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Journal of Bacteriology, December 2001, p. 6961-6964, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6961-6964.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Carboxy-Terminal Region Involved in Activity of
Escherichia coli TolC
Hiroyasu
Yamanaka,1
Hiroshi
Izawa,2 and
Keinosuke
Okamoto2,*
Department of Biochemistry, Faculty of
Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro,
Tokushima 770-8514,1 and Faculty of
Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama
700-8530,2 Japan
Received 29 June 2001/Accepted 10 September 2001
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ABSTRACT |
The Escherichia coli TolC acts as a channel tunnel in
the transport of various molecules across the outer membrane.
Partial-deletion studies of tolC revealed that the region
extending from the 50th to the 60th amino acid residue from the carboxy
terminus plays an important role in this transport activity of TolC.
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TEXT |
TolC, an outer membrane
protein of Escherichia coli, functions as a transporter in
multiple transport systems (3). For example, TolC
interacts with the HlyD/HlyB complex to secrete hemolysin
(20) and with the CvaA/CvaB complex to secrete colicin V
(6). In addition to being involved in the secretion of
these macromolecules, TolC functions in the export of small molecules such as antibiotics, sodium dodecyl sulfate (SDS), bile salts, and
organic solvents by interacting with complexes such as EmrA/EmrB and
AcrA/AcrB (2, 10, 12, 18).
These large and small molecules are secreted in a single step without
stagnation in the periplasm. Recently, Yamanaka et al. (24) and Foreman et al. (4) found that TolC
is also involved in the transportation of heat-stable enterotoxin I
(STI) and STII of E. coli. These STs are synthesized as
precursor proteins with an amino-terminal signal sequence and are
translocated across the inner membrane via the Sec machinery (8,
14). In contrast to the secretory molecules which cross two
membranes in a single step without stagnating in the periplasm, these
STs, after being released into the periplasm, remain for a short period
in this location (4, 13, 22). After being processed
in the periplasm, these STs translocate across the outer
membrane through the action of TolC. This suggests that
TolC can also pump periplasmic components out into the medium.
In addition to having a secretory function, TolC participates in the
uptake of colicin E1 (ColE1) from outside the cell into the periplasm
(16, 21). However, the regions of TolC involved in these
activities have not been established. In this study, we mutated the
gene encoding the carboxy terminus of tolC and examined the
efflux of STs and antibiotics and the influx of ColE1.
Function of mutant TolC.
The tolC gene carried by
pET11-STI-TolC (23) was mutated to delete amino acid
residues at the carboxy terminus from the protein product. We generated
a stop codon at target positions of TolC by PCR-based overlap extension
mutagenesis using appropriate primers (5). The targets
were the amino acid residues at positions 452, 442, 432, 422, and 412 of TolC. The mutations were verified by DNA sequencing. The mutant TolC
proteins produced from these genes had deletions of 20, 30, 40, 50, and 60 amino acid residues from the carboxy terminus,
respectively. The mutant plasmids were designated
pET11-STI-TolC(
C20), pET11-STI-TolC(C30),
pET11-STI-TolC(C40), pET11-STI-TolC(C50), and
pET11-STI-TolC(C60), respectively.
The function of the mutant TolCs was examined by determining the
sensitivity to acriflavine and novobiocin of the cells harboring these
plasmids. Both antibiotics are excreted from cells by pumps which are
composed of several proteins, including TolC (11). BL21-2,
a derivative of BL21 whose tolC gene was mutated
(24), was used as the host strain. Sensitivity was
determined by an agar plate diffusion assay. Approximately
107 cells were spread on an L agar plate containing
ampicillin (50 µg/ml). Sterile blank disks (6.4 mm in diameter) were
placed on a lawn. A 20-µl solution of novobiocin (1 mg/ml; Sigma, St.
Louis, Mo.) or acriflavine (1 mg/ml; Sigma) was pipetted onto each
disk. The plates were incubated overnight at 37°C. The sensitivity of the cells to the substances was classified according to the size of the
growth inhibition zone.
BL21-2 transformed with pET11-STI, which does not contain
tolC (
23), was sensitive to these inhibitors.
In contrast, BL21-2
transformed with pET11-STI-TolC was tolerant (Table
1).
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TABLE 1.
Sensitivities to antimicrobial agents of strains of an
E. coli BL21 tolC mutant (BL21-2)
harboring the indicated plasmids
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The cells transformed with pET11-STI-TolC(
C20),
pET11-STI-TolC(
C30), pET11-STI-TolC(
C40), and
pET11-STI-TolC(
C50) were
tolerant to the inhibitors,
indicating that a deletion of less
that 50 amino acid residues does not
affect the activity of TolC.
In contrast, the cells transformed with
pET11-STI-TolC(
C60) were
sensitive to both inhibitors (Table
1),
indicating that the region
extending from the 50th to the 60th amino
acid from the carboxy
terminus is necessary for TolC to excrete the
inhibitors.
The sensitivity of the transformed cells to ColE1 was also examined by
the disk assay. The concentration of ColE1 (Sigma)
used was 100 µg/ml. As shown in Table
1, truncations at the 20th,
30th, 40th, and
50th amino acid residues did not affect ColE1
sensitivity, but the
truncation at the 60th residue induced a
complete loss of ColE1
sensitivity.
Assembly of mutant TolCs and association with the outer
membrane.
The native TolCs associate with the outer membrane and
assemble to form a trimer (7). To examine whether
TolC(C60)s form trimers and associate with the outer membrane, we
did cross-linking and membrane fractionation experiments.
BL21-2 cells harboring the plasmids were gently sonicated, and 300 µl
of the sonicated suspension containing 5 mg of protein
was removed to a
new tube. One hundred microliters of 25 mM dimethyl
suberimidate (DMS),
a cross-linking reagent (
19), was added
to the tube, which
was then incubated at 37°C for 10 min. The
reaction was quenched by
the addition of Tris-HCl (pH 7.4) to
a final concentration of 50 mM.
The samples were separated by
SDS-polyacrylamide gel electrophoresis
(PAGE) (
9), and the
TolC on the gel was detected by
immunoblotting using the anti-TolC
antiserum, which was prepared by the
injection of a peptide (ELRKSAADRDAAFEK),
corresponding to
residues 16 to 30 from the amino-terminal end
of TolC, into a
rabbit.
The sample from BL21-2/pET11-STI-TolC was placed in lanes 1 and 2 of
the gel shown in Fig.
1 and analyzed. A
51-kDa band was
detected in the sample not treated with DMS
(lane 1). The calculated
molecular weight of TolC is 51,454. In the
sample treated with
DMS (lane 2), a band of 155 kDa, presumably
representing TolC
trimers, appeared.
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FIG. 1.
Cross-linking of TolC exists in cells. Cells of E. coli BL21-2, the tolC mutant strain, transformed with
the indicated plasmids were grown to the exponential phase in L broth
at 37°C. The cultured cells were collected by centrifugation and
suspended in phosphate-buffered saline. They were then disrupted by
sonication. The solution containing the cell debris was treated with
DMS to generate cross-linkages between associated proteins. The sample
obtained was separated by SDS-PAGE, and the TolCs on the gel were
detected by immunoblotting as described in the text. The bands
indicated by black arrows are monomers of TolC, and those indicated by
open arrows are trimers. Numbers along the right side indicate
molecular masses in kilodaltons.
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|
The TolC(
C60) sample not treated with DMS (lane 5) migrated to the
45-kDa position. TolC(
C60) treated with DMS (lane 6)
produced a band
of 135 kDa. This result showed that the mutant
TolC(
C60)s associated
to form a
trimer.
To examine the association of TolC(
C60) with the outer membrane, the
crude membrane fractions of BL21-2 harboring pET11-STI-TolC(
C60)
were centrifuged through sucrose density gradients spanning 24
to 70%.
A previous study showed that the outer membrane and inner
membrane were
recovered from the fractions containing 50 and 30%
sucrose,
respectively (
15).
After centrifugation, the solution in the centrifugation tube was
divided into fractions. All fractions were examined for
the presence of
TolC by immunoblotting. Both TolC(
C60) and wild-type
TolC were
located in tubes containing 50 to 58% sucrose (data
not
shown).
Secretion of STs through the mutant TolCs.
Secretion of STs
into the medium through the actions of mutant TolCs was examined by
pulse-labeling (17). BL21-2 cells transformed with the
derivatives of pET plasmids (Fig. 2) were
labeled for 3 min with [35S]cysteine. After further
incubation for 3 min (chase period), the cells were separated by
centrifugation. The periplasmic fractions were prepared by treatment
with polymyxin B (23). The culture supernatant and
periplasmic fractions obtained were resolved by SDS-PAGE, and STs in
the gels were detected by autoradiography.
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FIG. 2.
Secretion of STI and STII from cells. The secretion of
STs from cells was examined by the labeling method. Exponentially
growing cells of BL21-2 harboring the indicated plasmid were treated
with IPTG (isopropyl- -D-thiogalactopyranoside) and then
labeled for 3 min with L-[35S]cysteine. The
labeling was terminated by the addition of cold cysteine to the
culture. Incubation was continued for 3 min (chase period), and then
the culture supernatant was obtained by centrifugation. The periplasmic
fractions of the cells were prepared by treatment with polymyxin B. These prepared fractions were treated with an SDS-dye solution and
heated at 100°C for 10 min. The samples thus prepared were resolved
by SDS-PAGE. Radioactive bands were detected by image analysis.
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STI synthesized by the TolC-deficient cells transformed with pET11-STI
was not released into the external medium and remained
in the periplasm
(Fig.
2A, lanes 1 and 5). In contrast, almost
all STIs synthesized by
the cells transformed with pET11-STI-TolC
were secreted into the medium
and the amount of STIs remaining
in the periplasm was very small (lanes
2 and 6). Deletion of 50
amino acid residues at the carboxy terminus of
TolC did not affect
the secretion of STI (lanes 3 and 7). However,
deletion of 60
residues markedly reduced the amount of STI secreted
into the
culture supernatant and the amount of STI remaining in the
periplasm
was very large (lanes 4 and
8).
The secretion of STII was also examined. The mutant plasmid
pET11-STII-TolC(
C60) was obtained by mutagenesis from
pET11-STII-TolC,
which was prepared by inserting
tolC into
the
HindIII-
EcoRI site
of pET11-STII
(
13). STII was not secreted from
BL21-2/pET11-STII-TolC(
C60),
and the STIIs remained in the periplasm
(Fig.
2A, lanes 11 and
14).
Effect of replacement of Leu-412.
The 60th amino acid residue
from the carboxy-terminal end of TolC is leucine at position 412. We
replaced the bases encoding Leu-412 of appropriate plasmids with those
encoding proline and examined the properties of the mutant TolC
[TolC(L412P)].
As shown in lane 4 of Fig.
1, TolC(L412P) formed trimers. Association
of TolC(L412P) with the outer membrane was also confirmed
by
ultracentrifugation through a sucrose density gradient (data
not
shown).
E. coli BL21-2 transformed with the plasmid was sensitive to
acriflavine and novobiocin and tolerant to ColE1 (Table
1).
The
activity of the mutant cells for the secretion of STI and
STII into the
culture supernatant was low compared with that of
the wild type (Fig.
2B, lanes 3 and 8). A large amount of STs
remained in the periplasm
(Fig.
2B, lanes 6 and
10).
In this study, we showed that the region extending from the 50th to the
60th amino acid from the carboxy-terminal end is indispensable
to TolC
for expressing its export-import activity, although it
is
not required for the formation of the TolC
trimer.
The TolC molecule can be divided into three domains: a
-domain, an
-domain, and a mixed
-
domain (
1,
7). The
-domain
is embedded in the outer membrane. In contrast, the
-domain penetrates
the periplasm and forms a 12-stranded
antiparallel
-barrel. Some
side chains of residues constituting the
mixed
-
domain interact
with the
-domain through hydrogen
bonds and van der Waals contacts.
The region extending from the 50th to
the 60th amino acid from
the carboxy terminus is a part of the mixed
-
domain that interacts
with two strands of the
-domain (H3
and H4) in the periplasm.
Our results suggest that this interaction may
be important for
TolC to express its
activity.
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ACKNOWLEDGMENTS |
This study was supported in part by a grant-in-aid for scientific
research from the Ministry of Education, Science, Sports and Culture of Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Pharmaceutical Sciences, Okayama University, Tsushima-naka,
Okayama, Okayama 700-8530, Japan. Phone: 81-86-251-7945. Fax:
81-86-251-7927. E-mail: okamoto{at}pheasant.pharm.okayama-u.ac.jp.
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Journal of Bacteriology, December 2001, p. 6961-6964, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6961-6964.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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