1
Characterization of the periplasmic domain of MotB and 2
implications for its role in the stator assembly of the bacterial 3
flagellar motor 4
5
Seiji Kojima1§
*, Yukio Furukawa1, Hideyuki Matsunami
1, 6
Tohru Minamino1,2
and Keiichi Namba1,2
7
8
9
Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan1 10
and Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 11
565-0871, Japan 2 12
13
§Present address: Division of Biological Science, Graduate School of Science, Nagoya 14
University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan 15
16
Running title: Characterization of the periplasmic domain of MotB 17
18
Key word: Salmonella; flagellar motor, stator complex, peptidoglycan binding, motility 19
20
21
*Corresponding author. Mailing address: Division of Biological Science, Graduate School of 22
Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.; Tel: 23
+81-52-789-2992; Fax: +81-52-789-3001; E-mail: [email protected] 24
ACCEPTED
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01710-07 JB Accepts, published online ahead of print on 29 February 2008
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
2
ABSTRACT 1
MotA and MotB are integral membrane proteins that form the stator complex of the 2
proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and 3
couples proton flow with torque generation. The stator must be anchored to an appropriate 4
place of the motor, which is believed to occur through a putative peptidoglycan-binding 5
(PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed 6
and characterized an N-terminally truncated variant of Salmonella enterica serovar 7
Typhimurium MotB consisting of residues 78 through 309 (MotBC). MotBC significantly 8
inhibited motility of wild-type cells when exported into the periplasm. Some point mutations 9
in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant 10
MotBC(∆197-210) showed a significantly reduced inhibitory effect. Wild-type MotBC and its 11
point mutant variants formed stable homodimer while the deletion variant was monomeric. A 12
small amount of MotB was co-isolated only with the secreted form of MotBC-His6 by Ni-NTA 13
affinity chromatography, suggesting that the motility inhibition results from MotB-MotBC 14
heterodimer formation in the periplasm. However, the monomeric mutant variant 15
MotBC(∆197-210) did not bind to MotB, suggesting that MotBC is directly involved in the 16
stator assembly. We propose that the MotBC dimer domain plays an important role in targeting 17
and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor. 18
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
3
INTRODUCTION 1
Many bacteria swim by means of flagella, a filamentous organelle extended from the cell 2
surface. The flagellum consists of at least three parts: the filament (helical propeller), the hook 3
(universal joint), and the basal body (rotary motor) (27). The flagellar motor is fueled by the 4
proton or sodium motive force across the cell membrane and can rotate both clockwise (CW) 5
and counterclockwise (CCW) (3, 20, 47). A recent high-resolution observation of flagellar 6
motor rotation revealed a fine stepping motion of the motor rotation (39). The flagellar motor 7
is an elaborate molecular nanomachine that converts electrochemical potential energy to 8
mechanical work. The energy coupling mechanism, however, is still not known. 9
Intensive genetic and biochemical studies of the flagellum have been conducted in 10
Salmonella and E. coli, and now more than 50 gene products are known to be involved in 11
flagellar assembly and function (26). Among them, only five proteins are responsible for 12
torque generation. Three of them are rotor proteins FliG, FliM, and FliN (45), which are 13
mounted on the cytoplasmic face of the membrane-embedded MS ring made of FliF and form 14
the C ring structure (15). The FliG/FliM/FliN complex is also called “the switch complex” 15
because mutations in these proteins cause defects in switching the CCW/CW rotation in 16
response to environmental conditions (45). Crystal structures have been reported for these 17
rotor proteins (10, 11, 24, 30), and disulfide crosslinking experiments using structural 18
information obtained from those crystal structures revealed subunit arrangements in the MS-C 19
ring structure (25, 30, 32, 33). The other two proteins responsible for flagellar motor rotation 20
are integral membrane proteins MotA and MotB (14, 40). MotA and MotB have four and 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
4
single transmembrane segments, respectively (12, 49), and four copies of MotA and two 1
copies of MotB form the stator complex (8, 22, 36, 37, 48), which functions as a proton 2
channel to couple proton flux with motor rotation (4, 41). Each motor contains more than ten 3
MotA/MotB complexes around the MS-C ring (5, 7, 34). Conserved charged residues, Arg90 4
and Glu98, which are located in the cytoplasmic loop of E. coli MotA, interact with the 5
conserved charged residues of C-terminal domain of FliG (50), suggesting that these 6
electrostatic interactions are important for torque generation. MotB has an absolutely 7
conserved and functionally critical aspartic acid residue in its single transmembrane segment. 8
This Asp (Asp32 in E. coli MotB) is believed to function as a proton-binding site in the 9
channel for the motor function (51). Charge-neutralizing mutations of this residue cause a 10
conformational change in the cytoplasmic domain of MotA containing Arg90 and Glu98 (21), 11
providing a plausible hypothesis that protonation of this Asp residue may trigger a 12
conformational change of the stator complex that acts on the rotor to drive its rotation. 13
MotB has a large periplasmic domain, which contains a putative 14
peptidoglycan-binding (PGB) motif (13, 19) that is well conserved among proteins such as 15
OmpA, Pal and MotY, which are outer membrane proteins that interact with the peptidoglycan 16
layer non-covalently. The PGB motif of MotB is believed to associate with the peptidoglycan 17
layer to anchor the MotA/MotB stator complex around the rotor (12, 29, 44). However, the 18
stators appear to be replaced frequently even in the steadily rotating motor, as demonstrated 19
by abrupt and stepwise drops and restorations of the rotation speed of the motor (5, 7, 39), 20
which presumably reflects dynamic dissociation and association of the stator to the rotor. This 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
5
suggests that the association of the PGB motif of MotB with the peptidoglycan layer is also 1
highly dynamic. Hosking et al (17) identified a segment of MotB that acts as a plug to prevent 2
premature proton flow through the MotA/MotB complex and proposed that interaction of the 3
MotA/MotB complex with the flagellar basal body could trigger both opening of the proton 4
channel and unmasking of the peptidoglycan-binding domain of MotB. Although 5
high-resolution structural information of a few peptidoglycan-binding proteins are now 6
available (16, 31), still little is known as to how the stator is targeted to the rotor and how the 7
PGB motif of MotB associates with the peptidoglycan layer near the basal body. 8
In this study, we have carried out genetic and biochemical characterization of an 9
N-terminally truncated Salmonella MotB fragment missing the N-terminal 77 residues 10
(MotBC). We show here that MotBC forms stable homodimer as well as a small amount of 11
MotBC-MotB heterodimer, inhibits wild-type motility when exported to the periplasmic space, 12
and that this negative dominance effect is still retained albeit significantly reduced by a 13
deletion variant of MotBC that stays as monomer. We discuss possible roles of MotBC in the 14
assembly of the functional motor. 15
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
6
MATERIALS AND METHODS 1
Bacterial strains, plasmids, and mutagenesis. Bacterial strains and plasmids used in this 2
study are listed in Table 1. To design N-terminally-truncated fragments of Salmonella MotB, 3
we used a web-based secondary structure prediction program, PSIPRED (18), and chose a 4
surface exposed, unstructured region for the N-termini of the fragments. Mutations in the 5
peptidoglycan-binding motif of MotB were generated in the plasmid pNSK7 or pNSK6 using 6
the QuikChange 1-Day Site-Directed Mutagenesis method as described (Stratagene). In-frame 7
deletions were generated as described by Toker et al. (43). DNA sequencing was done by an 8
ABI PRISM 377 DNA sequencer (Applied Biosystems). 9
10
Preparation of whole cell and periplasmic fraction. Cells were grown exponentially at 11
37ºC in 5 ml LB medium (1% (w/v) tryptone, 0.5% (w/v) Yeast Extract, 0.5% (w/v) NaCl) 12
containing 100 µg/ml ampicillin. After addition of 0.1 mM IPTG, culture was continued at 13
37ºC for another 1 hour. The cells were harvested and resuspended in the spheroplast buffer 14
(50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 M Sucrose) at a cell concentration equivalent to 15
an OD660 of 5. This cell suspension was diluted 10 times by water and used as the whole cell 16
sample. To prepare the periplasmic fraction, 100 µl of cell suspension was diluted 5 times in 17
the spheroplast buffer and incubated at room temperature for 20 min. After centrifugation 18
(17,000 g, 5 min), cells were carefully resuspended in 500 µl of 0.5 mM MgSO4 and placed 19
on ice for 10 min. After centrifugation (17,000 g, 5 min), the periplasmic fractions were 20
collected. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
7
1
Motility assays. Swarming motility of SJW1103 transformed with appropriate plasmids was 2
analyzed on TB soft agar plates (1% (w/v) tryptone, 0.5% (w/v) NaCl, 0.28% (w/v) 3
bacto-agar) containing 100 µg/ml ampicillin at 30ºC typically for 6 hours. IPTG was added as 4
needed to a final concentration of 1 mM or 0.1 mM. To measure the swimming speed, the 5
cells were cultured in TB (1% (w/v) tryptone, 0.5% (w/v) NaCl) at 30ºC until log phase. If 6
necessary, IPTG was added in the beginning of culture, or at the log phase in final 7
concentration of 0.1 mM or 1 mM. The culture media were then diluted 1:10 in fresh TB to 8
observe the motility of the cells under a phase-contrast microscope. The swimming speed of 9
the cells was measured as described previously (2). 10
11
Purification of MotB fragments. E. coli BL21(DE3) cells transformed with pNSK6 were 12
collected by centrifugation and resuspended in buffer A (20 mM Tris-HCl pH 8.0, 100 mM 13
NaCl) containing 1 tablet of Complete protease inhibitor cocktail (Roche Diagnostics). The 14
cells were disrupted and soluble fraction was isolated by ultracentrifugation (186,000 g, 30 15
min), then supernatants were collected and loaded to a set of tandem columns of HiTrapSP 16
(GE Healthcare), HiTrapQ (GE Healthcare) and HisTrap (GE Healthcare) connected in this 17
order. MotBC-His6, which flowed through the HiTrapSP and HiTrapQ column but bound to 18
HisTrap column, was eluted by a linear gradient of imidazole, collected, and further purified 19
by a Sephacryl S-300 size exclusion column (GE Healthcare). Mutant variants of MotBC-His6 20
were purified in the same way. For purification of MotBC fragments without His-tag, we used 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
8
a plasmid pNSK11. The soluble fraction of BL21(DE3) carrying pNSK11 was loaded to the 1
HiTrapSP-HiTrapQ columns connected in this order and equilibrated with buffer B. 2
Flow-through fractions containing MotBC were collected by adding (NH4)2SO4 to 45% 3
saturation. The pellet was suspended in 10 ml of buffer B, and then MotBC was further 4
purified by the Sephacryl S-300 size exclusion column as described above. 5
6
Analytical size exclusion column chromatography. Analytical size exclusion 7
chromatography was performed with a Superdex 75 HR10/30 column (GE Healthcare) 8
connected to an AKTA system (GE Healthcare). The column was equilibrated with buffer B 9
and run at a flow rate of 0.7 ml/min. BSA (67 kD), ovalbumin (44 kD), and 10
chymotrypsinogen (25 kD) were used for size markers. 11
12
Analytical Ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation 13
was carried out using a Beckman Optima XL-A analytical ultracentrifuge with an AnTi 60 14
rotor as described previously (28). The purified samples of MotBC and 15
MotBC(∆197-210)-His6 were dialyzed against 20 mM Tris-HCl (pH8.0) buffer solutions 16
containing 100 mM and 300 mM NaCl, respectively, which were also used as the blank. 17
Measurements were done at 20ºC at 20,000, 22,000 and 24,000 rpm on the MotBC fragment 18
and at 24,000, 26,000 and 28,000 rpm on MotBC(∆197-210)-His6 using charcoal-filled Epon 19
and quartz windows. Concentration profiles of the samples were monitored by absorbance at a 20
wavelength of 280 nm and recorded at a spacing of 0.001 cm in the step mode, with 20 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
9
averages per step, for 10, 16 and 22 hours after each rotor speed was reached. Equilibrium 1
data were analyzed using a Beckman OptimaTM
XL-A/XL-I data analysis software, version 2
4.0, provided as an add-on to Origin Version 4.1 (MicroCal Inc.). A global, single-species fit 3
over two different loading absorbance (0.2 and 0.3 at 280 nm) and three rotor speeds as 4
described above were calculated. The partial specific volume, 0.730 ml/g for MotBC and 5
0.731 ml/g for MotBC(∆197-210)-His6, used for analysis were based on the amino acid 6
composition of each protein. 7
8
Antibodies and immunoblot. Purified MotBC-His6 was used to raise an anti-MotB antibody 9
in rabbits (MBL Co., LTD). After the proteins in each fraction were separated by SDS-PAGE, 10
immunoblotting with the polyclonal anti-MotB and anti-Pal antibodies was carried out as 11
described previously (21). Detection was performed with the SuperSignal West Pico 12
chemiluminescent procedure (Pierce). 13
14
Pull-down assay. The E. coli motA-motB deletion strain RP6894 was transformed with two 15
plasmids, one encoding His-tagged MotBC with or without PelB leader sequence at their 16
N-termini (pNSK7 or pNSK8) and the other encoding both non-tagged MotA and MotB 17
(pNSK31). A monomeric mutant variant, MotBC(∆197-210), was also expressed from 18
pNSK7-∆(197-210) in place of MotBC. For the opposite His-tag combination, we used 19
plasmid pNSK28 (non-tagged MotBC) and pNSK32 (MotA/MotB-His8). Overnight culture 20
was inoculated into fresh 10 ml LB containing 100 µg/ml ampicillin and 25 µg/ml 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
10
chloramphenicol and incubated at 37ºC until log phase. After adding 0.1 mM IPTG, 1
incubation was continued at 37ºC for another 1 hour. The cells were harvested and 2
resuspended in 1 ml of the spheroplast buffer, sonicated and centrifuged (3,000 g, 5 min). 3
After centrifugation (16,000 g, 15 min), the membrane fraction was suspended in 1 ml of 4
buffer C (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM imidazole, 10% (v/v) glycerol), and 5
then a detergent, dodecylphosphocholine (DPC) (Anatrace Inc.), was added to a final 6
concentration of 0.05% (w/v). After gentle shaking for 20 min at 30 ºC, insoluble materials 7
were removed by centrifugation (16,000 g, 15 min). The soluble fraction was mixed with 50 8
µl of a Ni-NTA agarose resin (Qiagen) pre-washed with buffer C containing 0.05% (w/v) 9
DPC. After gently mixing at 30 ºC for 20 min, the unbound materials were removed by brief 10
centrifugation (ca. 5 sec). The resin was washed twice with buffer C with 0.03% (w/v) DPC 11
(1 ml/wash) and then three times with buffer C containing 60 mM imidazole and 0.03% (w/v) 12
DPC (1 ml/wash). After incubation for 1 min at room temperature, proteins were eluted with 13
100 µl buffer C containing 500 mM imidazole and 0.03% (w/v) DPC. Eluted materials were 14
mixed with SDS loading buffer and boiled. 15
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
11
RESULTS 1
Multicopy effect of MotBC on motility. We constructed several N-terminally truncated 2
variants of MotB that lack the transmembrane segment, and placed them under the 3
IPTG-inducible trc promoter. These MotB fragments were fused to a PelB leader sequence 4
(PelBL) at the N termini to direct their location to the periplasmic space. A His6 tag was also 5
attached to these fragments at the C terminus to facilitate protein purification (Fig. 1A). We 6
transformed the wild-type Salmonella strain SJW1103 with these constructs, prepared the 7
whole cell and periplasmic fractions from the resulting transformants, and analyzed them by 8
immunoblotting with the polyclonal anti-MotB antibody (Fig. 1B). One of these constructs 9
consisting of residues from 78 through 309 (PelBL-MotBC-His6) (expressed from pNSK7) was 10
stably expressed and detected in the periplasmic fraction (Fig. 1B, lane 3). In contrast, 11
MotBC-His6, which does not have the PelB signal sequence at its N-terminus, was not 12
detected in the periplasm (Fig. 1B, lane 2). 13
Expression of PelBL-MotBC-His6 with 1 mM IPTG resulted in a severely impaired 14
swarming motility of wild-type cells on soft-agar plates, while expression of MotBC-His6 did 15
not show any notable effect on motility (Fig. 1C), indicating that the periplasmic location of 16
MotBC is critical for motility inhibition. The results were essentially the same when the IPTG 17
concentration in the plate was reduced to 0.1 mM,. Therefore, we used 0.1 mM IPTG for 18
motility assay thereafter. 19
To investigate how flagellar motor rotation is affected by the periplasmic location of 20
MotBC-His6, we measured the swimming speed of SJW1103 carrying pNSK7 or pNSK8 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
12
cultured to the log phase in the presence of 0.1 mM IPTG. The swimming speed of SJW1103 1
carrying pTrc99A or pNSK8 was not affected by IPTG induction (about 27 µm/s for both 2
strains). In contrast, the swimming speed of SJW1103 carrying pNSK7 was 18 µm/s and 11 3
µm/s in the absence and presence of IPTG, respectively. Thus, the swimming speed was 4
reduced significantly (to 40% of wild-type) by the addition of IPTG. Even in the absence of 5
IPTG, the speed was reduced to about 60% of the control strains, probably because of the 6
leakiness of the MotBC expression from the plasmid pNSK7. 7
8
Multicopy effect of mutant variants of MotBC-His6 on motility. Blair et al (6) have 9
identified many point mutations in E. coli motB that give mot (paralyzed flagella) phenotype. 10
Most of them are located within the periplasmic domain of MotB including the putative PGB 11
motif (Fig. 2A) (13, 19, 29). These mutant variants also exhibit a negative dominance effect 12
on motility of the wild type, probably reflecting the displacement of functional MotB by 13
nonfunctional one. To investigate if these dominant-negative mutations affect the multicopy 14
effect of MotBC on motility, we introduced five of these point mutations lying in the PGB 15
motif into MotBC-His6. We also constructed two in-frame deletion variants, MotBC(∆197-210) 16
missing residues 197 - 210 and MotBC(∆211-226) lacking residues 211 - 226 (Fig. 2A). These 17
mutant variants were fused to the PelB signal sequence at their N-termini. The plasmids 18
containing these mutations were introduced into SJW1103, and the level of negative 19
dominance on motility by these mutant versions of MotBC-His6 was assayed on soft agar 20
plates with or without 0.1 mM IPTG (Fig. 2B, upper two panels). All of the five point mutant 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
13
variants still exhibited negative dominance. The D198N, S215F and R223H mutant variants 1
inhibited motility almost at the wild-type MotBC levels (see pNSK7). The T197I and R218W 2
mutants impaired motility more strongly than the wild-type MotBC fragment, as also shown 3
even in the absence of IPTG (Fig. 2B, upper left panel). The (∆197-210) deletion mildly 4
inhibited the motility, but this dominance effect was much weaker than that of wild-type 5
MotBC. The (∆211-226) deletion did not exert any inhibitory effect (Fig. 2B, upper right 6
panel). 7
We next examined whether the T197I and R218W mutations show an additive 8
effect on motility inhibition (Fig. 2B, lower two panels). The T197I/R218W double mutant 9
variant exhibited much stronger negative dominance on motility of wild-type cells than either 10
of the single mutant variants as clearly shown in the absence of IPTG (Fig. 2B, lower left 11
panel). 12
To examine the level of protein expression and periplasmic localization of these 13
mutants, we prepared the whole cell and periplasmic fractions from SJW1103 carrying the 14
plasmids and carried out immunoblotting with the anti-MotB antibody (Fig. 3). All the point 15
mutant variants, the (∆197-210) deletion and the double mutation variant were expressed and 16
exported into the periplasm at wild-type levels. However, only a small amount of the 17
(∆211-226) deletion was detected in the periplasm although expressed at a wild-type level 18
(Fig. 3A, lane 8). Therefore, no inhibitory effect on motility by this deletion fragment was 19
probably due to the defect in its periplasmic localization. 20
21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
14
Dimerization of MotBC. To investigate the oligomerization property of MotBC, we carried 1
out sedimentation equilibrium analytical ultracentrifugation and analytical size exclusion 2
chromatography. We constructed the plasmid pNSK11, which overproduces MotBC without 3
the PelB leader sequence and the His6 tag, to avoid complexity in interpreting the results of 4
these measurements (35). Untagged MotBC was purified and subjected to analytical size 5
exclusion column chromatography with a Superdex 75 HR 10/30 column (Fig. 4A and 4B). 6
MotBC eluted from the column at a volume of 8.7 ml, which is close to the elution position of 7
BSA (67 kD), indicating that the size of MotBC in solution is much larger than its deduced 8
size of a MotBC monomer (25.7 kD) and suggesting that MotBC forms an oligomer in solution. 9
To measure the molecular size of the oligomer more precisely, we performed sedimentation 10
equilibrium analytical ultracentrifugation, which can determine the molecular mass of 11
particles in solution independent of their shape. The same batch of the MotBC sample was 12
used for the measurements at three different protein concentrations, and the results were 13
basically the same. The manufacturer’s software was used to test several models to fit the 14
obtained profiles, and a single species model produced the best fit in terms of low residuals 15
(Fig. 4C). The calculated molecular mass was 50.6 kD, which corresponds almost exactly to 16
that of the dimer of MotBC. 17
To test if the C-terminal His-tag affects the dimer formation of MotBC, we purified 18
MotBC-His6 from the periplasmic fraction of wild-type cells carrying pNSK7 by HisTrap 19
affinity chromatography and ran it on a Superdex 75 HR 10/30 column. MotBC-His6 was 20
eluted at a volume of 9.0 ml from the column (data not shown), indicating that MotBC-His6 in 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
15
periplasm, which exerts an inhibitory effect on flagellar motor rotation, also forms dimer. 1
2
Dimerization of mutant MotBC-His6 fragments. To examine if the mutations within the 3
PGB motif affect dimerization of MotBC, we purified His-tagged versions of these mutant 4
fragments and analyzed by analytical gel filtration chromatography with a Superdex 75 HR 5
10/30 column (Fig. 5A). 6
Purified MotBC(R218W) eluted at the same volume as the wild type (8.9 ml), 7
indicating that the size and shape of the R218W mutant variant are essentially the same as 8
those of wild-type MotBC. However, MotBC(∆197-210) eluted at a volume of 9.9 ml. We 9
performed sedimentation equilibrium analytical ultracentrifugation to precisely determine the 10
molecular mass of the particle that this deletion variant forms in solution (Fig. 5B). The model 11
fitting of the obtained profiles gave a molecular mass of 24.0 kD, which is close to the 12
deduced molecular mass of a MotBC monomer (25.0 kD). 13
We also purified MotBC(T197I/R218W) and analyzed its molecular size by 14
analytical gel filtration chromatography. This double mutant protein was eluted at a volume of 15
9.0 ml (data not shown), indicating that this also forms dimer in solution. 16
These results show a clear positive correlation between dimerization of MotBC 17
fragments and the level of motility inhibition: Wild-type MotBC, MotBC(R218W) and 18
MotBC(T197I/R218W), which form stable dimer, all impaired motility, whereas 19
MotBC(∆197-210), which is monomeric, showed a reduced inhibition effect, although the 20
level of inhibition is still significant. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
16
1
Association of MotBC with MotB. MotBC forms dimer in solution, but full-length MotB is 2
also known to form a homodimer in the stator complex (8, 9), raising the possibility that the 3
multicopy inhibitory effect of MotBC on motility may be caused by titration of endogenous 4
full-length MotB protein by the plasmid-borne MotBC fragment in the periplasm, interfering 5
with the formation of the functional MotA/MotB complex to be installed in the motor. To 6
investigate this possibility, we analyzed the association of MotBC with MotB by pull-down 7
assays (Fig. 6). If MotB associates with MotBC exported into the periplasm, it should be 8
co-purified with a His-tagged variant of MotBC by Ni-NTA affinity chromatography. 9
MotBC-His6 with or without the PelB leader sequence was co-expressed with MotA and MotB 10
in the E. coli ∆motA-B double null mutant. Both MotBC and MotB were expressed at similar 11
levels (Fig. 6, top panel, lanes 3 and 4). Although most of MotBC were in the soluble fraction 12
(data not shown), small amounts of MotBC were found in the insoluble membrane fractions, at 13
almost the same level for non-secreted and secreted variants of MotBC (Fig. 6, middle panel, 14
lanes 3 and 4). These membranes were solubilized by dodecylphosphocholine (DPC) and then 15
mixed with a Ni-NTA resin. After washing the resin, MotBC-His6 was eluted by a high 16
concentration of imidazole (Fig. 6, bottom panel, lanes 3 and 4). A significant amount of 17
MotB was co-isolated with MotBC-His6 exported to the periplasm (lane 3), but little with that 18
in the cytoplasm (lane 4). We also tested the binding of MotBC(∆197-210), a monomeric 19
variant of MotBC, to MotB. MotBC(∆197-210)-His6 was detected in the membrane fraction, 20
but only little amount of MotB was co-isolated (Fig. 6, bottom panel, lane 5). 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
17
Alternatively, the His8-tag was attached to MotB instead of MotBC, and then 1
pull-down assays were carried out. Again, MotBC was found in the membrane fraction, but no 2
MotBC was co-isolated with MotB-His8 (Fig. 6, bottom panel, lane 6). 3
These results suggest that MotBC can associate with MotB in the periplasm, 4
probably contributing to the motility inhibition to some extent. However, the amount of the 5
MotBC-MotB heterodimer is much smaller than that of the MotBC homodimer as shown in 6
Fig. 6. Note that there is always some non-specific association of MotBC with the membrane 7
even in the absence of MotA and MotB regardless of whether or not MotBC was exported to 8
the periplasm (Fig. 6, middle panel, lanes 1 and 2). The faint bands of MotB co-isolated with 9
MotB fragments by Ni-NTA affinity chromatography are also likely to be the results of 10
non-specific binding of MotB to the resin. 11
12
DISCUSSION 13
Asai et al. have reported that a chimeric stator complex consisting of PomA and a chimeric 14
protein PotB, a fusion of the N-terminal transmembrane segment of V. alginolyticus PomB 15
and the C-terminal periplasmic segment of E. coli MotB, is functional in E. coli flagellar 16
motor while the wild-type PomA/PomB complex is not (1), suggesting that an appropriate 17
periplasmic domain of the stator complex is required for association of stators with the basal 18
body to form a functional motor. In this study, to investigate the roles and mechanisms of the 19
periplasmic domain of MotB for the stator assembly, we have analyzed the C-terminal 20
periplasmic domain of MotB (MotBC) and obtained evidence that MotBC forms stable dimer 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
18
in the periplasm and inhibits flagellar motor rotation. 1
MotBC consisting of residues 78 through 309 inhibited swarming motility of 2
wild-type cells on soft agar plates when it was exported to the periplasmic space (Fig. 1). In 3
agreement with this, the swimming speed of wild-type cells expressing and exporting MotBC 4
to the periplasm was significantly reduced. The T197I and R218W variants exhibited stronger 5
negative dominance effects than wild-type MotBC while MotBC(∆197-210), an in-frame 6
deletion mutant in the PGB motif, exhibited a reduced dominance effect (Fig. 2B). Wild-type 7
and the point mutant variants of MotBC formed stable homodimer while the deletion variant 8
was monomeric (Figs. 4 and 5), suggesting that dimerization of MotBC strengthens the 9
negative dominance effect. 10
It has been estimated that there are at least 11 copies of the stator complex around 11
the rotor of the flagellar motor (34). Decrease in the number of functional stators in the motor 12
slows down the rotation speed (39). Therefore, the motility inhibition caused by 13
overexpressed MotBC in the periplasm is likely to be due to a decrease in the number of 14
functional stators. The question is how MotBC interferes with assembly of functional stators 15
into the motor. There are three possibilities: (i) MotBC has a motif to bind to the stator binding 16
sites of the flagellar basal body and occupies them, strongly in the dimer form and weakly in 17
the monomer form; (ii) MotBC forms heterodimer with endogenous MotB and thereby 18
interferes with formation of functional stators; (iii) MotBC dimers compete with endogenous 19
MotB in binding to MotA and thereby interfere with formation of functional stators. 20
The results of the co-isolation experiments using His-tagged MotBC and full-length 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
19
MotB (Fig. 6) supports the possibility (ii). A small but significant amount of MotBC-His6 was 1
found in the membrane fraction and co-purified with full-length MotB by Ni-NTA affinity 2
chromatography only when MotBC-His6 was exported into the periplasm. Since the motility 3
inhibition was seen only when MotBC was located in the periplasm, the MotB-MotBC 4
interaction in the periplasm might be responsible for the motility inhibition. 5
The amount of the MotB-MotBC heterodimer, however, is quite small compared to 6
that of the MotBC homodimer (Fig. 6). The majority of MotBC forms stable homodimer (Fig. 7
4 and 5). The stability of the MotBC homodimer is also supported by our NMR measurements 8
of MotBC at three different temperatures (30ºC, 40ºC and 50ºC) (Y. Sudo and C. Kojima, 9
personal communication). Moreover, even a monomeric variant of MotBC shows motility 10
inhibition albeit relatively weak. These results support the possibility (i). The MotBC fragment 11
might actually contain the targeting signal to drive the installation of the stator complex into 12
the flagellar motor. To test this, in vivo imaging of MotBC behavior using a fluorescent protein 13
such as “mCherry” (38) would be a good approach. 14
Although dominant-negative mot mutations within the putative PGB motif (6) seem 15
to interfere with the association of the PGB motif with PG, these mutations enhanced the 16
motility inhibition by MotBC (Fig. 2). These apparently conflicting results cannot simply be 17
explained by the possibility (i) because the number of MotBC dimers or monomers occupying 18
the stator binding sites would be decreased if the binding of the PGB motif with PG was 19
weakened. However, since no structural data are available for the interaction between the 20
PGB motif and PG, it would also be possible that the mutations strengthened the binding in 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
20
the case of MotBC, which has much more positional and orientational freedom than MotB in 1
binding to the basal body. More detailed analyses are required. 2
MotA was not detected in the elution fraction of the MotB-MotBC heterodimer by 3
immunoblotting with the polyclonal anti-MotA antibody (data not shown), suggesting that the 4
MotB-MotBC heterodimer may not form a complex with MotA. Alternatively, MotA may be 5
dissociated from the MotB/MotBC heterodimer by stringent washes during purification. To 6
test these possibilities, we need to examine whether MotA could be co-isolated with 7
MotBC-His6 in the absence of MotB. 8
9
It has been reported that MotB forms dimer at its single transmembrane segment (9). 10
In this study, we showed that the periplasmic domain of MotB alone suffices to form stable 11
dimer in the periplasm. Taken together, two MotB molecules are likely to associate with each 12
other in its entire length. The MotBC dimer domain of the MotA4MotB2 complex may play an 13
important role in targeting the complex to its binding site and anchoring it to the motor to be 14
the stator. In the stator resurrection experiments (5, 7, 39) abrupt drops of the rotation rate 15
were observed rather frequently, and it may reflect dissociation or turnover of stators from the 16
motor. In fact, a recent study using the fluorescence photobleaching technique has shown a 17
turnover of GFP-fused MotB between the membrane pool and the motor (23) suggesting that 18
the interactions between the MotA/B complex and its target site on the motor are dynamic and 19
that MotB does not always associate with PG even though MotB has a highly conserved 20
PG-binding motif. Our preliminary PG-binding assay showed that neither MotB nor MotBC 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
21
was found in the PG-associated fraction, while Pal, a PG-binding protein, showed strong 1
association with PG (data not shown), suggesting that the PG-binding site of MotB is opened 2
and activated only upon binding of the MotBC dimer domain to the basal body. So far, there 3
have been no reports showing evidence for specific stator binding sites on and around the 4
basal body. The crystal structure of the C-terminal domain of RmpM (RmpM_Cter) indicates 5
that RmpM_Cter may exist as dimer with two putative PGB sites located on the opposite site 6
to each other, suggesting that an RmpM dimer could simultaneously bind to two glycan 7
chains (16). The MotBC dimer domain may bind to PG in a similar manner, since remarkable 8
structural similarities are found among PG binding proteins (16, 31). However, as the 9
association of the MotA/MotB complex with the flagellar motor is highly dynamic, the 10
association of MotB with PG would presumably be more transient and dynamic than that of 11
those outer membrane proteins such as RmpM. Therefore, it would be quite interesting to see 12
how the MotBC dimer domain of the MotA/B complex behaves in vivo and how it associates 13
with the peptidoglycan layer after the MotA/B complex is installed into the motor. To address 14
these questions, MotBC would be a useful tool. Further efforts to understand the motility 15
inhibition mechanism by MotBC and to establish PG-association assay are ongoing, together 16
with crystallization screening of MotBC to obtain structural insight into the stator anchoring 17
mechanism. 18
19
ACKNOWLEDGMENTS 20
We thank Michio Homma and David Blair for critically reading the manuscript and 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
22
stimulating discussion. We acknowledge Sandy Parkinson for a gift of the strain RP6894, 1
Gillian Fraser for a gift of the pACTrc vector, and Hajime Tokuda for a gift of the polyclonal 2
anti-Pal antibody, Sachi Tatematsu for technical assistance, Yuki Sudo and Chojiro Kojima for 3
communicating unpublished results. We also thank Kelly Hughes for suggestions and 4
discussion, and Fumio Oosawa and Sho Asakura for continuous support and encouragement. 5
This work was partially supported by Grant-in-Aid for Scientific Research from the Ministry 6
of Education, Culture, Sports, Science and Technology of Japan (T.M. and K.N.). 7
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
23
REFERENCES 1
1. Asai, Y., T. Yakushi, I. Kawagishi, and M. Homma. 2003. Ion-coupling 2
determinants of Na+-driven and H
+-driven flagellar motors. J. Mol. Biol. 327:453-463. 3
2. Atsumi, T., Y. Maekawa, T. Yamada, I. Kawagishi, Y. Imae, and M. Homma. 1996. 4
Effect of viscosity on swimming by the lateral and polar flagella of Vibrio 5
alginolitycus. J. Bacteriol. 178:5024-5026. 6
3. Berg, H. C. 2003. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 7
72:19-54. 8
4. Blair, D. F., and H. C. Berg. 1990. The MotA protein of E. coli is a 9
proton-conducting component of the flagellar motor. Cell 60:439-449. 10
5. Blair, D. F., and H. C. Berg. 1988. Restoration of torque in defective flagellar motors. 11
Science 242:1678-1681. 12
6. Blair, D. F., D. Y. Kim, and H. C. Berg. 1991. Mutant MotB proteins in Escherichia 13
coli. J. Bacteriol. 173:4049-4055. 14
7. Block, S. M., and H. C. Berg. 1984. Successive incorporation of force-generating 15
units in the bacterial rotary motor. Nature 309:470-472. 16
8. Braun, T. F., L. Q. Al-Mawsawi, S. Kojima, and D. F. Blair. 2004. Arrangement of 17
core membrane segments in the MotA/MotB proton-channel complex of Escherichia 18
coli. Biochemistry 43:35-45. 19
9. Braun, T. F., and D. F. Blair. 2001. Targeted disulfide cross-linking of the MotB 20
protein of Escherichia coli: evidence for two H+ channels in the stator Complex. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
24
Biochemistry 40:13051-13059. 1
10. Brown, P. N., C. P. Hill, and D. F. Blair. 2002. Crystal structure of the middle and 2
C-terminal domains of the flagellar rotor protein FliG. EMBO. J. 21:3225-3234. 3
11. Brown, P. N., M. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair. 2005. Crystal 4
structure of the flagellar rotor protein FliN from Thermotoga maritima. J. Bacteriol. 5
187:2890-2902. 6
12. Chun, S. Y., and J. S. Parkinson. 1988. Bacterial motility: membrane topology of the 7
Escherichia coli MotB protein. Science 239:276-278. 8
13. De Mot, R., and J. Vanderleyden. 1994. The C-terminal sequence conservation 9
between OmpA-related outer membrane proteins and MotB suggests a common 10
function in both gram- positive and gram-negative bacteria, possibly in the interaction 11
of these domains with peptidoglycan. Mol. Microbiol. 12:333-334. 12
14. Dean, G. D., R. M. Macnab, J. Stader, P. Matsumura, and C. Burks. 1984. Gene 13
sequence and predicted amino acid sequence of the motA protein, a 14
membrane-associated protein required for flagellar rotation in Escherichia coli. J. 15
Bacteriol. 159:991-999. 16
15. Francis, N. R., G. E. Sosinsky, D. Thomas, and D. J. DeRosier. 1994. Isolation, 17
Characterization and Structure of Bacterial Flagellar Motors Containing the Switch 18
Complex. J. Mol. Biol. 235:1261-1270. 19
16. Grizot, S., and S. K. Buchanan. 2004. Structure of the OmpA-like domain of RmpM 20
from Neisseria meningitidis. Mol. Microbiol. 51:1027-1037. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
25
17. Hosking, E. R., C. Vogt, E. P. Bakker, and M. D. Manson. 2006. The Escherichia 1
coli MotAB proton channel unplugged. J. Mol. Biol. 364:921-937. 2
18. Jones, D. T. 1999. Protein secondary structure prediction based on position-specific 3
scoring matrices. J. Mol. Biol. 292:195-202. 4
19. Koebnik, R. 1995. Proposal for a peptidoglycan-associating alpha-helical motif in the 5
C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 6
16:1269-1270. 7
20. Kojima, S., and D. F. Blair. 2004. The bacterial flagellar motor: structure and 8
function of a complex molecular machine. Int. Rev. Cytol. 233:93-134. 9
21. Kojima, S., and D. F. Blair. 2001. Conformational change in the stator of the 10
bacterial flagellar motor. Biochemistry 40:13041-13050. 11
22. Kojima, S., and D. F. Blair. 2004. Solubilization and purification of the MotA/MotB 12
complex of Escherichia coli. Biochemistry 43:26-34. 13
23. Leake, M. C., J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. 14
Armitage. 2006. Stoichiometry and turnover in single, functioning membrane protein 15
complexes. Nature 443:355-358. 16
24. Lloyd, S. A., F. G. Whitby, D. F. Blair, and C. P. Hill. 1999. Structure of the 17
C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor. 18
Nature 400:472-475. 19
25. Lowder, B. J., M. D. Duyvesteyn, and D. F. Blair. 2005. FliG subunit arrangement in 20
the flagellar rotor probed by targeted cross-linking. J. Bacteriol. 187:5640-5647. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
26
26. Macnab, R. M. 2003. How bacteria assemble flagella. Annu. Rev. Microbiol. 1
57:77-100. 2
27. Minamino, T., and K. Namba. 2004. Self-assembly and type III protein export of the 3
bacterial flagellum. J. Mol. Microbiol. Biotechnol. 7:5-17. 4
28. Minamino, T., Y. Saijo-Hamano, Y. Furukawa, B. Gonzalez-Pedrajo, R. M. 5
Macnab, and K. Namba. 2004. Domain organization and function of Salmonella 6
FliK, a flagellar hook-length control protein. J. Mol. Biol. 341:491-502. 7
29. Muramoto, K., and R. M. Macnab. 1998. Deletion analysis of MotA and MotB, 8
components of the force-generating unit in the flagellar motor of Salmonella. Mol. 9
Microbiol. 29:1191-1202. 10
30. Park, S. Y., B. Lowder, A. M. Bilwes, D. F. Blair, and B. R. Crane. 2006. Structure 11
of FliM provides insight into assembly of the switch complex in the bacterial flagella 12
motor. Proc. Natl. Acad. Sci. U S A 103:11886-11891. 13
31. Parsons, L. M., F. Lin, and J. Orban. 2006. Peptidoglycan recognition by Pal, an 14
outer membrane lipoprotein. Biochemistry 45:2122-2128. 15
32. Paul, K., and D. F. Blair. 2006. Organization of FliN subunits in the flagellar motor 16
of Escherichia coli. J. Bacteriol. 188:2502-2511. 17
33. Paul, K., J. G. Harmon, and D. F. Blair. 2006. Mutational analysis of the flagellar 18
rotor protein FliN: identification of surfaces important for flagellar assembly and 19
switching. J. Bacteriol. 188:5240-5248. 20
34. Reid, S. W., M. C. Leake, J. H. Chandler, C. J. Lo, J. P. Armitage, and R. M. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
27
Berry. 2006. The maximum number of torque-generating units in the flagellar motor 1
of Escherichia coli is at least 11. Proc. Natl. Acad. Sci. U S A 103:8066-8071. 2
35. Saijo-Hamano, Y., T. Minamino, R. M. Macnab, and K. Namba. 2004. Structural 3
and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral 4
membrane component of the type III flagellar protein export apparatus in Salmonella. 5
J. Mol. Biol. 343:457-466. 6
36. Sato, K., and M. Homma. 2000. Functional reconstitution of the Na+-driven polar 7
flagellar motor component of Vibrio alginolyticus. J. Biol. Chem. 275:5718-5722. 8
37. Sato, K., and M. Homma. 2000. Multimeric structure of PomA, a component of the 9
Na+-driven polar flagellar motor of Vibrio alginolyticus. J. Biol. Chem. 10
275:20223-20228. 11
38. Shaner, N. C., R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E. Palmer, and 12
R. Y. Tsien. 2004. Improved monomeric red, orange and yellow fluorescent proteins 13
derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22:1567-1572. 14
39. Sowa, Y., A. D. Rowe, M. C. Leake, T. Yakushi, M. Homma, A. Ishijima, and R. 15
M. Berry. 2005. Direct observation of steps in rotation of the bacterial flagellar motor. 16
Nature 437:916-919. 17
40. Stader, J., P. Matsumura, D. Vacante, G. E. Dean, and R. M. Macnab. 1986. 18
Nucleotide sequence of the Escherichia coli MotB gene and site-limited 19
inccorporation of its product into the cytoplasmic membrane. J. Bacteriol. 20
166:244-252. 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
28
41. Stolz, B., and H. C. Berg. 1991. Evidence for interactions between MotA and MotB, 1
torque-generating elements of the flagellar motor of Escherichia coli. J. Bacteriol. 2
173:7033-7037. 3
42. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving 4
the sensitivity of progressive multiple sequence alignment through sequence 5
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids 6
Res. 22:4673-4680. 7
43. Toker, A. S., M. Kihara, and R. M. Macnab. 1996. Deletion analysis of the FliM 8
flagellar switch protein of Salmonella typhimurium. J. Bacteriol. 178:7069-7079. 9
44. Yakushi, T., N. Hattori, and M. Homma. 2005. Deletion analysis of the 10
carboxyl-terminal region of the PomB component of the Vibrio alginolyticus polar 11
flagellar motor. J. Bacteriol. 187:778-784. 12
45. Yamaguchi, S., S. Aizawa, M. Kihara, M. Isomura, C. J. Jones, and R. M. 13
Macnab. 1986. Genetic evidence for a switching and energy-transducing complex in 14
the flagellar motor of Salmonella typhimurium. J. Bacteriol. 168:1172-1179. 15
46. Yamaguchi, S., H. Fujita, K. Sugata, T. Taira, and T. Iino. 1984. Genetic analysis 16
of H2, the structural gene for phase-2 flagellin in Salmonella. J. Gen. Microbiol. 17
130:255-265. 18
47. Yorimitsu, T., and M. Homma. 2001. Na+-driven flagellar motor of Vibrio. Biochim. 19
Biophys. Acta. 1505:82-93. 20
48. Yorimitsu, T., M. Kojima, T. Yakushi, and M. Homma. 2004. Multimeric structure 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
29
of the PomA/PomB channel complex in the Na+-driven flagellar motor of Vibrio 1
alginolyticus. J Biochem (Tokyo) 135:43-51. 2
49. Zhou, J., R. T. Fazzio, and D. F. Blair. 1995. Membrane topology of the MotA 3
protein of Escherichia coli. J. Mol. Biol. 251:237-242. 4
50. Zhou, J., S. A. Lloyd, and D. F. Blair. 1998. Electrostatic interactions between rotor 5
and stator in the bacterial flagellar motor. Proc. Natl. Acad. Sci. U S A 95:6436-6441. 6
51. Zhou, J., L. L. Sharp, H. L. Tang, S. A. Lloyd, S. Billings, T. F. Braun, and D. F. 7
Blair. 1998. Function of protonatable residues in the flagellar motor of Escherichia 8
coli: a critical role for Asp 32 of MotB. J. Bacteriol. 180:2729-2735. 9
10
11
12
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
30
Figure legends 1
Fig.1. Multicopy effect of MotBC on motility of wild-type cells. (A) The primary structure of 2
Salmonella MotB protein and an N-terminally truncated fragment missing the N-terminal 77 3
residues, MotBC. MotB has a single transmembrane domain (black) in the N-terminal region 4
and a putative peptidoglycan-binding motif (gray) in the large periplasmic domain. Plasmid 5
pNSK7 encodes the MotBC fragment (residue 78 to the C-terminus 309) fused to a PelB 6
leader sequence (PelBL, 22 a. a., hatched) and a His6 tag at its N-and C-termini, respectively. 7
Plasmid pNSK8 does not contain PelBL at the N-terminus. (B) Periplasmic localization of the 8
MotBC-His6 fragment. Immunoblotting using the polyclonal anti-MotB antibody of whole cell 9
proteins (whole cell) and periplasmic fractions (periplasm) prepared from SJW1103 10
transformed with pTrc99A (vector control), pNSK8 or pNSK7. (C) Swarming motility assay 11
of SJW1103 carrying pTrc99A, pNSK8 or pNKS9 on soft-agar plates with or without 1 mM 12
IPTG. Plates were incubated at 30ºC for 6 hours. 13
14
Fig. 2. Dominance properties of various mutant variants of MotBC-His6. (A) Multiple 15
alignments of bacterial proteins containing a PGB motif and mutations generated in 16
MotBC-His6. Sequences aligned are: StMotB, Salmonella typhimurium MotB; EcMotB, 17
Escherichia coli MotB; VaPomB, Vibrio alginolyticus PomB; VaMotY, Vibrio alginolyticus 18
MotY; HiPal, Haemophilus influenzae Pal; EcOmpA, Escherichia coli OmpA. Multiple 19
sequence alignment was done by the ClustalW software (42). Asterisks indicate residues 20
mutated in this study. Residues shown in the black (or gray) box with white letter are 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
31
completely conserved (or well-conserved) among these six proteins. The point mutations are 1
those originally identified by Blair et al. (6) to produce dominant-negative mot- phenotype in 2
E. coli. ∆197-210 and ∆211-226 are deletion mutants that lack residues 197 - 210 and 211 - 3
226, respectively. (B) Swarming motility assay of the wild-type strain SJW1103 transformed 4
with the following plasmids: pTrc99A [vector control], pNSK8 [MotBC-His6], pNSK7 5
[PelBL-MotBC-His6], T197I [PelBL-MotBC(T197I)-His6], D198N 6
[PelBL-MotBC(D198N)-His6], S215F [PelBL-MotBC(S215F)-His6], R218W 7
[PelBL-MotBC(R218W)-His6], R223H [PelBL-MotBC(R223H)-His6], ∆197-210 8
[PelBL-MotBC(∆197-210)-His6), ∆211-226 [PelBL-MotBC(∆211-226)-His6), and 9
T197I/R218W [PelBL-MotBC(T197I/R218W)-His6]. Cells were inoculated onto the same 10
positions of soft agar plates with or without 0.1 mM IPTG. The strain names are indicated 11
only on the left plate. Plates were incubated at 30ºC for 6 hours. 12
13
Fig. 3. Periplasmic localization of mutant variants of MotBC. (A) MotBC with single 14
mutations or deletions. (B) MotBC with a T197I/R218W double mutation. Immunoblotting 15
using the anti-MotB antibody of whole cell proteins and periplasmic fractions of SJW1103 16
transformed with WT, pNSK7; T197I, pNSK7(T197I); D198N, pNSK7(D198N); S215F, 17
pNSK7(S215F); R218W, pNSK7(R218W); R223H, pNSK7(R223H); ∆197-210 , 18
pNSK7(∆197-210); ∆211-226, pNSK7(∆211-226); and T197I/R218W, 19
pNSK7(T197I/R218W). 20
21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
32
Fig. 4. Hydrodynamic properties of MotBC. (A) Elution profile of purified MotBC by 1
analytical size exclusion chromatography using a Superdex 75 HR 10/30 column. Arrows 2
indicate elution peaks of marker proteins: BSA (67 kD), ovalbumin (44 kD), and 3
chymotrypsinogen (25 kD) at 8.8 ml, 9.5 ml, 11.7 ml, respectively. (B) SDS-PAGE of elution 4
fractions from (A). Fraction numbers 7 to 10 correspond to the volume from around 7.5 ml to 5
10 ml. (C) Sedimentation equilibrium analytical ultracentrifugation of MotBC. Open circles 6
are data points, and the continuous line is a model fit. A concentration profile for MotBC 7
(initial absorbance of 0.2 at 280 nm, measured at 24,000 rpm) is shown in the lower panel. 8
For data fitting, we performed a global fit to six data sets from two different protein 9
concentrations and three rotor speeds. The residuals due to deviation of the data from this line 10
are shown in the upper panel. The obtained molecular mass was 50.6 kD, indicating that 11
MotBC is dimer. Measurements were done at room temperature. 12
13
Fig. 5. Hydrodynamic properties of mutant MotBC. (A) Elution profiles of purified 14
MotBC-His6 fragments by analytical size exclusion chromatography using a Superdex 75 HR 15
10/30 column. Black line, wild type MotBC (peak at 8.9 ml); Blue line, the MotBC(R218W) 16
mutant (peak at 8.9 ml); Red line, the MotBC(∆197-210) mutant (peak at 9.9 ml). Arrows 17
indicate elution peaks of size marker proteins: BSA (67 kD), ovalbumin (44 kD), and 18
chymotrypsinogen (25 kD) at 8.6 ml, 9.4 ml, 11.6 ml, respectively. (B) Sedimentation 19
equilibrium analytical ultracentrifugation of MotBC(∆197-210)-His6. Open circles are data 20
points, and the continuous line is a model fit. A concentration profile for 21
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
33
MotBC(∆197-210)-His6 (initial absorbance of 0.2 at 280 nm, measured at 28,000 rpm) is 1
shown in the lower panel. For data fitting, we performed a global fit to six data sets from two 2
different protein concentrations and three rotor speeds. The residuals due to deviation of the 3
data from this line are shown in the upper panel. The obtained molecular mass was 24.0 kD, 4
indicating that MotBC(∆197-210)-His6 is monomer. Measurements were done at room 5
temperature. 6
7
Fig. 6. Co-isolation assay of MotBC variants with MotB. MotBC-His6 designed to be exported 8
into the periplasm (expressed from plasmid pNSK7, lane 1) and expressed in the cytoplasm 9
(pNSK8, lane 2) were co-expressed with MotA and MotB (expressed from compatible 10
plasmid pNSK31) (lane 3 and 4). The expression levels of MotB and MotBC were similar in 11
both cases. Then, the membranes were isolated, solubilized by the detergent 12
dodecylphosphocholine (DPC), and mixed with a Ni-NTA resin. MotBC-His6 and its 13
associated proteins were eluted by imidazole, and samples prepared from each step were 14
analyzed by immunoblotting with the anti-MotBC antibody. A monomeric variant of MotBC 15
(MotBC(∆197-210), expressed from pNSK7-∆(197-210)) was also examined in the same way 16
(lane 5). An alternative combination (MotA/MotB-His8 and tag-less MotBC, expressed from 17
pNSK32 and pNSK28 respectively) was also examined (lane 6). Top panel, whole cell 18
extracts; middle panel, membrane fraction; bottom panel, eluted products. 19
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
34
Table 1. Strains and plasmids used in this study. 1
Strains or plasmids Relevant properties Source or
ref.
E. coli
Novablue recipient for cloning experiments Novagen
BL21(DE3) host for overexpression from the T7 promoter Novagen
RP6894 ∆motAB J. S.
Parkinson
Salmonella
SJW1103 wild-type for motility and chemotaxis (46)
Plasmids
pTrc99A cloning vector Pharmacia
pHMK11 pTrc expression vector This study
pACTrc pTrc promoter, p15A replication origin, lacIq,
Cmr
G. M. Fraser
pET19b T7 expression vector Novagen
pET22b T7 expression vector Novagen
pHMK1609 pHMK11/MotA+MotB-His8 This study
pNSK6 pET19b/MotBC-His6 This study
pNSK7 pHMK11/PelBL::MotBC-His6b This study
pNSK8 pTrc99A/MotBC-His6 This study
pNSK11 pET19b/MotBC This study
pNSK28 pHMK11/PelBL::MotBCb This study
pNSK31 pACTrc/MotA+MotB This study
pNSK32 pACTrc/MotA+MotB-His8 This study
pNSK6-R218W pET19b/MotBC(R218W)-His6 This study
pNSK6-∆(197-210) pET19b/MotBC(∆197-210)-His6 This study
pNSK6-T197I/R218W pET19b/MotBC(T197I/R218W)-His6 This study
pNSK7-T197I pHMK11/PelBL::MotBC(T197I)-His6b This study
pNSK7-D198N pHMK11/PelBL::MotBC(D198N)-His6b This study
pNSK7-S215F pHMK11/PelBL::MotBC(S215F)-His6b This study
pNSK7-R218W pHMK11/PelBL::MotBC(R218W)-His6b This study
pNSK7-R223H pHMK11/PelBL::MotBC(R223H)-His6b This study
pNSK7-∆(197-210) pHMK11/PelBL::MotBC(∆197-210)-His6b This study
pNSK7-∆(211-226) pHMK11/PelBL::MotBC(∆211-226)-His6b This study
pNSK7-T197I/R218W pHMK11/PelBL::MotBC(T197I/R218W)-His6b This study
a MCS, multi cloning site;
b MotB fragment coding for residues from 78 to the C-terminus 2
309 (MotBC) was fused to a PelB leader sequence derived from pET22b at the N-terminus, 3
and fused to His6 at the C-terminus. 4
5
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
pTrc99A
pNSK8
pNSK7
whole cell
periplasm
1 2 3
MotBc
- IPTG +1mM IPTG
pNSK7
pNSK8
pTrc99A(vector)
1 30 50 197 226 309
N CMotB
His 6
78 309
pNSK8
pNSK7
His 6
78 309
(A)
(B)
(C)
transmembranedomain
peptidoglycanbinding motif
Kojima et al., Fig.1.
PelB leader
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
PGB motifCNMotB
MotBc-His6 78 -His6
StMotB 197 TDDFPYANGEKGYSNWELSADRANASRREL 226EcMotB 196 TDDFPYASGEKGYSNWELSADRANASRREL 225VaPomB 231 TDNRPLDS-ELYRSNWDLSSQRAVSVAQEM 259VaMotY 218 LVATYTDSTDGKSASQSLSERRAESLRDYF 247
EcOmpA 261 TDRIGSDA-----YNQGLSERRAQSVVDYL 285HiPal 70 TDERGTPE-----YNIALGQRRADAVKGYL 94
** * * *
- IPTG + 0.1 mM IPTG
pTrc99A
pNSK7
pNSK8
T197ID198N
S215F R218W R223H
∆197-210 ∆211-226
(B)
(A)
pTrc99A
pNSK8
pNSK7
T197I/R218W
R218W
T197I
Kojima et al., Fig.2.
∆197-210
∆211-226
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
32.5
32.5
WT
T197I
D19
8N
S215F
R21
8W
R22
3H
∆197
-210
∆211
-226
Whole cell
Periplasm
kDa
1 2 3 4 5 6 7 8
(A)
Kojima et al., Fig.3.
vector
WT
whole cell
periplasm
1 2 3
T197I
/R21
8W(B)
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
0
25
50
75
100
5 7.5 10 12.5 15
67 44 25
OD
280
(mA
U)
Elution volume (ml)
8.7 mlMotBC
2 3 4 5 6 7 8 9 10 1112 13 14 15
Fractions
MotBc
(A)
(B)
(C)
Kojima et al., Fig.4.
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
0
20
40
60
80
5 7.5 10 12.5 15
OD
280
(mA
U)
∆197-210
R218W
WT
67 44 25
8.9 ml
8.9 ml 9.9 ml
Elution volume (ml)
(A)
(B)
Kojima et al., Fig.5.
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
32.5
1 2 3 4 5 6
32.5
3 4 5 6
32.5
1 2 3 4 5 6
Whole cell
Membrane
Elution
Kojima et al., Fig.6.
MotB
MotB
MotB
MotBC-His6MotBC
MotBC(∆197-210)-His6
MotBC-His6MotBC
MotBC(∆197-210)-His6
MotBC-His6MotBC
MotBC(∆197-210)-His6
ACCEPTED
on February 8, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
