(Received for publication, July 17, 1995)
From the
To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for
resistance to high concentrations of ethidium bromide. Recombinant
strains were found to be resistant not only to ethidium bromide but
also to daunomycin and tetraphenylphosphonium. The drug resistance is
conferred by the lmrP gene, which encodes a hydrophobic
polypeptide of 408 amino acid residues with 12 putative
membrane-spanning segments. Some sequence elements in this novel
membrane protein share similarity to regions in the transposon
Tn10-encoded tetracycline resistance determinant TetA, the multidrug
transporter Bmr from Bacillus subtilis, and the bicyclomycin
resistance determinant Bcr from E. coli. Drug resistance
associated with lmrP expression correlated with
energy-dependent extrusion of the molecules. Drug extrusion was
inhibited by ionophores that dissipate the proton motive force but not
by the ATPase inhibitor ortho-vanadate. These observations are
indicative for a drug-proton antiport system. A lmrP deletion
mutant was constructed via homologous recombination using DNA fragments
of the flanking region of the gene. The L. lactis (lmrP) strain exhibited residual ethidium extrusion
activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of
the functional drug-proton antiporter LmrP, L. lactis is able
to overexpress another, ATP-dependent, drug extrusion system. These
findings substantiate earlier studies on the isolation and
characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W.,
Poolman, B., Driessen, A. J. M., and Konings, W. N.(1994) J.
Bacteriol. 176, 6957-6964).
For many years antibiotics have been found effective in the
treatment of several infectious diseases caused by various pathogens.
The occurrence of antibiotic resistance, however, transformed many of
the up to now readily treatable diseases to a new threat to public
health (Cullinton, 1992; Nikaido, 1994). One of the mechanisms
underlying antibiotic resistance involves the extrusion of the
compounds by an efflux pump or carrier (Tennent et al., 1989;
Levi, 1992; Bolhuis et al., 1994; Midgley, 1987, 1989;
Miyauchi et al., 1992). For most (micro)organisms, it is not
clear whether efflux is mediated by one multispecific system or by
several, more or less specific systems (Hächler et al., 1991). The most intriguing mechanisms of drug
extrusion are those that can handle a wide variety of structurally
unrelated compounds (antibiotics, drugs, etc.), and those are often
referred to as multidrug resistance (MDR) ()transporters.
The bacterial MDR type transporters as well as several closely related
specific drug extrusion systems (SDR) can be divided into four groups
on the basis of their relatedness in the primary sequences, similarity
in the global molecular structure, and/or mechanism of energy coupling
(Nikaido, 1994). The first and largest group consists of secondary
transporters that are characterized by the presence of either 12 or 14
putative transmembrane spanning segments (Paulsen and Skurray, 1993;
Lewis, 1994; Marger and Saier, 1993; Lomovskaya and Lewis, 1992; Rouch et al., 1990). The second group, often referred to as the
Staphylococcal multidrug resistance (Smr) family, comprises drug-proton
antiporters that are about 100 amino acids long and are composed most
likely of four transmembrane
-helices (Yerushalmi et al.,
1995; Grinius and Goldberg, 1994). The third group of secondary drug
extrusion transporters is formed by the resistance-nodulation division
(RND) family, found in Gram-negative bacteria (Saier et al.,
1994). The resistance-nodulation division systems require an accessory
protein that spans the periplasm and most likely interacts with an
outer membrane pore. The accessory proteins are required for the
transport of the substrates to the external medium. The fourth group of
efflux systems consists of ATP binding cassette transporters (Fath and
Kolter, 1993; Higgins, 1993; Molenaar et al., 1992) and is
best exemplified by the mammalian P-glycoprotein (MDR1). Recently, also
a bacterial homolog of MDR1 was identified in Lactococcus
lactis. (
)
Although a number of MDR- and specific
drug resistance-type of transporters have been identified in bacteria,
their mechanism of action and energy coupling to transport have not
been studied in great detail. The resistance to high concentrations of
ethidium bromide (Eth), daunomycin (Dau
), and
rhodamine 6G (Rho
), of three independently isolated mutants
of L. lactis MG1363, suggested that at least two different
transport mechanisms are involved in multidrug resistance in this
organism (Bolhuis et al. 1994). One of the mechanisms is
dependent on the proton motive force (
p), while the other is
ATP-dependent. The
p-dependent system, termed LmrP, is the first
multidrug transporter for which both the membrane potential
(
), and the proton gradient (
pH), has been shown to
function as driving force for drug extrusion. In this paper, we
describe the gene cloning, characterization, mutant construction, and
functional analysis of LmrP.
Figure 1:
A, restriction map and open
reading frames (arrows) of the 3.2-kb HindIII
fragment of L. lactis. Flanking regions corresponding to the
multiple cloning site of pBluescript SKII are also
shown. B, nucleotide sequence of the multidrug transporter (lmrP) and the flanking regions. Putative ribosomal binding
site (rbs), promoter elements (-10/-35), and
terminator sequence (
) are indicated. The deduced amino
acid sequence of LmrP is shown below the DNA sequence (residue numbers
between brackets). The predicted membrane spanning
-helices are underlined and shown in boldface.
Figure 2: Secondary structure model of LmrP. The model is based on the hydropathy-profile of the amino acid sequence and the distribution of the arginine and lysine residues (+) according to the ``positive inside rule'' by Von Heijne(1986). The residues constituting the conserved sequence motif A in the first cytoplasmic loop (CL1) and motif B in transmembrane helix V are shaded.
Figure 3:
Resistance of E. coli CS1562 to
toxic compounds with and without expression of the lmrP gene.
Cells carrying pSKLMR3.2 () or pBluescript SKII
(
), were grown semianaerobically at 30 °C on Luria
Broth, supplemented with glucose (25 mM), carbenicillin (50
µg/ml), and ethidium (A), daunomycin (B), or
TPP
(C) at various concentrations. The
relative growth rate is plotted as a function of the drug
concentration. The growth rates in the absence of added drug
corresponded to 0.54 and 0.63 h
for E. coli CS1562(pSKLMR3.2) and E. coli CS1562(pBluescript
SKII
), respectively.
Figure 4: Ethidium efflux from L. lactis MG1363 (wild-type) and L. lactis/pGKLMR3.2. Ethidium was added to the cell suspensions (0.1 mg of protein/ml) at a final concentration of 10 µM, 10 min prior to energization with 25 mM of glucose (Glc). Valinomycin (Val) and nigericin (Nig) were added to final concentrations of 1 µM each, when indicated. The fluorescence intensity after the addition of ethidium and before the addition of the energy source was normalized to 100%.
Figure 6:
Ethidium uptake in L. lactis MG1363 (wild-type), L. lactis MG1363/pGKLMR3.2, and L. lactis MG1363/lmrP. Cells were preincubated
for 10 min in a phosphate-free buffer (50 mM HEPES, 25
mM K
SO
, 5 mM MgSO
; pH 7.4) in the presence (+) or
absence(-) of 0.5 mMortho-vanadate. L-Arginine (10 mM) was added as source of metabolic
energy to allow ortho-vanadate to be taken up. The assay was
started upon addition of 10 µM of ethidium bromide to the
cell suspension.
Figure 5:
PCR analysis of the lmrP mutation in L. lactis. Chromosomal DNA of L. lactis MG1363 (wild-type lmrP; lanes 3 and 4)
and L. lactis MG1363(
lmrP; lane 1 and 2) was used as template DNA for the PCR reaction. Two
different sets of primers (A), LMR28/LMR2 (lanes 1 and 3) and LMR28/LMR29 (lanes 2 and 4)
were used to distinguish between the wild-type and the deletion mutant
of lmrP. The PCR products were separated by agarose (1%)
gel-electrophoresis (B).
To
analyze the drug resistance phenotype of L. lactis MG1363(lmrP), the mutant strain was studied further
in growth and transport assays. Within the ethidium bromide
concentration range tested (0-25 µM), the L.
lactis deletion mutant MG1363(
lmrP) was only
slightly more sensitive than L. lactis MG1363(wild-type),
whereas L. lactis MG1363/pGKLMR3.2 was more resistant to
ethidium bromide than the wild-type (data not shown). Since L.
lactis possesses an ATP-dependent efflux activity in addition to
the
p-driven efflux activity, it is possible that alterations in
the expression of the ATP-dependent system could (partially) have
masked the drug resistance phenotype of L. lactis MG1363(
lmrP) and MG1363/pGKLMR3.2. Indeed, in the
presence of ortho-vanadate, the inhibitor of the ATP-dependent
efflux activity, the
lmrP strain accumulated more
ethidium than the wild-type strain (Fig. 6, +).
Overexpression of LmrP from pGKLMR3.2 reduced the intracellular
ethidium levels to the same extent irrespective of whether or not the
cells were preincubated with ortho-vanadate. (Fig. 6;
±).
In this paper, we report the isolation of a chromosomal DNA
fragment from L. lactis, which effects an increased resistance
to ethidium bromide, daunomycin, and TPP when
expressed in E. coli. This DNA fragment contains an ORF that
specifies an integral membrane protein (LmrP) with 12 putative
-helical membrane spanning segments. When the amino acid sequence
of the predicted LmrP polypeptide was compared with proteins in various
data bases, the search did not reveal significant similarity to any
known protein. However, when, the primary sequence of LmrP was directly
compared with known drug transporters and other transport proteins,
using the COMPARE program of PCGENE, which is based on the Dayhoff
MDM-78 comparison matrix (Schwartz and Dayhoff, 1987), some homologous
proteins were identified. The sequence comparisons revealed similarity
(19-24% identical residues) between LmrP and the multidrug
transporters Bmr of Bacillus subtilis (Neyfakh et
al., 1991) and NorA of Staphylococcus aureus (Yoshida et al., 1990) as well as the transporters involved in
tetracycline resistance and bicyclomycin resistance in E. coli (Bentley et al., 1993). Multiple alignment of these
proteins revealed two conserved sequence motifs (Fig. 2, A and B) that are located at similar positions within the
putative secondary structure of the proteins. Motif A is identical to
the consensus sequence GXXXDRXGR(K/R) (Henderson,
1991), which is found in various (unrelated) secondary transport
proteins and is present in the cytoplasmic loop of LmrP between
transmembrane segments 2 and 3. It has been suggested that this loop is
of structural importance to the polypeptide, but it might also be
involved in the conformational change required for the opening and
closing of the transport channel (Paulsen and Skurray, 1993).
Functional studies of site-directed mutants of the transposon
Tn10-encoded metal-tetracycline/proton antiporter (Tn10-Tet) showed
that the negatively charged aspartate (Asp
) and the
positively charged arginine (Arg
) are essential for
transport (Yamaguchi et al., 1993). Motif B has residues in
common with the conserved sequence GpilGPvlGG found at the end of
transmembrane segment 5, which, so far, has only been observed in drug
extrusion systems (Lewis, 1994; Paulsen and Skurray, 1993). Alignment
of the carboxyl-terminal half of LmrP with its amino-terminal half
displays about 18% identical residues. Among the identical residues are
an aspartate (Asp
) and a lysine (Lys
) in
the cytoplasmic loop between transmembrane segment 8 and 9, which align
with Asp
and Lys
of the
GXXXDRXGR(K/R) consensus sequence.
Although lmrP was originally cloned from an ethidium-resistant strain
of L. lactis ML3, the nucleotide sequence was identical to
that of the wild-type and the other multidrug-resistant mutants of L. lactis MG1363. Also, the promoter regions upstream of the
structural gene were identical, and DNA hybridization experiments
indicated that the observed increase in drug resistance and drug efflux
is not due to amplification of the lmrP gene on the
chromosome. As shown previously (Bolhuis et al., 1994), the
MDR phenotype of the drug-resistant mutants Eth,
Dau
, and Rho
resulted from increased drug
extrusion from the cell. These mutant strains have a comparable drug
resistance spectrum, but differ with respect to their mode of energy
coupling to active drug efflux. Whereas ethidium extrusion from the
Eth
strain is mainly driven by an ATP-dependent transport
activity, a significant contribution of both the ATP-dependent and the
p-driven transport systems was observed in the ethidium extrusion
from Dau
and Rho
(Bolhuis et al.,
1994). Since the nucleotide sequence of lmrP in the MDR
strains is identical, the increased resistance in these strains must be
due either to an increased expression of lmrP, possible
affected by mutation of a transcription factor, or to an alternative
drug extrusion activity. It has been shown that expression of the
homologous tetracycline-metal/drug antiporter TetA is repressed by the
repressor TetR in the absence of tetracycline (Hillen and Berens,
1994). Regulation of transcription of the operon for the EmrAB
multidrug resistance determinant from E. coli is mediated by
the repressor EmrR (Lomovskaya et al., 1995). Regulation of
gene expression by transcription activation is found for the B.
subtilis MDR transporter Bmr (Ahmed et al. 1994). The
possible involvement of similar regulatory proteins in the enhanced
drug resistance in L. lactis will be the subject of future
research.
The lmrP gene was cloned in E. coli HB101 by selection for resistance to high concentrations of
ethidium bromide. In the hypersensitive E. coli CS1562,
expression of lmrP not only increased resistance to ethidium
but also to unrelated hydrophobic compounds like TPP and daunomycin. Increased ethidium resistance was also observed
when lmrP was present on a plasmid in L. lactis MG1363, but the effect was less pronounced. The lower
LmrP-dependent ethidium resistance in L. lactis is most likely
the result of the relatively low copy number of the pGK13 vector in L. lactis as compared with the vector used in E. coli (Kok et al., 1984). Transport studies in L. lactis showed that LmrP catalyzes the energy-dependent efflux of ethidium
and daunomycin. The energy-dependent efflux of ethidium from the
wild-type strain and L. lactis MG1363(pGKLMR3.2) was
completely inhibited upon dissipation of the
p but unaffected by
the ATPase inhibitor ortho-vanadate. These observations are
consistent with a drug-proton antiport mechanism for LmrP. Deletion of
part of the lmrP gene from the chromosome of L. lactis MG1363 (wild-type strain) affected drug extrusion when the cells
were pretreated with ortho-vanadate. However, the
lmrP mutation did not result in significant changes in
the drug resistance phenotype. These results suggest an increased
expression (or activity) of the ATP-dependent drug transporter (Bolhuis et al., 1994). It is possible that this increased expression
is essential to survive the otherwise lethal effect of the deletion of lmrP. The occurrence of efflux-mediated resistance to various
hydrophobic toxic compounds and antibiotics in both pathogens and
nonpathogens implicates a general mechanism and an important
physiological function. This function could involve a general
detoxification mechanism against naturally occurring hydrophobic
compounds (Gottesman and Pastan, 1993; Higgins, 1993).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89779[GenBank].