JOURNAL OF BACTERIOLOGY, June 2006, p. 4190–4197
0021-9193/06/$08.00⫹0 doi:10.1128/JB.01896-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 188, No. 12

Role of the Intramolecular Disulfide Bond in FlgI, the Flagellar
P-Ring Component of Escherichia coli
Yohei Hizukuri,1 Toshiharu Yakushi,1* Ikuro Kawagishi,1,2 and Michio Homma1
Division of Biological Science, Graduate School of Science,1 and Institute for Advanced Research,2Nagoya University, Furo-Cho, Chikusa-Ku, Nagoya 464-8602, Japan
Received 13 December 2005/Accepted 4 April 2006

The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The
intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in
Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bondformation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved inother bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cyseliminated FlgI derivative formed a P ring that complemented the flagellation defect of our ⌬flgI strain when
it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring
assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type
FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of ⌬dsbB cells.
Additionally, the low level of FlgI observed in the ⌬dsbB strain increased in the presence of L-cystine, anoxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the
FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient selfassembly into the P ring.
Many motile bacteria swim by rotating one or more helicalflagella like a screw. Each flagellum consists of three substructures: a helical filament extending from the cell body, a motorthat is embedded in the cell envelope, and a flexible hook thatconnects these two structures. In gram-negative bacteria suchas Escherichia coli and Salmonella spp., the flagellar motor
consists of several ring structures surrounding a rod that penetrates the cell envelope (for reviews, see references 20 and21). The L, P, and MS rings are located in the outer membrane, the peptidoglycan layer, and the cytoplasmic membrane, respectively, whereas the C ring lies on the cytoplasmicside of the MS ring. The P and L rings together form a stiff
cylindrical structure that acts as a bushing to hold the rotatingrod (2). The MS ring/FliG complex is thought to rotate due totorque-generating units (the MotA/MotB complexes), whichsurround the rotor in a circular array. The rod acts as a driving
shaft, transmitting the rotary power of the motor to the flagellar filament (for a review, see reference 6).
The P ring consists of approximately 26 copies of a single
protein, FlgI. The precursor form of FlgI, which has a cleavable N-terminal 19-amino-acid signal sequence, is translocated
via the Sec apparatus to the periplasmic space (13, 14). The
Dsb system facilitates the formation of an intramolecular disulfide bond in the mature form of FlgI (8; see below). FlgA,a periplasmic chaperone, assists the copies of FlgI as theyassemble into the P ring surrounding the growing rod (15, 19,
22, 31). Because flagellar assembly is a highly ordered process,flagellar morphogenesis fails to continue beyond the assemblyof the P ring in the flgI mutant (19).
Many proteins require the formation of disulfide bonds forproper protein folding, stability, and function. In E. coli, disulfide bond formation in periplasmic proteins is catalyzed by theDsb system (for a review, see reference 7); after the periplasmic protein DsbA oxidizes a newly exported target protein, thereduced form of DsbA is reoxidized by DsbB, a cytoplasmic
membrane protein. Dailey and Berg (8) reported that when it
was grown on M63 minimal medium, an E. coli dsbB strain
which was not able to form disulfide bonds in periplasmic
proteins showed a defect in flagellation that was similar to theone observed in the flgI strain. The LP-ring structure was notdetected in the flagellar assemblies isolated from dsbB cells
grown on M63 minimal medium, whereas it was seen when thecells were grown in the presence of L-cystine, an oxidative
agent. Furthermore, during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the mobilityof the FlgI protein from dsbB⫹ cells was changed by the addition of ␤-mercaptoethanol, a reducing agent. From these lines
of evidence, the authors argued that the intramolecular disulfide bond in FlgI is essential for P-ring assembly. The mechanism by which the loss of this disulfide bond in FlgI results in
defective P-ring assembly, however, has remained unclear.
In this study, we assessed the role of the disulfide bond in
FlgI. Surprisingly, a Cys-eliminated FlgI variant formed a P
ring that complemented the flagellation defect of our ⌬flgI
strain, suggesting that the disulfide bond in FlgI is not essential
for P-ring assembly. In the periplasmic space, the Cys-eliminated FlgI variants were more susceptible to degradation thanwild-type FlgI. The level of the FlgI protein in ⌬dsbB cells was
relatively low but was elevated in the presence of L-cystine. The
motility defect of the ⌬dsbB strain was suppressed by overpro-* Corresponding author. Present address: Department of Bioscienceand Biotechnology, Faculty of Agriculture, Shinshu University, 8304
Minamiminowa, Nagano 399-4598, Japan. Phone: 81-265-77-1611.
Fax: 81-265-77-1629. E-mail: alfista@shinshu-u.ac.jp.
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VOL. 188, 2006

ROLE OF THE DISULFIDE BOND IN FlgITABLE 1. Strains and plasmids

Strain orplasmid

Strains
W3110
JM109
BL21(DE3)
HK295
HK320
RP3098
RP437
YZ1
Plasmids
pBAD24
pET-3apLysS
pYZ201
pYZ202
pYZ203
pYZ204pYZ220

Genotype or descriptiona

Wild type
recA1 endA1 gyrA96 thi hsdR17 supE44
relA1 ␭⫺ ⌬(lac-proAB) (F⬘ traD36
proAB lacIq lacZ⌬M15)
T7 expression host
MC1000 ⌬ara-714 leu⫹
HK295 ⌬dsbB⌬(flhD-flhA)4F⫺ thi thr leu his met eda rpsL; wild
type for chemotaxis
RP437 ⌬flgI::cat
PBAD araC Ampr
T7 expression vector, Ampr
T7 lysozyme, Cmr
pBAD24 flgI
pYZ201 C254A
pYZ201 C338A
pYZ201 C254A/C338A
pET-3a flgI (mature FlgI)

Referenceor source

33
32
16
16
29
25
This work
11
Novagen32
This work
This work
This work
This work
This workaAmpr, ampicillin resistant; Cmr, chloramphenicol resistant; PBAD, araBAD
promoter.

duction of the wild-type FlgI protein. Taking these findings
together, we propose that intramolecular disulfide bond formation in FlgI is not absolutely required for P-ring assembly
but is important to prevent the degradation of the protein.
MATERIALS AND METHODS
Bacterial strains, growth conditions, and media. The strains used in this work
are listed in Table 1. The HK295 (dsb⫹) and HK320 (⌬dsbB) strains were kindly
supplied by Hiroshi Kadokura and Jon Beckwith (Harvard Medical School). The⌬flgI::cat strain YZ1 was constructed by deleting the chromosomal flgI gene using
the method described by Datsenko and Wanner (9) and the following primers:⌬flgI(⫹) (5⬘-CTGGTTGCAGCGTTTCTTCCTTAACCTGTCGCCAATGTAACATATGAATATCCTCCTTAG-3⬘) and ⌬flgI(⫺) (5⬘-ATGATTTCCAGTTTTGCCCGCAGACATCCCGCACTTTGCAGTGTAGGCTGGAGCTGCTTC-3⬘). The constructed ⌬flgI::cat region contained the complete flgH open reading frame and an
approximately 40-bp region upstream of the initiation codon of flgJ, which presumably contained the ribosome-binding sequence of this gene. The ⌬flgI::catregion of the chromosome was transduced into the RP437 strain by P1 phage(27). E. coli cells were cultured at 37°C in LB medium (1% Bacto tryptone, 0.5%
yeast extract, and 0.5% NaCl), at 30°C in TG medium (1% Bacto tryptone, 0.5%
NaCl, and 0.5% [wt/vol] glycerol), or in M63 minimal medium supplemented
with 0.4% (wt/vol) glycerol as the sole source of carbon (27). When necessary,
ampicillin and chloramphenicol were added to final concentrations of 50 ␮g/ml
and 25 ␮g/ml, respectively.
Construction of the plasmids. Routine DNA manipulations were carried out
according to standard procedures (26). The plasmids used in this work are listed
in Table 1. The flgI gene was PCR amplified from purified chromosomal DNA of
the W3110 strain using appropriate primers. The PCR product was digested with
KpnI and SphI, and a 1.4-kb flgI fragment was inserted into the correspondingsites of the pBAD24 vector plasmid to obtain pYZ201. To construct pYZ220, theplasmid that encoded a mature form of FlgI under the control of a T7 promoter,
we used the PCR and the NdeI and BamHI sites of the pET-3a vector plasmid(Novagen). The 5⬘ sequence of the insert was 5⬘-CAT(ATG)GAG(CGT)-3⬘,
which encodes the initiator Met-Glu1-Arg2 (the NdeI site is underlined and thesuperscript numbers represent the amino acid residue positions in the matureform of FlgI), and the 3⬘ sequence was 5⬘-ATC(ATC)TAA(GGA)TCC-3⬘, which
encodes Ile345-Ile346-ochre (the BamHI site is underlined). Site-directed mutagenesis was performed as described previously (18).
Motility assays. The fraction of swimming cells was measured as follows. M63
minimal medium with 0.4% (wt/vol) glycerol was inoculated at a 100-fold dilution

4191

with an overnight culture (grown on LB medium at 37°C) and cultured at 30°C.
When necessary, 25 ␮g/ml L-cystine was added at the time of inoculation, and
0.04% L-arabinose was added 2 h after the inoculation. At the exponential
growth phase, the cultures were observed under a dark-field microscope. The
swimming fraction was determined by counting the swimming cells among thetotal cells. The data in this report are the average values from at least threeframes for each set of conditions.
For the swarm assay, a single colony from an LB plate was used to inoculatea soft-agar T broth plate (1% Bacto tryptone, 0.5% NaCl, and 0.27% Bacto agar)
containing 50 ␮g/ml ampicillin and 0.04% L-arabinose. The plates were incubated at 30°C for 11 h.
Detection of FlgI and flagellin. TG medium or M63 glycerol medium was
inoculated at a 100-fold dilution with an overnight culture (grown on LB medium) and cultured at 30°C. When necessary, 50 ␮g/ml ampicillin was added at
the time of inoculation and 0.04% L-arabinose was added 2 h after the inoculation. At the exponential growth phase, the cells from 50 ␮l of the cultures (opticaldensity at 660 nm [OD660], 1.0) were harvested by centrifugation (9,500 ⫻ g, 5min). Otherwise, trichloroacetic acid (TCA) was added to the cultures to a final
concentration of 10% (wt/vol), and the insoluble material was recovered bycentrifugation, washed with acetone, and dried. The centrifuged cells or the
TCA-insoluble material was suspended in sodium dodecyl sulfate (SDS) loading
buffer containing 5% (vol/vol) ␤-mercaptoethanol and then sonicated and heatedat 100°C for 5 min. The proteins were separated by SDS-PAGE. Immunoblotting
was performed using anti-FlgI (see below) or antiflagellin (laboratory stock)antibodies as described previously (34).
Antibodies. The anti-E. coli FlgI antibodies (FlgI346) were raised against themature form of FlgI. Mature FlgI was purified from the BL21(DE3)/pLysS strain
that was harboring plasmid pYZ220. Cells were harvested by centrifugation,
washed with buffer A (10 mM Tris-HCl, 150 mM NaCl, and 0.5 mM phenylmethylsulfonyl fluoride at pH 7.5), and resuspended in buffer A containing 5 mM
MgCl2, 10 ␮g/ml DNase I, and 1% (wt/vol) Triton X-100. The cell suspension
was twice passed through a French press at 500 kg/cm2. After the soluble fraction
was removed by centrifugation (7,000 ⫻ g for 10 min), the insoluble fraction was
suspended in buffer A containing 5 mM MgCl2, 10 ␮g/ml DNase I, and 1%(wt/vol) Triton X-100; sonicated for 1 min on ice; and centrifuged again. The
insoluble fraction was then homogenized in buffer A containing 8 M urea andgently shaken at room temperature overnight. Urea-insoluble material was removed by ultracentrifugation (100,000 ⫻ g for 1 h at 25°C), and the remaining
soluble material was passed through an anion-exchange column (Amersham).
Partially purified mature FlgI was separated by SDS-PAGE and stained with
Coomassie blue R250. The band corresponding to FlgI was excised and used toinoculate rabbits. Anti-FlgI rabbit antibodies were produced and affinity purified
by Biogate Co., Ltd. (Gifu, Japan).
NaN3 treatment. TG medium with 50 ␮g/ml ampicillin was inoculated at a100-fold dilution with an overnight culture (grown on LB medium) and cultured
at 30°C. Four hours later, 0.04% L-arabinose was added to the culture. The cells
were incubated for 10 min at 30°C, and then 2 mM NaN3 was added to one of two
aliquots. After a subsequent 10-min incubation, whole-cell extracts from 50 ␮l ofthe culture (OD660 ⫽ 1.0) were collected by TCA precipitation and analyzed byimmunoblotting using anti-FlgI antibodies as described above.
Pulse-induction experiment. TG medium with 50 ␮g/ml ampicillin was inoculated at a 100-fold dilution with an overnight culture (grown on LB medium)and cultured at 30°C. L-Arabinose (0.04%) was added 2 and 3 h after the
inoculation for the RP3098 and YZ1 strains, respectively. The cells were incubated for 30 min and divided into two aliquots. Each aliquot was centrifuged(2,500 ⫻ g, 5 min, 25°C) to harvest the cells and then suspended in TG medium
with or without 0.04% L-arabinose. Both samples were then incubated for 20 min
at 30°C, and whole-cell extracts from 50 ␮l of the cultures (OD660 ⫽ 1.0) werecollected by TCA precipitation as described above. The YZ1 strain was subjected
to an additional 40-min cultivation. All samples were analyzed by immunoblotting using anti-FlgI antibodies as described above.
Isolation of flagellar structures. The isolation of the flagellar structures was
carried out as described previously (1, 10) with several modifications. TG medium was inoculated at a 100-fold dilution with an overnight culture (grown on
LB medium) and cultured at 30°C. Arabinose was added to a final concentrationof 0.04% 2 h after the inoculation. After a subsequent 3-h cultivation, the cells
were harvested and converted into spheroplasts by adding lysozyme and EDTA
to the final concentrations of 0.1 mg/ml and 2 mM, respectively. The spheroplasts
were lysed by adding Triton X-100 to a final concentration of 1% (wt/vol). To
reduce the viscosity of the samples after lysis was complete, MgSO4 and DNase
I were added to the suspension to the final concentrations of 5 mM and 0.1
mg/ml, respectively. After the viscosity of the solution decreased, EDTA wasadded to a final concentration of 5 mM. Unlysed cells and cellular debris were4192

HIZUKURI ET AL.

J. BACTERIOL.

TABLE 2. Numbers of Cys residues in FlgI proteins
No. of Cys residues
Organism(s)dEscherichia coli
Salmonella spp.
Yersinia pestis
Pseudomonas putida
Vibrio cholerae
Vibrio parahaemolyticus a
Aeromonas hydrophilaa,bAquifex aeolicus
Rhodobacter sphaeroides
Agrobacterium tumefaciens
Caulobacter crescentus
Sinorhizobium meliloti
Helicobacter pylori
Thermotoga maritima

Full-length
FlgI

Predicted
mature FlgI

2
2
2
1
0
0
4
1
1
0
2
1
0
1

2
2
2
0
0
0
2
0
0
0
1
0
0
1

Identity to E. coli
FlgI (%)cshown), and had no flagella, as was confirmed by electron
microscopy (data not shown). The swarming (Fig. 1A) andswimming (data not shown) abilities of the bacteria were restored upon expression of wild-type FlgI from the pYZ201
plasmid. To address the importance of the disulfide bond, we

92.3
78.4
48.8
47.8
46.7
44.4
44.1
43.9
43.9
43.7
43.4
36.5
33.9

a

FlgI of polar flagella.
The cleavage site of the signal sequence was predicted by multiple-alignment
analysis.
c
The identity between the full-length FlgI protein and E. coli FlgI was calculated with GENETYX version 10 (GENETYX).
d
UniProt accession numbers for the FlgI proteins are as follows: E. coli,
P75941; Salmonella spp., P15930; Y. pestis, Q8ZFB1; P. putida, Q52082; V.
cholerae, Q9KQ14; V. parahaemolyticus, Q9X9J4; A. hydrophila, Q8GLP1; A.
aeolicus, O67608; R. sphaeroides, P58204; A. tumefaciens, Q44340; C. crescentus,
P33979; S. meliloti, Q52948; H. pylori, O25028; and T. maritima, Q9X1M5.
bremoved by centrifugation at 8,000 ⫻ g for 20 min. Polyethylene glycol 6000 and
NaCl were added to the lysate to the final concentrations of 3% and 0.15 M,
respectively. The samples were then incubated at 4°C overnight to form bundled
flagella (17). The suspension was centrifuged at 27,000 ⫻ g for 30 min at 4°C, andthe pellet was resuspended in 3 ml of TET buffer (10 mM Tris-HCl, 5 mM
EDTA, and 0.1% [wt/vol] Triton X-100 at pH 8.0). To remove cellular debris, thesolution was centrifuged at 1,000 ⫻ g for 15 min at 4°C. The supernatant wascentrifuged at 100,000 ⫻ g for 30 min, and the pellet was resuspended in 300 ␮lof TET buffer. To dissociate the flagellar filaments into monomeric flagellin, thepreparation was diluted 10-fold in 50 mM glycine-HCl (pH 2.5) and shaken for
30 min at room temperature. The suspension was centrifuged at 150,000 ⫻ g for
40 min at 25°C, and the pellet was resuspended in 20 ␮l of TET buffer.
Electron microscopy. The isolated flagellar structures were negatively stainedwith 2% phosphotungstic acid (pH 7.2) and observed with a JEM-1200EX
electron microscope (JEOL, Japan).

RESULTSThe Cys residues in FlgI are not conserved in other bacterial
species. If the disulfide bond is essential for the structure andfunction of FlgI, the two Cys residues are likely to be conserved
among various species. To address this, we analyzed the primary sequences of the FlgI proteins from various bacterial
species that rely on flagellar motility (Table 2). Some FlgI
proteins of the other species had either one or zero Cys residues in the predicted mature form of FlgI. Thus, the two Cys
residues in E. coli FlgI are not conserved in most of the othermotile bacteria.
Cys-eliminated FlgI variants can restore motility in the⌬flgI strain. In order to confirm the importance of the two Cys
residues in E. coli FlgI, we replaced them and characterized
the resulting mutant proteins. First, we constructed the YZ1
flgI deletion strain of E. coli (⌬flgI::cat) using the gene disruption procedure described by Datsenko and Wanner (9). The⌬flgI strain lost the ability to swarm in semisolid agar (Fig. 1A),
was nonmotile under a dark-field microscope (data not

FIG. 1. Motility and the production of the flagellar structures by
the ⌬flgI strains producing Cys-eliminated FlgI. (A) Swarms of YZ1(⌬flgI) cells harboring pYZ201 (wild-type [wt] FlgI), pBAD24 (vector),or the pYZ201 derivatives (C254A, C338A, and C254A/C338A substitutions). Soft-agar T broth containing L-arabinose was inoculated withsingle colonies and incubated at 30°C for 11 h. (B) Flagellin producedin RP437 or YZ1 cells harboring pBAD24, pYZ201, or the derivativesof pYZ201 was detected by immunoblotting using antiflagellin antibodies. The cells were cultured in the absence or presence of L-arabinose, harvested, and suspended in SDS loading buffer with ␤-mercaptoethanol. The position of flagellin is indicated on the right side of thepanel. Ara, L-arabinose. (C) Electron micrographs of the flagellar
structures isolated from YZ1 cells producing wild-type FlgI (left) andfrom YZ1 cells producing FlgI C338A (right). A P ring stacked with an
L ring, an MS ring, and a hook can be seen. Bar, 20 nm. The defects
in the flagellation and P-ring formation of the ⌬flgI strain were complemented by the Cys-eliminated FlgI derivatives.

VOL. 188, 2006

then replaced the two Cys residues with Ala residues usingsite-directed mutagenesis. Hereinafter, these substitutions arereferred to as C254A, C338A, and C254A/C338A (the numbers correspond to the positions of the amino acid residues in
the mature form of FlgI). The ⌬flgI strain was transformed
with each of the plasmids encoding the Cys-eliminated FlgI
variants, and the transformants were examined for their ability
to spread in soft-agar medium. The ⌬flgI strain with the C338A
mutation was almost as motile as the strain that produced
wild-type FlgI, whereas the C254A and C254A/C338A mutation-containing strains displayed a limited ability to spread in
the agar (Fig. 1A). When the ⌬flgI strain expressed either the
C338A variant or wild-type FlgI, the fractions of cells that wereobserved to swim in liquid medium were roughly the same(data not shown).
The formation of flagella in the strains was examined byimmunoblotting analysis using antiflagellin antibodies. An intense band was detected in the whole-cell extract from cells
expressing the C338A variant or wild-type FlgI. On the other
hand, in the C254A and C254A/C338A preparations, significant but much weaker bands were detected (Fig. 1B). As
judged by electron microscopy, the average number of flagellaper ⌬flgI cell was slightly smaller in cells producing the C338A
variant than in cells producing wild-type FlgI (data not shown).
The C254A and C254A/C338A substitutions significantly decreased the number of flagella to approximately 10% of thenumber observed in wild-type bacteria and resulted in thefrequent occurrence of shorter filaments (data not shown).
These results show that the C338A substitution had a very
limited effect on flagellar formation but that C254A andC254A/C338A produced more-profound effects. Since flagellation without the LP ring in an flgI strain of E. coli has been
observed through an overproduction of the FlgE hook protein(23), we examined P-ring formation in the C338A variantproducing ⌬flgI strain. Isolated flagellar structures from ⌬flgI
cells producing wild-type FlgI or the C338A variant contained
P-ring structures (Fig. 1C). No rivet-like structures, i.e., flagellar structures missing the LP ring (19), were observed in these
preparations. These results strongly argue that the disulfide
bond in the FlgI protein is not essential for the formation of
the P ring by this protein.
The Cys-eliminated FlgI variants are degraded in the
periplasmic space. The mutant FlgI proteins expressed in the⌬flgI strain were examined by immunoblotting using anti-FlgI
antibodies (Fig. 2A). Although the C338A protein and wildtype FlgI each restored the motility of the ⌬flgI strain, theamount of the C338A protein was significantly less than theamount of wild-type FlgI (Fig. 2A, lanes 5 and 9). Additionally,
the levels of the C254A and C254A/C338A proteins were lower
than that of the C338A protein (Fig. 2A, lanes 7 and 11), whichis consistent with the swarm size and the degree of flagellin
and filament production observed for cells expressing these
proteins. These results suggest that the intramolecular disulfide bond in FlgI is involved in the accumulation of the
FlgI protein.
FlgI is synthesized as a precursor protein with a cleavable
19-amino-acid signal sequence at the N terminus before it istransported to the periplasmic space via the Sec apparatus. In
order to determine if the FlgI bands shown in Fig. 2A was themature form or the precursor form of the protein and how

ROLE OF THE DISULFIDE BOND IN FlgI

4193

FIG. 2. Levels of the mature and the precursor forms of FlgI in the⌬flgI strain producing Cys-eliminated FlgI. The FlgI proteins from
RP437 and YZ1 cells harboring pBAD24 (vector), pYZ201 (wild-type
FlgI), or the derivatives of pYZ201 (C254A, C338A, and C254A/
C338A substitutions) were detected by immunoblotting using anti-FlgI
antibodies. Whole-cell extracts were prepared by TCA precipitation.
(A) The position of FlgI is indicated on the right side of the figure.
Ara, L-arabinose. (B) A culture in the exponential growth phase was
supplemented with L-arabinose and incubated for 10 min. Then thealiquot was incubated for another 10 min in the absence or presence of
2 mM NaN3. The positions of the precursor (38-kDa band) and themature (36-kDa band) forms of FlgI are indicated on the right side of
the figure. Whereas the levels of the mature forms of the Cys-eliminated FlgI derivatives were lower than that of wild-type FlgI, those of
the precursor forms were unchanged.

much the precursor forms can be detected, ⌬flgI cells producing the Cys-eliminated FlgI variants were treated with NaN3, a
SecA inhibitor (24), and examined by immunoblotting (Fig.
2B). The mature form (36-kDa band) was the only band detected in all the preparations that did not receive the NaN3
treatment. On the other hand, an additional 38-kDa bandcorresponding to the precursor form appeared in the wild-type
and the derivative FlgI samples after the NaN3 treatment. The
accumulated amounts of the precursor forms of wild-type FlgIand the FlgI derivatives were comparable. These results suggest that like wild-type FlgI, the Cys-eliminated FlgI variants
were produced and translocated to the periplasmic space. The
levels of the mature FlgI variants significantly decreased in thepresence of NaN3 (Fig. 2B, compare the lanes with and without
NaN3 treatment), whereas wild-type FlgI levels were almost
unchanged (Fig. 2B, lanes 3 and 4). The levels of the matureforms after the NaN3 treatment were indicative of the proteinin the periplasmic space after the inhibition of the transloca-

4194

HIZUKURI ET AL.

FIG. 3. Degradation of the Cys-eliminated FlgI variants in flhDC
cells. The FlgI proteins from RP437 or RP3098 (flhDC) cells harboring
pBAD24 (vector), pYZ201 (wild-type [wt] FlgI), or the pYZ201 derivatives (C254A, C338A, and C254A/C338A substitutions) weredetected by immunoblotting using anti-FlgI antibodies. In the pulseinduction experiment, the second incubation lasted for 20 min. Wholecell extracts from RP437 cells at the exponential growth phase wereprepared as a positive control. For comparison, a fifth of a sample from
cells producing wild-type FlgI was loaded on the gel (lanes 3 and 8).
The position of FlgI is indicated on the right side of the figure. Ara@chase, L-arabinose in the medium for the second incubation. The
Cys-eliminated FlgI derivatives were depleted when the production of
the protein was arrested.

tion of newly synthesized FlgI. Thus, we speculate that in the
periplasmic space, the Cys-eliminated FlgI variants were degraded more quickly than wild-type FlgI.
We performed a pulse-induction experiment to examine thedegradation of the Cys-eliminated FlgI variants. Arabinose wasadded to the cell cultures at the exponential growth phase, andthe cultures were incubated for 30 min to induce protein synthesis. Then the medium was exchanged to one with or withoutarabinose and each culture was further incubated for 20 min,followed by immunoblotting analysis (Fig. 3). To avoid complexity, the flhDC strain RP3098 was used in this experiment.
Because this strain cannot express all the flagellar genes, theJ. BACTERIOL.

FlgI proteins expressed from the plasmid are not incorporated
into flagella. The Cys-eliminated FlgI variants were depleted
within 20 min when incubated in the absence of arabinose (Fig.
3, lanes 9 to 11), whereas wild-type FlgI remained (Fig. 3, lane
8). These lines of evidence suggest that FlgI protein that cannot form the intramolecular disulfide bond is susceptible todegradation in the periplasmic space.
Even without the disulfide bond, the FlgI protein is stabilized when incorporated into the flagellar structures. In the⌬flgI strain, some of the plasmid-derived FlgI protein is presumed to exist as the P rings incorporated into the flagella. To
address whether the FlgI derivatives incorporated into theflagella were still susceptible to degradation, we performed apulse-induction experiment with the ⌬flgI strain YZ1 (Fig. 4).
In the ⌬flgI strain, some of the C338A protein remained after
a 20-min incubation (Fig. 4, lane 15), or even after an additional 40-min incubation (Fig. 4, lane 20), although withoutarabinose, the Cys-eliminated FlgI variants were depleted
within 20 min in the flhDC strain (Fig. 3). Consistent with theseresults, after a 60-min incubation, the C338A protein-producing cells were still able to swim (data not shown). Although the
FlgI C338A protein was degraded in the flhDC strain, it remained in the ⌬flgI strain, indicating that some of the C338A
protein was not rapidly degraded, presumably due to incorporation into the flagella as the P rings. If this was the case,overproduction of the FlgI protein should suppress the motility
defect of dsb cells by allowing FlgI to accumulate to sufficientlevels in the periplasmic space for P-ring assembly, in spite of
the absence of the normal mechanism for disulfide bond formation.
Overproduction of the FlgI protein suppresses the motility
defect of the ⌬dsbB strain. It was previously proposed that the
intramolecular disulfide bond in the FlgI protein is essential
for P-ring assembly. Our experimental data, however, suggestthat it is instead required to prevent degradation of the proteinin the periplasmic space. To investigate the possibility that the
intramolecular disulfide bond in FlgI is involved in limiting thedegradation of the protein, we introduced the FlgI-encoding

FIG. 4. Levels of the Cys-eliminated FlgI proteins in ⌬flgI cells after the production of the proteins was halted. The FlgI protein from RP437
or YZ1 (⌬flgI) cells harboring pBAD24 (vector), pYZ201 (wild-type [wt] FlgI), or the pYZ201 derivatives (C254A, C338A, and C254A/C338A
substitutions) were detected by immunoblotting using anti-FlgI antibodies. The sample preparations were the same as those used for Fig. 3, withsecondary incubations of 20 min (lanes 2 to 6 and 12 to 16) or 60 min (lanes 7 to 11 and 17 to 21). For comparison, a fifth of a sample from cells
producing wild-type FlgI was loaded on the gel (lanes 3, 8, 13, and 18). The position of FlgI is indicated on the right side of the figure. The asterisk
denotes a nonspecific band. The chase time is the length of the second incubation in minutes. Ara @chase, L-arabinose in the medium during thesecond incubation. Some of the FlgI C338A derivative remained even 60 min after the production of the protein was arrested.

VOL. 188, 2006

ROLE OF THE DISULFIDE BOND IN FlgI

FIG. 5. Fraction of the ⌬dsbB cells overproducing FlgI that wasobserved to be motile. The fractions of HK295 (dsb⫹) cells, HK320(⌬dsbB) cells, and HK320 cells harboring pYZ201 (wild-type FlgI) thatwere observed to swim were measured. The cells were cultured in M63
glycerol minimal medium in the absence or presence of L-cystine andL-arabinose. Ara, L-arabinose. The motility defect of the ⌬dsbB strain
was suppressed by overproducing FlgI.

plasmid into HK320 cells, a ⌬dsbB strain that does not form
disulfide bonds in periplasmic proteins, and measured the fraction of the transformants that swam in liquid M63 minimal
medium (Fig. 5). As described previously (8), the ⌬dsbB mutation inhibited swimming ability, and the defect was rescuedby the addition of 25 ␮g/ml L-cystine. The level of FlgI undereach set of conditions was examined by immunoblotting analysis (Fig. 6). The level of FlgI decreased in the ⌬dsbB strain(Fig. 6, compare lanes 1 and 9) but increased after the additionof cystine (Fig. 6, compare lanes 9 and 11). This result indicatesthat the formation of the disulfide bond in the FlgI protein
confers resistance to degradation.
Even in the absence of cystine, ⌬dsbB cells overproducing
FlgI showed a nearly wild-type level of motility (Fig. 5). For an
unknown reason, the fraction of motile FlgI-overexpressing⌬dsbB cells slightly decreased upon the addition of cystine. We
examined whether each strain was able to spread in M63 softagar plates. The ⌬dsbB strain overproducing FlgI swarmed ata significantly higher rate than the ⌬dsbB strain did (data notshown). These results show that overproduction of FlgI suppresses the motility defect of the ⌬dsbB strain. This supports
the idea that the disulfide bond in FlgI is not required for
P-ring assembly. The overproduction of FlgI considerably increased the amount of FlgI in the ⌬dsbB strain (Fig. 6, compare lanes 9 and 14). The increased level of the FlgI protein
may account for the suppression of the motility defect of the⌬dsbB strain.
DISCUSSION
We propose that the intramolecular disulfide bond in FlgI is
not absolutely required for P-ring assembly. In flgI cells, theflagellar structures are constructed up to the rivet that contains
the MS ring and the rod (19). Since the FlgI chaperone FlgA
is present in the ⌬flgI strain, the FlgI protein derived from the

4195

FIG. 6. Level of FlgI in the ⌬dsbB strain. The FlgI proteins from
HK295 (dsb⫹) and HK320 (⌬dsbB) cells harboring pBAD24 (vector)
or pYZ201 (wild-type FlgI) were detected by immunoblotting using
anti-FlgI antibodies. The cells were cultured in M63 glycerol minimal
medium in the absence or presence of L-cystine and L-arabinose. The
whole-cell extracts were prepared by TCA precipitation and suspended
in SDS loading buffer with ␤-mercaptoethanol. The position of FlgI is
indicated on the right side of the figure. Ara, L-arabinose. The low levelof the FlgI protein in the ⌬dsbB strain increased in the presence ofL-cystine.

plasmid is able to form a P ring around the preexisting rod.
Although the FlgI C338A protein cannot form the intramolecular disulfide bond, it was able to form an apparently normal
P-ring structure (Fig. 1C) and restored flagellation and motility
in the ⌬flgI strain when overproduced, as wild-type FlgI did(Fig. 1A and B). We therefore concluded that a P ring constructed with FlgI that did not have the intramolecular disulfide bond was structurally and functionally normal. However,the disulfide bond might somehow participate in the efficient
self-assembly of FlgI into the P ring. To address such an issue,
we have to develop some methods for assessing the self-assembly process.
The intramolecular disulfide bond in FlgI likely reduces thesusceptibility of the protein to degradation in the periplasmic
space. In the present study, we showed that the levels of themature forms of the Cys-eliminated FlgI variants were significantly lower than that of wild-type FlgI (Fig. 2), and theCys-eliminated FlgI variants were more susceptible to degradation than wild-type FlgI (Fig. 3 and 4). Additionally, theamount of FlgI in the ⌬dsbB strain increased in the presence ofL-cystine (Fig. 6). It is generally postulated that in dsb strains,most of the substrates of the Dsb proteins are not able to form
disulfide bonds, resulting in unstable proteins that aggregate orare proteolyzed. For example, the PhoA protein, which is oneof the most characterized substrates of DsbA, has four Cys
residues that form two disulfide bonds. Under reducing conditions or in the absence of the Dsb system, the PhoA protein
is unable to form the disulfide bonds and susceptible to protease in vivo and in vitro (3, 5). Additionally, the reduced formof the OmpA protein is also susceptible to protease treatment
in vitro (4, 5). Recently, Hiniker and Bardwell systematicallycompared the proteome of the dsbA strain with that of wildtype E. coli to identify in vivo periplasmic substrates of DsbA
(12). Those authors found that the levels of 10 periplasmic
proteins that contained at least two Cys residues decreased in
the dsbA strain. These results support the assumption that theabsence of the folding catalyst DsbA causes its substrates to beunstable. Thus, we propose that the disulfide bond in FlgI is
involved in forming a rigid structure that reduces the susceptibility of the protein to degradation.

4196

HIZUKURI ET AL.

Dailey and Berg proposed that the two Cys residues of FlgI
form an intramolecular disulfide bond, which is essential for
P-ring assembly (8). Because a pulse-labeling experiment using
minicells showed that L-cystine had no effect on the level of
FlgI, the authors argued against the possibility that the disulfide bond is involved in the stability of the FlgI protein. Theseobservations appear inconsistent with the present study. Suchan inconsistency might be explained by the properties of minicells. Because minicells do not have a chromosome, proteins
are not produced via chromosomal expression. The levels ofproteins that have a rapid turnover rate under reducing conditions (28) (e.g., DegP [HtrA], a periplasmic protease/chaperone) would decrease in minicell preparations. Therefore, it ispossible that some of the factors involved in degrading incorrectly folded FlgI proteins are missing in minicells.
Spontaneous oxidation of the FlgI protein may account forthe suppression of the motility defect of the ⌬dsbB strain by
FlgI overproduction (Fig. 5). In fact, approximately 10% of⌬dsbB cells were motile in the absence of L-cystine under our
experimental conditions. It has been reported that disulfide
bond formation in OmpA, an outer membrane protein, andPhoA, a periplasmic protein, takes place spontaneously but
slowly in the absence of the DsbA/DsbB system (4, 5), presumably due to the oxidative environment of the periplasmic space.
Therefore, to assess spontaneous oxidation in our experiments,
we examined the motility of ⌬dsbB cells producing the Cyseliminated FlgI variants. Overproducing FlgI C338A restored
the swimming ability of the ⌬dsbB cells to almost the samelevel as wild-type FlgI, whereas overproduction of both FlgIC254A and FlgI C254A/C338A partially rescued the motility
defect of these cells (data not shown). These results suggestthat the effects of spontaneous oxidation were negligible in our
overproduction experiments.
Because DsbA immediately acts on newly translocatedperiplasmic proteins (30), the disulfide bond forms in newlytranslocated FlgI as it enters the periplasmic space. With theassistance of FlgA (22), the folded monomers are assembled
into the P ring around the growing rod. In ⌬dsbB strains,
however, disulfide bond formation and therefore the folding of
the FlgI monomers should be the rate-limiting step in the
P-ring formation pathway. In these strains, most of the translocated FlgI proteins likely aggregate or are degraded, with
only a small fraction of FlgI folding appropriately without thedisulfide bond. It is plausible that in strains that are deficient indisulfide bond formation, the chromosomal expression level of
FlgI is insufficient to allow P-ring assembly due to the limited
amount of the required building blocks. Overproduction froma plasmid is necessary to construct the P ring with FlgI proteinsthat do not have the intramolecular disulfide bond.
We found that the Cys-eliminated FlgI variants were rapidly
degraded in the flhDC strain (Fig. 3), but some of the FlgI
C338A protein remained in the ⌬flgI strain even after FlgI
production was arrested for 60 min (Fig. 4). These results
indicate that the FlgI protein even without the disulfide bondis resistant to degradation in the presence of the other flagellar
proteins. This resistance could be due to P-ring formation,
because it can be postulated that the C338A proteins thatremained after the 60-min incubation were incorporated into
the flagellum as a P ring. Another possibility is that FlgA
associates with the FlgI protein to protect it from degradation

J. BACTERIOL.

(22). The first possibility is supported by several lines of evidence. A previous study reported that the LP ring is fairly
stable; several chemical treatments failed to disassemble thestructure in vitro (2). In this study, no flagellar structures without a P ring were observed by electron microscopy in theflagellar preparations from the cells expressing the C338A
protein (data not shown), suggesting that P rings made up of
FlgI C338A were not disassembled during the preparation
processes. Also, a P ring made with the C338A protein must be
moderately stable in vivo, because ⌬flgI cells with the C338A
mutation retained their swimming ability after the synthesis of
the C338A protein was arrested for 60 min. Thus, we suggestthat once FlgI is incorporated into the flagellar basal body, theprotein becomes resistant to degradation even without the
intramolecular disulfide bond.
ACKNOWLEDGMENTS
We express thanks to Kazuhiro Kutsukake (Okayama University)
for kindly providing the anti-Salmonella FlgI antibodies, which were
useful in the beginning of this study, and to Hiroshi Kadokura and Jon
Beckwith (Harvard Medical School) for the dsbB strains and plasmids.
We thank Hiroyuki Terashima, Akari Shinohara, Teppei Morita, andToshifumi Inada (Nagoya University) for technical assistance. We are
grateful to Mike Manson (Texas A&M University), Chi Aizawa (Hiroshima Prefectural University), and Hideyuki Matsunami (Osaka University) for their invaluable suggestions about the manuscript.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan;
from the Japan Science and Technology Corporation (to M.H., I.K.
and T.Y.); and from the Soft Nano-Machine Project of the Japan
Science and Technology Agency (to T.Y. and M.H.).
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