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Journal of Bacteriology, January 2004, p . 262-265, Vol . 186,
No . 1
An H+-Coupled
Multidrug Efflux Pump, PmpM, a Member of the MATE Family of Transporters, from
Pseudomonas aeruginosa
Gui-Xin He, Teruo Kuroda, Takehiko Mima, Yuji Morita, Tohru
Mizushima, and Tomofusa Tsuchiya*
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama
University, Tsushima, Okayama, 700-8530, Japan
Received 18 July 2003/ Accepted 24 September 2003
We cloned the gene PA1361 (we designated the gene pmpM), which
seemed to encode a multidrug efflux pump belonging to the MATE
family, of Pseudomonas aeruginosa by the PCR method using the
drug-hypersensitive Escherichia coli KAM32 strain as a host .
Cells of E . coli possessing the pmpM gene showed elevated
resistance to several antimicrobial agents . We observed
energy-dependent efflux of ethidium from cells possessing the pmpM
gene . We found that PmpM is an H+-drug antiporter, and
this finding is the first reported case of an H+-coupled
efflux pump in the MATE family . Disruption and reintroduction of the
pmpM gene in P . aeruginosa revealed that PmpM is
functional and that benzalkonium chloride, fluoroquinolones, ethidium
bromide, acriflavine, and tetraphenylphosphonium chloride are
substrates for PmpM in this microorganism .
Pseudomonas aeruginosa is an opportunistic pathogen and a leading
cause of nosocomial infections . A major problem in the treatment
of patients infected with P . aeruginosa is that this bacterium
shows intrinsic and acquired resistance against many antibiotics
and disinfectants, including most ß-lactams, fluoroquinolones,
tetracycline, chloramphenicol, erythromycin, and benzalkonium
chloride (4, 7, 11,
12, 13, 15,
20, 27) . Several mechanisms for
drug resistance are known, such as (i) inactivation of drugs by
degradation or modification, (ii) alteration of the target, (iii)
emergence of an alternative pathway, and (iv) active efflux of the
drug . Among these mechanisms, active efflux, especially multidrug
efflux, has been recognized as a major mechanism for multidrug
resistance . Once a bacterium acquires a gene for a certain multidrug
efflux pump or if a silent or weak gene for a multidrug efflux pump
is activated, the cell instantly becomes resistant to many
antimicrobial agents because multidrug efflux pumps extrude many
structurally unrelated antimicrobial agents from cells . Thus,
multidrug efflux pumps play important roles in multidrug resistance
in bacteria . Multidrug resistance in P . aeruginosa has been
attributed mainly to the activity of several multidrug efflux pumps .
MexAB-OprM (11, 15), MexCD-OprJ
(20, 23), MexEF-OprN (7),
MexXY-OprM (15, 16), MexJK-OprM (4),
MexHI-OpmD (27), MexVW-OprM (13),
and EmrE (12) have been characterized and are
potent multidrug efflux pumps in P . aeruginosa . MexAB-OprM is
the only constitutive Mex pump in wild-type P . aeruginosa (11,
15) . MexCD-OprJ (20, 23)
and MexXY-OprM (15, 16) are inducible
pumps, and MexEF-OprN (7), MexJK-OprM (4),
MexHI-OpmD (27), and MexVW-OprM (13)
are silent pumps in the wild-type strain .
The genome sequence of P . aeruginosa (29; http://www.Pseudomonas.com)
suggests the presence of about 34 multidrug efflux pumps in this
microorganism . Twenty of these pumps belong to the major facilitator
superfamily, 10 or 12 belong to the resistance nodulation cell
division family, 6 belong to the small multidrug resistance (SMR)
family (12), 2 or 3 belong to the multidrug and toxic
compound extrusion (MATE) family, and 1 belongs to the ATP binding
cassette superfamily . Multidrug efflux pumps of the major facilitator,
resistance nodulation cell division, and SMR families utilize
an electrochemical potential of H+ across the cytoplasmic membrane
as the driving force for drug extrusion . Pumps of the ATP binding
cassette superfamily utilize ATP as an energy source . The driving
force in the MATE family is unique . Pumps of this family utilize
an electrochemical potential of Na+ across the cytoplasmic
membrane as the driving force (2, 19,
21, 22) .
We are especially interested in multidrug efflux pumps belonging
to the MATE family because of its unique energy coupling . So far, we
have characterized several MATE family pumps, such as NorM (18)
and VmrA (2) of Vibrio parahaemolyticus, YdhE of
Escherichia coli (18), and VcmA (22)
and VcrM (21) of Vibrio cholerae non-O1 . We
have shown that all of them utilize an electrochemical potential of
Na+ across membranes as the driving force (2,
18, 21, 22) .
Here, we report that PmpM (a product of the PA1361 gene) (http://www.Pseudomonas.com)
of P . aeruginosa is a unique multidrug efflux pump belonging
to the MATE family that utilizes H+, but not Na+, as
the coupling ion for drug extrusion .
Cloning of pmpM. We cloned the open reading frame
PA1361 (http://www.Pseudomonas.com)
by the PCR method using chromosomal DNA of P . aeruginosa PAO1
as a template . Chromosomal DNA was prepared by the procedure
described by Chen (3) . The primers used were forward primer
1 (F1), 5'-CTACGGAATTCCCCTGCCCAGACAAGGAC-3' (containing an EcoRI
site), and reverse primer 1 (R1), 5'-TCCTCGCCTCGGTCGACACTACCCTCAG-3'
(containing a SalI site) . The primer F1 carries the Shine-Dalgarno
sequence of the pmpM gene but not a putative promoter of the
gene . We designated the gene pmpM (Pseudomonas MATE family
efflux pump) . The vectors used were pSTV28 and pUCP20T, and the
resulting hybrid plasmids, pPBE2 and pUPBE2T, respectively, carry the
pmpM gene but not its original promoter . The cloned pmpM
gene is located downstream from the lactose promoter in the two
plasmids . The addition of an inducer of the lactose operon (isopropyl
ß-D-thiogalactopyranoside) did not have a
significant effect on the level of drug resistance when cells were
grown in L broth, which may contain a natural inducer . Thus, we
investigated drug resistance without the addition of an inducer .
Drug susceptibility. To investigate the role of PmpM in drug
resistance, plasmid pPBE2, carrying the pmpM gene, was
introduced into cells of drug-hypersensitive E . coli KAM32 ( acrB
ydhE
Hsd-) (2) . We compared the MICs of
various antimicrobial agents for E . coli KAM32/pPBE2 and E .
coli KAM32/pSTV28 (control) (Table 1) . Elevated MICs
of fluoroquinolones, fradiomycin, benzalkonium chloride, chlorhexidine
gluconate, ethidium bromide, tetraphenylphosphonium chloride
(TPPCl), and rhodamine 6G were observed for KAM32/pPBE2 . Thus, the
pmpM gene is responsible for conferring multidrug resistance . It
seems that the disinfectant benzalkonium chloride is a good substrate
for PmpM when expressed in E . coli cells .
| TABLE 1 . MICs of various antimicrobial agents for E . coli
KAM32/pSTV28 and E . coli KAM32/pPBE2
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Drug transport via PmpM and cation coupling. PmpM is a member
of the MATE family of efflux pumps . Members of this family, such as
NorM (18, 19) and VmrA (2)
of V . parahaemolyticus and YdhE of E . coli (18),
are Na+-coupled efflux pumps . Thus, it was anticipated
that PmpM would also be a Na+-coupled efflux pump . If this
were the case, then ethidium efflux would be very weak in the absence
of Na+ and greatly enhanced by the addition of Na+
to the assay mixture . Thus, we tested the effect of NaCl on ethidium
efflux . First, we prepared energy-starved and ethidium-loaded cells (17,
22) . Then, lactate was added to energize the cells,
and ethidium efflux was measured in the absence or presence of
NaCl . Ethidium efflux energized by lactate was observed with cells of
E . coli KAM32/pPBE2 but not with cells of KAM32/pSTV28 (Fig.
1) . Addition of NaCl resulted in no significant change
in the efflux . We added various concentrations of NaCl ranging
from 1 to 100 mM and observed no significant effect (data not shown) .
Addition of NaCl prior to the addition of lactic acid gave no
significant effect . We also tested the effect of LiCl, because Li+
can replace Na+ as a coupling cation in the case of NorM (18) .
However, no significant effect of LiCl was observed (data not shown) .
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FIG . 1 . Ethidium transport assays . Cells (E . coli KAM32/pSTV28
and KAM32/pPBE2) were grown in L broth (10) under
aerobic conditions at 37°C . The cells were harvested, washed twice with
0.1 M MOPS (morpholinepropanesulfonic acid)-tetramethylammonium
hydroxide (pH 7.0) containing 2 mM MgSO4, and suspended in
the same medium containing 25 µM ethidium bromide and 40 µM carbonyl
cyanide m-chlorophenylhydrazone (16,
21) . The cells were incubated at 37°C for 1 h to
starve cellular energy and to load with ethidium (16,
21) . The cells were washed three times with the same
medium (no carbonyl cyanide m-chlorophenylhydrazone) and
resuspended in the same medium . This cell suspension was preincubated at
37°C for 5 min, and lactic acid (the pH was adjusted to 7.0 with
tetramethylammonium hydroxide) was added to the assay mixture at 40 mM .
The change in the fluorescence intensity was measured . Where indicated,
NaCl was added at 10 mM to test the effect of NaCl.
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If the coupling ion is Na+, we should be able to observe Na+
efflux from cells elicited by influx of substrate, as observed
with NorM (18, 19) and VmrA (2) .
We measured Na+ flux by using a Na+ electrode .
No Na+ flux was detected when substrates of the PmpM pump
were added to a suspension of Na+-loaded cells of E .
coli KAM32/pPBE2, although Na+ efflux was observed with
cells of E . coli KAM32/pMVP36 (data not shown) . pMVP36 carries
norM, the gene for a Na+-coupled multidrug efflux pump,
NorM, belonging to the MATE family (18,
19) . Thus, it is highly likely that H+
instead of Na+ is the coupling cation for PmpM .
If the coupling cation in PmpM is an H+, then it must be an
H+-substrate antiporter . One convenient method to test this
possibility is to measure the flux of H+ caused by a substrate
of the pump involved . A previous study has reported evidence
for H+-chloramphenicol antiport in a multidrug efflux pump,
Cmr (MdfA), by measuring fluorescence quenching of quinacrine (17) .
Therefore, we measured fluorescence quenching in everted membrane
vesicles prepared from cells of E . coli KAM32/pSTV28 and
KAM32/pPBE2 (Fig . 2) . Fluorescence quenching due to inwardly
directed H+ transport by the respiratory chain was elicited
by the addition of lactate in both types of everted membrane
vesicles . We chose benzalkonium chloride as the substrate for the
assay because some other substrates of PmpM had some unfavorable
effects on the fluorescence of quinacrine . Addition of benzalkonium
chloride to the assay mixture caused efflux of H+, indicating
that H+-benzalkonium chloride antiport took place . Thus, we
conclude that the coupling ion in PmpM is H+ but not Na+ .
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FIG . 2 . Benzalkonium chloride-H+ antiport activity in
membrane vesicles . Cells (E . coli KAM32 and KAM32/pPBE2) were
grown in L broth supplemented with 20 mM potassium lactate, and everted
membrane vesicles were prepared by passing cells through a French press
(9) . Benzalkonium chloride-H+ antiport was
measured by quinacrine fluorescence quenching (the final concentration
of quinacrine was 0.5 µM) . At the time point indicated by the arrow,
potassium lactate (5 mM) was added to initiate respiration . After the
fluorescence quenching reached a steady state, benzalkonium chloride
(BC; the final concentration was 0.0004%) was added to the assay
mixture . Finally, at the time point indicated by the arrow, Triton X-100
was added at a concentration of 0.0125% to collapse the H+
gradient.
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We compared the amino acid sequences of Na+-coupled pumps (NorM,
YdhE, VmrA, VcmA, and VcrM) and H+-coupled PmpM and tried to
find residues or regions important for recognition of ions .
Unfortunately, we have not succeeded in locating such residues or
regions, so far . It would be interesting to isolate or construct
mutant-type PmpMs (or NorMs) which show differences in ion recognition
in order to gain an insight into the mechanism of ion recognition .
Previously, other studies identified residues important for ion
recognition in the melibiose transport protein by a similar strategy
(5, 8) .
We prepared a dendrogram using representatives of the MATE family
of efflux pumps or putative pumps (data not shown) . There are several
subfamilies within the MATE family . NorM of V . parahaemolyticus
(18), YdhE of E . coli (19),
and VcmA of V . cholerae (22) belong to one
subfamily . It seems that PmpM belongs to this subfamily but is a
little apart from these three . VmrA of V . parahaemolyticus (2)
and VcrM of V . cholerae (21) belong to another
subfamily . All of the MATE family pumps so far characterized are Na+-coupled
efflux pumps . Since PmpM is an H+-coupled pump, it is likely
that there are other H+-coupled pumps in the MATE family . It
seems that PA5294 of P . aeruginosa is a member of the first
subfamily, although PA5294 has not previously been recognized
as a drug efflux pump . Thus, PA5294 may be a multidrug efflux pump
and may be an H+-coupled pump because it is apart from the
Na+-coupled NorM, YdhE, and VcmA pumps in the dendrogram .
Deletion of the pmpM gene and role of the PmpM pump in P .
aeruginosa. So far, several P . aeruginosa mutants lacking
genes for Mex multidrug efflux pumps have been constructed (20,
27) . P . aeruginosa YM64 lacks four major
Mex pumps, MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY (20) .
Recently, a mutant PMX52 lacking an additional pump, MeHI-OpmD, was
constructed from YM64 (27) . The pmpM gene
from the chromosome of PMX52 was deleted .
Chromosomal DNA from P . aeruginosa PAO1 was used as a template
for PCR . We cloned a longer DNA region containing the pmpM gene
for disruption of the gene, because longer fringe regions are
better for recombination to take place . The primers used were forward
primer 2 (F2), 5'-AGATAATTCACCGGGCTCTTCG-3' (containing an EcoRI
site), and reverse primer 2 (R2), 5'-CCTTGCCCGGTACCCTGGAAATGG-3'
(containing a KpnI site) . The PCR product with the length of
2.2-kbp was digested with EcoRI and KpnI, the EcoRI-KpnI
fragment containing the pmpM gene was ligated into vector
pSTV29 (TaKaRa Co., Kyoto, Japan), and the resulting pPBEA29 plasmid
was obtained . The pPBEA29 plasmid was digested with StuI, and
a 1.9-kbp SmaI-SmaI fragment from pPS858 (6)
containing a gentamicin-resistance marker sandwiched by two FRT sites
was ligated to the StuI-StuI sites, which are present
in the pmpM gene of pPBEA29 . The resulting recombinant plasmid
was designated pPBEA29G . Plasmid pPBEA29G contains a disrupted
pmpM gene . The length of the deleted StuI fragment is 796
bp . Finally, a 4.7-kbp SspI-SspI fragment from plasmid
pPBEA29G was ligated to the SmaI site of pEX100T (30),
which contains a sacB gene (6), to construct
plasmid pPBEA29GS . The pmpM gene was removed from the
chromosomal DNA of strain PMX52 by replacing the pmpM region
with the corresponding deleted region of pPBEA29GS by a Flp-FRT
recombination system, as previously reported (1,
20, 24, 25,
26, 28), to obtain strain PMX6 . Disruption
of the pmpM gene in PMX6 was confirmed by PCR methods .
We compared the MICs of various antimicrobial agents for P .
aeruginosa PMX52, PMX6, and PMX6/pUPBE2T in order to evaluate the
role of the PmpM pump in P . aeruginosa (Table 2) .
Deletion of the pmpM gene from the chromosome of PMX52
resulted in a decrease in the MICs of benzalkonium chloride, ethidium
bromide, acriflavine, and TPPCl . Thus, we conclude that PmpM is
functional in the parental cell PMX52 . Since PMX52 is a deletion
derivative of wild-type PAO1, it seems that PmpM is also functional
in wild-type PAO1 .
| TABLE 2 . MICs of various antimicrobial agents for P . aeruginosa
PMX52, PMX6, and PMX6/pUPBE2T
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Seven Mex multidrug efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN,
MexXY-OprM, MexJK-OprM, MexHI-OpmD, and MexVW-OprM) (4,
7, 11, 12,
13, 15, 16,
20, 23, 27) and one SMR
pump (12) have been characterized in P .
aeruginosa so far . Benzalkonium chloride is an inducer and
substrate of MexCD-OprJ (20) . It has been reported
that cells of P . aeruginosa that were adapted to benzalkonium
chloride showed resistance to other membrane-active agents (14) .
One reason for this adaptive resistance might be that the MexCD-OprJ
multidrug efflux pump is induced by benzalkonium chloride . It
is also possible that another membrane-related mechanism(s) is
involved in this adaptive resistance (14) . The PmpM pump
is also responsible for resistance to benzalkonium chloride .
The MIC of benzalkonium chloride for P . aeruginosa PAO1 was 64
µg/ml . Deletion of the mexCD-oprJ operon reduced the MIC to 20
µg/ml . Further deletion of the pmpM gene reduced the value to
5 µg/ml (Table 2) . It seems that these two pumps
are major systems for extrusion of benzalkonium chloride in P .
aeruginosa .
The MICs of fluoroquinolones, fradiomycin, chlorhexidine, and
rhodamine 6G, which are thought to be substrates of PmpM in E .
coli cells (Table 1), were not changed by pmpM gene
disruption in P . aeruginosa . On the other hand, introduction
of the pmpM gene into the pmpM-deleted PMX6 cell
resulted in an increase in the MICs not only of benzalkonium
chloride, ethidium bromide, acriflavine, and TPPCl but also of
fluoroquinolones (norfloxacin, ciprofloxacin, and ofloxacin) . The
observed MICs of benzalkonium chloride, ethidium bromide, and TPPCl
for PMX6/pUPBE2T were about twofold higher than those for PMX52,
perhaps due to a gene dosage effect . The increase in the MICs of
fluoroquinolones for PMX6/pUPBE2T may also be due to the gene dosage
effect .
We thank M . Varela of Eastern New Mexico University for critical
reading of the manuscript prior to submission .
This work was supported by a grant-in-aid for Scientific Research
on Priority Areas (C) "Genome Biology" from the Ministry of
Education, Culture, Sports, Science and Technology of Japan .
* Corresponding author . Mailing address: Department of
Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima,
Okayama, 700-8530, Japan . Phone and fax: 81-86-251-7957 . E-mail: tsuchiya@pharm.okayama-u.ac.jp.
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