doi:10.1016/j.jmb.2004.03.070 J. Mol. Biol.

(2004) 339, 423–435

In Vitro Characterization of FlgB, FlgC, FlgF, FlgG,

and FliE, Flagellar Basal Body Proteins of Salmonella
Yumiko Saijo-Hamano1,2, Naoko Uchida3, Keiichi Namba1,2,4 and
Kenji Oosawa1,5*
1
Protonic NanoMachine Project
ERATO, JST, 1-7 Hikaridai
Seika, Kyoto 619-0237 Japan
2 shaft that transmits torque through the hook to the filament to propel the
Dynamic NanoMachine
Project, ICORP, JST, 1-7
Hikaridai, Seika, Kyoto
619-0237 Japan
3
Department of Biosciences
Teikyo University, 1-1
Toyosatodai, Utsunomiya
Tochigi 320-8551 Japan
4
Graduate School of Frontier
Biosciences, Osaka University
3-4 Hikaridai, Seika, Kyoto
619-0237 Japan
5
Graduate School of
Engineering, Gunma
University, 1-5-1 Tenjin, Kiryu
Gunma 376-8515, Japan
*Corresponding author
The bacterial flagellar basal body is a rotary motor. It spans the cyto-
plasmic and outer membranes and drives rapid rotation of a long helical
filament in the cell exterior. The flagellar rod at its central axis is a drive
bacterial locomotion. To study the structure of the rod in detail, we have
established purification procedures for Salmonella rod proteins, FlgB,
FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an
Escherichia coli expression system. While FlgF was highly soluble, FlgB,
FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in
solutions at neutral pH or below, at high ionic strength, or at high protein
concentration. These aggregates were characterized to be b-amyloid
fibrils, unrelated to the rod structure formed in vivo. Under non-aggrega-
tive conditions, no protein –protein interactions were detected between
any pairs of these five proteins, suggesting that their spontaneous,
template-free polymerization is strongly suppressed. Limited proteolyses
showed that FlgF and FlgG have natively unfolded N and C-terminal
regions of about 100 residues in total just as flagellin does, whereas FlgB,
FlgC and FliE, which are little over 100 residues long, are unfolded in
their entire peptide chains. These results together with other data indicate
that all of the ten flagellar axial proteins share structural characteristics
and folding dynamics in relation to the mechanism of their self-assembly
into the flagellar axial structure.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: bacterial flagellum; rod protein; b-amyloid; natively unfolded;
self-assembly

Introduction multiple copy number ranging from several to

The bacterial flagellum is an organelle for
motility. It is made of three parts: the basal body, a
rotary motor that generates torque; the hook, a
universal joint that transmits the motor torque to
the long helical propeller in its different orien-
tations; and the filament, whose thin helical struc-
ture and rapid rotation propels the cell locomotion
in viscous environments. The flagellum rotates at
around 300 Hz, driven by the rotary mechanism of
the basal body.1,2 The basal body is a large complex
made of more than 20 kinds of proteins, each in
Abbreviations used: MALDI-TOF MS, matrix-assisted
laser desorption, ionization time-of-flight mass
spectrometry.
E-mail address of the corresponding author:
kenji@nms.gunma-u.ac.jp
several tens, and spans both the cytoplasmic and
outer membranes. It can be divided into five parts
according to their functions (from the cytoplasmic
to the distal side): the C ring (cytoplasmic ring),
which regulates the reversal frequency of the
motor rotation; the type III protein export appar-
atus, which selectively translocates flagellar axial
proteins into the central channel of the flagellum;
the MS ring (inner-membrane ring), which is the
rotor and the base for the entire flagellar assembly;
the LP ring (outer-membrane and peptidoglycan-
layer ring), which functions as the bushing; and
the rod, which is the drive shaft that connects the
MS ring with the hook and transmits the motor
torque to the helical filament though the hook
(Figure 1).
The rod is composed of four distinct proteins:
FlgB, FlgC, FlgF and FlgG,3,4 and the stoichiometry

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
424
Figure 1. Schematic diagram of the bacterial flagellum
showing the relative size and position of its
substructures. OM, outer membrane; PG, peptidoglycan
layer; CM, cell membrane. The rod portion is shaded.
has been estimated to be about 6, 6, 6 and 26 sub-
units per basal body, respectively.5 An analysis of
the structure and protein components of the
flagellum detached from the basal body by a
mutation in FliF, the sole component of the MS
ring, has shown that FlgG forms the distal part of
the rod and the other three probably form the cell-
proximal portion.6
The rod can also be regarded as the most
proximal portion of the flagellar axial structure,
which continues to the hook made of hook protein
(FlgE), the hook –filament junction made of HAP1
(FlgK) and HAP3 (FlgL), the filament made of
flagellin (FliC), and the filament cap made of
HAP2 (FliD).7 – 10 It is well established that the
filament and the hook are helical assemblies of
flagellin and hook protein, respectively.11,12
Therefore, it has been thought that the rod is also
a helical assembly of rod proteins with the helical
symmetry similar to those of the hook and the
filament. The three-dimensional image recon-
struction of the basal body showed that the thin
proximal portion of the rod inserts into the
cylindrical hollow that extends distally from the S
ring.13 The domain forming this cylindrical hollow
is a distal portion of the FliF ring and because of
this additional feature, we use the term “FliF ring”
in place of “MS ring” throughout this work. The
FliF ring has a circular symmetry, and since it is
difficult to join substructures tightly together over
symmetry mismatch, it is believed that a relatively
large area of molecular interface is present in this
connection.
FliE is also a component of the basal body4,14 and
its stoichiometry was estimated to be about nine
subunits per basal body.14 Minamino et al.15 have
provided both genetic and biochemical evidence
that FliE physically interacts with FlgB, suggesting
Characterization of Flagellar Rod Proteins
that FliE could be a protein acting as a structural
adapter between the FliF ring and the rod.
Although there is much genetic and biochemical
information available for these five proteins, the
structure of the rod has never been visualized in
detail by electron microscopy because the LP ring
and the FliF ring mostly cover the surface of the
rod (Figure 1). Many interesting pieces of infor-
mation are still missing, such as the order of FlgB,
FlgC, and FlgF along the rod, precise stoichiometry
of each subunit protein, and the regulatory mech-
anism that determines the rod length. Here, as the
first step of the structural characterization of the
rod in vitro, we constructed Escherichia coli over-
expression systems for Salmonella FlgB, FlgC, FlgF,
FlgG and FliE, established their purification
procedures, and studied their self and cross-
association behavior under various solution
conditions and domain organizations by limited
proteolysis. We discuss these results in comparison
with available data for other flagellar axial proteins
and also in relation to the self-assembly mechan-
ism of the flagellum. For convenience, the term
“rod components” is used throughout this work to
indicate any of five proteins we studied, namely,
FlgB, FlgC, FlgF, FlgG and FliE.
Results
Purification of over-expressed rod components
As described in Materials and Methods in detail,
we over-expressed five rod components of
Salmonella typhimurium in E. coli and established
purification procedures. Four of the five, FlgB,
FlgC, FlgG, FliE, all form an inclusion body in the
cell, and therefore its solubilization by 6 M to 8 M
urea was necessary for further purification. FlgF,
on the other hand, did not form the inclusion
body, but it was rapidly degraded by intrinsic
protease as soon as the cells were disrupted. There-
fore, the disruption of the cells and the initial puri-
fication of FlgF were carried out in a solution
containing 8 M urea to suppress the protease
activity.16 We combined ion-exchange and gel-
filtration column chromatography to establish the
purification procedures, which now produce
individual rod components in a highly pure form
and sufficiently large amounts for their physico-
chemical and structural characterizations (Figure 2).
To obtain a single species of each rod com-
ponent, we carried out gel-filtration chromato-
graphy with a Superdex 75 (26/60) column at the
final purification step. Because most of the proteins
showed a strong tendency to aggregate in the
presence of salt, as described in more detail below,
gel-filtration chromatography was performed in
appropriate buffer solutions without NaCl to
avoid aggregation. Each SDS-PAGE pattern in
Figure 2 shows that the main peak fraction eluted
from the gel filtration column contains only FlgB,
FlgC, FlgF, FlgG or FliE.
Characterization of Flagellar Rod Proteins 425

Figure 2. Elution profile of gel-filtration chromatography using a Superdex 75 (20/60) column. (a) FlgB, (b) FlgC,
(c) FlgF (d) FlgG and (e) FliE. Inset: SDS-PAGE pattern of the fractions with fraction numbers above and protein
maker positions on the left.

The molecular mass of FlgB, FlgC, FlgF, FlgG
and FliE deduced from the elution volume of the
main peak fraction corresponded to about 31 kDa,
30 kDa, 52 kDa, 51 kDa and 20 kDa, respectively.
Comparing with the molecular mass of each pro-
tein deduced from the amino acid sequence (see
Table 1), these results suggested that all of them
formed a dimer complex. However, the molecular
mass estimated by sedimentation velocity
measurements by analytical ultracentrifugation
426
Table 1. Mass analysis of purified rod components
Detected molecular mass Deduced molecular mass
of purified protein from of mature protein isolated
Protein E. coli expression system from the flagellum
FlgB 15,129.9 15,128.1
FlgC 13,852.7 13,850.7
FlgF 26,095.0 26,102.3
FlgG 27,773.6 27,769.9
FliE 10,946.5 10,947.3
The mature proteins of FlgB, FlgF and FlgG isolated from of
S. typhimurium have their N-terminal methionine, whereas
those of FlgC and FliE have their N-terminal methionine
removed.5,14 There is an error in the amino acid sequence of
FlgC in the Swiss-Prot database: the residue 111 is not Thr ðMr ¼
101Þ but Ser ðMr ¼ 87Þ (Komatsu et al., unpublished results). To
check if the rod components of Salmonella expressed in E. coli
are the same as those of mature proteins isolated from the
Salmonella flagellum, we measured their molecular masses by
MALDI-TOF MS (the second column in the Table). The
molecular masses of mature proteins (the third column) were
calculated from their DNA sequences,3 with other information
such as N-terminal processing and sequence correction all
taken into account.
indicated that all of these proteins are present as
the monomer (data not shown). These results
suggest that these proteins all have a relatively
elongated shape.
A typical yield of protein after purification was
about 25 mg, 20 mg, 20 mg, 45 mg and 40 mg per
liter of culture for FlgB, FlgC, FlgF, FlgG and FliE,
respectively. The molecular masses of the purified
proteins measured by matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) were all identical within experi-
mental errors with those deduced from the amino
acid sequence of mature proteins isolated from the
S. typhimurium flagellum (Table 1), indicating that
the same post-translational processing occurs in
the E. coli over-expression system and in chromo-
somal expression in S. typhimurium.
Characterization of rod components in solution
While trying to establish the purification pro-
cedure for each of the five rod components, we
found that all of them except FlgF tend to self or
cross-aggregate and precipitate under a wide
range of solution conditions: at neutral pH or
lower; at relatively high ionic strength; and at rela-
tively high protein concentration. FliE showed a
particularly strong tendency to aggregate. As
shown in Figure 2(e), when FliE in 8 M urea was
loaded onto a gel-filtration column, two peaks con-
taining FliE were eluted: one in the void fraction
(, 120 ml); and the other at , 182.5 ml. Since FliE
is a small protein (11 kDa), this suggested that FliE
started aggregating as soon as urea was removed.
This aggregation was also promoted in the
presence of NaCl or at lower pH (data not
shown). When FliE eluted at , 182.5 ml was stored
for a week in a solution containing 20 mM Tris –
HCl (pH 9.0) it completely precipitated.
In the case of FlgB and FlgC, which were
Characterization of Flagellar Rod Proteins
co-overproduced, when the inclusion body was
suspended in 6 M urea and dialyzed against a buf-
fer solution at pH 8.5 or lower to remove urea,
these two proteins co-precipitated. To avoid this
undesirable precipitation, we isolated FlgB and
FlgC from each other in a solution containing 6 M
urea at pH 8.5 by using a Q column. FlgB alone
was soluble in a buffer at pH 8.0 or higher, but pre-
cipitated in buffer solutions containing 0.5 M or
higher concentration of NaCl. FlgC alone was
soluble in buffer solutions at pH 9.0 or higher in
the absence of NaCl, even after urea was removed
by dialysis, but it precipitated in the same buffer
solutions in the presence of 150 mM or higher con-
centrations of NaCl. Even in the absence of salt,
after storage in a buffer at pH 9.0 for a month, the
FlgC solution showed some sign of precipitation,
indicating that FlgC aggregated, although slowly.
In the case of FlgG, after the inclusion body was
suspended in 6 M urea and dialyzed against buffer
solutions to remove urea, FlgG was soluble in a
buffer at pH 8.0 or higher, but precipitated in a
buffer solution containing 0.5 M or higher concen-
trations of NaCl.
Regardless of pH within a range from pH 5 to 10
and ionic strength up to 1 M NaCl, FlgB, FlgC,
FlgG and FliE precipitated within a few days at a
protein concentration of 0.3 mM (4.5 mg/ml),
0.2 mM (2.8 mg/ml), 0.3 mM (8.4 mg/ml) and
0.2 mM (2.2 mg/ml), respectively. Below these
protein concentrations, these proteins were soluble
for a week at pH 8.0 or higher and in the absence
of NaCl. In contrast, FlgF was soluble in the
presence of NaCl up to a concentration of 1 M in a
buffer solution at pH 8.0, and it was soluble even
at a protein concentration as high as 0.3 mM
(7.8 mg/ml) in the absence of NaCl.
Characterization of protein aggregates
As described above, FlgB, FlgC, FlgG and FliE
self-aggregate, and FlgB and FlgC co-aggregate
under a wide range of solution condition. We
examined the products of aggregation by electron
microscopy and the results are shown in Figure 3.
FlgB, FlgB/FlgC, and FliE were polymerized into
relatively flexible filamentous aggregates
(Figure 3(a) – (c), respectively). The diameter of the
FlgB filaments, the FlgB/FlgC filaments and the
FliE filaments were about 110 Å, 100 Å, and 60 Å,
respectively, with a relatively small range of distri-
bution. FlgG formed thin and thick filaments with
diameters of about 50 Å and 120 Å, respectively
(Figure 3(d)). The thick filament appears to be
relatively straight, whereas the thin filament is
very flexible. All the thin filaments branched out
from the thick filament, suggesting that the thick
filament is formed as a bundle of many thin proto-
filaments. FlgC polymerized into relatively straight
filamentous aggregates, which formed a kind of
paracrystalline sheet (Figure 3(e)).
The flagellar rod is a short cylindrical structure
and has a diameter of about 180 Å for the distal
Characterization of Flagellar Rod Proteins 427

Figure 3. Electron micrographs of fibrils formed by rod components. (a) Aggregates of FlgB. Purified FlgB was dia-
lyzed against 20 mM Tris – HCl (pH 8.0) containing 1 M NaCl. After centrifugation, precipitates were suspended and
observed. (b) Co-aggregates of FlgB and FlgC. Isolated inclusion bodies of co-overproduced FlgB and FlgC were sus-
pended in 8 M urea and dialyzed against 20 mM Tris – HCl (pH 8.0). After centrifugation, precipitates were suspended
and observed. (c) Aggregates of FliE. After FliE in 8 M urea was loaded onto a gel-filtration column, two peaks contain-
ing FliE were eluted. One was in the void fraction and the other at , 182.5 ml. FliE in the void fraction was observed.
(d) Aggregates of FlgG. Purified FlgG was dialyzed against 20 mM Tris – HCl (pH 8.0) containing 1 M NaCl. After cen-
trifugation, precipitates were suspended and observed. (e) Aggregates of FlgC. Purified FlgC was dialyzed against
20 mM Tris – HCl (pH 9.0) containing 150 mM NaCl. After centrifugation, precipitates were suspended and observed.
The scale bar represents 100 nm.

portion6 and about 80 Å for the proximal portion.13 logically different from the rod structure. When
Also, it does not grow longer than approximately these samples were stained with Congo red, they
300 Å.13 Therefore, these filamentous aggregates of all showed a birefringence under a polarization
the rod proteins formed in solution are morpho- microscope (data not shown). X-ray diffraction
428 Characterization of Flagellar Rod Proteins

Figure 4. Time-course of digestion by limited proteolysis of FlgF and FlgG. Aliquots of the reaction mixture were
mixed with trichloroacetic acid to a final concentration of 5% (v/v) to inhibit proteolytic activity at the indicated
times, and then subjected to SDS-PAGE. Arrowheads indicate cleavage products of FlgF and FlgG for which molecular
masses were determined by MALDI-TOF MS (Table 2). Numbers in parentheses are the positions of the N and C
termini in each protein sequence. The positions of molecular mass markers are indicated on the left.

patterns from the filamentous aggregates of FlgC
showed a sharp reflection at a spacing of 4.7 Å,
indicating the presence of extensive b-structures
(data not shown). These data suggest that these
filamentous aggregates are b-amyloid fibrils and
unrelated to the rod structure.
Limited proteolysis
The flagellar structure that grows in the cell
exterior is made of five proteins, hook protein,
HAP1, HAP3, flagellin and HAP2, arranged in
this order from the proximal to the distal end of
the cell. Flagellin,17,18 hook protein,19,20 HAP3,21
and HAP222 have been found to contain unfolded
terminal regions in their monomeric form or free
pentamer cap form in solution.23,24 HAP1 has also
been found to have unfolded terminal regions
(Furukawa et al., unpublished results). The
unfolded terminal regions are known to play
essential roles in regulating their self-assembly
process of flagellin,18 hook protein,20 and HAP2.22
They are all essential for the formation of stably
assembled structures under physiological con-
ditions. Now, FlgB, FlgC, FlgF and FlgG, exhibit
strong sequential homologies with hook protein in
their N and C-terminal regions,7 and this suggests
that the terminal regions of the rod proteins are
also unfolded in their monomeric forms.
To characterize the domain organizations and
terminal chain conformations of the five rod com-
ponents in their monomeric form, we carried out
limited proteolyses under mild digesting conditions,
monitored the time-course of digestion patterns, and
analyzed the mass of major fragments by MALDI-
TOF MS to determine the cleavage site.
FlgB, FlgC and FliE, of which the molecular mass
is relatively small (, 15 kDa), were rapidly
degraded, and the MALDI-TOF MS analyses of
reaction mixtures at 15 minutes of these proteins
showed complete digestion into small peptides
(results not shown). This means that all the
cleavage sites are exposed, suggesting that the
entire chains of these relatively small proteins are
unfolded.
The results on FlgF and FlgG are partly shown in
Figure 4 and summarized in Table 2. FlgF was
degraded into a relatively stable fragment by
clostripain (Figure 4(a), top panel), a metastable
and a more stable fragment by V8 protease
Characterization of Flagellar Rod Proteins 429

Table 2. Major products by limited proteolyses of FlgF and FlgG

Most
Detected significantly
molecular Corresponding fragment protected Unfolded terminal
Protein Protease mass (deduced molecular mass) domain regions
FlgF Clostripain 17,564 T55-R225 (17,566); FlgF17k T61-E207 M1-R54, M229-S251
V8 protease 21,221 M1-E207 (21,219)
CPY 21,478 M1-N210 (21,477)
FlgF17k V8 protease 15,668 T55-E207 (15,680) T61-E207 M1-R54, M229-S251
API 16,996 T61-R225 (16,994)
CPY 17,038 T55-M220 (17,041)

FlgG Clostripain 16,713 L80-R238 (16,717); FlgG16k G84-E220 M1-E42, L230-L260

V8 protease 17,786 Q59-E229 (17798)
FlgG16k V8 protease 14,691 L80-E220 (14,691) G84-E220 M1-E42, L230-L260
15,637 L80-E229 (15,635)
API 16,256 G84-R238 (16252)
CPY 15,968 L80-N232 (15,961)

(Figure 4(a), middle panel), and a relatively stable
fragment by CPY (results not shown). Examination
by MALDI-TOF MS of fragments produced by
clostripain or V8 protease showed that these pro-
teases first cleaved off a C-terminal region and
then an N-terminal region. We purified a fragment,
FlgF17k (Thr55 –Arg225), which is the most stable
fragment produced by clostripain digestion, by
using a Resource Q column and carried out further
limited proteolysis by V8 protease (Figure 4(a),
bottom panel), API or CPY (results not shown).
The fragments were identified as Thr55 – Glu207
by V8 protease, Thr61 –Arg225 by API and Thr55-
Met220 by CPY. Interestingly, while digestion of
full-length FlgF by CPY produced a fragment
Met1-Asn210 (see Table 2), the C-terminal chain of
FlgF17k was digested only up to Met220. This
suggests that the C-terminal region of FlgF17k is
somehow more protected than that of FlgF.
FlgG was degraded into two metastable frag-
ments and one stable fragment by clostripain
(Figure 4(b), top panel), and one metastable and
one stable fragment by V8 protease (Figure 4(b),
middle panel). As in the case of FlgF, proteases
first cleaved off a C-terminal region and then an
N-terminal region. We purified a fragment, FlgG16k
(Leu80 – Arg238), which is the most stable fragment
produced by clostripain, by using a Resource Q
column and then further digested it by V8 protease
(Figure 4(b), bottom panel). The size of the
digestion products appears bigger than that of
FlgG16k on an SDS-PAGE gel, but it is probably
influenced by removed charges. Two fragments
were identified in the product and they are
Leu80 – Glu229 and Leu80 – Glu220. Digestion of
FlgG16k by API or CPY produced fragments
Gly84 – Arg238 and Leu80 – Asn232, respectively.
By taking all these results into account, the
region that forms the most stable core domain of
FlgF, which consists of 251 amino acid residues, is
identified to be Thr61 – Glu207, and that of FlgG,
which consists of 260 amino acid residues, to be
Gly84 – Glu220. However, considering the rapid
digestion patterns monitored by SDS-PAGE, the
unfolded terminal regions are 1– 54 and 2292 251
for FlgF and 1– 42 and 230 – 260 for FlgG.
Effect of terminal truncation of FlgG on self-
aggregation
To characterize the role of these unfolded
terminal regions in the aggregation process to
form b-amyloid fibrils, we constructed over-
expression systems for the FlgG(59 – 229) (18 kDa)
and FlgG(80 – 229) (16 kDa) fragments (Figure 4(b),
middle and bottom panels), which are missing
both N and C-terminal unfolded regions. We
purified these fragments by the same protocol as
we used for FlgF and examined their aggregation
behaviors. The results are shown in Figure 5.
While full-length FlgG precipitated in a solution
containing 1 M NaCl at pH 8.0, neither of these
FlgG fragments precipitated. Even at a protein con-
centration of 1 mM (16 – 18 mg/ml), these two frag-
ments were soluble at pH 8.0 in the absence of
NaCl (data not shown). The precipitated sample
of FlgG (Figure 5, lane 6) was examined by electron
microscopy, and thin and thick filaments were
Figure 5. Aggregation behaviors of FlgG fragments
missing terminal regions. FlgG(59 – 229), FlgG(80 – 229) and
FlgG were dialyzed against 20 mM Tris – HCl (pH 8.0)
with 1 M NaCl overnight at 4 8C and centrifuged at
100,000g for 30 minutes at 4 8C. s, supernatants; p,
precipitates. The positions of molecular mass makers
are indicated on the left.
430
observed just as shown in Figure 3(d). These
results clearly demonstrate that the unfolded
terminal regions of FlgG are responsible for self-
aggregation of FlgG into b-amyloid fibrils. There-
fore, it is likely that self-aggregation of FlgB, FlgC
and FliE and cross-aggregation of FlgB/FlgC into
b-amyloid fibrils are also caused by the unfolded
terminal regions.
The two FlgG fragments were analyzed by
analytical gel-filtration chromatography with a
Superdex 75 (10/30) column equilibrated with
20 mM Tris – HCl (pH 8.0), 150 mM NaCl.
FlgG(59 – 229) and FlgG(80 – 229) were eluted at an elution
volume of , 11.54 ml and , 11.97 ml, respectively,
both of which corresponded to their monomeric
size (data not shown). This indicates that FlgG
was eluted at the elution volume corresponding to
the size of its dimer because the unfolded terminal
chains are largely extended, making the shape of
the highly elongated molecule. The same would
be true for the other rod component proteins.
Trial of in vitro reconstitution of the
rod structure
In trying to reconstitute the flagellar rod struc-
ture with rod component proteins in vitro, we
systematically examined pairwise associations of
five rod components at a protein concentration of
10 mM (0.10 – 0.26 mg/ml), in a pH range from 5 to
10, and in a range of NaCl concentration from 0 M
to 1 M. However, we did not detect any signs of
cross-association under these conditions. As
observed for flagellin25 and other flagellar axial
proteins,20,21,26,27 spontaneous self-assembly into
the rod structure may be inhibited by the unfolded
terminal regions, which are supposed to fold up
and form the inner core of the axial structure
when these proteins are properly assembled.
In vivo assembly of most flagellar proteins is
known to proceed only in the presence of template
structures. As a preliminary experiment, we used
the FliF ring complex as a template for the rod
protein assembly. The FliF ring complex was
prepared as a soluble form in a solution containing
50 mM Tris –HCl (pH 8.0), 0.1% (w/v) n-dodecyl-
b-D -maltoside (Nagashima et al., unpublished
results), and each of the three proximal rod com-
ponents, FliE, FlgB, and FlgC, was mixed with the
FliF ring complex. The binding was monitored by
co-sedimentation assay by ultracentrifugation. How-
ever, we did not detect any signs of cross-association.
Discussion
The flagellar rod is located at the proximal end
of the flagellar axial structure and plays an import-
ant role of transmitting the motor torque to the
helical propeller for bacterial locomotion. It has
been thought that the rod is a helical assembly of
rod component proteins that form a continuous
axial structure extending to the hook and the fila-
Characterization of Flagellar Rod Proteins
ment, of which helical structures have been well
characterized. A certain level of amino acid
sequence homology identified in the terminal
regions of all the flagellar axial proteins including
flagellin and hook protein7 also suggests that the
rod is a helical assembly of subunit proteins. The
FliF ring, to which the rod is presumed to bind
directly, however, has a circular symmetry, and
therefore the mechanically tight binding between
the FliF ring and the rod has to be achieved over
symmetry mismatch. In order for the rod to freely
rotate inside the LP ring, which is another struc-
ture with a circular symmetry,28 the symmetry mis-
match may be used to achieve the smooth rotation
with a small friction.29,30
Binding mechanism over symmetry mismatch
also plays an important role at the other end of
the flagellar axial structure, where the filament
cap made of the HAP2 pentamer complex binds
stably to the distal end of the filament and
promotes the efficient assembly of flagellin
molecules exported through the narrow central
channel.31 The structure of the cap –filament com-
plex revealed the active use of symmetry mismatch
in the rotary cap mechanism for the promotion of
flagellin self-assembly in which flexible movement
of five leg domains of the cap is essential for stable
binding to and rotation against the tip of flagellum.
However, the rod structure may be designed to
overcome or use the symmetry mismatch in differ-
ent ways. The rod must be bound tightly to the
FliF ring to transmit the torque, and the rod struc-
ture must be rigid as well. The smooth and
mechanically stable rotation of the rod within the
LP ring would require those two structures to be
relatively rigid in their backbones and only surface
side-chains to be flexible. In order to understand
this physically as well as physiologically interest-
ing problem based on the molecular structure and
the assembly behavior of component proteins, we
established the purification procedures for five
rod component proteins overproduced by E. coli
over-expression systems.
Filaments formed by rod components in vitro
While establishing the purification procedures,
we found that the rod components tend to aggre-
gate under a wide range of solution conditions.
Examination by electron microscopy revealed that
the aggregated products were all long fibers, either
flexible or relatively rigid and straight, with rela-
tively well-defined diameters of about 120 Å for
the thick filaments and 50 Å for the thin filaments.
Observation of these products stained with Congo
red and X-ray diffraction data indicated that these
fibrils contain extensive b-structures. Fibrils
formed by FlgG showed a relatively straight and
rigid appearance like the rod, but revealed a
twisted ribbon-like feature in magnified images
(Figure 3(d), lower panel). These filaments also
had many thin, flexible filaments branched out
from them (Figure 3(d), upper panel).
Characterization of Flagellar Rod Proteins 431

On the other hand, the rod is a short cylindrical
structure of high mechanical stability and has a
diameter of about 180 Å for the distal portion4 and
about 80 Å for the proximal portion.13 The amino
acid sequence of FlgB, FlgC, FlgF and FlgG all
have extensive heptad repeats of hydrophobic
side-chains in their terminal regions, indicating
that these regions form a-helical coiled coils.3 This
feature is shared by almost all of the terminal
regions of flagellar axial proteins including flagel-
lin. The structure of the flagellar filament, which
has recently been visualized in atomic detail by
electron cryo-microscopy and helical image
analysis,32 shows that the terminal chains of flagel-
lin form an a-helical coiled coil in the inner core
of the filament with its axis approximately parallel
with the filament axis. Hydrophobic interactions
between the a-helical coiled coils of neighboring
subunits are identified to be the key factor to
build the mechanically stable filament structure in
aqueous environments. It is therefore likely that
the terminal chains of rod proteins also form
a-helical coiled coils to construct the mechanically
rigid and stable structure of the rod. Taking all
these into consideration, the relatively thin and
long filaments formed by rod proteins in solution
do not appear to be related to the flagellar rod
structure.
Many different proteins have been found to form
b-amyloid fibrils if some portion of the proteins are
natively unfolded or under conditions where the
proteins are partially unfolded.33,34 Among the
flagellar axial proteins, HAP3 has been found to
form b-amyloid fibrils in solutions of relatively
high ionic strength or high protein concentration,
and its unfolded terminal chains are responsible
for b-amyloidosis.21 The characteristic features of
rod components revealed here, such as the aggre-
gation behavior into fibrils, unfolded terminal
regions, and the structural features of their
filamentous aggregates, thus strongly indicate that
the filaments formed in vitro are b-amyloid fibrils.
The filament made of FlgG showed a twisted rib-
bon-like feature made of thin protofilaments,
which is also similar to the structural feature of
Asp67His lysozyme amyloid fibrils observed by
electron cryo-microscopy.35
We identified that the unfolded terminal regions
are responsible for the fibril formation. In the cyto-
plasm, these fibril formations would not occur,
where FliJ appears to act as a chaperone preventing
aggregation of the flagellar proteins until they are
exported by the type III protein export
apparatus.36,37 FliJ would bind to and protect
unfolded terminal chains, so that toxic b-amyloid
fibrils would not form in the cytoplasm.

Figure 6. Unfolded regions in the amino acid sequences of flagellar axial proteins. Continuous lines and open boxes
indicate unfolded regions and compactly folded domains, respectively, as determined by limited proteolyses
(flagellin,17,18 hook protein,19,20 HAP2,22 HAP321). Asterisks indicate the positions of the heptad repeat of hydrophobic
residues: those on FlgB, FlgC, FlgF, FlgG, hook protein, HAP1 and HAP3 are based on heptad analysis;7 those on
flagellin are based on its atomic structure;32 and those on HAP2 and FliE are based on our examination of the amino
acid sequences.
432
Common features of unfolded terminal regions
The flagellar axial proteins have a common motif
in their amino acid sequences of the terminal
regions, namely heptad repeats of hydrophobic
amino acid residues, suggesting that these terminal
regions all form a-helical coiled coils.7 It has also
been found that the terminal regions of flagellin,17
hook,20 HAP222 and HAP321 are unfolded in their
monomeric form in solution. As mentioned in the
previous section, it has been confirmed by the
structure analysis of the filament34 that the terminal
regions of flagellin that are unfolded in its mono-
meric state form an a-helical coiled coil as the
inner core domain of the filament. Hydrophobic
interactions between subunits in this region are
responsible for the mechanically stable filament
structure in aqueous environments. Truncation of
these regions actually results in significant
reduction in the polymerization ability and
stability of the filament.18 The total numbers of
amino acid residues of unfolded terminal regions
of these flagellar axial proteins are all within a
range from 85 to 111 (Figure 6), suggesting that
the size of the inner core domain responsible for
polymerization into the stable flagellar axial struc-
ture is well conserved in all of the axial proteins.
Here, limited proteolysis showed that FlgF and
FlgG have unfolded terminal regions within this
common size range, while FlgB, FlgC and FliE are
almost completely unfolded. These results are
reasonable if the common size range of unfolded
terminal regions also applies for the five rod com-
ponents. The amino acid chains of FlgF and FlgG
are 251 and 260 residues long, respectively, which
are long enough to have compactly folded domains
as well as unfolded terminal regions, whereas
FlgB, FlgC and FliE are only 138, 133 and 103
residues long, respectively, which are all too short
to have a compactly folded domain (Figure 6). All
these data strongly indicate that the inner core
domains of the flagellar axial proteins are all in
a-helical coiled coil conformation formed by
terminal chains of similar lengths, and form a con-
tinuous axial structure throughout the length of
the flagellum, making it mechanically stable in
physiological environments by intersubunit hydro-
phobic interactions.
Assembly of rod components in the flagellum
The growth of the long axial structure of the
flagellum proceeds at its distal end by self-
assembly of flagellar axial proteins transported
from the cytoplasm through the central
channel.4,38 – 40 The self-assembly of adding com-
ponents always requires an appropriate structural
template, and the growing structures play this role
in vivo. In vitro reconstitution studies of flagellin25
and hook protein26,27 show that monomers do not
assemble spontaneously in solution of physiologi-
cal ionic strength. The distal end structure of the
filament or the hook was necessary as a template
Characterization of Flagellar Rod Proteins
for self-assembly of monomeric proteins. In vitro
reconstitution of the flagellar axial structure start-
ing from the hook also demonstrated that only
appropriate template structures can promote incor-
poration of HAPs and flagellin at the distal ends of
growing structures.40 We examined cross-assembly
between five rod components in all pairs in
solution, but did not find any signs of interactions.
There still remains a possibility that the protein
concentration was not high enough to detect any
cross-associations. It is likely, however, that those
stable associations are inhibited by unfolded
terminal chains in the absence of appropriate
template structures.
We do not yet understand why the FliF ring
complex did not act as the template for the
assembly of rod components. The structure of the
FliF ring complex may be designed in such a way
that only rod proteins exported through the central
channel of the FliF ring can bind properly to the
ring; or, some other factors may be necessary to
promote the binding. Further studies on the
binding assay are under way.
Materials and Methods
Construction of over-expression system
Plasmid pKK1427 carries a PvuII-ClaI fragment of the
S. typhimurium chromosome that contains flgBCD.3 An
AluI fragment carrying flgB of pKK1427 was inserted
into a SmaI site of pHSG398, giving pKOT160. A HpaI
fragment carrying flgC of pKK1427 was inserted into a
SmaI site of pHSG398, giving pKOT161. BglI-BamHI
fragments of pKOT160 and pKOT161 were ligated with
a BamHI fragment of pHSG397, giving pKOT162.
A BamHI fragment carrying flgBC of pKOT162 was
inserted into a BamHI site of pET3a, giving pKOT167.
After introducing a new NdeI site at the start codon of
flgB by mutagenesis on pKOT167, giving pKOT190, the
small NdeI fragment of pKOT190 was removed and the
rest was religated, giving pKOT191, which is a plasmid
co-overproducing FlgB and FlgC.
Plasmid pMH64 carries a ClaI-EcoRI fragment of the
S. typhimurium chromosome that contains flgEFG.41
A PstI-FspI fragment carrying flgFG of pMH 64 was ligated
with a PstI-HincII fragment of pHSG398, giving pNUT1.
A SalI-FspI fragment of pMH64 was ligated with a SalI-
XbaI fragment (the XbaI site was treated with Klenow
enzyme to create a blunt end) of pHSG398, giving
pNUT2. A SacI-BglII fragment from pNUT1 and a
BglII-EcoRI fragment from pNUT2 were ligated with a
SacI-EcoRI fragment of pHSG398, giving pNUT3. The
BamHI fragment of pNUT3 was inserted into a BamHI
site of pET3b, giving pNUT6, which is a plasmid
co-overproducing FlgF and FlgG. A BamHI-PstI frag-
ment of pNUT6 was ligated with a BclI-PstI fragment
of pNUT6, giving pNUT8 which is a plasmid overpro-
ducing FlgF alone. An AluI-KpnI fragment of pMH64
was ligated with a HincII-KpnI fragment of pHSG397,
giving pNUT9. A ClaI-EcoRI fragment of pNUT9 was
ligated with a ClaI-EcoRI fragment of pHSG396, giving
pNUT10. A BglII-HindIII fragment of pNUT2 was
ligated with a BglII-HindIII fragment of pNUT10, giving
pNUT11. A BamHI fragment of pNUT11 was inserted
Characterization of Flagellar Rod Proteins
into a BamHI site of pET3b, giving pNUT12 which is a
plasmid overproducing FlgG alone.
The fliE of pMM100115 carried on an NdeI-BamHI
fragment was ligated with an NdeI-BamHI fragment of
pET3c to create pYSN1 which is a plasmid over-
producing FliE.
The deletion mutant genes of flgG were amplified with
GeneAmp PCR System 9700 (Applied Biosystems) from
pNUT12 using 2.5 units of TaKaRa Ex Taq (Takara) and
synthetic primers (Amersham-Pharmacia) in Ex Taq
buffer (Takara) containing 0.2 mM each dNTP. The DNA
fragment PCR-amplified with primer GN80, 50 -
ACGCATATGCTGCACAGTCAGGGG-30 with an NdeI
recognition site (underlined) containing the starting
codon, and primer GC229, 50 -CATGGATCCTTACTCTT
CCGCCACGTT-30 with a BamHI recognition site (under-
lined) downstream of the stop codon, was purified using
QIAquick PCR purification kit (QIAGEN), and digested
with NdeI and BamHI. The NdeI-BamHI fragment was
ligated with an NdeI-BamHI fragment of pET3c to create
pYSN7 which is a plasmid overproducing FlgG(80 – 229).
The DNA fragment PCR-amplified with primer GN59,
50 -TCGCATATGCAGACGACGCTGC-30 with an NdeI
recognition site (underlined) at starting codon, and
primer GC229 was digested with NdeI and BamHI. This
NdeI-BamHI fragment was ligated with an NdeI-BamHI
fragment of pET3c to create pYSN8, which is a plasmid
overproducing FlgG(59 – 229).
The pKOT191, pNUT8, pNUT12, pYSN1, pYSN7 and
pYSN8 were introduced into E. coli strain BL21 (DE3)
pLysS.
Overproduction of rod proteins
Overnight culture of BL21 (DE3) pLysS carrying the
appropriate plasmid was diluted 100 times into fresh
Luria-Bertani (LB) broth (1% (w/v) tryptone, 0.5%
(w/v) yeast extract, 1% (w/v) NaCl) containing 50 mg/l
of ampicillin and 30 mg/l of chloramphenicol, and
grown at 37 8C to an A600 of 0.5 – 0.6. Protein production
was induced with addition of isopropyl-b-D -thiogalacto-
pyranoside to a final concentration of 1 mM. After
further growth for two hours, the cells were harvested
by centrifugation. The harvested cells were stored at
280 8C. Co-overproduced FlgB and FlgC, overproduced
FlgG and overproduced FliE formed inclusion bodies.
The harvested cells were suspended in TE buffer
(50 mM Tris – HCl (pH 8.0), 1 mM EDTA) and sonicated
(ASTRASON, model XL2020 sonicator; Misonix Inc.) on
ice. After centrifugation at 10,000g for ten minutes at
4 8C, the inclusion bodies obtained as a precipitate were
suspended in TE buffer containing 2% (w/v) n-octyl-b-
D -glucopyranoside (OG). The suspension was sonicated
on ice and centrifuged at 10,000g for ten minutes at 4 8C.
This procedure of washing in TE buffer containing OG
(suspension, sonication and centrifugation) was repeated
twice or three times, and then the inclusion bodies were
washed finally in TE buffer without OG. Washed
inclusion bodies were stored as a pellet at 280 8C.
Purification of FlgB and FlgC
Inclusion bodies of FlgB and FlgC from 1.25 liters of
culture medium were dissolved in 10 ml of solution A
(6 M urea, 20 mM Tris –HCl (pH 8.5)) and sonicated on
ice. After centrifugation at 100,000g for 30 minutes at
4 8C, the supernatant was loaded onto a Q-Sepharose
HP column (26/10) (Pharmacia) equilibrated with sol-
433
ution A. After washing with solution A, the Q column
was developed with a linear gradient from 0 M to
0.15 M NaCl. FlgC and FlgB were eluted at , 40 mM
and , 80 mM NaCl, respectively. FlgB was precipitated
by adding ammonium sulfate (40% saturation) and
collected by centrifugation at 50,000g for 30 minutes at
4 8C. The precipitate was suspended in 8 ml of 8 M urea,
around pH 8.0, and loaded onto the Superdex 75 gel
filtration column (26/60) (Pharmacia) equilibrated with
20 mM Tris – HCl (pH 9.0), at a flow rate of 2.5 ml/
minute. A peak containing the FlgB was eluted at
,175 ml. FlgC pooled from the Q column was further
purified by the same protocol as for FlgB.
Purification of FlgF
We have reported the purification method for over-
produced proteins that are sensitive to endogenous pro-
teases, such as FlgF.16 In this study, the procedure was
modified for purification of large amounts of FlgF. The
frozen cells from 450 ml of culture medium were sus-
pended in 8 ml of 8 M urea, around pH 8.0, and soni-
cated on ice. After centrifugation at 100,000g for
20 minutes at 4 8C, the supernatant was loaded onto an
SP-Sepharose HP column (26/10) (Pharmacia) equili-
brated and washed with solution B (6.4 M urea, 50 mM
acetic acid – NaOH (pH 5.0)). FlgF was eluted in the
flow-through fraction. The fractions containing FlgF
were dialyzed against solution C (50 mM NaCl, 20 mM
Tris – HCl (pH 8.0)) and loaded onto a Q-Sepharose HP
column (26/10) (Pharmacia) equilibrated with solution
C. After washing with two column volumes of solution
C, the column was developed with a linear gradient
from 0 M to 0.5 M NaCl and FlgF was eluted at
,0.25 M NaCl. FlgF was precipitated by adding
ammonium sulfate (to 40% saturation) and collected by
centrifugation at 50,000g for 30 minutes at 4 8C. The pre-
cipitate was suspended in 8 ml of 20 mM Tris –HCl (pH
8.0), and loaded onto a Superdex 75 gel-filtration column
(26/60) equilibrated with 20 mM Tris –HCl (pH 8.0) at a
flow rate of 2.5 ml/minute. A sharp peak containing
FlgF was eluted at , 155 ml.
Purification of FlgG
Inclusion bodies of FlgG from 1.25 l of culture
medium were dissolved in 8 ml of solution B. After cen-
trifugation at 100,000g for 30 minutes at 4 8C, the super-
natant was loaded onto an SP-Sepharose HP column
(26/10) (Pharmacia) equilibrated with solution B. FlgG
was eluted in the flow-through fraction and was precipi-
tated by adding ammonium sulfate (to 40% saturation).
After centrifugation, the precipitate was suspended in
8 ml of 8 M urea, around pH 8.0, and loaded onto a
Superdex 75 gel-filtration column (26/60) equilibrated
with 20 mM Tris – HCl (pH 8.0) at a flow rate of 2.5 ml/
minute. A sharp peak containing FlgG was eluted at
,157 ml.
Purification of FliE
FliE was purified by the purification protocol for FlgG
as described above. After the FliE fraction was loaded
onto a Superdex 75 gel-filtration column (26/60) equi-
librated with 20 mM Tris –HCl (pH 9.0) at a flow rate of
2.5 ml/minute, two peaks containing FliE were eluted.
One was in the void fraction (at , 120 ml) and the other
434
at , 182.5 ml. FliE in the peak at ,182.5 ml was stored
for further experiments.
Cross-association assay
The individual rod components were prepared in
solution at a protein concentration of 10 mM, and then
pairwise mixture was dialyzed against buffer solutions
at 4 8C for overnight, at various pH values from pH 5 to
10 and NaCl concentration from 0 M to 1 M. After the
mixture was centrifuged at 200,000g for 30 minutes at
4 8C, proteins in pellets and supernatants were analyzed
by SDS-PAGE. The mixture solution was also analyzed
by analytical gel-filtration chromatography.
Limited proteolysis
Limited proteolysis was performed at room tempera-
ture and protein concentrations of 0.4– 0.8 mg/ml.
Proteolysis by clostripain (SIGMA) was performed at a
clostripain/protein ratio of 1 : 200 (w/w) in 20 mM
Tris – HCl (pH 8.0) containing fresh 2.5 mM dithio-
threitol. Proteolysis by endprotease Glu-C (V8 protease)
(Roche Diagnostics) was performed at a V8 protease/
protein ratio of 1 : 200 (w/w) in 50 mM phosphate buffer
(pH 7.0). Proteolysis by aminopeptidase I (API) (SIGMA)
was performed at an API/protein ratio of 1 : 20 (w/w) in
20 mM Tris – HCl (pH 8.0). Proteolysis by carboxy-
peptidase Y (CPY) (WAKO) was performed at a CPY/
protein ratio of 1 : 50 (w/w) in 20 mM Tris – HCl (pH
8.0). After the start of the proteolytic reactions, aliquots
of the reaction mixture were removed at various times
and trichloroacetic acid was added to a final concen-
tration of 5% (v/v) to inhibit the proteolytic activity.
After centrifugation, precipitates were suspended in
1 M Tris – HCl (pH 8.0) and analyzed by SDS-PAGE.
Purification of FlgF17k and FlgG16k
FlgF and FlgG from S. typhimurium consist of 251 and
260 amino acid residues and have the molecular masses
of 26.1 kDa and 27.8 kDa, respectively. Digestion by
clostripain produced a FlgF fragment of approximately
17 kDa (FlgF17k) and a FlgG fragment of approximately
16 kDa (FlgG16k). After digestion by clostripain for four
hours under the condition of limited proteolysis
described above, the digestion mixture was loaded onto
a Resource Q column (6 ml) (Pharmacia) equilibrated
with 20 mM Tris – HCl (pH 8.0). Elution from the column
was done with a linear gradient from 0 M to 0.5 M NaCl.
FlgF17k and FlgG16k were eluted at , 0.2 M NaCl.
Mass spectrometry
Unless mentioned otherwise, proteins and their frag-
ments were analyzed by means of MALDI-TOF MS as
described.16
For MALDI-TOF MS, the fragments digested by API
and CPY were separated by SDS-PAGE from the diges-
tion mixture. After portions of the gel containing main
fragments were cut out, fragments were eluted out of
the gel by electrophoresis and concentrated with Zip-
Tip-C18 (Millipore).
Electron microscopy
Samples were negatively stained in 3% phospho-
Characterization of Flagellar Rod Proteins
tungstic acid (pH 7.0) on the carbon-coated copper grids
and observed with a JEM-1010 electron microscope
(JEOL, Tokyo, Japan).
Acknowledgements
We thank K. Kutsukake, M. Homma &
T. Minamino for providing plasmids pKK1427,
pMH6 and pMM1001, respectively. We also thank
S. Maki-Yonekura for preliminary observations of
fibrils by electron microscopy; K, Imada, F. A.
Samatey, J. Tame & Y. Furukawa for their technical
help in the analysis of rod proteins by
sedimentation velocity ultracentrifugation; and
S. Nagashima and H. Suzuki for advice in purifi-
cation of the FliF ring complexes.
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Edited by M. Moody