-ring structure can be divided into
five domains (C, P, S, M, and R), and discussed functions of each domain. The
near-axial C and P domains are involved in the protein export and thought tochange its conformation by the association with the export apparatus and open
a channel through the protein. The C domain associates with the mostproximal side of the rod and thereby the rod begins its assembly on the C
domain. Although the S and M domains form the two disks characteristic of
the MS ring, their functions are not clear. The R domain that forms cylinderlike part attaches to the outer face of the rod. It was speculated that theinteraction between the rod and R domain is important for the rod–MS
ring junction to release the twisting stress and symmetry mismatch. Thomas
et al. (2006) has greatly improved resolution of images, by classifying particles
according to size and applying the averaging procedures appropriate for eachsymmetry class. This improvement allows us to see for the first time thedetailed feature of the C ring, not just a dumbbell-like structure. Their
images revealed that the symmetry of individual M rings varies from 24-foldto 26-folds, whereas that of the C rings varies from 32-fold to 36-fold, with noapparent correlations between the symmetries of the two rings. The resolutionof these improved EM images is now good enough to allow the crystal
structures solved for the C-ring components to be docked into the map.
The LP ring is an extraordinarily rigid structure and can maintain the ringmorphology even under stringent condition such as 7.5 M urea (Akiba et al.,1991). Single particle analysis revealed that the P ring looks like a dumbbell andseems to contact with a part of the rod, whereas the L ring seems not to contact
the rod (Stallmeyer et al., 1989).
MotX and MotY, which are required for the rotation of the polar
flagellar motor of V. alginolyticus, are associated with the basal body andform an additional ring structure beneath the LP ring, termed T ring
(Terashima et al., 2006). Partial T-ring structures were observed in the
DmotX strain but not in the DmotY and DmotXDmotY strains, suggestingthat MotX associates with the basal body via MotY to form a complete
T-ring structure. Stoichiometry of the MotX and MotY proteins in the Tring has not been determined yet. Further detailed observations of the Tring to reconstruct the three-dimensional images will be informative tounderstand their function.

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Hiroyuki Terashima et al.

2.2. Export apparatus
Most of the flagellar substructures are constructed beyond the cytoplasmic
membrane. Therefore, protein components, synthesized in the cytoplasm,
must be exported across the inner and outer membranes to be assembled at
the appropriate final destinations. This is achieved by the flagellum-specificexport apparatus that resides inside the MS-ring structure (Macnab, 2004).
This system has a character in common with the needle complex that works
for the secretion of virulence factors by pathogenic bacteria (Cornelis,2006). Morphology of these two secretion machineries is quite similar toeach other, and they are now classified in the type III export pathway(Hueck, 1998). The flagellum-specific export apparatus exports protein
substrate in order without signal peptide cleavage. Most of the studies forthe export apparatus have been carried out in Salmonella, and here we
describe its general overview.
The core of the export apparatus is composed of six transmembraneproteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, that form an export
channel complex inside the MS-ring structure. Three cytosolic proteins
FliH, FliI, and FliJ are required for flagellum-specific export and interact
with the channel, thereby contributing a part of the export apparatus.
Recent study revealed that flagellar C-ring protein FliN is also involvedin the export, so it is a part of the apparatus (Brown et al., 2005; GonzalezPedrajo et al., 2006; McMurry et al., 2006; Paul et al., 2006). FlhA and FlhB
have a large cytoplasmic domain at their C-termini, where soluble components interact with. The export apparatus (or a part of it) has been visualized
as a protrusion inside the C ring, by the freeze-fracture image (Katayama
et al., 1996). However, only FliP and FliR have been detected in the basal
body preparations so far (Fan et al., 1997). FliI is an ATPase and its sequenceshows similarity to the b subunit of the FOF1-ATPase, a rotary motor thatdrives chemical reaction of ATP synthesis (Fan and Macnab, 1996).
Recently, crystal structure of FliI is solved at 2.4 Å, and revealed that its
similarity is not only in the sequence (29% identity) but also in the structurallevel (Imada et al., 2007). FliI is a member of the Walker-type ATPasefamily, and it is thought to form a ring-shaped hexamer for protein export(Claret et al., 2003; Minamino et al., 2006). These lines of evidence lead to
an attractive model that FliI hexamer functions as a motor, unfolding andthreading export substrates thought its central channel by cooperative
conformational changes of subunits, just as speculated for AAA ATPase
complexes. ATPase activity of FliI is negatively regulated by binding to
FliH, in a complex of FliH2FliI1 stoichiometry (Minamino and Macnab,
2000a). FliH also binds to the hydrophobic patch of FliN, and theirinteraction would mediate efficient localization of FliI near the C-ring
complex. FliJ is a general chaperone, preventing aggregation of export
substrates presumably by interacting with them (Fraser et al., 2003;

Structure and Function of Flagellar Motor

53

Minamino et al., 2000a). Interactions among export components have been
investigated by far-western method (affinity blotting), and it was found thatcytosolic components interact with each other and probably forming FliH/
FliI/FliJ complex in some point, and the cytoplasmic domain of membranecomponents FlhA and FlhB interacts with all soluble components(Minamino and Macnab, 2000b). Affinity blotting experiments also indicatethat cytoplasmic domain of FlhA and FlhB interact with substrates, suggesting that these two proteins are involved in protein translocation. From theseresults, the outline of export is emerging: protein substrate is captured by
FliH/FliI/FliJ complex without denaturation and transferred to the export
apparatus. In the same time, FliI docks to the cytoplasmic face of theapparatus, forming hexameric ring structure that has an enhanced ATPase
activity released from FliH inhibition. Then the substrates are exported
through a central channel of apparatus, probably formed mainly by transmembrane segments (TMs) of FlhA and FlhB. After crossing the inner
membrane, substrates are assembled at the appropriate position in orderedfashion, from the innermost structure to the external ones.
How is this ordered export achieved? As discussed in Section 1.2,
coupling of flagellar gene expression to the stages of flagellar assembly
makes it possible for the ordered export. But there is one moremechanism operating in the flagellum-specific export apparatus: thesubstrate-specificity switching (Kutsukake et al., 1994). The apparatus has
two export substrate-specificity states, the rod/hook type and the filament
type (Minamino and Macnab, 1999). Therefore, proteins forming the rod
and hook structures are exported prior to the proteins required for filament
formation. When the hook structure reaches a certain length (ca 55 nm),
FliK and FlhB sense this state, and the substrate specificity of the apparatus
switches from the rod/hook type to the filament type, causing the export ofa new substrate class (Ferris and Minamino, 2006; Moriya et al., 2006;
Shibata et al., 2007; Waters et al., 2007). In addition, this switching leads
to FlgM export, followed by expression of the class 3 operons (Hughes et al.,
1993; Kutsukake, 1994). It is reported that this switching is irreversible(Minamino et al., 1999).

2.3. Switch complex
The switch complex functions in the rotation/switching/assembly of theflagellum and is composed of the three kinds of proteins, FliG, FliM, andFliN (Fig. 2.6). Null mutants of each protein exhibit Fla phenotype (nonflagellate), and point mutations give rise to Fla, Che (defective in chemotaxis), and Mot (defective in motility) phenotypes (Yamaguchi et al., 1986a,b). Stoichiometry of the FliG, FliM, and FliN in the C ring have beenreported to be 26, 34, and more than 100 copies, respectively (Francis et al.,
1992; Suzuki et al., 2004; Thomas et al., 1999). Three-dimensional

54

Hiroyuki Terashima et al.

Vibrio cell

Salmonella cell

H+-type

Hook
Na+-type

L ring (FlgH)
T ring (MotX, Y)

P ring (Flgl)

Rod

MotB

PomB

MotA
FliG
C ringFliM, N

PomA
FliG
FliM, NMS-ring (FliF)

Export apparatus
ca. 50 nm

Figure 2.6 Cell body and flagellar basal structure of the proton- and sodium-driven
type. The components emphasized by darker drawing are essential components fortorque generation. The stator of flagellar motor consists of MotA and MotB of Hþdriven motor in Escherichia coli and of PomA and PomB of Naþ-driven motor in Vibrio
alginolyticus. For the Naþ-driven motor, additional components of MotX and MotY,
which form a T ring in the basal body, are also essential. Ion flow through MotAB or
PomAB complex is believed to be coupled with torque generation by the interaction
between the stator component of MotA or PomA and the rotor component of FliG.
Electron micrograph of osmotically shocked Salmonella cell to visualize flagellar baseis shown in left photograph. The flagellum of V. alginolyticus cell is observed andvisualized by the electron microscopy in right photograph.

reconstructions from electron cryomicrographs of the rotor revealed that the
C ring displays 34-fold symmetry and the MS ring shows about 25-foldsymmetry (Thomas et al., 2006), and confirmed that most of the C-ring
structure was composed of FliM and FliN. It was known that the FliF binds
to FliG, and N-terminal 46 residues of FliG are required for the binding(Oosawa et al., 1994). Recent studies revealed more detailed FliG–FliM
interactions, and it will be discussed later (Brown et al., 2007). In addition,
spontaneous mutants of FliF–FliG fusion were found to be functional(Francis et al., 1992). Therefore, the FliF:FliG ratio should be 1:1, and it isStructure and Function of Flagellar Motor

55

consistent with FliG stoichiometry in C ring and the symmetry of MS ring as
described earlier. By direct comparison of the ring structure made of FliF and
FliF–FliG fusion protein obtained by electron cryomicroscopy and singleparticle image revealed that a part of the FliG occupies the outer rim and faceof the M ring, whereas the remaining part is likely a part of the C ring (Suzuki
et al., 2004). FliM interacts with FliG and FliN but not with FliF, and FliN
does not interact with FliF (Oosawa et al., 1994). Therefore, FliM and FliN
are probably located at the central and bottom position of the C ring,
respectively.
Structural studies of the switch proteins have been undertaken by using
the thermophilic bacteria Thermotoga maritima as a protein source, and crystal
structures of the functional domains have been determined (Fig. 2.7). For
FliG, middle and C-terminal domains of the protein (residues 104–335,
termed FliGMC) correspond to two-third of the full-length protein (Brown
et al., 2002; Lloyd et al., 1999). The middle domain (residues 115–190,
termed FliGM) has a conserved surface patch formed by the residues
EHPQ125–128 and R160 (the EHPQR motif ), which is important forbinding to FliM (Brown et al., 2007). The FliGC (residues 198–335) has aconserved surface hydrophobic patch, which is also important for thebinding to FliM (Brown et al., 2007). A region near the C-terminus has
five well-conserved charged residues that interact with those in the stator
protein MotA, and electrostatic interaction of these charged residues at therotor–stator interface is important for torque generation (Lloyd and Blair,
1997; Zhou et al., 1998a). Structure showed that charged residues of FliG
are clustered on the prominent ridge of FliGC where two subsets of thecharged residues are aligned as a ‘‘V’’-like shape. FliGM is connected to the
FliGC by an a-helix and short linker including well-conserved two consecutive Gly residues (termed Gly–Gly motif ). This Gly–Gly motif seems to be
involved in the motion of both domains, acting as a flexible hinge mediating
relative movements of two connecting domains. These structural featuresof FliG suggest that FliG is likely to have two orientations that bringdifferent subset of charged residues into alignment around the edge of therotor, where they could interact sequentially with charged residues ofthe stator protein MotA. Therefore, such an arrangement of charged residues may be important for the switching of the rotational direction. Yeast
two-hybrid method was used to investigate the interaction between switch
proteins, and revealed two distinct binding sites for FliM in FliG: theEHPQR motif of the FliGM and the hydrophobic patch of the FliGC(Marykwas et al., 1996). Consistent with this, tryptophan replacements inresidues that participate in these binding sites influenced the binding to FliM
and also the assembly of the flagellum (Brown et al., 2007). The hydrophobic patch in FliGC is positioned adjacent to the Gly–Gly linker and opposite
to charge-bearing ridge. Because the structure of the N-terminal domain
has not been determined yet, it is not clear how FliG binds to FliF.

56

Hiroyuki Terashima et al.

2B subunits
Ions(Na+ or H+)

Ions(Na+ or H+)

4A subunits
FliF

Conserved
charge residues
FliGC-terminal
regionGly-Gly motifhydrophobic patch

FliGN-terminal
regionEHPQR motif

FliM

FliN

Figure 2.7 The structures of stator and rotor.The stator is formed by the MotA4MotB2
or PomA4PomB2 complex. The B subunit has a peptidoglycan-binding motif (protruded ball) and the A subunit has large cytoplasmic domain, containing the conserved
charged residues important for flagellar rotation, between second and third transmembrane segments. The crystal structure of the FliG middle and the C-terminal domain,
the FliM middle domain, and the two-third of FliN of Thermatoga maritima are shownusing the PDB data, 1l kv, 2hp7, and 1yab, respectively. The C-terminal region of FliG
has a ridge containing the important charged residues, which are believed to interact
with the charged residues of the cytoplasmic domain of MotA, for the flagellar rotation.
FliN is depicted as a homotetramer composed of dimer-of-dimers. FliG, FliM, and FliN
form a switch complex. FliF forms the MS ring of the basal body.

Structure and Function of Flagellar Motor

57

However, as noted above, since FliF–FliG fusion protein is functional, theC-terminal end of FliF and N-terminal end of FliG are likely to be inproximity.
Crystal structure of the middle domain of FliM (residues 44–226, termed
FliMM) from T. maritima, which corresponds to two-third of the full-length
protein, has been determined (Park et al., 2006) (Fig. 2.7). FliM is involvedin changing of the rotational direction triggered by the binding of CheY-P
to FliM, a chemotactic signaling molecule (Sockett et al., 1992; Welch et al.,1993). The phosphorylation state of CheY is regulated by a kinase (CheA)
and phosphatase (CheZ, CheC, or CheX), which are involved in chemotactic signaling pathway (Parkinson, 2003). The structure of FliM reveals
structural similarity to these phosphatases. Disulfide cross-linking experiments using Cys-substituted FliM variants designed by using the structural
information revealed the interface of FliM protein responsible for its
self-association in the C-ring structure. Based on this arrangement of FliM
subunits, about 33–35 copies of FliM subunits form a ring with a diameterof 44 nm, consistent with its rotational symmetry (34-fold) in the C ringobserved in electron cryomicroscopy (Thomas et al., 2006, 1999). CheY-P
binds to N-terminal portion of FliM, which is not present in the structure.
Truncation of the N-terminal 43 residues is required for crystal growth of
FliMM. It is probably disordered when extracted from the C-ring structure.
On the other hand, crystal structure of CheY protein in complex with FliM
peptide including N-terminal 16 residues has been reported (Dyer andDahlquist, 2006; Lee et al., 2001). The CheY-FliM peptide structure
revealed the allosteric communication between the phosphorylation site
and the target-binding surface of CheY protein. The C-terminal part of
FliM has been shown to be important for the binding to FliN (Marykwas
et al., 1996). A GGXG motif involved in the interaction with FliG exists inthe opposite side of the FliN-binding region (Mathews et al., 1998; Tanget al., 1996; Toker and Macnab, 1997). However, weak electron density inthis region discerns only the first two glycine residues.
The structure of FliN corresponding to the two-third of full-length
protein (residues 68–154) from T. maritima (Brown et al., 2005) has been
determined. FliN, that mostly contributes to form the C-ring structure, isfound to be a tightly intertwisted dimer composed mostly of b-sheet(Fig. 2.7). Structure also revealed a hydrophobic patch, formed by several
well-conserved hydrophobic residues, on the surface of the FliN dimer.
Mutations in these residues in the patch give rise to Fla or Che phenotype, indicating that it is important for the flagellar assembly and the switching. Motility of these mutants is partially restored by the overexpression of
FliI and FliH (Paul et al., 2006), soluble components of export apparatus,suggesting that FliN is involved in the secretion of flagellar proteins. This is
not surprising, since a temperature-sensitive FliN mutant was unable toregrow flagellar filament after shearing it at the restrictive temperature

58

Hiroyuki Terashima et al.

(Vogler et al., 1991). FliN has been known to involve the export of filament
subunits or capping proteins. In addition, FliN has a homology to the export
apparatus for virulence factors of pathogenic bacteria (Tang et al., 1995).
Therefore, although FliN is involved in switching and motility, it is more
directly involved in flagellar assembly, probably as a part of flagellumspecific export apparatus. Consistent with this idea, recent biochemical
studies showed that FliN associates with FliH (McMurry et al., 2006; Paulet al., 2006), and a five-protein complex consisting of FliG, FliM, FliN,
FliH, and FliI can be isolated (Gonzalez-Pedrajo et al., 2006). These lines ofevidence suggest that FliN in C ring provides a docking site for export
substrate via FliH to efficiently deliver them to the apparatus, and FliN–
FliH interaction involves the hydrophobic patch of FliN. Contribution of
FliN to the rotational switching is also involved in the hydrophobic patch.
Mutations giving Che phenotype are mapped around the hydrophobic
patch, and defects could be partially rescued by overexpression of the CheY,
suggesting that FliN may contribute to the binding site of CheY-P (Paulet al., 2006). As describe earlier, FliN occupies most part of the C ring, and it
is located at the bottom of the ring. Analytical ultracentrifugation of purified
FliN of T. maritima showed that the FliN exists as a dimer in solution, andFliM and FliN together form the stable FliM1/FliN4 complex (Brown et al.,2005). E. coli FliN exists as a stable homotetramer in solution. These results
are consistent with the stoichiometry of FliM to FliN in the C ring.
Targeted disulfide cross-linking studies of FliN suggested that FliN isorganized in doughnut-shaped tetramers, whose shape is closely matched
for the bottom of C ring in the reconstructed electron microscopic image(Paul and Blair, 2006).

2.4. Motor complex
The MotA/MotB or PomA/PomB complex that acts as the torquegenerating unit exists in the cytoplasmic membrane and assembles aroundthe rotor (Kojima and Blair, 2004a; Yorimitsu and Homma, 2001). MotA
and MotB form the torque-generating unit of the Hþ-driven flagellar
motor, whereas PomA and PomB form that of the Naþ-driven flagellar
motor of Vibrio spp. and are orthologues of MotA and MotB (Asai et al.,
1997; Dean et al., 1984; Stader et al., 1986) (Figs. 2.7 and 2.8). MotA and
PomA have four TMs (Asai et al., 1997; Zhou et al., 1995). A large
cytoplasmic loop between the second and third TMs contains conserved
charged residues that have been shown to interact with the conserved
charged residues in the rotor protein FliG, and their electrostatic interactions are important for torque generation (discussed also in Section 3.1;
Lloyd and Blair, 1997; Zhou and Blair, 1997; Zhou et al., 1998a) (Fig. 2.8).
On the other hand, periplasmic loops between first and second TMs(loop1–2) and third and fourth TMs (loop3–4) are very short. MotB and59

Structure and Function of Flagellar Motor

A

H+-driven flagellar motor
MotB(stator)
C
Periplasm

FliG(rotor)IM−+ R297
D289
N−+ P173
R90
D288 −
Electrostatic− E98
interaction
R281 +

K264 +

C

N

P222

D32

MotA(stator)

Cytoplasm

B

MotY

Na+-driven flagellar motor
PomB(stator)

MotX
C

Periplasm
FliG(rotor)IM+ R317
NP151− D309+R88
D308 −+
K89(Electrostatic?)−
interaction
R301 +
E96 − −E97 E99
Cytoplasm

K284 +

−
C

N
D24

P199

PomA(stator)

Figure 2.8 Charged residues of the putative interaction surface between stator androtor. The Vibrio alginolyticus PomA R88 and E96 correspond to R90 and E98 of the
Escherichia coli MotA, and K284, R301, D308, D309, and R317 of V. alginolyticus FliG
correspond to K264, R281, D288, D289, and R297 of E. coli FliG, respectively.The aspartic
acid residue in B subunit (D32 in MotB and D24 in PomB) is believed to be an
ion-binding site.

PomB have a single TM at its N-terminus (Asai et al., 1997; Chun andParkinson, 1988). This TM contains an absolutely conserved negatively
charged residue (Asp32 in MotB of E. coli and Asp24 in PomB of
V. alginolyticus), which is critical for motor rotation and predicted to be
the ion-binding site in the stator complex (Zhou et al., 1998b). Most of
MotB and PomB proteins are located in the periplasmic space. C-terminal
portions of MotB and PomB contain the putative peptidoglycan-binding(PGB) motif that is well conserved among proteins such as OmpA and Pal,

60

Hiroyuki Terashima et al.

which are outer membrane proteins that interact with the peptidoglycan
layer noncovalently (De Mot and Vanderleyden, 1994; Koebnik, 1995).
The MotA/MotB and PomA/PomB complexes are also called the stator,
the nonrotating part of the motor. The stator complex forms a heterohexamer composed of four A subunits (MotA or PomA) and two B subunits(MotB or PomB) (Kojima and Blair, 2004b; Sato and Homma, 2000a,b;
Yorimitsu et al., 2004). The MotA/MotB stator functions as a Hþ channel
and the PomA/PomB stator functions as an Naþ channel (Blair and Berg,
1990; Sato and Homma, 2000a; Stolz and Berg, 1991).
Although structural information is critical to understand the mechanism
for torque generation, currently there are no high-resolution structural dataon the stator complex because its strongly hydrophobic nature hinders to
obtain crystals. Instead, arrangements of the TMs of MotA and MotB from
E. coli (18 segments total in a complex) have been investigated by systematic
disulfide cross-linking studies. The results uncovered initial picture of the
MotA/MotB stator complex: a symmetric dimer of MotB segments is at thecenter of the complex, and TMs of four MotA molecules are arranged
around the MotB dimer (Braun and Blair, 2001; Braun et al., 2004). Cys
residues introduced in the third and fourth TMs (TM3 and TM4) of MotA
can form disulfide bridges between those introduced in the single TM of
MotB that contains the proton-accepting Asp residue, suggesting that
proton-conducting channel is formed by these three segments. Arrangement of the MotB dimer that fits to the cross-linking results revealed thatthe critical Asp32 residues of two MotB molecules are positioned on theseparate surface of the MotB dimer, so possibly there exist two protonconducting channels in a MotA4/MotB2 stator complex. This arrangement
seems to fit into the PomA/PomB Naþ-conducting stator complex: theTM3 of PomA positions very near the TM of PomB (Yakushi et al., 2004).
Phenamil, a potent inhibitor for eukaryotic epithelial sodium channel, also
specifically inhibits the rotation of Naþ-driven motor (Atsumi et al., 1990).
Mutations that allow motor to rotate in the presence of phenamil (Kojima
et al., 1997) were mapped near the cytoplasmic face of the TM3 of PomA
(D148Y) and TM of PomB (P16S) ( Jaques et al., 1999; Kojima et al., 1999),
and when replaced to Cys, these two residues can form a disulfide bridge(Yakushi et al., 2004). These results suggest that residues in TM3 of PomA
and TM of PomB (including critical Asp24) may participate in forming an
Naþ-binding site near the cytoplasmic face. Systematic Cys replacement of
the residues located in the periplasmic loops of PomA revealed that Pro172,
which locate in the loop3–4, forms cross-linked dimer under the oxidation
condition, so loop3–4 of two PomA molecules are positioned side by side ina PomA/PomB complex (Yorimitsu et al., 2000).
The number of the stator complexes assembled around the rotor wasmeasured by various methods. The stepwise increase of the rotation speed
that was dependent on the expression of the stator proteins (Blair and Berg,

Structure and Function of Flagellar Motor

61

1988; Block and Berg, 1984) and the fluorescence intensity change of green
fluorescent protein (GFP)-MotB measured by fluorescence recovery after
photobleaching (FRAP) method indicate that at least 11 stator complexes
are estimated in a single motor (Leake et al., 2006; Reid et al., 2006). The
electron cryotomography of whole cells of Treponema primitia showed in situ
structure of the complete flagellar motor at the 7 nm resolution (Murphy
et al., 2006). The image indicates that the stator assembly possessed 16-foldrotational symmetry, within the range of the stator number described above.
Recently, the purified PomA/PomB complexes reconstituted into theproteoliposome have been observed by cryo-electron microscopy andrough image of stator was reported (Yonekura et al., 2006). Rod-shaped
objects protruded out from both sides of the lipid bilayer. Its diameter was
20 Å, and length of a longer rod and a shorter rod were 70 and 35 Å,respectively. The PomA/PomB complex with truncated C-terminus of
PomB, which lacks PGB motif, was also observed, and the longer rod isfound to be the C-terminal domain that contains the PGB motif.

3. Torque Generation
In this chapter, we overview the current knowledge of the mechanismof torque generation, based on genetic and biochemical studies. Many
hypotheses of torque generation have been proposed, and were extensively
reviewed elsewhere (Berg, 2003; Kojima and Blair, 2004a). It is noteworthy
that some interesting motor models were recently published (Schmitt, 2003;
Xing et al., 2006).

3.1. Interaction between stator and rotor
It is believed that the rotational force of the flagellar motor is generated by
the interaction between the cytoplasmic loop region of the stator component, MotA or PomA, and the C-terminal domain of the rotor component,
FliG. The rotor–stator interaction is coupled to the Hþ or Naþ flow
through the stator powered by the electrochemical gradient across thecytoplasmic membrane. Domains of MotA (or PomA) and FliG responsible
for the rotor–stator interaction contain the conserved charged residues
important for the torque generation (Fig. 2.8) (Lloyd and Blair, 1997;
Yorimitsu et al., 2002; Zhou and Blair, 1997). In E. coli motor, they were
Arg90 and Glu98 of MotA, and Lys264, Arg281, Asp288, Asp289, andArg297 of FliG (Fig. 2.8A). No single residue is critical for rotation, butthey function collectively. Charge neutralization or inversion of theseresidues disrupts motor rotation, but certain combinations of MotA mutations with FliG mutations show strong synergism (e.g., MotA-R90A and62

Hiroyuki Terashima et al.

FliG R281A) or suppression (e.g., MotA-R90E and FliG D289K), whichsuggests that the charged residues of MotA interact with those of FliG
(Zhou et al., 1998a). Therefore, such electrostatic interactions between
rotor and stator are important for the torque generation. As discussed in
Section 2.3, crystal structure of the FliGC revealed that these charged
residues are clustered on the prominent ridge of the FliGC, where two
subsets of the charged residues (R281/D288/K264 and R281/D289/
R297) are aligned as a ‘‘V’’-like shape (Lloyd et al., 1999). Structure andgenetic evidence lead to the rotational switching model that switching may
result from distinct combination of electrostatic interactions between rotor
and stator.
In the case of Naþ-driven motor of V. alginolyticus, these charged
residues are also conserved in PomA and FliG (R88 and E96 of PomA
and K284, R301, D308, D309, and R317 of FliG) (Fig. 2.8B). However,when these residues and neighboring additional three charges (K89, E97,
and E99) of PomA were all neutralized, PomA was still functional(Yorimitsu et al., 2002). Likewise, single or all possible combinations ofcharge-neutralizing mutations in five conserved charged residues did notaffect the motility. Inversion of charge in PomA or FliG barely gave
nonmotile or slow-motile phenotype (Yorimitsu et al., 2002, 2003).
These results suggest that Naþ-driven motor may require additionalcharged residues in PomA and/or FliG for complete electrostatic interaction(s). Alternatively, electrostatic interaction between rotor and stator are
not important for the torque generation in V. alginolyticus. It was found thatchimeric FliG protein consisting of the N-terminal two-third fromV. alginolyticus and the C-terminal one-third from E. coli (termed FliGVE)is functional in the fliG strain of V. alginolyticus, and its opposite variant ofchimeric protein (N-terminal two-third from E. coli and C-terminal onethird from V. alginolyticus; FliGEV) is functional in the fliG strain of E. coli
(Yorimitsu et al., 2003). Likewise, the MotA/MotB stator from E. coli isfunctional in the DpomAB strain of V. alginolyticus and its motor is driven byproton motive force (PMF) (Asai et al., 2003). Chimeric protein PotB,
consisted of the N-terminal TM of PomB from V. alginolyticus and the
C-terminal periplasmic segment from E. coli MotB, is functional in the
DmotAB strain of E. coli, whose motor is driven by sodium motive force(Asai et al., 2003) (Fig. 2.9A). These results indicate that certain parts of the
motor responsible for rotation can be interchangeable between species, andgeneral mechanism of the motor rotation is quite similar regardless of thecoupling ion. To understand the role of charged residues in V. alginolyticus
for motor rotation, conserved charged residues of V. alginolyticus with orwithout mutations were introduced into the hybrid E. coli motor composedof chimeric rotor (FliGEV) and stator proteins (PomA/PotB), and Naþdriven motility of the cells containing these motor proteins were investigated (Yakushi et al., 2006). It was revealed that the charged residues in the

63

Structure and Function of Flagellar MotorA

Na+-driven motor in E. coli

E. coliNa+

H+

V. alginolyticus
Na+
MotX MotY

MotB

PotB

PomB

MotA

PomA

PomA

MotA

PomA

PomA

MotB

PotB

PomB

B
4000

Torque (pN nm)

V. alginolyticus
3000
Chimera
2000

1000

0

E. coli

0

200

400
600
Speed (Hz)800

Figure 2.9 Hybrid and chimeric motors with the Naþ- and the Hþ-driven components. (A) The gray and the open part show the regions of the Hþ- (MotA or MotB) andthe Naþ-driven components (PomA or PomB), respectively. The horizontal lines show
the outer membrane, peptidoglycan layer, and the cytoplasmic membrane. (B) The
torque^speed relationships of different flagellar motors. Measured speeds and estimatedtorques of chimeric motors are represented by circles, the Hþ-driven Escherichia coli
motor (squares) using filament drag coefficients (Inoue et al., 2008). Dotted and dashedline are reported torque^speed relationships for Hþ-driven E. coli motor (Chen andBerg, 2000b) and Naþ-drivenVibrio alginolyticus motor (Sowa et al., 2003), respectively.

V. alginolyticus rotor and stator proteins were found to require for motor
rotation when they were engineered in the E. coli motor, although thesynergism and suppression in rotor–stator double mutants were weaker thanthose seen in E. coli. Therefore, basically rotor–stator interaction occurs inthe V. alginolyticus motor in the same way as in E. coli, but the rotor–stator
interface is more robust in V. alginolyticus. Additional charged residues of
PomA may contribute such robustness (Obara et al., 2008), but otherfactors, probably MotX and MotY, may enhance the motor function in
V. alginoltyticus. The flagellar motor of S. meliloti rotates only CW direction,and its speed was modulated by the tactic stimuli (Gotz and Schmitt, 1987;
Schmitt, 2002). Like E. coli and V. alginolyticus, electrostatic interactions

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Hiroyuki Terashima et al.

between conserved charged residues in rotor (R294 and E302 of FliG) andstator (R90, E98, and E150 of MotA) of Sinorhizobium are important for
torque generation. Initially, it was expected that different charge distribution at the rotor–stator interface, due to the absence of several conserved
charged residues in FliG (only two are conserved), might be the basis for thedifferent modes of motor rotation. However, mutational analyses revealed
that unlike E. coli, E150 is essential for torque generation, whereas R90 andE98 are crucial for chemotaxis-controlled modulation of rotation speed(Attmannspacher et al., 2005). Therefore, it was proposed that MotA
E150 interacts with FliG R294 to achieve fast rotation, but conformationalchanges in FliG triggered by CheY-P binding to FliM lead to the a newrotor–stator alignment that places E150 of MotA adjacent to D302 of FliG,
so that electric repulsion between them might result in lower torque andslower rotation.
Biochemical analysis of the interaction between stator and rotor has notproceeded as compared to the genetic analysis. Only a study has beenreported so far that demonstrated MotA–FliG or MotA–FliM interactions
by pull-down assay (Tang et al., 1996). Then what is the nature of torquegeneration? Although the structure of the stator complex has not beensolved and physical property of the stator complex have been still unclear,systematic mutational studies indicated that MotB Asp32 of E. coli, which
exists in the single TM of MotB, is the only protonatable residue in the
motor proteins responsible for the flagellar rotation, suggesting that protonation of this critical aspartate in the stator complex triggers conformationalchanges in the stator complex that drive rotor (Zhou et al., 1998b). To test
this idea, protease susceptibility of MotA in complex with MotB was
examined, and it was revealed that replacement of the critical aspartate(Asp32) of MotB to asparagine or any other small neutral amino acid caused
a conformational change in MotA, that could be detected as a change ofprotease susceptibility (Kojima and Blair, 2001). MotA conformation is also
restricted by the well-conserved prolines located at the cytoplasmic face of
TM3 and 4 (Pro173 and Pro222 in E. coli). In a way that disrupts a-helix,
forming b-turn, proline greatly affects secondary and tertiary structures ofthe protein. Mutations in Pro173 and Pro222 abolished or severely
impaired motility, and mutant proteins exhibit strong dominant-negative
effect on motility. Therefore, these residues might regulate the conformation of the MotA/MotB complex and/or control conformational changes(Braun et al., 1999; Zhou and Blair, 1997).

3.2. Ion-binding site
As discussed in Section 3.1, an ion-binding site of the stator channel has
been believed to be MotB D32 in E. coli and PomB D24 in V. alginolyticus(Yorimitsu and Homma, 2001). Hþ or Naþ associates with the negatively

Structure and Function of Flagellar Motor

65

charged side chain of the acidic amino acid in the stator, although pKa ofcarboxyl group in these residues in the physiological condition has not beenmeasured yet. As described in Section 3.1, when the conserved acidic orbasic amino acid residues in MotA, MotB, FliG, FliM, and FliN, those ofwhich are important for torque generation, were replaced to Ala, all themutants except D32A of MotB still retained motility (Zhou et al., 1998b).
Replacement of Asp at position 32 of MotB to various amino acids showedthat all but glutamate at that position were nonfunctional. Therefore, D32
of MotB is likely to serve as the proton-binding site in the stator complex.
Likewise, PomB D24 probably functions as the binding site for Naþ. It isnoteworthy that an Naþ binds to the functionally critical glutamate residue(E139) in the crystal of NtpK protein, the channel component of anotherNaþ-translocating rotary motor, VOV1-ATPase from Enterococcus hirae(Murata et al., 2005). Recently determined crystal structures of Naþ-coupledtransporters revealed that not a single but several side chains including
backbone carbonyl group(s) participate in forming an Naþ-binding site
(Hunte et al., 2005; Yamashita et al., 2005). Therefore, in the case of a
PomA/PomB complex, there still may exist additional residues involved information of an Naþ-binding site, possibly including D148 of PomA andP16 of PomB, those of which affect phenamil resistance when mutated. Also,
it is possible that charged residues at the channel entrance or exit or the main
chain carbonyl group(s) of the hydrophobic residues lining in the channel
may play important roles for the selective ion influx (Kojima et al., 2000).
It has been known that cell growth and motility are still normal even if
the MotA/MotB complex is overexpressed more than 50 times over thewild-type level, suggesting that overproduced complexes are inactive for
proton traslocation (Wilson and Macnab, 1988, 1990). When the
N-terminal 60 residues of MotB that includes its single TM was fused tothe unrelated polypeptide, consisted of 50 residues encoded by a part of
TetA (MotB60-TetA), its overproduction together with MotA impairs thecell growth (Stolz and Berg, 1991), and this growth impairment was
abolished by the mutation in D32 of MotB (Zhou et al., 1998b). These
results led to the proposal that a part of the complex (possibly the periplasmic domain of MotB) blocks proton flow through the MotA/MotB channel making it inactive. This model is supported by the mutational study
demonstrating that a deletion of the segment just C-terminal to the TM of
MotB (D51–70) or substitution of the residues in that region (I58, Y61, F62,
and P52/P65) causes a strong growth impairment when overproduced(Hosking et al., 2006). Detailed analyses of this region brought a modelthat the segment (termed ‘‘plug’’) consists of an amphipathic a-helix and isinserted into the cell membrane parallel with its periplasmic face to interferewith channel formation. Interaction of a MotA/MotB complex with aflagellar basal body triggers movement of the plug from membrane andopening of the proton channel. Therefore, this plug may play a central role

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in regulating the open-closed states of the stator channel to prevent premature proton flow.

3.3. Ion specificity
So far, two types of the motor classified by the coupling ion have been
identified: the Hþ-type and the Naþ-type. Most motile bacteria including
E. coli, S. typhimurium, B. subtilis, R. sphaeroides, and Pseudomonas aeruginosahave Hþ-driven motor and V. alginolyticus and alkalophilic Bacillus haveNaþ-driven motor (Berry and Armitage, 1999; Doyle et al., 2004; Imae andAtsumi, 1989; Yorimitsu and Homma, 2001). It seems that since V. alginolyticus lives in the sea where Naþ is abundant, it may have evolved to acquireNaþ-conducting activity through the stator complex.
Which part of the stator proteins determines ion specificity? The Hþdriven stator of the Rhodobacter sphearoides is composed of MotA (RsMotA)
and MotB (RsMotB), and RsMotA are found to be remarkably similar to
the Naþ-driven stator of PomA from V. alginolyticus (Asai et al., 1999; Shahand Sockett, 1995). Both of them are composed of 253 amino acids andtheir identity is more than 40% over their entire length. RsMotB has
similarity only to the transmembrane region of PomB. When RsMotA
was expressed in the pomA strain of V. alginolyticus, the motor was functionaland driven by Naþ-motive force. On the other hand, expression of
RsMotB together with RsMotA in the pomB strain of V. alginolyticus, cells
are nonmotile (Asai et al., 1999). Therefore, determinants of ion specificityof stator should exist in the PomB (and MotB). A series of chimeric
proteins, consisting of N-terminal RsMotB and C-terminal PomB (termed
MomB) (Asai et al., 2000), was constructed. Some of the MomB constructs,
whose entire TM was derived from RsMotB, functioned as Naþ-type statorwhen coexpressed with RsMotA in the DpomAB strain of V. alginolyticus.
In this case, four proteins RsMotA, MomB, MotX, and MotY are involvedin the Naþ-driven rotation. Interestingly, a MomB construct, whose junction is between F33 and V34 of PomB, functions better as Liþ-driven motor
than as Naþ-driven motor. Later, it was found that a chimeric protein of
PotB, a fusion of N-terminal TM of PomB and the C-terminal periplasmic
segment of E. coli MotB, functions as an Naþ-type stator in E. coli only
when coexpressed with PomA (Asai et al., 2003). In this case, just two
proteins PomA and PotB are required for Naþ-driven rotation (Fig. 2.9A).
These lines of evidence indicate that cytoplasmic and transmembrane
domains of PomA/PomB complex are sufficient for Naþ-driven motility,and periplasmic C-terminal part of B subunit (MotB or PomB) determines
the requirement of MotX and MotY for function. Probably, the size ofchannel pore is varied in these chimeric stator complexes, altering the ion
specificity. More precise descriptions of ion specificity in the stator will be
provided by the high-resolution atomic model of the stator complex.

Structure and Function of Flagellar Motor

67

3.4. Assembly of functional motor
Whereas the assembly mechanism of the axial flagellar structure is now wellunderstood (see Section 1.3), the mechanism of stator assembly is still not clear.
Early experiments showed that controlled expression of motA or motB in theirdefective strains demonstrated stepwise restoration of the rotation rate withequal speed increment that saturated at eight steps, suggesting that eight
independently functioning MotA/MotB stator units are incorporated into
the wild-type motor (Blair and Berg, 1988; Block and Berg, 1984). Recent
studies with higher resolution reported that as much as 11 stator units can beincorporated (Reid et al., 2006). This number is in proximity to the observed
particles that surround the rotor by freeze-fracture images (Khan et al., 1988).
MotB has a putative PGB motif that is well conserved among proteins such as
OmpA and Pal, which are outer membrane proteins that interact with the
peptidoglycan layer noncovalently (De Mot and Vanderleyden, 1994;
Koebnik, 1995). The PGB motif of MotB is believed to associate with the
peptidoglycan layer to anchor the MotA/MotB stator complex around therotor. It is not clear how and when MotA/MotB stator complexes are targeted
and anchored at the appropriate position around the rotor.
In V. alginolyticus and V. parahaemolyticus, MotX and MotY have been
identified as essential components for the rotation of Naþ-driven polar
flagellar motors (McCarter, 1994a,b; Okabe et al., 2001; Okunishi et al.,1996). As described in Section 2.1, they attach to the basal body to form a ring
structure (termed T ring) that can be observed beneath the LP ring byelectron microscopy (Terashima et al., 2006). Biochemical studies haveshown that MotX directly interacts with MotY, and affects membrane
localization of the PomA/PomB complex and of the PomB alone,suggesting an interaction between MotX and PomB (Okabe et al., 2005).
In addition, when MotX or MotY is absent, GFP-fused PomA or GFP-fused
PomB in complex with their partner subunit does not localize at theflagellated cell pole, suggesting that MotX and MotY in the T ring are
involved in the incorporation of PomA/PomB complex into the flagellar
motor (Terashima et al., 2006). Like MotB and PomB, MotY possesses
peptidoglycan-binding motif and a recently solved MotY structure showedremarkably similar structure to the Pal and RmpM (OmpA homologue),well-known peptidoglycan-binding protein (Kojima et al., 2008). Detailed
functions of MotX and MotY are still unclear, but they are speculated to be
involved in ion specificity, or in extremely rapid rotation (1100 Hz)
approximately four times faster than that of E. coli (300 Hz).
In S. meliloti, MotC and MotE have shown to be involved in the motility
and regulation of the rotation speed of the flagllar motor (Eggenhofer et al.,
2004; Platzer et al., 1997). MotC is the periplasmic protein and regulates therotation speed by acting on MotB. MotE is the specific chaperone for MotC
and controls the rotation speed indirectly by regulating amount of the68

Hiroyuki Terashima et al.

MotC proteins in periplasm. These proteins do not show similarity to
MotX and MotY. In some bacteria, two kinds of stator might be assembled
to a single flagellar base. Pseudomonas aeruginosa, which has a single polar
flagellum, has dual sets of motA and motB genes, motAB and motCD, as wellas another gene, motY. All these five genes contribute to Hþ-driven motility(Doyle et al., 2004). Function of MotA/MotB stator requires MotY, andthese three proteins are important for the surface swarming. On the other
hand, MotC/MotD does not require MotY for its function, and they areimportant for the swimming in liquid. Furthermore, noncognate pairs like
MotA/MotD and MotB/MotC can work together to generate torque:mutants that contain motA/motD or motB/motC double mutations still retain
motility. Therefore, Pseudomonas cells seem to choose two types of statorsfor motility depending on the surrounding environment (swimming or
surface swarming) (Doyle et al., 2004; Toutain et al., 2005). Similarly,
when swarming on the surface, V. parahaemolyticus as well as V. alginolyticus
cells induced multiple lateral flagella that are driven by proton-type motor(Atsumi et al., 1992; McCarter et al., 1988). The stator for lateral flagellar
motors contains LafT and LafU, orthologues of MotA and MotB, andsomehow its function requires a second set of MotY, MotYL (Stewart andMcCarter, 2003). The reason why MotYL is necessary for rotation of thelateral flagellar motor is not clear.
Another example is the motor of B. subtilis: it has an Naþ-driven MotP/
MotS stator and a proton-driven MotA/MotB stator, but a single set offlagellar rotor proteins (Ito et al., 2004). Like the hybrid stator of P.
aeruginosa, hybrid stators like MotP/MotB and MotA/MotS are functional
in B. subtilis and ion specificities of these motors depend on the B subunit(MotA/MotS for Naþ-type and MotP/MotB for Hþ-type) (Ito et al.,2005). These results are consistent with the observation of hybrid motors
with chimeric proteins using stator proteins of V. alginolyticus and R.
sphaeroides. Since cells carrying MotP/MotS and MotP/MotB stator canswim faster in liquid with high viscosity, MotP function is suggested to beimportant for the optimal function in elevated viscosity. In order to achieve
optimal behavior in variable environments, these bacteria seem to evolve touse distinct sets of stator complexes or have additional components besides
conventional stator proteins to fully exert motor rotation.

4. Molecular Physiology of Motor
4.1. Torque–speed relationshipTo understand the mechanism of motor rotation, we need to know its
basic properties: the power input and output, and their relationships.
Measuring these properties of the motor involves technical difficulties, soStructure and Function of Flagellar Motor69

understanding of the motor physiology has been accompanied by technicaldevelopment in the measurement system. Although the studies of power
input for a single motor have still been hindered by such difficulties, one canmeasure the power output as the ‘‘rotation’’ by using a quite simple methodthat has been employed since the discovery of flagellar rotation. Cells areattached to a coverslip by a single flagellar filament and the rotation of eachsingle motor is monitored by the resulting rotation of the cell body
(Silverman and Simon, 1974). Such tethered cells rotate slowly (around
10 Hz) because of the large viscous load. Measurement of rotation of lightly
loaded motors in swimming cells was achieved by a light scattering method
under the microscope (Lowe et al., 1987), and values obtained by these two
measurements were used for the initial estimation of a torque–speed relationship, an essential feature that characterizes the flagellar motor. The
results showed a linear relationships: that as the speed increases, the torquelinearly decreases.
To cover a wide range of the speed by the measurement of a single
motor, the electrorotation method was developed (Berg and Turner, 1993;
Iwazawa et al., 1993). In this technique, a tethered cell was spun under arotating electric field that enabled motors to rotate up to several hundreds ofHertz. Measurement of the motor rotation has been carried out by thismethod, and revealed characteristic feature of the torque–speed relationships for the flagellar motor: for forward rotation, motor torque remained
approximately constant up to speeds of about 60% of the zero-torque speed,then the torque dropped linearly with speed, crossed zero, and reached aminimum (Berg and Turner, 1993). In yet another approach, the cell bodywas fixed to the glass surface and a polystyrene bead was attached to a stub of
one of its flagellar filaments (Ryu et al., 2000). By using beads of different
sizes and changing the viscosity of external medium, one can measure motor
torque under a wide range of speeds. The results confirmed the previous
measurement by electrorotation method, showing the essentially constanttorque and then linear decline as the speed increases (Chen and Berg,
2000b). The latter method allows us to measure the rotation at various
conditions, such as different temperatures and using D2O instead of H2O
for the effect of solvent isotope (Chen and Berg, 2000a). The results showedthat in the low-speed regime, torque was independent of temperature, andsolvent isotope effects were relatively small.
In the high-speed regime, torque was strongly dependent on temperature, as seen by a downward shift in the ‘‘knee’’ value, the region of thetransition from constant torque to declining torque, at lower temperature.
Also, solvent isotope effects were large. These results were consistent withprevious studies for artificially energized cells of Streptococcus (Khan andBerg, 1983; Manson et al., 1980). Therefore, torque–speed relationship ofthe flagellar motor indicates that at low speeds, the motor operates nearthermodynamic equilibrium, where rates of movement of the internal

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Hiroyuki Terashima et al.

mechanical components or translocation of protons are not rate-limiting,and that at high speeds, the rate-limiting step is proton transfer event, which
results in the decline in torque at high speed. As discussed by Berry andBerg, a torque–speed relationship with this shape can be interpreted in thecontext of a simple three-state kinetic model and it suggests a rotation
mechanism with a power stroke, in which motor rotation and dissipationof the energy available from proton transit occur synchronously (Berry andBerg, 1999). Fung and Berg (1995) found that when the motor operates in
the low-speed regime near stall, its speed is proportional to the voltage
applied across the inner cell membrane. Moreover, Gabel and Berg (2003)
reported a linear relationship between speed (0–270 Hz) and PMF for lightload on the motor. If we assume tightly coupled motor in which thetranslocation of a fixed number of protons drives each rotation, the linear
speed–PMF relation is indicative of a simple voltage-gated proton channel.
Comparative studies have been reported for the Naþ-driven polar
flagellar motor of V. alginolyticus (Sowa et al., 2003). It has been shownthat the torque–speed curve had the same shape as those of the wild-type
E. coli motor and the hybrid motor with PomA/PotB chimeric statorin E. coli with full expression of the stator proteins: the torque is approximately constant (at 2200 pN nm) from stall up to a ‘‘knee’’ speed of
420 Hz, and then falls linearly with speed, extrapolating to zero torque at910 Hz (Inoue et al., 2008) (Fig. 2.9B). The overall shape of the torque–
speed curve is quite similar to the Hþ-driven E. coli motor describedearlier, but the effect of the concentrations of the coupling ion on
torque–speed curves of V. alginolyticus was different from those of E. coli.
The curves for E. coli did not change even when the external environment
had a pH value in the range of 4.7–8.8. However, when external NaCl
concentrations were changed, the generated torque was changed over awide range of speeds for Vibrio motor. This difference seems to be derived
from not the mechanism of flagellar motors but the cell homeostasis when
the concentration of the external coupling ion is varied. The Naþ-driven
motor is likely to generate torque using basically the same mechanism as theHþ-driven motor.

4.2. Steps in rotation of motor
The rotation of the motor occurs by a sequence of discrete molecular
events, presumably including the stages of ion binding in the stator followedby conformational changes in the stator that drive the rotor. Therefore, one
can imagine that the motor behaves like a stepping motor. Since thediscovery of flagellar rotation, such a step has been investigated but until
recently it has not clearly demonstrated. The difficulty in observing rotational steps is due to the presence of an elastic linkage, the hook, between
the tethered filament and the cell body, which smoothes the observedStructure and Function of Flagellar Motor

71

rotation, and is also due to multiple independently functioning torquegenerating units (stator complexes) per motor. One way to avoid thisdifficulty is to examine variations in rotation period. Such a stochastic
analysis by assuming that intervals between steps follow a Poisson distribution led to an estimation of about 400 steps per revolution (Samuel andBerg, 1995). A similar analysis was done with motors containing only one ora few torque-generating units, and showed that the individual units step
independently (Samuel and Berg, 1996).
Recently, direct observation of steps in the motor has been achieved bymaking a motor rotate as slow as possible in the work of Sowa et al (2005).
The hybrid E. coli motor equipped with chimeric Naþ-driven stator complex PomA/PotB was used under the expression control of inducible
promoter. Therefore, it was able to establish a motor with a small initial
number of stator units, possibly one, by growing cells with low inducer
level. Furthermore, by using the Naþ-type motor, the rotation rate can be
reduced by lowering external Naþ concentration. Motor rotation was
detected by back-focal-plane (BFP) interferometry of 500-nm diameterbeads attached to spontaneously sticky flagellar filament stub or by highspeed video recording of 200-nm diameter fluorescent beads attached in thesame way. By using these experimental setups, the stepping rotations wereobserved at speeds below 7 and 40 Hz in BFP and fluorescence experiments, respectively. Interestingly, both backward and forward steps wereobserved at all speeds in both experiments, with higher probability at lower
speeds. In this experiment, a cheY strain was used so that motor never rotates
CW, thus backward steps represent microscopic reversibility rather thanmotor switching. Similar steps have been detected in the ATP-driven
molecular motors. The step size was analyzed from these observations,and it was revealed that there is a peak at 13.7 (26 per revolution) and10.3 (35 per revolution) for forward and backward steps, respectively.
Stepping motion in the ATP-driven molecular motors reflects both thediscrete nature of the input energy and the periodicity of the ‘‘track’’ along
which the motor runs (Mehta et al., 1999; Schnitzer and Block, 1997;
Yasuda et al., 1998). Likewise, observation of 26 steps per revolution in
the flagellar motor is consistent with the periodicity of the ring of FliG
protein (see Section 2.3), suggesting that the motor steps along each FliG
molecule in the rotor. Then how many ions can be translocated during one
step? Because the simultaneous measurement of a single motor rotation andthe ion translocation through the stator complex has not yet been established, we can only estimate it from the published parameters. It wasreported that a wild-type E. coli cell with a PMF of about 150 mV drives
a 1 mm bead with an estimated 280 pN nm per unit (Ryu et al., 2000),
suggesting that maximal step size for one ion in one unit would be 5 if all
the input energy was consumed for the torque generation. Thus, there may
be smaller substeps, or alternatively mechanical step may be coupled to72

Hiroyuki Terashima et al.

several ion translocations. As discussed in Section 2.4, the structural modelfor the stator complex proposed by the disulfide cross-linking study
suggested two distinct channels per stator complex (Braun and Blair,
2001; Braun et al., 2004), indicative of the latter possibility. In any case,the atomic model of the stator complex and establishment of simultaneous
measurement system to investigate input–output relationships will be
awaited for solving this problem.

4.3. Fluorescent imaging of motor
Recent development of single-molecule measurements of enzyme activities
by using fluorescent microscopic methods makes it possible to reveal muchabout fundamental mechanisms of these protein machines. Toward thesimultaneous measurement of the single motor rotation and ion flux
through the stator complex, several attempts of fluorescent imaging of
motor components have been undertaken. Our laboratory has used GFP
for such an imaging in vivo (Fukuoka et al., 2007, 2005). GFP was fused tothe stator proteins, PomA and PomB of V. alginolyticus, and their behaviors
in vivo were observed under the fluorescent microscope (Fukuoka et al.,2005). By using this system, first, it was confirmed that GFP-PomA or GFPPomB proteins were actually localized at the flagellated cell pole of Vibrio.
Their polar localization requires the partner subunit (PomB or PomA) andthe C-terminal domain of PomB that contains PGB motif required foranchoring the stator complexes to the motor. The polar localization of
GFP-fused stator proteins was not observed in the absence of the polar
flagellum. Finally, cells that express the PomA/GFP-PomB stator exhibited
motility, although it was significantly reduced as compared with wild-typestrain. Therefore, this is the initial success in imaging the functional stator
proteins in vivo. Since the PomA/PomB stator complex assembles into thesingle polar flagellum of Vibrio, assembly of the stator complex at theflagellated cell pole can be observed as the polar localization of the GFPfused stator proteins.
When we observed their behavior in the absence of Naþ, the coupling
ion for polar flagellar motor of Vibrio, the GFP-fused stator proteins were
not localized at the flagellated cell pole (Fukuoka et al., submitted for
publication). This effect appears to be Naþ-dependent and reversible,
since addition of Naþ in the medium restores the polar localization of the
GFP-fused stators. The mutations of PomB D24, which is the binding site
for Naþ, affected the localization. Therefore, stator assembly seems to be
regulated by the energy source of the motor, the Na-motive force. Thisfinding also suggested more dynamic behavior of the stator proteins thanpreviously expected. These results are consistent with the previous reports

Structure and Function of Flagellar Motor

73(Armitage and Evans, 1985; Evans and Armitage, 1985) and the recent
report by Leake et al. (2006), demonstrating simultaneous measurement of
the single motor rotation and the number and dynamics of GFP-fused
MotB molecules in the motor in single molecule level by total internalreflection fluorescence (TIRF) microscopy. They tethered cells expressing
GFP-MotB, and observed them under the TIRF or brightfield microscopy.
TIRF images of tethered cells showed spots at the center of the cell rotation
measured from brightfield images, indicating functional assembly of thestator consisting of GFP-MotB and MotA. Then they carried out stepwise
photobleaching of single GFP-MotB in the motor. Counting fluorophores
revealed that each motor contains around 22 copies of GFP-MotB, consistent with 11 stators reported by the recent resurrection experiment. They
also reported by the analyses using FRAP and fluorescence loss in photobleaching methods that (1) there is a membrane pool of 200 GFP-MotB
molecules diffusing at 0.008 mm2 s1, and (2) turnover of GFP-MotB
between the membrane pool and motor was observed with a rate constantof the order of 0.04 s1. Therefore, the static image of the ‘‘stator’’ is in needof change, and we need to understand its function under the considerationof more dynamic behavior. The stator complex appears to be transiently
anchored around the rotor to generate torque, and rapidly exchanged when
it worn or by responding to the change in energetic environment in the cell.
Fluorescent imaging of the rotor protein has also been attempted, andwe were able to visualize functional GFP-FliG molecules in the center ofthe rotating tethered cell (Fukuoka et al., 2007). We are expecting thatappropriate combination of functional GFP-fused stator and rotor proteinin the motor may enable us to carry out measurement of FRET between
stator and rotor components to demonstrate real-time imaging of rotor–
stator interaction in vivo.
Imaging of the energy input into the motor is another big challenge.
Recently, using a fluorescent probe for Naþ, Sodium Green, intracellularNaþ concentration in single E. coli cells has been measured (Lo et al., 2006).
This method requires low-light electron-multiplying charge-coupleddevice camera and laser fluorescence microscopy, and makes it possible tomeasure intracellular Naþ of a series of 50 single cells, without any detectable effect on the flagellar motor. The values obtained are consistent withthe ones measured by other methods. Likewise, fluorescence technique tomeasure membrane potential in single cell has been established by using thedye tetramethylrhodamine methyl ester (Lo et al., 2007). These fluorescence
techniques to measure input energy (membrane potential and ion gradient)
together with the rotation analysis in the single molecule level finally open
the new era for motor physiology that would make it possible to visualize
input–output relationships in real time and in vivo.

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Hiroyuki Terashima et al.

5. Conclusion
In addition to the proton-driven motor, the Na+-driven motor has
been studied extensively and many important data have accumulated. Using
these insights, a genetic manipulation of the Na+-driven E. coli hybrid
motor with chimeric stator led us to a recent breakthrough to observe
directly the steps in rotation of a single motor, the basic process of the
motor. From now on, we can expect to elucidate the rotation mechanism
by discussing the input and output relations of the energy during a single
step in a rotation. Moreover, the technology of single-molecule fluorescentobservation has been introduced, and it will be able to visualize a dynamic
interaction between rotor and stator. To understand the mechanism ofenergy conversion that changes the ion flux into the mechanical power,
the crystal structures of the membrane motor proteins are also needed. We
would like to learn the biological nature from the tiny nanomachine of thebacterial flagella.

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