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Journal of Bacteriology, October 2001, p. 5639-5644, Vol. 183, No. 19
Faculty of Pharmaceutical Science, Osaka
University, Suita, Osaka 565-0871,2 and
Department of Cell Membrane Biology, Institute of
Scientific and Industrial Research, Osaka University,
Ibaraki,1 and CREST, Japan Science
and Technology Corporation,3 Osaka 567-0047, Japan
Received 12 March 2001/Accepted 10 July 2001
In the Escherichia coli genome, five putative open
reading frame (ORF) clusters, mdlAB,
ybjYZ, yddA, yojHI, and yhiH,
have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these
ORFs in multicopy plasmids and investigated the drug
resistance of drug-supersensitive host cells lacking constitutive
multidrug efflux transporter genes acrAB. Among them, only
ybjYZ gave significant erythromycin resistance and
significantly decreased the accumulation of
[14C]erythromycin. Therefore, ybjYZ was
renamed macAB (macrolide-specific ABC-type efflux carrier).
Plasmids carrying both the macA and -B genes
conferred resistance against macrolides composed of 14- and
15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA
sequence suggests that MacB is an integral membrane protein with
four transmembrane segments and one nucleotide-binding domain, while
MacA belongs to a membrane fusion protein (MFP) family with a
signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB
required TolC for its function in a way similar to that of most of the
MFP-dependent transporters in E. coli. MacB is thus a novel
ABC-type macrolide efflux transporter which functions by cooperating
with the MFP MacA and the multifunctional outer membrane channel TolC.
This is the first case of an experimentally identified ABC antibiotic
efflux transporter in gram-negative organisms.
ATP-binding cassette (ABC)
transporters are the major drug efflux transporters in mammalian cells
that cause multidrug resistance of cancer cells (5). On
the other hand, ABC transporters are involved mainly in the uptake of a
wide range of molecules (13), protein export
(13), and the efflux of toxic metal ions, such as an
arsenite (23), in bacteria. LmrA in a gram-positive
bacterium, Lactococcus lactis (30), has been
the only example of an experimentally identified bacterial ABC
multidrug exporter. A small number of ABC single-drug exporters for
macrolides (22) and daunomycin (21) are also
known in gram-positive bacteria, whereas no functional ABC drug
exporter has been definitely established to exist in gram-negative
bacteria yet (32). The efflux-based multidrug resistance
of gram-negative bacteria is most often conferred by RND
(resistance-nodulation-cell division) family transporters (9,
15), in addition to some SMR (small multidrug resistance) transporters (18). Efflux-based single-drug resistance of
gram-negative bacteria is usually due to plasmid-encoded
MFS (major facilitator superfamily) transporters such as
tetracycline-H+ antiporters (11, 31).
Complete genomic DNA sequences have been determined for various
microorganisms, including Escherichia coli (2).
On the basis of these genomic DNA sequences, all of the possible open reading frames (ORFs) can be determined (25), and possible
drug exporter genes have been determined (19). As a
result, it was found that the E. coli chromosome contains
five ORF clusters for possible ABC drug efflux genes (19),
i.e., mdlAB, ybjYZ, yddA, yojHI, and yhiH. Recently, we cloned all of these
putative ABC transporter genes in multicopy plasmids
(14a), and their drug resistance was investigated in
drug-supersensitive strain E. coli KAM3 (12),
which lacks constitutive multidrug efflux transporter genes
acrAB (13). We found that only the
ybjYZ gene products confer macrolide-specific drug
resistance, and the genes were renamed macAB. In this study,
we investigated the macAB gene products and revealed that
they comprise a macrolide efflux transporter system. This is the first
case of an ABC drug efflux transporter being experimentally established
in a gram-negative bacterium.
Materials and bacterial strains.
[14C]erythromycin (1.85 GBq/mmol) was purchased from NEN
Life Science Products, Inc. Erythromycin, clarithromycin, rokitamycin, azithromycin, oleandomycin, josamycin, and leucomycin were kindly provided by Takeshi Nishino (Kyoto Pharmaceutical University, Kyoto,
Japan). All other materials were of reagent grade and were obtained
from commercial sources. E. coli AcrAB-deficient strain KAM3
(12) and TolC-deficient strain ZK796 (33)
were kindly provided by Tomofusa Tsuchiya and Yuji Morita (Okayama
University, Okayama, Japan) and by Hiroshi Nikaido (University
of California, Berkeley), respectively. All of the bacterial strains
and plasmids used in this study are listed in Table
1.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5639-5644.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Novel Macrolide-Specific ABC-Type Efflux Transporter in
Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Cloning and expression of ORFs encoding putative ABC transporters. Chromosomal DNA was isolated from E. coli W3104 cells as previously described (14). The DNA fragments including putative ABC transporter ORFs and their upstream regions containing the natural promoters were amplified by the PCR method using primers containing the restriction enzyme site that exists in the multicloning sites of pUC119. The digested DNA fragments containing ORFs were ligated into the multicloning sites of pUC119 in the same direction as the lac promoter (Table 1), and then E. coli KAM3 cells were transformed with these recombinant DNAs.
pUCmacAB carries the 502-bp upstream region containing the native promoter, in addition to the entire macA and macB ORFs. The macA and macB ORFs were individually subcloned into pUC119 as follows. pUCmacA was constructed by subcloning the SmaI fragment of pUCmacAB, in which about one-half of the macB ORF was removed while the 500-bp upstream region and the entire macA ORF were left. For pUCmacB, the macA ORF was removed from pUCmacAB by means of crossover PCR; as a result, the 502-bp upstream region was directly connected to the macB ORF. In order to investigate the intracellular localization of MacA and MacB, the macA and macB genes were also individually subcloned into plasmid pQE70 (Table 1). Plasmid pQE70 carries an optimized promoter-operator element consisting of the phage T5 promoter, which is recognized by E. coli RNA polymerase, and two lac operator sequences. In addition, a six-His tag coding sequence was attached at the 3' terminus of the cloned gene.Drug resistance determination. The drug resistance of cells was determined by means of a sequential twofold dilution method on yeast extract-Bacto Tryptone agar plates as previously described (14) without induction, and the results were expressed as MICs. E. coli KAM3 cells carry the lacIq gene; thus, the cloned genes are not expressed from the lac promoter without induction. Expression of the putative ABC transporter genes was expected from the native promoters.
Assay of [14C]erythromycin accumulation in E. coli cells. E. coli KAM3/pUCmacAB and its host cells were grown in 30 ml of Luria-Bertani broth at 37°C until they reached an optical density at 600 nm of 0.6. The cells were then harvested and washed twice with 50 mM potassium phosphate buffer (pH 7.0). The cells were then finally suspended in 7.2 ml of the same phosphate buffer. The cell suspension (500 µl) was preincubated with 25 mM glucose at 37°C for 1 min. Erythromycin uptake was started by addition of 5 µl of [14C]erythromycin (1.53-µg/ml solution). At the indicated times, aliquots (250 µl) were withdrawn and filtered through Millipore filters (pore size, 0.45 µm). The filters were washed twice with 700 µl of potassium phosphate buffer, and then the radioactivity was measured with a liquid scintillation counter.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Drug resistance phenotypes produced by ORFs encoding putative ABC drug efflux transporters. Five putative ABC transporter genes, mdlAB, macAB, yddA, yojHI, and yhiH, were cloned into pUC119 under the control of the native promoters as described in Materials and Methods. Drug-supersensitive strain E. coli KAM3 (12), which lacks constitutive multidrug efflux transporter AcrAB, was transformed with these plasmids. The resistance phenotypes of the cells carrying these plasmids against 22 different drugs and toxic compounds were determined without induction. The MICs (in parentheses in micrograms per milliliter) of the following 21 compounds were unaltered in KAM3 containing mdlAB, macAB, yddA, yojHI, and yhiH: chloramphenicol (0.39), tetracycline (0.39), minocycline (0.20), nalidixic acid (0.78), norfloxacin (0.025), enoxacin (0.05), vancomycin (200), fosfomycin (1.56), doxorubicin (3.13), novobiocin (1.56), rifampin (6.25), trimethoprim (3.13), acriflavine (12.5), crystal violet (1.56), ethidium bromide (12.5), methylviologen (100), tetraphenylphosphonium bromide; (6.25), carbonyl cyanide m-chlorophenylhydrazone (6.25), benzalkonium (3.13), sodium dodecyl sulfate (50), and deoxycholate (1,250). For cells carrying the macAB genes, the MIC of erythromycin was 25 µg/ml, which was eight times higher than the MIC for the host cells (3.13 µg/ml). Plasmids carrying the other four ORF clusters produced no alteration in the MIC of erythromycin.
Amino acid sequences of MacA and MacB.
The DNA sequences of
the macAB genes were determined by means of the dideoxy
sequencing method with an ABI PRISM310 Genetic Analyzer. The ORFs of
macA and macB partially overlap; i.e., the sequence ATGA at the end of macA contains the stop codon TGA
for macA and the initiation codon ATG for macB
(Fig. 1A). There is only one promoter
sequence in the upstream region of macA, indicating that
macA and macB form one operon. The DNA sequences
of the macAB region cloned in this study are the same as
those in the reported genome sequence of E. coli
(2). MacA has a signal-like sequence composed of 40 amino
acid residues at its N terminus, which is rich in positively charged
residues in the N-terminal half and hydrophobic residues in the
C-terminal half. There is no hydrophobic cluster in the MacA sequence
except in this region (Fig. 1B). On the other hand, MacB
contains four putative transmembrane (TM) regions in its C-terminal
half (Fig. 1C). The N-terminal half of MacB includes a
nucleotide-binding cassette domain containing Walker A and B and
signature motifs.
|
Contributions of MacA, MacB, and the multifunctional outer
membrane channel TolC to resistance against macrolide
antibiotics.
In order to examine the contributions of MacA and
MacB to drug resistance, the macA and macB genes
were separately subcloned in the pUC119 vector under the
control of the natural promoter. The resistance of cells carrying
pUCmacAB, pUCmacA, and pUCmacB against
various macrolide antibiotics is shown in Table
2. Compared with the MIC for the host
KAM3 cells, neither cells carrying pUCmacA nor those carrying
pUCmacB showed an increase in resistance.
|
acrAB) and ZK796 (Intracellular localization of MacA and MacB.
In order to
reveal the intracellular localization of MacA and MacB, the
macA and macB genes were separately
subcloned into vector pQE70 to attach polyhistidine tags at their C
termini. The expression of MacA and MacB was induced with
isopropyl-
-D-thiogalactopyranoside (IPTG). Cells carrying pUCmacAB,
pQEmacA, and pQEmacB were disrupted by brief sonication, and then
supernatants (S) and membrane fractions (P) were obtained by
ultracentrifugation. After sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, total proteins were visualized by Coomassie brilliant
blue (CBB) staining and MacA and MacB were visualized by Western
blotting with anti-polyhistidine serum (Fig.
2A). Both MacA (40 kDa) and MacB (70 kDa)
bands were observed only for the membrane fractions (Fig. 2A, lanes 4 and 5). Both bands were also detected for the membrane fraction
of cells carrying pUCmacAB on CBB staining (Fig. 2A,
lane 3).
|
Uptake of [14C]erythromycin by cells carrying
pUCmacAB.
In order to determine whether or not macrolide
resistance is based on the active efflux of drugs, we compared the
uptake of [14C]erythromycin by E. coli KAM3
cells with and without the pUCmacAB plasmid (Fig.
3). [14C]erythromycin was
taken up by the host KAM3 cells. In cells carrying pUCmacAB,
uptake was significantly inhibited, indicating active efflux of
this drug out of the cells.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we showed that the MacAB complex confers
TolC-dependent macrolide resistance via active drug efflux. We examined the resistance pattern against 22 different drugs and toxic compounds which contain the typical compounds exported by multidrug transporters in bacteria and mammals. As a result, MacAB was revealed to confer macrolide-specific resistance. As shown in Fig.
4, MacB is a half-type ABC protein
having four putative TM segments and one nucleotide-binding cassette. There is a large hydrophilic loop region between TM1 and TM2,
which is probably located on the periplasmic surface and may interact
with MacA. MacA is a peripheral membrane protein that belongs to the
membrane fusion protein (MFP) family (4). According
to the sequence characteristics of the signal-like sequence and the
positive-inside rule of protein topogenesis (20), most of
the MacA molecule seems to be secreted and attached on the periplasmic surface (Fig. 4).
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A BLAST search revealed that there are homologues of macAB in gram-negative organisms. Putative ABC transporter proteins NMA0729 (16) and NMB0548 (29) in Neisseria meningitidis, PA2389 in Pseudomonas aeruginosa (26), and CAB75243.1 in Campylobacter jejuni (17) exhibit sequence identity with MacB, i.e., 48, 48, 43, and 41%, respectively. They show sequence similarity not only in the ATP-binding domains but also in the TM domains. In contrast, the sequence similarity between MacB and putative ABC transporters such as MsrA (24) in gram-positive organisms is restricted to the ATP-binding cassette region. The putative ABC transporter genes in the gram-negative organisms mentioned above include genes for MacA homologues in the same operon. Therefore, these genes can be classified into the same subfamily of ABC transporters in gram-negative organisms.
In eukaryotes, most ABC transporters are involved in multidrug resistance. However, in bacteria, the majority of drug exporters are drug-proton antiporters (8), including MFS-, SMR-, and RND-type transporters, while ABC transporters are usually involved in the uptake of a wide range of molecules (6). On the basis of the results of genome analysis, a number of putative ABC drug exporters are predicted (19), while the only ABC multidrug exporter experimentally identified in a bacterium is LmrA in Lactococcus lactis (30). In gram-positive organisms, some macrolide resistance genes code for ABC-type efflux transporter proteins (22, 24). Among them, MsrAB in Staphylococcus species has two nucleotide-binding domains and probably acts as an efflux system in cooperating with some other integral membrane proteins (10), while its characteristics as a drug exporter have not been fully revealed. As for macrolide exporters, some major facilitator-type exporters are known in gram-positive organisms (3, 28), such as MefA, which, similarly to MacAB, confers resistance against macrolides with 14- and 15-membered lactones.
A half-type ABC transporter is usually expected to have six TM segments (1, 30). MacB appears to have four TM segments. In addition, the N-terminal ABC on an ABC transporter is also unusual. Among the ABC transporters hitherto reported, only the ABCG subfamily in mammalian cells shows an N-terminal ABC (7). MacB requires the MFP MacA and the outer membrane channel TolC for drug efflux function. Although some bacterial ABC transporters such as HlyB (34) accompany an MFP and TolC, it is quite interesting that ABC-type drug efflux transporters depend on an MFP and TolC, as seen with RND-type multidrug efflux transporters (9, 13). In short, MacAB represents a quite unique and novel bacterial subfamily of ABC transporters.
Very recently, Sulavik et al. (27) constructed 22 E. coli strains with deletions of putative drug efflux transporters and outer membrane channels. The strain with a deletion of the macAB (ybjYZ) genes showed no change in drug susceptibility. However, since the macAB deletion strain carried the acrAB genes, the effect of macAB deletion might have been masked. The tolerance of E. coli cells to macrolide antibiotics is conferred mainly by AcrAB (35). Expression cloning of an individual gene into an AcrAB-deficient strain may be necessary to discover a potential drug efflux transporter gene.
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ACKNOWLEDGMENTS |
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This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. K. Nishino is supported by a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists.
K. Nishino and N. Kobayashi contributed equally to this work, and both should be considered first authors.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan. Phone: 81-6-6879-8545. Fax: 81-6-6879-8549. E-mail: akihito{at}sanken.osaka-u.ac.jp.
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Journal of Bacteriology, October 2001, p. 5639-5644, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5639-5644.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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