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Journal of Bacteriology, August 2000, p. 4264-4267, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cross-Linked Complex between Oligomeric Periplasmic
Lipoprotein AcrA and the Inner-Membrane-Associated Multidrug Efflux
Pump AcrB from Escherichia coli
Helen I.
Zgurskaya and
Hiroshi
Nikaido*
Department of Molecular and Cell Biology,
University of California, Berkeley, California
Received 18 February 2000/Accepted 15 May 2000
 |
ABSTRACT |
In Escherichia coli, the intrinsic levels of resistance
to multiple antimicrobial agents are produced through expression of the
three-component multidrug efflux system AcrAB-TolC. AcrB is a
proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the
periplasm and the outer membrane, respectively. In this study, these
three proteins were expressed separately, and the interactions between
them were analyzed by chemical cross-linking in intact cells. We show
that AcrA protein forms oligomers, most probably trimers. In this
oligomeric form, AcrA interacts specifically with AcrB transporter
independently of substrate and TolC.
| |
INTRODUCTION |
AcrAB is a major, constitutively
expressed, multidrug efflux system from Escherichia coli
(8). It recognizes and expels from the cells an unusually
broad range of antimicrobial agents (9). The
acrAB operon codes for two proteins, the efflux transporter AcrB (110 kDa) and the periplasmic lipoprotein AcrA (41 kDa without the
attached lipid moiety), both of which are essential for drug efflux.
Genetic studies have provided evidence that the multifunctional outer
membrane channel TolC is the third component of this transporter, since
mutations in tolC completely abolish the AcrAB-dependent multidrug resistance phenotype of E. coli (2).
AcrAB-TolC was suggested to be similar in structure to the
well-characterized tripartite complexes such as hemolysin A transporter
HlyBD-TolC (5): the inner-membrane-associated
transporter AcrB is thought to form a complex with the
periplasmic component AcrA, which is anchored to the outer surface of
the inner membrane through its lipid moiety and with the outer membrane
channel TolC. Such organization is believed to provide the structural
means for the transport of substrates from the cells directly into the
medium, bypassing the periplasmic space (10). However, no
evidence was presented for the interaction between these three components.
Chemical cross-linking in intact cells has been used extensively to
define specific interactions in the membrane transport complexes
(1, 13, 15). In this study, we have used such cross-linking
along with the immunological detection to provide evidence for an
interaction between AcrA and AcrB.
| |
MATERIALS AND METHODS |
Bacterial strains, growth conditions, and plasmids.
All strains and plasmids used in this study are listed in Table
1. E. coli strains were grown
at 37°C in L broth (10 g of Difco Yeast Extract, 10 g of Difco
Tryptone Peptone, and 5 g of NaCl per liter). Antibiotics were
added, when required, to the following final concentrations: kanamycin,
34 µg/ml; ampicillin, 100 µg/ml; and chloramphenicol, 15 µg/ml.
Plasmid pAB was constructed by cloning an
NdeI-
NsiI fragment, encoding
acrA and
acrB, from pUC151A (
8) into
NcoI and
PstI
sites of the polycloning region of pBAD/Myc-HisA
(Invitrogen).
Plasmid pAB expressed the AcrA and AcrB proteins
under the dosage-dependent,
arabinose-inducible promoter
P
BAD. Because
NsiI cuts downstream
from the
termination codon of
acrB, an intact AcrB without the
Myc-His tag was expressed from the vector. To construct plasmid
pOA,
the
SphI-
BglI fragment from pBAD/Myc-HisA
containing the
activator gene
araC, the promoter
P
BAD, and polycloning region
was cloned into
SphI and
HincII sites of the polycloning region
of pUC18, generating plasmid pARA. Then the
NcoI-
BlpI fragment
from pEZ4 (
16)
encoding
ompA-acrA-his6 was inserted into
NcoI
and
BglII sites of pARA. The resulting
plasmid, named pARAOmpA-AcrA,
served as a donor of
SphI-
SmaI DNA fragment to be cloned into
SphI and
HincII sites of pACYC184. The resultant
plasmid pOA expressed
the AcrA protein, whose own signal sequence was
replaced by the
OmpA signal peptide and which in addition contained a
C-terminal
fusion of six histidine residues, under the control of
P
BAD promoter.
Chemical cross-linking in intact cells.
In general, E. coli cells were grown in 5 ml of Luria-Bertani medium supplemented
with appropriate antibiotics to an optical density at 600 nm of 0.7 to
1.0, harvested by centrifugation, and washed with sodium phosphate
buffer (pH 7.5). Cells were resuspended in 0.5 ml of the same buffer,
and dithiobis(succinimidylpropionate) (DSP; Pierce) or disuccinimidyl
glutarate (DSG; Pierce) was added to 0.4 or 0.2 mM, respectively.
Cross-linking was carried out for 30 min, with DSP at 37°C or with
DSG on ice, and was terminated with the addition of Tris-HCl (pH 7.5)
to 20 mM (15). Cells were harvested by centrifugation and
sonicated on ice for 1 to 2 min using the microtip of a Gallenkamp
Soniprep 150 sonicator. Membranes were collected by centrifugation at
100,000 × g for 30 min.
Immunoblot analysis.
The membrane proteins were solubilized
at room temperature for 20 min in 1% sodium dodecyl sulfate (SDS)-1
mM EDTA-10 mM Tris-HCl (pH 8.0)-1 mM phenylmethylsulfonyl fluoride
and then separated by electrophoresis on an 8% polyacrylamide gel or 5 to 13% gradient polyacrylamide gel containing 0.1% SDS (SDS-PAGE).
The proteins were transferred to an Immobilon-P polyvinylidene
difluoride (PVDF) membrane (Millipore) overnight at 30 V, using the
transfer buffer described in reference 17. The PVDF
membranes were probed with the anti-AcrA and anti-AcrB antibodies as
described elsewhere (16, 17).
Purification and analysis of the AcrA-His6-containing
protein complexes.
After cross-linking with DSP,
membrane-associated AcrA-His6 was purified by metal
chelation chromatography using His-Bind Quick 300 cartridges (Novagen).
For this purpose, cell membranes were isolated as described above and
solubilized for 20 min on ice in loading buffer containing 100 mM
Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, 1% Triton X-100, and
0.2% sarcosyl. Insoluble material was removed by centrifugation at
100,000 × g for 30 min, and membrane extract was
loaded on His-Bind 300 cartridges. After extensive washing with loading
buffer supplemented with 30 mM imidazole, bound proteins were eluted
with solution containing 100 mM Tris-HCl (pH 8.0), 0.4 M imidazole, and
2% SDS.
To analyze the composition of cross-linked complexes, proteins eluted
from the His-Bind 300 cartridges were separated by SDS-PAGE
(5 to 13%
gradient gel) and then further resolved by the second-dimension
SDS-PAGE: a 1-cm lane of the first-dimension gel with the protein
complexes was excised, dipped into 1% mercaptoethanol to cleave
DSP
cross-links, and placed on the top of an SDS-10% polyacrylamide
gel
as described previously (
14). Proteins were visualized by
the Coomassie blue staining or by immunoblotting with an anti-AcrA
antibody.
| |
RESULTS AND DISCUSSION |
Major complexes containing both AcrA and AcrB are immunodetected
after cross-linking in intact cells.
The AcrA and AcrB proteins
were expressed in AcrAB-deficient E. coli strain AG100A
(11) from plasmid pAB carrying acrA and acrB under the tightly controlled arabinose-inducible
promoter PBAD. The expression of both AcrA and AcrB
proteins from this construct was dosage dependent and increased with
increasing concentrations of arabinose in the medium (Fig.
1A).
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FIG. 1.
Immunoblotting analysis of AcrB and AcrA produced from
pAB before (A) and after (B) cross-linking with DSP. Cross-linked
complexes (B) were separated by SDS-PAGE (8% gel) and analyzed by
Western blotting with anti-AcrB (top) and anti-AcrA (bottom)
antibodies. Positions of AcrB- and AcrA-specific complexes are
indicated by arrows. M, size markers.
|
|
To stabilize protein complexes, intact
E. coli cells were
treated with DSP, which has a spacer arm of 12 Å and a disulfide
bond
that can be cleaved with reducing agents. After treatment
with DSP,
membrane proteins from AG100A/pAB were analyzed by SDS-PAGE
followed by
immunoblotting with antibody against the AcrB protein
(
17)
(Fig.
1B). In addition to the AcrB protein band, we observed
two bands,
C1 and C2, which had apparent molecular weights greater
than that of
AcrB protein alone. Confirming the specificity of
these complexes,
their amounts paralleled the amounts of AcrB
protein (Fig.
1B, top),
and these bands disappeared upon treatment
of the membrane preparation
with dithiothreitol (100 mM) at 37°C
(data not shown). Parallel
experiments with antibody against AcrA
(
16) (Fig.
1B,
bottom) showed that two complexes with mobilities
very similar to those
of C1 and C2 were also present, suggesting
that these complexes
contained both AcrA and AcrB. From the Western
blots, the apparent
molecular masses of C1 and C2 were found to
be >250 and about 180 kDa,
respectively. In addition to complexes
C1 and C2, the anti-AcrA
antibody revealed two other complexes,
CA1 and CA2, with apparent
molecular masses of 132 and 100 kDa,
respectively.
AcrA forms oligomers.
The secondary structure predictions for
AcrA (7, 12) suggest that AcrA and its homologs from the
membrane fusion protein family have extended 
-helical regions with a
high probability to form coiled coils. Such structures are often
involved in protein oligomerization; at least for one membrane fusion
protein, HlyD, a trimeric structure was suggested based on
cross-linking experiments (15).
To assess the oligomeric state of AcrA, we used a similar approach.
Cross-linking was performed in intact cells with the noncleavable
cross-linker DSG, which has a spacer arm shorter (7.7 Å) than
that of
DSP and introduces cross-links only to proteins that are
very close to
each other. After cross-linking of AG100A/pUC151A
with DSG, the
membranes were purified and protein interactions
were analyzed by
SDS-PAGE and immunoblotting (Fig.
2A). In
addition
to the monomeric form of AcrA, migrating at 45 kDa, two
high-molecular-weight
forms of AcrA, migrating at 132 and 100 kDa, were
identified.
These AcrA forms were found also when the chromosomal copy
of
acrA gene was expressed in the AcrB-deficient
E. coli strain AG102MB
and the TolC-deficient strain ZK796 (
4,
6) (Fig.
2A). AcrA
thus might form a trimer when in association
with the inner membrane
of
E. coli, even in the absence of
the inner membrane transporter
AcrB and the outer membrane channel
TolC. The mobilities of these
complexes were identical to those of CA1
and CA2 in Fig.
1B, suggesting
that they also represent the trimeric
and dimeric forms of AcrA,
respectively. Finally, the results with
ZK796 and AG102MB show
that oligomerization occurs with the expression
of a chromosomal
copy of
acrA and that it is not an artifact
of
acrA overexpression.
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FIG. 2.
Inner-membrane-associated AcrA is a trimer. (A) DSG
cross-linking experiments demonstrating that oligomerization of AcrA is
independent of AcrB and TolC. AcrA was stained with anti-AcrA antibody.
(B) Cross-linked oligomeric AcrA after purification on an
Ni2+ affinity column. AcrA was expressed as
OmpA-AcrA-His6 fusion protein from pOA. After cross-linking
with DSP, AcrA-His6 was purified and analyzed by Western
blotting with the anti-AcrA antibody. Treatment with dithiothreitol
(lane 2), which reverses DSP cross-links, confirmed that the purified
AcrA forms are products of cross-linking. (C) Analysis of the
cross-linked AcrA by two-dimensional SDS-PAGE demonstrating that AcrA
(arrow 1) is the only component of complexes CA1 (arrow 3) and CA2
(arrow 2). The proteins were stained with Coomassie blue.
|
|
To identify the components of these complexes, we transformed
E. coli AG100A with plasmid pOA, which expresses
OmpA-AcrA-His
6 (
16) under the P
BAD
promoter. Here, the OmpA-AcrA-His
6 is a
fusion product of
the signal peptide from OmpA and the signal
peptide-deficient AcrA with
a hexahistidine tag at its C terminus.
After induction with 0.2%
arabinose, AG100A/pOA produced a large
amount of AcrA-His
6
protein, which was shown to be mostly located
in the periplasm
(
16). After cross-linking with DSP in intact
cells,
membrane-associated AcrA-His
6 was solubilized in 1% Triton
X-100-0.2% sarcosyl (Materials and Methods) and purified by metal
chelation chromatography. Three bands corresponding to monomeric
AcrA-His
6, CA1, and CA2 were detected by Coomassie blue
staining
and also by immunoblotting of the SDS-polyacrylamide gels with
the protein fractions eluted from the Ni
2+ column (Fig.
2B). All three forms of AcrA-His
6 were further analyzed
by
a second-dimension SDS-PAGE after reductive cleavage of DSP
cross-links
(Fig.
2C). Complexes CA1 and CA2 were found to contain
only one protein
with an apparent mobility of 45 kDa corresponding
to monomeric AcrA.
CA1 and CA2 thus correspond to the trimeric
and dimeric forms of AcrA.
We cannot rule out the possibility
that AcrA might exist in the cells
as a higher-order oligomer,
although the recovery of only the trimeric
and dimeric forms of
AcrA indicates that AcrA exists as trimers in
cells. We note also
that AcrA-His
6 does not contain the
N-terminal cysteine, the site
for lipid modification (
16).
Oligomerization of these lipid-deficient
AcrA derivatives indicates
that AcrA forms dimers and trimers
through specific interactions, not
through nonspecific aggregation
driven by the lipid moiety.
Furthermore, the AcrA-His
6 fraction,
which is apparently
not associated with the membranes and is recovered
in the
periplasmic/cytosolic fraction after the sedimentation
of membranes by
centrifugation at 100,000 ×
g, also appeared to
form
oligomers (data not shown). Thus, the interaction with the
membrane is
not required for AcrA
oligomerization.
Oligomeric AcrA forms complexes with the AcrB transporter in the
absence of the outer membrane channel TolC.
The molecular masses
of AcrA and AcrB calculated from the amino acid composition are 41 and
110 kDa, respectively, and the respective migration rates in SDS-PAGE
corresponded to 45 and 97 kDa. Accordingly, complexes C1 and C2
reacting with both anti-AcrA and anti-AcrB antibodies might correspond
to an AcrB monomer associated with three and two molecules of AcrA,
respectively. To test this hypothesis, cross-linking with DSP was
performed on intact cells of AG102MB
(acrB::kan). In this strain, complexes
C1 and C2 were not detected with the anti-AcrA antibody (Fig.
3, lane 2). Complementation experiments
were carried out with AG102MB transformed with plasmid pUCBPA, producing AcrB. The expression of AcrB from
pUCBPA restored the formation of complexes C1 and C2 on
Western blots (Fig. 3, lane 1). It is possible that a single AcrB
molecule is cross-linked to two or three monomeric AcrA molecules in
these complexes. However, the absence of a complex containing one AcrB
molecule and one AcrA molecule suggests that only oligomeric forms of
AcrA can interact with AcrB and that C1 and C2 are AcrB monomers
cross-linked to trimers and dimers, respectively, of AcrA. These
complexes were also identified in the TolC-deficient strain ZK796 (Fig. 3, lane 3), suggesting that TolC is not present in either C1 or C2 and
that these complexes are formed without overexpression of AcrA and
AcrB. Thus, AcrA and AcrB exist in a stable complex in association with
the inner membrane of E. coli, and this complex can assemble
independently of the outer membrane channel TolC.
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FIG. 3.
Immunoblotting analysis of AcrA in the AcrB- and
TolC-deficient cells cross-linked with DSP in intact cells. Western
blot analysis with the anti-AcrA antibody showed that complexes C1 and
C2 were absent in the acrB::kan strain
AG102MB but were present in the tolC mutant ZK796. This
finding is consistent with the conclusion that these complexes contain
both AcrA and AcrB but not TolC. M, size markers.
|
|
To identify complexes containing the TolC protein, we used a polyclonal
anti-TolC antibody, a gift from R. Misra. However,
even with the use of
a plasmid that produces an increased level
of TolC, so far we have not
been able to find cross-linked products
containing TolC. The reason for
this failure can be trivial. For
example, outer membrane proteins
usually contain few exposed lysine
residues and are usually difficult
to cross-link with reagents
that use the
-amino group of lysine as
targets. In fact, TolC
(471 amino acid residues) contains only 16 lysine residues. Another
reason could be the low avidity and low
specificity of the anti-TolC
serum used. Furthermore, the complex
containing three AcrA, one
AcrB, and one TolC is expected to have a
size of about 350 kDa,
and it may be difficult to transfer such a large
complex to PVDF
membranes by electrophoresis. On the other hand, the
reason for
the failure may be nontrivial. With the type I protein
secretion
system, the recruitment of TolC into the secretion complex
takes
place only after the substrate, the exported protein, has
assembled
the rest of the machinery, including the transporter and the
AcrA
homolog, HlyD (
15). Similarly, the recruitment of TolC
into
the efflux complex may take place on a transient basis, only when
the substrate molecule is about to be
exported.
In conclusion, we showed that the periplasmic lipoprotein AcrA
interacts specifically with the inner-membrane-associated efflux
pump
AcrB and that this complex is assembled even in the absence
of the
outer membrane component TolC. It appears that only oligomeric
forms of
AcrA form complexes with the AcrB protein. Oligomerization
of AcrA
itself, however, does not require
AcrB.
| |
ACKNOWLEDGMENT |
This study was supported in part by Public Health Service grant
AI-09644.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cell Biology, Room 229 Stanley Hall, University of
California, Berkeley, CA 94720-3206. Phone: (510) 642-2027. Fax: (510)
643-9290. E-mail: nhiroshi{at}uclink4.berkeley.edu.
| |
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Journal of Bacteriology, August 2000, p. 4264-4267, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
