Microbiology (2008), 154, 810–817

DOI 10.1099/mic.0.2007/013854-0

Systematic Cys mutagenesis of FlgI, the flagellar Pring component of Escherichia coli
Yohei Hizukuri, Seiji Kojima, Toshiharu Yakushi,3 Ikuro Kawagishi4
and Michio Homma
Correspondence
Michio Homma

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho,
Chikusa-Ku, Nagoya 464-8602, Japan

g44416a@cc.nagoya-u.ac.jp

Received 8 October 2007
Revised

24 November 2007

Accepted 26 November 2007

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the
peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring,which is located in the peptidoglycan layer, is thought to be required for smooth rotation and tofunction as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli
P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa
mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts offive FlgI variants were significantly decreased. Swarm assays showed that almost all of thevariants had nearly wild-type function, but five variants significantly reduced the motility of thecells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The fiveresidues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate
which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine
modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide,and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled.
Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues
known to be important for protein amount or motility. From these results and multiple alignments ofamino acid sequences of various FlgI proteins, the highly conserved region in the N terminus,
residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure
and the formation of the P ring by interacting with FlgI molecules and/or other flagellar
components.

INTRODUCTION
Bacteria swim by the rotation of flagella in a screw-likemanner. The flagellar motor is embedded in the cytoplasmic membrane and the rotational power generated by the
motor is transmitted to the helical flagellar filament
through the hook. The flagellar motor, which is composedof numerous proteins, is divided into two parts, the rotor
and the stator that surrounds the rotor. The rotor is mainlycomposed of the MS ring and the C ring, which is locatedon the cytoplasmic side of the MS ring. The stator is
3Present address: Department of Bioscience and Biotechnology,
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa,
Nagano 399-4598, Japan.
4Present address: Department of Frontier Bioscience, Faculty ofEngineering, Hosei University, 3-7-2 Kajino-Cho, Koganei, Tokyo 1848584, Japan.
Abbreviation: mPEG-maleimide, methoxypolyethylene glycol 5000
maleimide.
A supplementary figure showing multiple alignments of FlgI amino acidsequences derived from various bacteria is available with the online
version of this paper.

810

composed of the MotA/MotB complex, which contains atleast four molecules of MotA and two molecules of MotB
(Kojima & Blair, 2004), and functions as a proton channel(Blair & Berg, 1990; Stolz & Berg, 1991). About 10 of thesestator units surround the rotor, and interactions between
the stator and rotor are believed to generate the driving
force for rotation (Reid et al., 2006). The MotA/MotB
stators assemble around the flagellar basal body, and thefunctional flagellar motor is established. It is thought thatthe peptidoglycan binding motif of MotB is involved in theassembly. A recent study has revealed that in Escherichia
coli, the stator complexes incorporated into the motor are
exchanged frequently with the one that floats on thecytoplasmic membrane near the motor (Leake et al., 2006),
suggesting that the interaction between the rotor and thestator is weak and/or temporary.
The P ring is one of the components of the basal body. In
Gram-negative bacteria, it assembles around a proximal
part of the basal body, and is thought to be attached to the
peptidoglycan layer; it forms a stiff cylindrical structure to
hold the central rod with the L ring, which assembles at the
LPS (outer membrane) layer (Akiba et al., 1991). The P
2007/013854 G 2008 SGM Printed in Great Britain

Systematic Cys mutagenesis of FlgI

ring is a part of the basal body, but is believed to be a nonrotating component to hold the rod as a bushing. The P
ring is thought to consist of 26 copies of a single protein,
FlgI (Jones et al., 1990; Sosinsky et al., 1992), which isexpressed as a precursor form with a cleavable N-terminal
19 aa leader sequence, and exported to the periplasmic
space via the Sec apparatus (Homma et al., 1987; Joneset al., 1989), where it assembles into the P ring surrounding
the rod (Kubori et al., 1992). The flagellar structure isconstructed by a highly ordered process. First the MS ringis assembled at the cytoplasmic membrane as a base plate,then the C ring, the transport apparatus and the rod
structure are assembled in turn. Next, the P- and L-ring
structures are assembled around the rod, followed by thehook and the filament. Disruption of any flagellar
component causes the assembly of flagellar structure toarrest; a disruption in FlgI causes a motility defect because
the flagellar construction terminates at the rod structure.
Recently, we revealed that the intramolecular disulfide
bond formation in FlgI is not necessary for P-ring assembly
but is important to protect against degradation of theprotein (Hizukuri et al., 2006).
Various interactions have been speculated for the P-ringprotein FlgI in the flagellar basal body (Fig. 1). To expand
our knowledge of the P-ring structure and to understandthe spatial arrangement around the rod in the periplasmic
space, we constructed and characterized a series ofsystematically Cys-substituted E. coli FlgI variants.
Among 32 FlgI Cys variants constructed, the protein
amounts of five FlgI variants were significantly decreased,
and cells carrying five of the variants showed reduced
motility. We further characterized the variants using a
thiol-specific reagent to investigate which residue of theprotein was exposed to solvent on the protein surface.
Interestingly, this work showed that the residues of FlgI

that can be labelled never overlap with the residues foundto be important for protein stability or motility.

METHODS
Bacterial strains, growth conditions, and media. The E. coli

strains used in this work are listed in Table 1. To delete the cat gene
cassette of the DflgI : : cat strain YZ1 (Hizukuri et al., 2006), we used
the method described by Datsenko & Wanner (2000), and theconstructed strain was named YZ11. The DmotAB : : cat strain YS5 was
kindly provided by Yoshiyuki Sowa (Oxford University). To construct
a DflgI DmotAB : : cat triple deletion strain, the DmotAB : : cat region of
YS5 was transferred into strain YZ11 by using P1 phage (Silhavy et al.,1984), and the resultant strain was named YZ12-1. E. coli cells were
cultured at 37 uC in LB medium (1 % Bacto tryptone, 0.5 % yeast
extract, 0.5 % NaCl) or at 30 uC in TG medium (1 % Bacto tryptone,
0.5 % NaCl, 0.5 %, w/v, glycerol). When necessary, ampicillin andkanamycin were added to a final concentration of 50 mg ml21.
Construction of plasmids. Routine DNA manipulations werecarried out according to standard procedures (Sambrook et al.,1989). The plasmids used in this work are listed in Table 1. To
construct the pYZ301 plasmid, a 1.4 kb KpnI–SphI fragment
containing the E. coli flgI gene was cut out of pYZ201 (Hizukuri
et al., 2006) and inserted into the corresponding sites of the vector
pSU38. To obtain a series of plasmids expressing FlgI Cys variants, we
performed site-directed mutagenesis on pYZ301 by which a fulllength of plasmid DNA was amplified by PfuUltra High-Fidelity DNA
Polymerase (Stratagene) using a pair of complementary primers
carrying a mutagenized codon, and was then digested by DpnI. To
construct the pJN726 plasmid, a 2.4 kb SalI–SalI fragment containing
the motAB genes was cut out of pYA6022 (Asai et al., 2003) andinserted into the vector pBAD24.
Motility assays. Swarming motility was assayed as follows.
Overnight culture (2 ml) (grown on LB medium at 37 uC) was

dropped on a soft agar T broth plate (1 % Bacto tryptone, 0.5 % NaCl,
0.27 % Bacto agar) containing 50 mg ml21 each of ampicillin andkanamycin and 0.04 % L-arabinose. If necessary, 5 mM DTT wasadded to agar plates. The plates were incubated at 30 uC for the time
indicated for each experiment. Relative swarm size of the FlgI Cys
mutant was calculated by normalizing to the diameter of the swarm
ring of the wild-type FlgI-expressing cells after subtracting thediameter of the swarm ring of the vector-containing cells.
Detection of FlgI. Immunoblot analysis using anti-E. coli FlgI
antibodies (FlgI346) was performed as described previously (Hizukuri
et al., 2006).
Cysteine modification by methoxypolyethylene glycol 5000
maleimide (mPEG-maleimide). An overnight culture (grown on LB

Fig. 1. Information about the P-ring protein FlgI; possible
interactions between the P-ring protein FlgI and other components. In the left-hand image, cylindrically averaged flagellar
structure reported by DeRosier (1998) is outlined. The estimatedP-ring region is shaded. The drawing of the FlgI monomer
displayed in the right-hand box is an estimated shape. PG,
peptidoglycan.
http://mic.sgmjournals.org

medium at 37 uC) was inoculated at a 50-fold dilution into TG
medium containing 50 mg ml21 ampicillin and kanamycin and 0.04 %L-arabinose, and cultured at 30 uC. At the exponential growth phase,
300 ml of cultured cells (OD66051.0) was harvested by centrifugation(9500 g, 5 min). The cells were suspended in 1 ml Wash Buffer(10 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM
EDTA-K), centrifuged again and resuspended in 25 ml MLM Buffer(Wash Buffer containing 10 mM DL-lactate/KOH and 0.1 mM Lmethionine). The thiol-specific reagent mPEG-maleimide (Fluka) wassuspended in DMSO as a 40 mM stock solution and stored at 220 uC
in the dark. Twenty-five microlitres of mPEG-maleimide reaction
buffer (MLM Buffer containing 4 mM mPEG-maleimide, preparedfreshly before each experiment) was added to cell suspensions andmixed well, then incubated with shaking at 37 uC for 30 min. To
811

Y. Hizukuri and others

Table 1. Strains and plasmids
Strain or plasmid
Strains
RP437
YZ1
YZ11
YS5
YZ12-1
Plasmids
pBAD24
pSU38
pYZ201
pYZ301
pYA6022
pJN726

Genotype or description*

Reference or source

F2 thi thr leu his met eda rpsL wild-type forchemotaxis
RP437 DflgI : : cat
RP437 DflgI
RP437 DmotAB : : cat
RP437 DflgI DmotAB : : cat

Hizukuri et al. (2006)
This work
Yoshiyuki Sowa
This work

PBAD araC Ampr
Plac lacZa Kmr
pBAD24 flgI
pSU38 flgI
pSU41 motAB
pBAD24 motAB

Guzman et al. (1995)
Bartolome et al. (1991)
Hizukuri et al. (2006)
This work
Asai et al. (2003)
This work

Parkinson & Houts (1982)*Abbreviations: Ampr, ampicillin resistant; Kmr, kanamycin resistant; PBAD, araBAD promoter; Plac, lacpromoter.

terminate the reaction, 5 ml b-mercaptoethanol was added and then
5 ml 10 % SDS. The sample solution was boiled at 100 uC for 5 min
and mixed with 15 ml 56 SDS loading buffer containing bmercaptoethanol. An aliquot of 5 ml was used for SDS-PAGE.

RESULTS
Protein amounts and cross-linked products of
FlgI Cys variants
We constructed the E. coli DflgI DmotAB : : cat triple
deletion strain YZ12-1. The constructed strain showed no
motility in either liquid or soft agar medium, and noflagella were observed by electron microscopy (data notshown). When transformed with two plasmids, one
harbouring the flgI gene and the other the motAB genes,
YZ12-1 cell motility was almost the same as that of wildtype cells in liquid medium (data not shown). Hereafter,
we refer to this complemented strain as the wild-type FlgIexpressing strain. In the future we plan to investigate theinteraction between the P ring and the MotA/MotB stator
complex; however, we focused our current investigation on
the P-ring structure and its function.
We systematically constructed a series of FlgI Cys variants.
We designed cysteine substitutions every 10th residue inthe mature form of FlgI (the numbers correspond to thepositions of the amino acid residues in the mature form of
FlgI). Two additional mutants were also designed in which
Ile3 and Ile346 were substituted with cysteine (Fig. 6). FlgI
protein has two native cysteine residues, at positions 254
and 338, and they remained intact in our Cys-substituted
variants. From the 37 candidates designed, we obtained 32
FlgI Cys variants, but were unable to generate D41C, Q61C,
A131C, Q221C or L261C. We examined the protein
amounts of the FlgI Cys variants by immunoblot analysis
812

using anti-FlgI antibodies (Fig. 2). Most of the FlgI variants
showed slightly decreased amounts of product comparedto wild-type FlgI. However, the protein amounts of fivevariants, I3C, D111C, I181C, G241C and L251C, weresignificantly decreased compared to the other variants(Fig. 2, b-ME +, filled triangles). We have reported that
FlgI C254A, FlgI C338A and the double mutant seem to be
more susceptible to degradation (Hizukuri et al., 2006), so
we speculate that FlgI Gly241 and Leu251 (which is locatednear Cys254) affect the susceptibility of the protein todegradation when replaced with Cys. In FlgI E1C, an
additional band that was larger (by ~2 kDa) than theestimated monomer band was detected. The larger protein
may be a precursor form (38 kDa) of the mature form of
FlgI (36 kDa) because replacement of the residue next to asignal cleavage site probably affects the cleavage efficiency.
In the absence of the reductant b-mercaptoethanol,
disulfide cross-linked products were detected in most of
the FlgI Cys variants (Fig. 2, b-ME -). The apparent
molecular masses of the cross-linked products showed awave-like pattern with a peak at residues ~160–190. The
variants with Cys replacements at the N- or C-terminal
regions had the approximate estimated size of dimers(72 kDa). On the other hand, replacements near the central
region caused decreased mobility of the bands, probably
because the cross-linked dimers at the middle positions
formed aberrant shapes. It is worth noting that FlgI Y191C
showed a large number of cross-linked products, whereas
FlgI N171C and FlgI A321C showed almost no cross-linked
products.
Effects of the FlgI Cys variants on motility
Defects in FlgI cause a failure of P-ring assembly and resultin the termination of flagellar formation after rod
construction. To assess the effects of the Cys replacements
Microbiology 154

Systematic Cys mutagenesis of FlgI

Fig. 2. Detection of products of systematically
substituted FlgI Cys variants. The FlgI proteinsfrom YZ12-1 (DflgI DmotAB : : cat) cells harbouring pJN726 (MotAB) and pYZ301 (wt
FlgI), pSU38 (vec.) or the pYZ301 derivatives
containing a series of FlgI Cys variants weredetected by immunoblotting using anti-FlgI
antibodies. Upper panel, without b-mercaptoethanol (b-ME -); lower panel, with bmercaptoethanol (b-ME +). The arrowheads
on the right-hand side indicate the monomer ofthe mature form of FlgI (36 kDa). Filled
triangles below the lower panel indicate the
FlgI Cys variants for which the amount ofprotein was significantly lower than that of thewild-type.

in FlgI on P-ring assembly, we first examined swarming
ability in soft agar plates. We measured swarm ring sizes
after sufficient incubation and calculated the relativeswarm rates for each FlgI Cys mutant against that ofwild-type FlgI (Fig. 3). Most of the FlgI Cys mutantsretained swarming ability, but five mutants, FlgI I3C,
G21C, G51C, G81C and D111C, had significantly
decreased swarm rates (see also Fig. 4a, upper panel).
When we observed swimming ability in liquid medium
using dark-field microscopy, cells carrying each of these
five mutations except G21C were motile, although thefractions of motile cells were extremely low (,5 %); cells
carrying the G21C mutation completely lost swimming
ability. When we observed the cells by electron microscopy,
the mutant cells expressing FlgI G21C were shown to becompletely non-flagellate (data not shown), implying that
the Gly21 residue of FlgI is critical for P-ring assembly.
We investigated the effects of reducing agents such as DTT
on the motility of the weakly motile mutants FlgI I3C,
G51C, G81C and D11C, and the non-motile mutant G21C

(Fig. 4). In the absence of DTT, these five mutants showedpoor or no motility, as described above. On the other hand,when 5 mM DTT was added to the motility agar, the swarm
ring size of the wild-type cells was slightly decreased, but thefour weakly motile mutants had significantly restored
swarming abilities. The non-motile mutant G21C remainedcompletely non-motile. These results suggest that the
motility defects of the four weakly motile mutants werecaused by the formation of an incorrect disulfide bond by
the replacement Cys either within FlgI itself or with anotherCys-containing protein(s).
Thiol modification of the FlgI Cys variants by
mPEG-maleimide
To obtain structural information about FlgI, we examinedthe cysteine modification of the FlgI Cys variants using
mPEG-maleimide, which is a membrane-impermeable
thiol-specific reagent that carries an attached polyethylene
glycol and has a high molecular mass of ~5000 Da

Fig. 3. Motility of the FlgI Cys variant-expressing cells. The relative swarm size is shown of
YZ12-1 (DflgI DmotAB : : cat) cells harbouring
pJN726 (MotAB) and pYZ301 (wt FlgI),
pSU38 (vec.) or pYZ301 derivatives containing a series of FlgI Cys variants. A drop (2 ml)of overnight culture was inoculated on 0.27 %soft agar T broth plates, which were incubatedat 30 6C for an appropriate period of time. The
swarm assays were repeated three times
independently and the relative swarm sizes
calculated in each experiment were averaged.
$, Weakly motile mutant; #, non-motile
mutant.
http://mic.sgmjournals.org

813

Y. Hizukuri and otherssuggesting that the two native Cys residues of FlgI were notaccessed by mPEG-maleimide, probably because they
formed an intramolecular disulfide bond. On the other
hand, some of the FlgI Cys variants showed ~5 kDa shift ofthe monomer band (36 kDa), indicating that the additional Cys residue was accessible to mPEG-maleimide,
which means that the additional Cys residues are likely to
be exposed to solvent on the surface of the protein. To
analyse accessibility further, we quantified the labelling
efficiencies of each FlgI Cys variant, which are given
relative to the total amount of FlgI present (Fig. 5b). Basedon this analysis, we could classify these variants into threegroups: the high labelling efficiency group (.30 %, open
rectangles), FlgI G11C, G161C, Y191C and S211C; themoderate labelling efficiency group (.15 %, closed rectangles), FlgI D31C, T71C, T101C, N121C, Q301C, N311C
and Q331C; and the low labelling efficiency and nonlabelling group (,15 %), which contains the othermutants.
Alignment of the amino acid sequences of the Pring protein FlgI from various species

Fig. 4. Effect of DTT on motility of the FlgI Cys mutants. (a)Swarms of YZ12-1 (DflgI DmotAB : : cat) cells harbouring pJN726(MotAB) and pYZ301 (wt FlgI), pSU38 (vec.) or pYZ301
derivatives containing the FlgI Cys variants I3C, G21C, G51C,
G81C and D111C. A drop (2 ml) of overnight culture was
inoculated on 0.27 % soft agar T broth plates without (upper
panel) or with (lower panel) 5 mM DTT and plates were incubatedat 30 6C for 8 h. (b) Relative swarm sizes from (a). Grey bars,
without DTT; black bars, with 5 mM DTT. The swarm size of cells
producing wild-type FlgI was used for normalization of the mutants
under the equivalent conditions.

(Akiyama et al., 2004). We detected the reaction bymobility shifts of the bands on SDS-PAGE gels (Fig. 5a).
The band of wild-type FlgI did not shift in mobility,
814

The replaced residues that impaired cell motility in thiswork were localized around the N terminus of FlgI (Ile3,
Gly21, Gly51, Gly81 and Asp111; Fig. 3). To assess theimportance of the N terminus of FlgI, we aligned theamino acid sequences of FlgI obtained from various
flagellated bacteria (Supplementary Fig. S1). Multiplealignments revealed that the amino acid sequences of FlgI
were well conserved among various bacteria, except the Nterminal leader sequence, and in particular, the sequencesof the N-terminal region of FlgI (Arg2–Asp31 and Lys63–
Gly120) were extremely well conserved. Replacement of
Gly21 with Cys, which is in the most highly conserved
region, caused the most severe phenotype, non-motility.
The five residues whose mutation impaired cell motility
were all positioned around this N-terminal highly conserved region. From this alignment analysis and ourexperimental results, the N-terminal highly conserved
region of FlgI is suggested to play important roles invarious functions, such as maintenance of the structure of
FlgI and formation of an interface with other flagellar
proteins or with itself.

DISCUSSION
In this study, we characterized a series of FlgI Cyssubstituted mutants with respect to protein amount,motility of the cells, and modification by a thiol-specificreagent. The results of this work are summarized in Fig. 6.
Among the mutations, the well- or moderately labelledresidues (rectangles) never overlapped with the residues
that affected their protein amount (triangles) or the
motility of cells (circles). This may suggest that theimportant residues for protein folding or P-ring assembly
are not exposed to solvent on the surface of the protein.
Microbiology 154

Systematic Cys mutagenesis of FlgI

Fig. 5. Cysteine modification of the FlgI Cys
variants by mPEG-maleimide. (a) mPEG-maleimide-labelled FlgI proteins derived from
YZ12-1 (DflgI DmotAB : : cat) cells harbouring
pJN726 (MotAB) and pYZ301 (wt FlgI),
pSU38 (vec.) or pYZ301 derivatives containing the FlgI Cys variants were detected byimmunoblotting using anti-FlgI antibodies. For
the mPEG-maleimide modification, cultures inthe exponential growth phase were harvested,washed and incubated with 2 mM mPEGmaleimide at 37 6C for 30 min. After terminating the reaction, the samples were mixed
with SDS loading buffer containing b-mercaptoethanol. The arrowhead on the right-handside indicates the mature form of the FlgI
monomer (36 kDa) and the asterisk indicatesmPEG-maleimide-labelled FlgI, as judged by
the ~5 kDa shift of the monomer band. (b)mPEG-maleimide labelling efficiency was
obtained by dividing the intensity of the shiftedband by the total band intensity of FlgI. &,Well-labelled (.30 % labelling efficiency); h,moderately labelled (.15 % labelling efficiency).

This seems to be reasonable, because residues importantfor protein folding or assembly are likely to be locatedinside the protein (i.e. forming the core) or at theprotein–protein interface. FlgI is secreted to the periplasmic space and then assembled around the rod of theflagellar basal body to form the P-ring structure. FlgIinteracts with other FlgI molecules to form the P ring, with
the L-ring protein that is located above the P ring, with rod
proteins, with peptidoglycan (PG), and possibly with the
MotA/B stator complex or other components in theFig. 6. Profiles of the FlgI Cys mutants. The
grey bar at the N terminus indicates the leader
peptide, which is cleaved when the protein isexported to the periplasm. The white verticalbands indicate the residues substituted with
Cys in this study, while the grey vertical bands
indicate the substitutions that we were unable
to generate. C254 and C338 are shown above toindicate the positions of the native cysteine
residues in FlgI. Profiles are shown under thename of each variant: Amount, the residues forwhich the protein amount was decreased whensubstituted with Cys; Motility, the residues thatcaused decreased ($) or completely disrupted(#) motility of the cells; Modified, the residues
that were well- (h) or moderately (&) labelled
by mPEG-maleimide. The residues well- or
moderately labelled by mPEG-maleimide (rectangles) never overlap with the residues that
affect their protein amount (triangles) or the
motility of cells (circles).
http://mic.sgmjournals.org

815

Y. Hizukuri and others

periplasmic space. Speculative interactions of FlgI are
illustrated in Fig. 1. FlgI is predicted to form strong
interactions with FlgI itself or FlgH, the L-ring protein. In
Salmonella enterica serovar Typhimurium, the P and L
rings form an extremely stiff cylindrical structure. Only the
L–P ring complex remains after 7.5 M urea treatment,
during which all of the other flagellar components
dissociate (Akiba et al., 1991). Based on this evidence, it
has been inferred that the interactions of FlgI–FlgI and
FlgI–FlgH are probably very strong.
FlgI may also interact with FlgG, a distal rod protein. In theprocess of P-ring assembly, secreted FlgI is predicted torecognize the rod structure in the periplasm and associatewith the rod. The rod is a part of the rotor structure, but
the P ring is believed to be a part of the stator that supports
the rod as a bushing and allows the rotor to run smoothly.
Therefore, it has been predicted that the FlgI–FlgG
interaction is temporary and/or very weak. The P ring islocated in the peptidoglycan layer (and so is named the Pring), and it may interact with the peptidoglycan layer.
Considering the role of the P ring, it is very likely that the P
ring is fixed in the peptidoglycan layer to stabilize rotationof the motor.
The highly conserved region in the N terminus, residues 1–
120, of FlgI is suggested to play an important role, such as
in stabilizing the structure of FlgI or forming an interface
with other FlgI proteins or other flagellar components, i.e.
FlgH. The role of the conserved region is not known, but it
may be important to maintain FlgI structure, because there
are many Gly and Pro residues in this region. The G161C
variant was the most accessible to the cysteine modification
reagent. When the hook basal bodies (HBBs) isolated from
cells expressing FlgI G161C were treated with mPEGmaleimide, FlgI was labelled and showed band shifting(data not shown). In addition, FlgI Y191C showednumerous cross-linked products compared to othervariants (Fig. 2). These results may suggest that the central
region of FlgI is exposed to the outer surface of the P ring.
In our previous work, we reported that the replacement of
the native Cys residues of FlgI (Cys254 and/or Cys338) with
Ala has little effect on motility but results in a significantly
decreased amount of protein (Hizukuri et al., 2006). We
concluded that the intramolecular disulfide bond formed
between Cys254 and Cys338 is required to prevent thedegradation of protein. Here, we showed that the amountof FlgI protein is decreased for FlgI G241C or L251C, but is
not changed in FlgI Q331C or A341C. The FlgI C254A
mutation has a more severe effect on both flagellar motility
and protein amount than the FlgI C338A mutation(Hizukuri et al., 2006). These results may suggest that theamino acids around Cys254 are more important for protein
folding or protection against degradation than thosearound Cys338.
Recently, a novel structure, named the T ring, wasdiscovered in the flagellar basal body of Vibrio alginolyticus(Terashima et al., 2006). The T ring is located on the816

periplasmic side of the P ring and is composed of MotX
and MotY, which are essential proteins for motor function.
The T ring is proposed to interact with the PomA/PomB
stator complex, which is homologous to the MotA/MotB
complex, by the interaction between MotX and PomB. The
T ring has an important role in the incorporation andstabilization of the stator (Okabe et al., 2005; Terashima
et al., 2006). E. coli and Salmonella species do not have a Tring or protein homologues of MotX or MotY. We thinkthat the P ring of E. coli might have a similar role to thatof the T ring for incorporation or stabilization of the
MotA/MotB stator in the motor. The C-terminal peptidoglycan binding (PGB) motif of MotB is believed toanchor to the peptidoglycan layer via the central flexible
linker region of MotB and to stabilize the stator complex
during rotation. The P ring is also located in the
peptidoglycan layer; thus, the stator complex may be
associated with the P ring via the PGB motif of MotB when
it assembles and functions around the motor. In futurestudies, we will assess the possible interactions between the
P ring and MotB.

ACKNOWLEDGEMENTS
We thank Yoshinori Akiyama and Kayo Koide for invaluable advice
about the cysteine modification experiment. This work was supported
in part by grants-in-aid for scientific research from the Ministry of
Education, Science, and Culture of Japan (to M. H., S. K, I. K and
T. Y.), the Japan Science and Technology Corporation (to M. H.
and T. Y.), the Soft Nano-Machine Project of the Japan Science andTechnology Agency (to T. Y. and M. H.), and the Japan Society for the
Promotion of Science (to Y. H.).

REFERENCES
Akiba, T., Yoshimura, H. & Namba, K. (1991). Monolayer crystalliza-tion of flagellar L-P rings by sequential addition and depletion oflipid. Science 252, 1544–1546.
Akiyama, Y., Kanehara, K. & Ito, K. (2004). RseP (YaeL), an

Escherichia coli RIP protease, cleaves transmembrane sequences.
EMBO J 23, 4434–4442.
Asai, Y., Yakushi, T., Kawagishi, I. & Homma, M. (2003). Ion-

coupling determinants of Na+-driven and H+-driven flagellar
motors. J Mol Biol 327, 453–463.

Bartolome, B., Jubete, Y., Martinez, E. & de la Cruz, F. (1991).

Construction and properties of a family of pACYC184-derived
cloning vectors compatible with pBR322 and its derivatives. Gene
102, 75–78.
Blair, D. F. & Berg, H. C. (1990). The MotA protein of E. coli is
a proton-conducting component of the flagellar motor. Cell 60,
439–449.
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. Proc
Natl Acad Sci U S A 97, 6640–6645.
DeRosier, D. J. (1998). The turn of the screw: the bacterial flagellar

motor. Cell 93, 17–20.
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight

regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121–4130.
Microbiology 154

Systematic Cys mutagenesis of FlgI
Hizukuri, Y., Yakushi, T., Kawagishi, I. & Homma, M. (2006). Role ofParkinson, J. S. & Houts, S. E. (1982). Isolation and behavior of

the intramolecular disulfide bond in FlgI, the flagellar P-ringcomponent of Escherichia coli. J Bacteriol 188, 4190–4197.

Escherichia coli deletion mutants lacking chemotaxis functions. J
Bacteriol 151, 106–113.

Homma, M., Komeda, Y., Iino, T. & Macnab, R. M. (1987). The flaFIX

Reid, S. W., Leake, M. C., Chandler, J. H., Lo, C. J., Armitage, J. P. &Berry, R. M. (2006). The maximum number of torque-generating

gene product of Salmonella typhimurium is a flagellar basal bodycomponent with a signal peptide for export. J Bacteriol 169,
1493–1498.

units in the flagellar motor of Escherichia coli is at least 11. Proc Natl
Acad Sci U S A 103, 8066–8071.

Jones, C. J., Homma, M. & Macnab, R. M. (1989). L-, P-, and M-ringSambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:proteins of the flagellar basal body of Salmonella typhimurium:gene sequences and deduced protein sequences. J Bacteriol 171,
3890–3900.

a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory.

Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S. (1990).

Stoichiometric analysis of the flagellar hook-(basal-body) complexof Salmonella typhimurium. J Mol Biol 212, 377–387.
Kojima, S. & Blair, D. F. (2004). Solubilization and purification of the

MotA/MotB complex of Escherichia coli. Biochemistry 43, 26–34.
Kubori, T., Shimamoto, N., Yamaguchi, S., Namba, K. & Aizawa, S.
(1992). Morphological pathway of flagellar assembly in Salmonella

typhimurium. J Mol Biol 226, 433–446.
Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M. &Armitage, J. P. (2006). Stoichiometry and turnover in single,

functioning membrane protein complexes. Nature 443, 355–358.
Okabe, M., Yakushi, T. & Homma, M. (2005). Interactions of MotX

with MotY and with the PomA/PomB sodium ion channel complexof the Vibrio alginolyticus polar flagellum. J Biol Chem 280,
25659–25664.

http://mic.sgmjournals.org

Silhavy, T. J., Berman, M. L. & Enquist, L. W. (1984). Experiments with

Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.
Sosinsky, G. E., Francis, N. R., DeRosier, D. J., Wall, J. S., Simon, M. N.
& Hainfeld, J. (1992). Mass determination and estimation of subunit

stoichiometry of the bacterial hook–basal body flagellar complex of
Salmonella typhimurium by scanning transmission electron microscopy. Proc Natl Acad Sci U S A 89, 4801–4805.
Stolz, B. & Berg, H. C. (1991). Evidence for interactions between

MotA and MotB, torque-generating elements of the flagellar motor ofEscherichia coli. J Bacteriol 173, 7033–7037.
Terashima, H., Fukuoka, H., Yakushi, T., Kojima, S. & Homma, M.
(2006). The Vibrio motor proteins, MotX and MotY, are associatedwith the basal body of Na+-driven flagella and required for stator
formation. Mol Microbiol 62, 1170–1180.

Edited by: J. G. Shaw817