Vol 451 | 24 January 2008 | doi:10.1038/nature06449

LETTERSDistinct roles of the FliI ATPase and proton motive
force in bacterial flagellar protein export
Tohru Minamino1,2 & Keiichi Namba1,2

a

b
WT

W
T
∆f
liH
∆f
liI
∆f
liH
∆f I
lh
B

oligomerization and ATPase activity4,8. However, as FliI cannot
efficiently dock to the export gate when FliH is missing14, FliH ispostulated to provide a link between ATP hydrolysis by FliI andflagellar protein export, although it is not known how this is done.
We therefore investigated this by characterizing a Salmonella fliH fliI
double null mutant and by isolating gain-of-function mutants fromthis mutant.
A fliH null mutant was weakly motile whereas a fliI null mutant
was non-motile14,15. Notably, when both FliH and FliI were missing(Fig. 1b, lane 4), the cells formed swarms on soft agar plates after
prolonged incubation (Fig. 1a). Consistently, intact flagella as well as
immature flagellar structures were occasionally observed by electron
microscopy (data not shown). We then examined the effect of FlhB
deletion, which makes wild-type cells non-motile (Fig. 1a). The motility of the double null mutant was totally abolished by a flhB::Tn10
mutation (Fig. 1c), indicating that some flagella are produced at a low
probability even in the absence of FliH and FliI, but in a manner thatstill requires FlhB.

(kDa)
FliI

47.5
∆fliI

∆fliHI

∆flhB

FliH

25

30 °C, 8 h∆fliI

1∆fliHI fliHI

∆flhB

2

3

4

5

c
liH
I

∆fliH

∆f
liH
flh I
B:
:T
n1
0

∆fliH

∆f

Translocation of many soluble proteins across cell membranes
occurs in an ATPase-driven manner. For construction of the bacterial flagellum responsible for motility, most of the components
are exported by the flagellar protein export apparatus1,2. The FliI
ATPase is required for this export3, and its ATPase activity isregulated by FliH4; however, it is unclear how the chemical energy
derived from ATP hydrolysis is used for the export process. Here
we report that flagellar proteins of Salmonella enterica serovar
Typhimurium are exported even in the absence of FliI. A fliH fliI
double null mutant was weakly motile. Certain mutations in FlhA
or FlhB, which form the core of the export gate, substantially
improved protein export and motility of the double null mutant.
Furthermore, proton motive force was essential for the export
process. These results suggest that the FliH–FliI complex facilitates only the initial entry of export substrates into the gate, withthe energy of ATP hydrolysis being used to disassemble and release
the FliH–FliI complex from the protein about to be exported. The
rest of the successive unfolding/translocation process of the substrates is driven by proton motive force.
The flagellar export apparatus consists of six integral membraneproteins (FlhA, FlhB, FliO, FliP, FliQ and FliR) and three soluble
proteins (FliH, FliI and FliJ), and is thought to be located at the baseof the flagellum1,2. Export of bacterial flagellar proteins has characteristics in common with the type III secretion system (T3SS) of
virulence factors of pathogenic bacteria, and these two systems actually consist of many homologous component proteins5.
The soluble export component FliI is an ATPase3. Because mutants
lacking the ATPase activity cannot export flagellar proteins, FliI wasthought to convert chemical energy released by ATP hydrolysis intomechanical work necessary for the export3. FliI shows extensive similarity to the a/b subunits of proton-driven F0F1-ATPase for its entire
molecular structure6, although sequence similarity is limited to theirrespective ATPase domains7,8. Unlike F1-ATPase, however, whichforms the a3b3 hexameric ring, FliI self-assembles into a homohexamer9,10. When FliI oligomerization is suppressed by a small deletion in its amino-terminal region, flagellar protein export does notoccur efficiently, suggesting that FliI hexamerizes on docking to theexport gate made of the six integral membrane components, forwhich the cytoplasmic domains of FlhA and FlhB are thought toform the docking platform11. FliI binds to FlgN (chaperone) and toa FlgN–FlgK (HAP1) complex12, suggesting that FliI has a critical rolein substrate recognition as well. As Salmonella InvC—a virulence
T3SS homologue of FliI—binds to chaperone–effector complexes
and induces chaperone release from, and unfolding of, the effector
to be secreted in an ATPase-dependent manner13, FliI has beenthought to act in a similar manner.
FliH binds to the extreme N-terminal region of FliI to form
the FliH2–FliI complex in the cytoplasm, and suppresses FliI

30 °C, 24 h

30 °C, 24 h

Figure 1 | Characterization of a fliH fliI double null mutant. a, Swarming
motility of SJW1103 strain (wild type, WT), and MKM11 (DfliH), MKM30(DfliI), MMHI001 (DfliH fliI, indicated as DfliHI) and MKM50 (DflhB)
mutant strains on soft agar plates. b, Immunoblotting, using anti-FliH andanti-FliI antibodies, of whole cell proteins. Weak bands just below those of
FliI are due to nonspecific reactions. A slight downward shift of the FliI bandin lane 1 is caused by an invisible band of a large amount of FliC (51.5 kDa)
just above it. c, Effect of a flhB::Tn10 mutation on motility of the DfliH fliImutant.

1
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 2Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan.

485©2008 Nature Publishing GroupLETTERS

NATURE | Vol 451 | 24 January 2008

W
T
∆f
liH
∆f I
liH
W I, fl
T hB
*
∆f
liH
∆f I
liH
I,
flh
B*

FliH and FliI interact not only with FlhA and FlhB but also withflagellar chaperones such as FliJ and export substrates, leading to
the proposal that the FliH–FliI complex is responsible for deliveryof the substrates to the export gate4,11,12,14. To test this, gain-offunction mutants were isolated from the fliH fliI double null mutant
by streaking an overnight culture out on soft agar plates, incubating
at 30 uC for 2 days and looking for motility haloes emerging fromthe streak. In total 22 pseudo-revertants were obtained. When
we analysed the secretion of FliC (flagellin) into the culture supernatant by Coomassie brilliant blue staining, FliC was seen on SDS–
polyacrylamide gel electrophoresis gels from the MMHI0117 strain,
but not from the others (data not shown). Therefore, we decided tocarry out further characterization of MMHI0117. The motility of
MMHI0117 was significantly better than that of the parent mutant
although not as good as that of wild type (Fig. 2a). Most cells of thisstrain had a couple of long flagellar filaments, whereas wild-type cells

b

a
WT

Cell

∆fliHl,flhB*∆fliHl

Sup

(kDa)
32.5

FlgG

47.5
FlgE
32.5
30 °C, 8 h

c

25

FlgD

47.5

FliK

47.5

FliC

62

FlgK

32.5

FlgL
1 2 3 4 5 6d

FlhBTM

CM

33
N1
P28T

211
FlhBCN

FlhBCC

Cyto

Relative secretion levels

Peri

C383
FlgL

FlgK

FliC

FliK

FlgD

FlgE

FlgG

Figure 2 | Isolation of pseudo-revertants from the DfliH fliI mutant.
a, Motility of wild type, DfliH fliI mutant (DfliHI) and its pseudo-revertant(DfliH fliI, flhB(P28T), indicated as DfliHI, flhB*). b, Secretion analysis of
FlgG, FlgE, FlgD, FliK, FliC, FlgK and FlgL by immunoblotting. ‘Cell’ and‘Sup’ indicate whole cell proteins and culture supernatant fractions,
respectively. Relative secretion levels are normalized to the wild-type level ofeach protein. Black bar, wild type; grey bar, pseudo-revertant. c, Electron
micrographs of the flagella and basal bodies isolated from DfliH fliI,
flhB(P28T). Scale bar, 200 nm. d, Position of a suppressor mutation in FlhB.
CM, cytoplasmic membrane; cyto, cytoplasm; peri, periplasmic space.

produce five or more (data not shown). Most flagellar proteins weredetected in the culture supernatant of this mutant (Fig. 2b, lane 6).
The amounts of FlgD (hook cap protein) and FliK (hook-length
control protein) secreted by this pseudo-revertant were even larger
than those of the wild type, whereas the secretion levels of FlgG (distal
rod protein) and FlgE (hook protein) were threefold and tenfold
lower than wild-type levels, respectively. Consistently, flagellar basal
bodies, often with the hook and filament attached, were observed byelectron microscopy (Fig. 2c). The secretion levels of these proteins
were restored to wild-type levels by introduction of a plasmid encoding FliH and FliI into the mutant (Supplementary Fig. 1). These
results indicate that the export of FlgE and FlgG depends on the
FliH–FliI complex more strongly than the export of FlgD and FliK.
As all of these proteins belong to the same rod-type and hook-type
export class16, the FliH–FliI complex not only guides these proteins to
the export gate but also controls their export order and levels. The
cellular levels of FliC, FlgK and FlgL (HAP3) in the MMHI0117 strain
were much lower than those in the wild type because expression ofthese genes occurs only after hook completion17, which is evidently
retarded in this strain (Fig. 2b).
P22-mediated genetic mapping18 showed that the gain-of-function
mutation lies in the flhBAE operon (data not shown). DNA sequencing identified a missense mutation, P28T, near the N terminus of
FlhB, close to the interface with the first membrane span (Fig. 2d).
The position of this P28T mutation was very close to the positions of
the FliH-bypass flhB mutations isolated previously14. Therefore, we
tested whether the FliH-bypass flhB and flhA mutations can improve
motility of the fliH fliI double mutant. These mutations, as represented here by a flhA(V404M) mutation, considerably enhanced
both motility and flagellar protein export (Supplementary Fig. 2),
although their suppression abilities were much weaker than that of
the flhB(P28T) mutation.
Salmonella T3SS virulence factors are secreted by the flagellar
export apparatus in the absence of their chaperones19. As InvC andSsaN are the T3SS homologues of FliI in Salmonella20,21, there is apossibility that InvC and/or SsaN may complement the fliH fliI double null mutants for flagellar protein export, although SsaN is not
expressed under our experimental condition21. However, this was
ruled out by our observations that neither motility nor protein exportof the pseudo-revertant was abolished by InvC or SsaN deletions(Supplementary Fig. 3). Therefore, we conclude that the gain-offunction mutations in FlhA and FlhB increase the probability of entry
of flagellar proteins into the export gate, thereby increasing export
efficiency. An increased gate-opening probability for higher efficiency of protein entry could be deleterious to the cells owing toleakage of small solutes. However, the growth of the mutants was
normal (Supplementary Fig. 4a), suggesting that the export gate is
not always open. It is likely that the interaction of export substrates
with the gate, with or without the FliH–FliI complex, induces opening of the pore.
Unlike the fliH null and the fliH fliI double null mutants, the fliInull mutant was non-motile (Fig. 1a), indicating that FliH inhibits
flagellar protein export in the absence of FliI. To test whether theflhB(P28T) mutation would also suppress the fliI null mutation, we
analysed the motility of the pseudo-revertant transformed with aplasmid encoding FliH (Supplementary Fig. 5). The motility gainedby the flhB(P28T) mutation was significantly suppressed, suggestingthat the docking of free FliH to the FlhA–FlhB platform interfereswith the entry of export substrates into the export gate, even in thepresence of the flhB(P28T) mutation.
As it has been shown that Yersinia enterocolitica type III secretion is
inhibited considerably by treatment with the protonophore carbonyl
cyanide m-chlorophenylhydrazone (CCCP)22, and that flagellar
formation is also dependent on proton motive force (PMF)23, weinvestigated whether flagellar protein export is still dependent on
PMF in the gain-of-function mutant as well as wild-type cells. The
rate of cell growth decreased when CCCP concentration increased,

486©2008 Nature Publishing GroupLETTERS

NATURE | Vol 451 | 24 January 2008

and 25 mM CCCP immediately caused growth arrest (Supplementary
Fig. 4b). As expected, the flagellar motor rotation, which is driven by
the PMF1, was abolished by 25 mM CCCP treatment, indicating that
the PMF is significantly collapsed (data not shown). The intracellularlevels of FlgD were maintained even at 25 mM (Fig. 3); however, thelevels of FlgD secretion by both wild-type and mutant cells decreased
significantly above 10 mM CCCP and diminished at 25 mM (Fig. 3). In
agreement with previous data22, the intracellular ATP level did notchange on CCCP treatment within 1 h (Supplementary Fig. 4c).
These results indicate that PMF is absolutely essential for FlgD export
regardless of the presence or absence of FliH and FliI.
(1) The MotA–MotB complex acts as a proton conductive pathway tocouple proton influx with flagellar motor rotation1. Mutations in
MotA or MotB do not affect flagellar formation while the motor isparalized1. Consistently, deletions of these two proteins do not interfere with flagellar protein export even in the gain-of-function mutant(data not shown).
Both ATP and PMF seem to be required for pre-protein translocation in Escherichia coli, where ATP is essential for the initial step ofprotein translocation and PMF increases the rate of translocation24.
SecA is the ATPase that uses the energy of ATP binding and hydrolysis to drive pre-protein translocation into and across the cytoplasmic membrane24. Each catalytic cycle of SecA permits the stepwise
translocation of pre-proteins across the membrane25,26. In contrast,
we have shown that ATP hydrolysis by FliI is not absolutely required
for flagellar protein export. As the diameter of the central channel ofthe flagellum—which is the export path for flagellar proteins—is only
2 nm27, proteins to be exported must be largely unfolded for entry
into and translocation through the channel. Our observations—inparticular, levels of FlgD and FliK secretion by a gain-of-function

mutant that exceed wild-type levels (Fig. 2c)—suggest that the successive process of unfolding and translocation of export substrate isdriven by PMF. The role of the FliH–FliI complex for efficient export
would probably be to increase the initial docking efficiency of the
N-terminal segments of export substrates into the export gate
through specific interactions between the FliHx–FliI6 complex and
the FlhA–FlhB platform. As the binding of the FliHx–FliI6 complex
without ATPase activity inhibits the export process4, the energy of
ATP hydrolysis seems to be used to facilitate the release and disassembly of the FliHx–FliI6 complex from the export gate and theprotein about to be exported, in order for the subsequent PMFdriven, successive process of unfolding of the protein and translocation of the polypeptide chain through the gate by the export gate
complex to proceed without retardation, as well as for efficient use of
the FliH2–FliI complexes for the next cycle of initial docking (Fig. 4).
Because N-terminal segments of export substrates containing export
signals are all natively unfolded in the monomeric form of the proteins28, the energy of ATP hydrolysis would not be required for theprocess of initial docking and entry to the gate.
Considering the many similarities between the flagellar export
apparatus and F0F1-ATPase, such as almost identical structures of

Rod
Peri
PMF

MS ring

CM

H+

Cell
CCCP (µM)

Sup

5 10 25

0

0

5

C ring

Cyto(FliH)2(Flil)6
Export gate

10 25
Mostlyclosed(kDa)
WT

Docking andopen

PMF anddiffusion
N

FlgD

25

N
∆fliHI, flhB*

FlgD

25

H+
ATP

N
1

2

3

4

5

6

7

FliJ

8

C
Substrate
1.0

∆flil

Relative secretion level

WT

C

C

ADP + Pi

(FliH)2
Flil∆fliH∆fliH fliI

∆fliH fliI
flhB* or flhA*∆fliHI, flhB*

0.8

0.6
ATP
0.4

ADP + Pi

0.2

0

0

5

10

15

20

25

CCCP (µM)

Figure 3 | Effect of CCCP on FlgD secretion. a, Immunoblotting, using theanti-FlgD antibody, of whole cell proteins and culture supernatant fractions
prepared from wild-type (WT) and a gain-of-function mutant (DfliH fliI,
flhB*) grown at 30 uC in the presence of 0, 5, 10 and 25 mM CCCP. b, The
decay in the secretion levels shown by plotting band densities of the twoblots, which are normalized for the cellular FlgD levels. These data are theaverage of three independent experiments. The experimental errors arewithin 10%.

Figure 4 | Model for flagellar protein export. Top panel: The flagellar
protein export apparatus in the basal body. Middle panel: in wild type, theFliH2–FliI complex delivers export substrates to the export gate along withFliJ (left). Once the N-terminal segment of a substrate is docked byformation of the FliHx–FliI6 complex (middle), ATP hydrolysis induces
dissociation of the FliHx–FliI6 complex and successive unfolding andtranslocation of the substrates is driven by the PMF (right). Bottom panel:protein translocation is inhibited by FliH in the fliI mutant (left). The fliH
mutant retains the export function to some extent (middle left). The fliH fliI
double mutant also retains the export function, albeit at a low level (middle
right). Gain-of-function mutations increase the efficiency of substrate entry(right). Pi, inorganic phosphate.
487©2008 Nature Publishing GroupLETTERS

NATURE | Vol 451 | 24 January 2008

FliI and the a/b subunits6, the PMF-driven functions of the export
gate and the F0 portion, and sequence/functional similarities between
FliH and the d/b subunits29, these two remotely related systems may
be similar to each other for their entire structural architectures.
METHODS SUMMARY
Salmonella strains and plasmids used in this study are listed in Supplementary
Table 1. L-broth (LB) and soft tryptone agar plates were prepared as described30.
Ampicillin and tetracycline were added as needed at a final concentration of
100 mg ml21 and 15 mg ml21, respectively. Motility assays were carried out
on soft agar plates as described before4. Export assays were done as described
previously30.
The hook–basal bodies were purified and negatively stained with 3% phosphotungstic acid (pH 4.5) on carbon-coated copper grids. Micrographs wererecorded at a magnification of 325,000 with a JEM-1011 transmission electron
microscope (JEOL) operated at 100 kV.
The cellular levels of ATP were measured using the ATP bioluminescenceassay kit CLS II as described previously22.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 8 August; accepted 29 October 2007.
Berg, H. C. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19–54(2003).
2. Minamino, T. & Namba, K. Self-assembly and type III protein export of thebacterial flagellum. J. Mol. Microbiol. Biotechnol. 7, 5–17 (2004).
3. Fan, F. & Macnab, R. M. Enzymatic characterization of FliI: an ATPase involved inflagellar assembly in Salmonella typhimurium. J. Biol. Chem. 271, 31981–31988(1996).
4. Minamino, T. & Macnab, R. M. FliH, a soluble component of the type III flagellar
export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase
activity. Mol. Microbiol. 37, 1494–1503 (2000).
5. Cornelis, G. R. The type III secretion injectisome. Nature Rev. Microbiol. 4, 811–825(2006).
6. Imada, K., Minamino, T., Tahara, A. & Namba, K. Structural similarity between theflagellar type III ATPase FliI and F1-ATPase subunits. Proc. Natl Acad. Sci. USA 104,
485–490 (2007).
7. Vogler, A. P., Homma, M., Irikura, V. M. & Macnab, R. M. Salmonella typhimurium
mutants defective in flagellar filament regrowth and sequence similarity of FliI to
F0F1, vacuolar, and archaebacterial ATPase subunits. J. Bacteriol. 173, 3564–3572(1991).
8. Lane, M. C., O’Toole, P. W. & Moore, S. A. Molecular basis of the interaction
between the flagellar export proteins FliH and FliI from Helicobacter pylori. J. Biol.
Chem. 281, 508–517 (2006).
9. Auvray, F., Ozin, A. J., Claret, L. & Hughes, C. Intrinsic membrane targeting of theflagellar export ATPase FliI: Interaction with acidic phospholipids and FliH. J. Mol.
Biol. 318, 941–950 (2002).
10. Claret, L., Susannah, C. R., Higgins, M. & Hughes, C. Oligomerisation andactivation of the FliI ATPase central to bacterial flagellum assembly. Mol.
Microbiol. 48, 1349–1355 (2003).
11. Minamino, T. et al. Oligomerization of the bacterial flagellar ATPase FliI iscontrolled by its extreme N-terminal region. J. Mol. Biol. 360, 510–519 (2006).
12. Thomas, J., Stafford, G. P. & Hughes, C. Docking of cytosolic chaperone-substratecomplexes at the membrane ATPase during flagellar type III protein export. Proc.
Natl Acad. Sci. USA 101, 3945–3950 (2004).
13. Akeda, Y. & Galán, J. E. Chaperone release and unfolding of substrates in type III
secretion. Nature 437, 911–915 (2005).
1.

14. Minamino, T., González-Pedrajo, B., Kihara, M., Namba, K. & Macnab, R. M. The
ATPase FliI can interact with the type III flagellar protein export apparatus in theabsence of its regulator FliH. J. Bacteriol. 185, 3983–3988 (2003).
15. González-Pedrajo, B., Fraser, G. M., Minamino, T. & Macnab, R. M. Moleculardissection of Salmonella FliH, a regulator of the ATPase FliI and the type III flagellar
protein export pathway. Mol. Microbiol. 45, 967–982 (2002).
16. Hirano, T., Minamino, T., Namba, K. & Macnab, R. M. Substrate specificity classes
and the recognition signal for Salmonella type III flagellar export. J. Bacteriol. 185,
2485–2492 (2003).
17. Kutsukake, K., Ohio, Y. & Iino, T. Transcriptional analysis of the flagellar region of
Salmonella typhimurium. J. Bacteriol. 172, 741–747 (1990).
18. Yamaguchi, S., Fujita, H., Sugata, K., Taira, T. & Iino, T. Genetic analysis of H2, thestructural gene for phase-2 flagellin in Salmonella. J. Gen. Microbiol. 130, 255–265(1984).
19. Lee, S. H. & Galán, J. E. Salmonella type III secretion-associated chaperones confer
secretion-pathway specificity. Mol. Microbiol. 51, 483–495 (2004).
20. Eichelberg, K., Ginocchio, C. C. & Galán, J. E. Molecular and functional
characterization of Salmonella typhimurium invasion genes invB and invC:
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21. Deiwick, J., Nikolaus, T., Erdogan, S. & Hensel, M. Environmental regulation of
Salmonella pathogenicity island 2 gene expression. Mol. Microbiol. 31, 1759–1773(1999).
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motive force but not on the flagellar motor components MotA and MotB. Infect.
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23. Galperin, M. Y., Dibrov, P. A. & Glagolev, A. N. mH1 is required for flagellar growthin Escherichia coli. FEBS Lett. 143, 319–322 (1982).
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25. Schiebel, E., Driessen, A. J. M., Hartl, F.-U. & Wickner, W. DmH and ATP function
at different steps of the catalytic cycle of preprotein translocase. Cell 64,
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27. Yonekura, K., Maki-Yonekura, S. & Namba, K. Complete atomic model of thebacterial flagellar filament electron cryomicroscopy. Nature 424, 643–650(2003).
28. Namba, K. Roles of partly unfolded conformations in macromolecular selfassembly. Genes Cells 6, 1–12 (2001).
29. Pallen, M. J., Bailey, C. M. & Beatson, S. A. Evolutionary links between
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank A. Blocker, A. Veenendaal and K. Imada for criticalreading of the manuscript and comments; N. Moriya, S. Nakamura and K.-I.
Kazetani for technical help; K. Hughes for a gift of an ssaN::tetRA allele; andM. Macnab and F. Oosawa for continuous support and encouragement. This work
was partially supported by Grants-in-Aid for Scientific Research from the Ministryof Education, Culture, Sports, Science and Technology of Japan to T.M. and K.N.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should be
addressed to K.N. (keiichi@fbs.osaka-u.ac.jp).

488©2008 Nature Publishing Groupdoi:10.1038/nature06449

METHODSTransductional crosses and DNA manipulations. P22-mediated transductional crosses were carried out with p22HTint18. DNA manipulations were carried out as described before4.
Motility assay. Fresh colonies were inoculated on soft tryptone agar plates andincubated at 30 uC.
Export assay. Whole cells and culture supernatant fractions were prepared from
Salmonella cells grown at 30 uC in LB, as described previously30.
To test the effect of CCCP on flagellar protein export, the cells were grownwith shaking in 5 ml of LB at 30 uC until the cell density had reached an opticaldensity at 600 nm (OD600) of approximately 0.6–0.7. After washing twice with
LB, the cells were re-suspended in 5 ml LB with or without CCCP and incubatedat 30 uC for 1 h. Cultures were centrifuged to obtain the cell pellets and culture
supernatants. Cell pellets were re-suspended in the SDS-loading buffer, andnormalized to a cell density to give a constant amount of cells. Proteins in theculture supernatants were precipitated by 10% trichloroacetic acid, suspended in
the Tris/SDS loading buffer and heated at 95 uC for 5 min.
Immunoblotting with polyclonal anti-FlgG, anti-FlgD, anti-FlgE, anti-FlgK,
anti-FlgL, anti-FliK, anti-FliC, anti-FliH and anti-FliI antibodies was carried out
as described before30. Detection was performed with an ECL plus immunoblotting detection kit (GE Healthcare).
Preparation of the hook–basal bodies. The hook–basal bodies were prepared as
described previously31 with minor modifications. Samples were negatively
stained with 3% phosphotungstic acid (pH 4.5) on carbon-coated copper grids.
Micrographs were recorded at a magnification of 325,000 with a JEM-1011
transmission electron microscope (JEOL) operated at 100 kV.
Construction of a DinvC::tetRA strain. An invC null strain (DinvC::tetRA) wasconstructed using the l Red homologous recombination system, as described
before32–34.
Measurement of intracellular ATP levels. The cellular levels of ATP were measured using the ATP bioluminescence assay kit CLS II (Roche) as described
previously22. Cultures, 1.5 ml each, were centrifuged, and the pellets were resuspended in 100 mM Tris-HCl pH 7.75, 4 mM EDTA, with adjustment of
the OD600 of the cell suspensions to 1.0. The cell suspensions, 300 ml each, wereboiled for 2 min at 100 uC. Samples were centrifuged, and 100 ml of eachsupernatant was transferred to a microtitre plate that was kept on ice until
measurement. A 100 ml solution of luciferase reagent was added and thenbioluminescence was detected by a LAS-3000 luminescent image analyser(Fujifilm).
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