ARTICLE

Molecular Properties of Bacterial Multidrug Transporters

Monique Putman, Hendrik W. van Veen, Wil N. Konings
DOI: 10.1128/MMBR.64.4.672-693.2000

SUMMARY

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

The development and use of antibiotics has been one of the most important steps towards controlling infectious diseases in the 20th century. However, the subsequent appearance and spread of antibiotic resistance in pathogenic organisms have made many currently available antibiotics ineffective (160, 169). As a consequence, tuberculosis and other infectious diseases are reemerging, causing a serious public health problem (32, 38). To successfully fight the increasing numbers of drug-resistant and multidrug-resistant (MDR) bacteria, extensive knowledge of the molecular mechanisms underlying microbial antibiotic resistance is required.

Microorganisms have developed various ways to resist the toxic effects of antibiotics and other drugs (Fig. 1) (83, 169). One of these mechanisms involves the production of enzymes that inactivate antibiotics by hydrolysis or the formation of inactive derivatives (Fig. 1A) (40). Well-known examples are β-lactamases (29, 206) and enzymes that phosphorylate, adenylate, or acetylate aminoglycoside antibiotics (234). A second mechanism of resistance is target alteration (Fig. 1B). Cellular targets can be altered by mutation or enzymatic modification in such a way that the affinity of the antibiotic for the target is reduced (95, 239, 265, 277). A third, more general mechanism of resistance is the inhibition of drug entry into the cell (Fig. 1C). Due to the low permeability of the outer membrane of gram-negative bacteria (175, 176) and the exceptionally efficient barrier of the gram-positive mycobacteria (101), drug diffusion across the cell envelope is reduced. The permeability of the outer membrane can be further decreased by the loss of porins (77, 157, 177). These barriers, however, cannot prevent the drugs from exerting their toxic action once they have entered the cell, and the active efflux of drugs (Fig. 1D) is essential to ensure significant levels of drug resistance (122, 177).

Fig. 1.
Fig. 1.

Resistance mechanisms in bacteria comprise (A) drug inactivation, (B) target alteration, (C) prevention of drug influx, and (D) active extrusion of drug from the cell.

Some transporters, such as the tetracycline efflux proteins (209,237), are dedicated systems which mediate the extrusion of a given drug or class of drugs. In contrast to these specific drug transporters, the so-called multidrug transporters can handle a wide variety of structurally unrelated compounds (127, 178, 190,257). On the basis of bioenergetic and structural criteria, multidrug transporters can be divided into two major classes (Fig.2). Secondary multidrug transporters utilize the transmembrane electrochemical gradient of protons or sodium ions to drive the extrusion of drugs from the cell. ATP-binding cassette (ABC)-type multidrug transporters use the free energy of ATP hydrolysis to pump drugs out of the cell.

Fig. 2.
Fig. 2.

Schematic representation of the two major classes of multidrug transporters. (A) ABC-type multidrug transporters utilize the free energy of ATP hydrolysis to pump drugs out of the cell. (B) Secondary multidrug transporters mediate the extrusion of structurally unrelated drugs in a coupled exchange with protons or sodium ions.

Here we present a comprehensive review describing the currently known multidrug transporters in bacteria. Important issues addressed are (i) the functional reconstitution of purified multidrug transporters into proteoliposomes, which allows detailed molecular characterization of the proteins, (ii) the transcriptional regulation of genes encoding multidrug efflux systems, (iii) the structure-function relationships that dictate drug recognition and transport, (iv) the presence of multiple drug-binding sites, and (v) the mechanism of transport of both ATP-driven and secondary multidrug transporters.

SECONDARY MULTIDRUG TRANSPORTERS

Most bacterial multidrug efflux systems known to date are sensitive to agents that dissipate the proton motive force (PMF), indicating that they mediate the extrusion of toxic compounds from the cells in a coupled exchange with protons (15, 42, 84, 133,190). On the basis of size and similarities in the primary and secondary structure, these secondary multidrug transporters can be subdivided into distinct families of transport proteins: the major facilitator superfamily (MFS) (146), the small multidrug resistance (SMR) family (191), the resistance-nodulation-cell division (RND) family (216), and the multidrug and toxic compound extrusion (MATE) family (28). These families are not solely associated with multidrug export but include proteins involved in other PMF-dependent transport processes or other functions.

Major Facilitator SuperfamilyThe MFS consists of membrane transport proteins which are found from bacteria to higher eukaryotes and are involved in the symport, antiport, or uniport of various substrates, such as sugars, Krebs cycle intermediates, phosphate esters, oligosaccharides, and antibiotics (146). Hydropathy analysis and alignment of conserved motifs of the resistance-conferring drug efflux proteins revealed that these proteins can be divided into two separate clusters, with either 12 or 14 transmembrane segments (TMS) (Fig. 3and 4) (188). Table1 summarizes the multidrug transporters from both the 12-TMS and 14-TMS clusters found in gram-positive and gram-negative bacteria.

Fig. 3.
Fig. 3.

Structural model for the 12-TMS multidrug transporters of the MFS. The residues constituting the conserved sequence motifs are shaded.

Fig. 4.
Fig. 4.

Structural model for the 14-TMS multidrug transporters of the MFS. The residues constituting the conserved sequence motifs are shaded.

View this table:
Table 1.

MFS multidrug transporters

The 12-TMS cluster of multidrug transporters.TheStaphylococcus aureus NorA protein was first discovered in a quinolone- and methicillin-resistant clinical isolate (251). Sequencing and characterization of the norA gene revealed that NorA is a 388-amino-acid membrane protein that confers resistance to hydrophilic compounds and only low or no resistance to hydrophobic drugs (271, 278). Studies on the substrate specificity demonstrated that NorA is a true multidrug transporter, mediating resistance to a range of structurally dissimilar drugs (171,172).

A structural and functional homolog of NorA has been found in aBacillus subtilis strain selected with increasing concentrations of rhodamine 6G and named Bmr for bacterial multidrug resistance (170). Ahmed et al. (4) described a second multidrug transporter of B. subtilis. This protein, named Blt, is highly homologous to Bmr and confers resistance to a similar range of drugs. Despite the similarities between Bmr and Blt, their expression patterns are different. Under standard cultivation conditions, Bmr is expressed while the expression of Blt is undetectable. The differences in regulation of transcription and the operon organization suggest that Bmr and Blt have distinct physiological functions and that the two transporters may be involved in the transport of different natural substrates (4).

PMF-dependent drug extrusion in Lactococcus lactis was first observed in strains exposed to increasing stepwise concentrations of daunomycin and rhodamine 6G (20). The gene responsible for this activity, lmrP, encodes an integral membrane protein that shows 19 and 24% homology with the multidrug transporters Bmr and NorA, respectively (21). Transport studies in whole cells and inside-out membrane vesicles demonstrated that LmrP mediates the electrogenic export of lipophilic drugs from the inner leaflet of the membrane to the aqueous phase (23, 203) (see below).

The Escherichia coli transporter Bcr confers resistance to the diketopiperazine antibiotic bicyclomycin (18). Vedantam-Gayatri et al. (261) revealed that the sulfathiazole resistance determinant sur is identical tobcr, indicating that Bcr is involved in resistance to structurally unrelated antibiotics. EmrD, an E. coli homolog of Bcr, is involved in a low-energy shock adaptive response. Expression of EmrD protects the cell against the uncouplers CCCP (carbonyl cyanidem-chlorophenylhydrazone) and tetrachlorosalicylanilide, but does not affect resistance to bicyclomycin, quinolones, or chloramphenicol, suggesting that EmrD has a fairly narrow substrate spectrum (167). Another MDR transporter found in E. coli is MdfA (49), also known as Cmr (179) and CmlA (208), which was initially described as a membrane-associated efflux pump for chloramphenicol. However, detailed analysis of the substrate spectrum revealed that MdfA confers resistance to a wide range of unrelated neutral and positively charged drugs. Substitutions of E26 in the first transmembrane segment demonstrated that a single membrane-embedded negatively charged residue is critical for the recognition of positively charged drugs by MdfA (50). Surprisingly, overexpression of MdfA causes decreased resistance to spectinomycin (19). Thus, the activity of a multidrug pump can result in resistance to several toxic compounds and increased sensitivity to certain others.

The multidrug transporter Cmr of the nonpathogenic organismCorynebacterium glutamicum significantly increases resistance to several structurally unrelated antibiotics when expressed in E. coli but does not affect antibiotic resistance when expressed in C. glutamicum. The inability of Cmr to confer resistance in its original host could result from a lower expression level of Cmr (98).

Mycobacteria cause several diseases, including tuberculosis and leprosy. The treatment of these infections is often difficult because mycobacteria are intrinsically resistant to most commonly used antibiotics. The Tap multidrug efflux pump in Mycobacterium fortuitum and Mycobacterium tuberculosis confers resistance to aminoglycosides and tetracycline and makes it more difficult to control mycobacterial infections (5). The number of TMS could not be unambiguously determined from the primary sequence and was estimated to be close to 10. However, Aı́nsa et al. (5) suggested, on the basis of the homology between Tap and other multidrug transporters of the MFS, that Tap is a 12-TMS transporter.

Several studies suggested that a reserpine-sensitive multidrug transporter is involved in fluoroquinolone resistance inStreptococcus pneumoniae (15, 25, 26, 279). Recently, Gill et al. (63) cloned and characterized the efflux pump PmrA of S. pneumoniae. Analogous to NorA, Bmr, and LmrP, PmrA is inhibited by the plant alkaloid reserpine. Expression of PmrA increases resistance to acriflavine, ethidium, and the fluoroquinolones ciprofloxacin and norfloxacin.

The 14-TMS cluster of multidrug transporters.Resistance to antiseptic and disinfectant compounds, including intercalating dyes, quarternary ammonium compounds, and diamines, is widespread among multidrug-resistant strains of Staphylococcus aureus. One of the energy-dependent export mechanisms responsible for this resistance is encoded by the qacA gene (248). Hydropathy analysis (213) and topological analysis using alkaline phosphate and β-galactosidase fusions (192) indicated that the QacA protein contains 14 TMS. Whereas QacA confers resistance to monovalent and divalent organic cations, the closely related QacB only confers resistance to monovalent organic cations (192). It has been speculated that the extensive use in hospital environments of bacteriostatic divalent cations, such as chlorhexidine, resulted in the evolution ofqacA from qacB (193).

Sequencing of an E. coli locus for multidrug resistance revealed the presence of two open reading frames, designatedermA and emrB (135). TheemrB gene encodes a 513-amino-acid protein which is homologous to QacA. ErmA, a 42.7-kDa membrane fusion protein with a single TMS and a large C-terminal periplasmic domain, is thought to form a continuous channel between the inner and outer membrane (126, 135). EmrAB confers resistance to hydrophobic uncouplers and antibiotics (59, 135). Tanabe et al. (246) characterized the E. coli emrKY operon. Transcription of emrKY is growth phase dependent and induced by tetracycline. EmrK and EmrY are 50 and 63% identical to EmrA and EmrB, respectively, suggesting a similar function. However, the involvement of EmrKY in multidrug efflux remains to be demonstrated.

Bmr3, a third B. subtilis multidrug transporter, shows moderate homology to EmrB (25% identity). Bmr3 confers resistance to the antibiotics puromycin, tosufloxacin, and norfloxacin. Northern hybridization analysis revealed that expression of Bmr3 is decreased in the late log phase (181).

Shortly after the introduction of fluoroquinolones for the treatment of tuberculosis, resistant clinical isolates of M. tuberculosiswere described. From the rapidly growing Mycobacterium smegmatis, which was used as a model genetic system for M. tuberculosis, the lfrA gene, which confers low-level fluoroquinolone resistance when present on a multicopy plasmid, was isolated. LfrA confers resistance to hydrophilic quinolones but not to the more hydrophobic quinolones (134). Hybridization analysis showed that LfrA homologs are present in M. tuberculosis and Mycobacterium avium (244).

Vibrio cholerae, an important gram-negative enteric pathogen, is the causative agent of the severe diarrheal disease cholera. Colmer et al. (37) identified a multidrug resistance pump, VceAB, that provides V. cholerae with resistance to several toxic compounds, including deoxycholate and the antibiotics nalidixic acid and chloramphenicol. VcreA and VceB show significant homology to EmrA and EmrB, respectively.

Small Multidrug Resistance FamilyMultidrug transporters of the SMR family (Table2, Fig. 5), the smallest secondary drug efflux proteins known, are typically about 107 amino acid residues in length. Hydropathy analysis (191), gene fusions with alkaline phosphatase and β-galactosidase (179), α-periodicity analysis (51), cysteine accessibility scanning (242), and Fourier transform infrared spectroscopy (11) support a model of a tightly packed four-helix antiparallel bundle. Due to the small size of the multidrug transporters of the SMR family, it has been proposed that they may function as homooligomeric complexes (187,189).

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Table 2.

SMR multidrug transporters

Fig. 5.
Fig. 5.

Structural model for multidrug transporters of the SMR family. The residues constituting the conserved sequence motifs are shaded.

The first gene encoding a multidrug transporter of the SMR family was detected on both conjugative and nonconjugative plasmids from clinical isolates of S. aureus and other staphylococci (120,121, 132). This gene, first described as qacC and also known as qacD (132) and ebr(221), has been renamed smr (71). Grinius and Goldberg (72) demonstrated that the purified and reconstituted Smr protein mediates electrogenic drug/proton antiport. In staphylococci isolated from the food industry, plasmids pST94 and p2H6 were found. Sequence analysis of these plasmids revealed two new members of the SMR family, designated QacG and QacH (84,85). QacG and QacH have 69 and 76% identity to Smr, respectively. Both transporters confer resistance to ethidium bromide and quaternary ammonium compounds.

The E. coli multidrug transporter EmrE (also known as MvrC) has been identified and cloned on the basis of its ability to confer resistance to ethidium and methyl viologen (162, 202). Yerushalmi et al. (275) demonstrated that resistance is caused by PMF-dependent extrusion of drugs. Negative dominance studies indicated that EmrE functions as a homooligomer, probably composed of three monomers (276). Studies using single cysteine mutants of EmrE showed that none of the residues in the putative transmembrane helices react with N-ethyl-maleimide (NEM), a small membrane-permeant sulfhydryl reagent. As the reaction with NEM requires water, the results imply very tight packing of the protein without a continuous aqueous domain. Based on these findings, it was proposed that EmrE translocates its substrates through a hydrophobic pathway (242).

The antiseptic resistance genes qacE and qacEΔ1are located on an integron, a potentially mobile element. As a consequence, these genes are widely spread among gram-negative bacteria (108). The qacEΔ1 gene but not theqacE gene was detected in clinical isolates of gram-positive bacteria (109). The qacE gene was originally found on the Klebsiella aerogenes plasmid R751 (187). The qacEΔ1 gene probably represents a disrupted form of qacE that evolved as a result of the insertion of a DNA segment near the 3′ end of the qacE gene. Ethidium transport experiments indicated that the mechanism of resistance mediated by QacE is active export driven by the PMF. Neither QacE nor QacΔE1 is inhibited by reserpine, a potent inhibitor of multidrug transporters such as Bmr, NorA, and LmrP (187).

The multidrug transporter Mmr of M. tuberculosis confers resistance to tetraphenyl phosphonium (TPP+), ethidium bromide, erythromycin, acriflavine, safarin O, and pyronin Y (42). TPP+ accumulation experiments showed that Mmr actively extrudes TPP+, using the PMF as the driving force. The presence of mmr-like genes in otherMycobacterium species (M. simiae, M. gordonae, M. marinum, and M. bovis) was demonstrated by Southern hybridization (42).

The Bacillus subtilis genome encodes seven SMR-type proteins (194). Surprisingly, six of these proteins are encoded from gene pairs in three distinct operons, ebrAB,ykkCD, and yvdRS. Masaoka et al. (154) demonstrated that neither EbrA nor EbrB is sufficient for conferring drug resistance. However, coexpression of the two proteins causes an MDR phenotype in both E. coli and B. subtilis. A similar result was obtained for the YkkCD pair. Only when both theykkC and ykkD genes were expressed together was resistance observed against a broad range of drugs, including cationic dyes and neutral and anionic antibiotics (97). In contrast to the SMR-type multidrug transporters, which appear to function as homooligomers, EbrAB and YkkCD are composed of two dissimilar but homologous subunits. One member of each pair is short (105 to 106 amino acids), while the other is longer (111 to 117 amino acids). This difference is due to a C-terminal hydrophilic extension (97).

The tehA gene of E. coli, which was initially identified as part of an operon associated with resistance to potassium tellurite, encodes a 36-kDa protein with 10 TMS (247). Surprisingly, a region of TehA which includes TMS 2 through 5 was found to be homologous to members of the SMR family (250). Expression of TehA increased the resistance to monovalent cations, such as tetraphenylarsonium and ethidium bromide, but decreased the resistance to the divalent cations dequalinium and methyl viologen (250). Thus, like MdfA of E. coli, TehA confers resistance to one group of toxic compounds and hypersensitivity to another group. Since deletion of the C-terminal region of TehA (TMS 8 to 10) did not decrease transport significantly, TMS 2 to 5 may be primarily responsible for the activity of TehA (250). Although TehA contains only one of the three SMR signature sequences (191) and is larger than the reported SMR-type multidrug transporters, it may represent a distantly related member of the SMR family.

Resistance-Nodulation-Cell Division FamilyMultidrug transporters belonging to the RND family (Table3) interact with a membrane fusion protein (MFP) and an outer membrane protein to allow drug transport across both the inner and outer membrane of gram-negative bacteria. The secondary structure of RND-type efflux proteins (Fig.6) was proposed to consists of 12 TMS, with two large loops between TMS 1 and 2 and TMS 7 and 8 (190,216). Membrane topology analysis of the Pseudomonas aeruginosa RND protein MexB recently verified this model (75). The MFP proteins, which contain a single N-terminal TMS and a large C-terminal periplasmic domain, are thought to induce fusion of the inner and outer membrane (45, 216) or form a channel-like structure that spans the periplasmic space (281).

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Table 3.

RND multidrug transporters

Fig. 6.
Fig. 6.

Structural model for multidrug transporters of the RND family. The residues constituting the conserved sequence motifs are shaded.

The acrA locus of E. coli has long been known to be involved in resistance to basic dyes, detergents, and antibiotics (166, 167). Characterization of this locus revealed the presence of two genes, acrA and acrB (formerlyacrE), which are located within a single operon (141). AcrA is a highly asymmetric protein that belongs to the MFP family, while AcrB is a member of the RND family (45,281). Insertion mutations showed that both AcrA and AcrB are required for drug resistance (143). A study by Fralick (58) suggested that the outer membrane protein TolC is the third component of the AcrAB efflux system. Recently, the 2.1-Å crystal structure of TolC has been determined (118). Three TolC promoters assemble to form a continuous, solvent-accessible “tunnel” that spans the outer membrane and periplasmic space. This tunnel may transiently open up upon the interaction with an energized inner membrane transporter and establish a conduit between the cytosol and the external environment. E. coli contains two AcrAB homologs, AcrEF (formerly known as EnvCD [110]) and YhiUV (formerly called OrfAB), as well as an AcrB homolog, AcrD, which is not associated with a gene for an MFP (142). Inactivation of AcrEF decreased the resistance to a broad range of drugs, including dyes, detergents, and various classes of clinically important antibiotics, indicating that AcrEF functions as a multidrug transporter (141). Mutations in the yhiUV genes result in drug hypersusceptibility, suggesting that YhiUV also plays a role in antibiotic resistance (142, 143). Direct multidrug transport, however, has not yet been demonstrated.

P. aeruginosa is a clinically important opportunistic pathogen characterized by relatively high intrinsic resistance to a variety of antimicrobial agents. This property is now recognized to result from the synergy between low outer membrane permeability and at least four RND efflux systems. The mexAB-oprM operon was first thought to play a role in the export of the siderophore pyoverdine (198). Further characterization of the operon, however, showed that overexpression of MexAB-OprM increased the resistance of P. aeruginosa to various antibiotics (199). A direct assay of drug accumulation showed that MexAB-OprM indeed pumps out various antibacterial agents in an energy-dependent fashion (129). MexA and MexB are members of the MFP and RND families, respectively. The third gene of the operon was originally identified as oprK, but Gotoh et al. (64) demonstrated that the outer membrane component of this efflux system is OprM rather than OprK. Results obtained by Evans et al. (52) suggested that the quorum-sensing-related homoserine lactone PAI-1 is a substrate for MexAB-OprM and that exclusion of PAI-1 limits the production of virulence factors, such as the blue-green pigment pyocyanin. Examination of P. aeruginosa strains showing widely different levels of intrinsic resistance suggested the presence of more than one endogenous multidrug efflux system (128). Indeed, Poole and coworkers (200) identified a second efflux operon in P. aeruginosa, designated MexCD-OprJ. The third antibiotic efflux system of P. aeruginosa is composed of the MFP MexE, the RND protein MexF, and the outer membrane protein OprN (115). Similar to MexAB-OprM, MexEF-OprN expression correlates inversely with the production of the virulence factor pyocyanin (115). In contrast to MexAB-OprM, MexCD-OprJ and MexEF-OprN do not confer resistance to β-lactam antibiotics (65, 115). Subunit swapping experiments demonstrated that the inner membrane efflux components of the MexAB-OprM transporter are responsible for the β-lactam specificity of the multidrug efflux pumps in P. aeruginosa (66, 155, 240). Recently, two new genes mediating resistance to quinolones, aminoglycosides, and macrolide antibiotics were cloned from the chromosome of P. aeruginosa(6, 158). These genes, designated mexX andmexY, show significant similarity to mexAB,mexCD, and mexEF. No open reading frame corresponding to an outer membrane protein was found downstream ofmexXY. Mine et al. (158) demonstrated that TolC or OprM is required for MexXY activity in E. coli. It has been reported that OprM contributes to antibiotic resistance inP. aeruginosa independent of MexAB (283). The substrate specificity of this OprM-dependent and MexAB-independent system corresponds to that of the MexXY system, suggesting that OprM functions as the outer membrane protein of the MexXY drug efflux pump in P. aeruginosa (158).

The resistance of Neisseria gonorrhoeae to hydrophobic agents, including detergent-like fatty acids and bile salts, is conferred by the MtrRCDE efflux system. The mtrRCDE region of the gonococcal chromosome contains a transcriptional regulator gene (mtrR) and three tandemly linked genes (mtrC,mtrD, and mtrE), encoding proteins of the MFP, RND, and outer membrane protein families, respectively (80). Insertional inactivation of either the mtrC (80) or mtrE (41) gene resulted in a significant decrease in resistance to erythromycin, penicillin, Triton X-100, and fatty acids, suggesting that both proteins are required in the efflux process.

Genome sequencing of Haemophilus influenzae has identified a three-gene cluster, consisting of HI0893, HI0894, and HI0895, which is homologous to the acrRAB cluster of E. coli(57). Disruption of either HI0894 (an acrAhomolog) or HI0895 (an acrB homolog) caused hypersusceptibility to erythromycin, rifampin, novobiocin, sodium dodecyl sulfate, and cationic dyes. Erythromycin accumulation experiments indicated that the HI0894 and HI0895 proteins contribute to the resistance of H. influenzae through PMF-dependent drug efflux (220).

Burkholderia pseudomallei, a motile gram-negative rod, is the causative agent of melioidosis. Successful treatment of melioidosis patients is difficult because B. pseudomallei is intrinsically resistant to a variety of antibiotics, including β-lactams, aminoglycosides, and macrolides. Recently, theamrAB-OprA operon was identified, which encodes a multidrug efflux system in B. pseudomallei which confers resistance to both aminoglycoside and macrolide antibiotics. AmrA and AmrB show strong homology to MexC and MexB, respectively, of P. aeruginosa (161).

Multidrug and Toxic Compound Extrusion FamilyNorfloxacin accumulation experiments revealed that Vibrio parahaemolyticus contains an energy-dependent efflux system, designated NorM (163). NorM and its E. colihomolog YdhE mediate resistance to dyes, hydrophilic fluoroquinolones, and aminoglycosides. NorM contains 12 putative TMS, and on this basis it has been suggested to be a member of the MFS (163). NorM and YdhE, however, do not have sequence similarity with any members of the MFS and do not exhibit any of the signature sequences (see below). Brown et al. (28) found that NorM and YdhE are members of a previously unidentified family which contains more than 30 proteins, termed the multidrug and toxic compound extrusion (MATE) family. Phylogenetic analysis of the MATE family revealed the presence of three distinct clusters, of which the first includes NorM, YdhE, and hypothetical multidrug efflux proteins from H. influenzaeand B. subtilis (28).

Electrogenic Drug/Proton AntiportDrug transport by multidrug transporters of the MFS, SMR, RND, and MATE families is driven by the transmembrane electrochemical gradient of protons or possibly sodium ions. The PMF is composed of a chemical proton gradient (ΔpH, inside alkaline) and an electrical potential (ΔΨ, inside negative). The ionophores nigericin and valinomycin, which can selectively dissipate the ΔpH and ΔΨ, respectively, are valuable tools for the study of the energetics of secondary multidrug transporters. Ng et al. (174) demonstrated that NorA-mediated norfloxacin uptake in inside-out membrane vesicles was completely abolished by nigericin. In contrast, addition of valinomycin resulted in a slight increase in the uptake of norfloxacin. These results indicate that the ΔpH is the major driving force for NorA. Grinius et al. (73) reported that NorA-mediated drug transport in proteoliposomes is an electrogenic drug/proton antiport process. Transport experiments in the presence of valinomycin and/or nigericin showed that both the ΔpH and the ΔΨ are driving forces of LmrP-mediated transport of trimethyl ammonium-diphenyl hexatriene (TMA-DPH) (22) and Hoechst 33342 (203), consistent with an electrogenic exchange of one lipophilic cation for two or more protons. Mitchell et al. (159) investigated which component of the PMF is involved in energizing QacA-mediated drug efflux. It was found that both the ΔpH and the ΔΨ drive the electrogenic transport of monovalent and divalent cations by QacA. The involvement of both components of the PMF as the driving force in Smr-mediated drug transport was demonstrated by studies using purified Smr reconstituted into proteoliposomes. A ΔΨ (positive inside) superimposed on ΔpH (acidic inside) resulted in additional ethidium uptake via Smr (72). An electrogenic drug/proton antiport reaction by Smr was further supported by the observation that Smr increased the resistance of E. coli cells to ethidium in alkaline medium, when the PMF predominantly consists of a ΔΨ (72). The electrogenic drug/nH+(n ≥ 2) antiport mechanism observed for LmrP, NorA, QacA, and Smr appears to be a general feature of multidrug transporters of the MFS and SMR families and may also be valid for the RND- and MATE-type multidrug transporters. The substrates used in the studies on the energetics of secondary multidrug transporters are monovalent cations. However, several multidrug transporters, such as QacA, MdfA, and YkkCD, also mediate resistance to divalent cations, neutral drugs, and anionic compounds. An analysis of the relative contribution of the ΔΨ and the ΔpH to the driving force for transport of these cations can provide further insight into the drug/proton ratio of the export process.

Conserved Sequences in Secondary Multidrug TransportersMultiple-sequence analysis of transporters within the various clusters of solute and drug transporters of the MFS revealed a substantially larger sequence similarity in the N-terminal halves of these proteins than in their C-terminal halves (70, 146, 188,213). It has been hypothesized that the N-terminal domain is involved in proton translocation, while the C-terminal domain of the transporters is primarily involved in determining the substrate specificity (70, 213). On the other hand, the significant sequence similarity observed between the N- and C-terminal halves of transporters of the MFS suggests that they have evolved from a duplication of a common ancestor with six TMS (122, 145, 215,274). The 14-TMS cluster of the MFS may have evolved via a similar gene duplication event and the acquisition of two additional TMS (70, 188, 217). The homology between the first and second halves of RND proteins suggests that these proteins also may have arisen via an intragenic duplication event during evolution (75, 216).

The multiple alignments of amino acid sequences further revealed a number of strongly conserved amino acid sequence motifs (Table4 and Fig. 3, 4, 5, and 6) throughout the multidrug transporters of the MFS, SMR, and RND families (188,190, 191, 213, 216). The conservation of these motifs suggests that they play an important structural or functional role in the transporters. Motif A in the cytoplasmic loop between TMS 2 and 3 in transporters of the MFS, which is predicted to contain a β-turn structure, may be involved in the reversible conformational change required for the opening and closing of a transport channel (102,188, 272, 273). The absolute conservation of the basic arginine residue suggests that motif B in TMS 4 of MFS transporters may be involved in proton transfer (188). Interestingly, motif C in TMS 5 is found in multidrug transporters and specific drug efflux pumps, but not in symporters of the MFS. This suggests that motif C dictates the direction of transport (70, 188). The GP dipeptide of this motif causes a bend in helix 5, and the repeating pattern of glycine residues forms a pocket devoid of side chains. These structures may determine the orientation of the unoccupied substrate-binding site(s) and, consequently, the direction of transport (261). The C-terminal motifs F and G represent partial duplications of motif C and may have a similar function. Site-directed mutagenesis studies can provide valuable information on the structure and mechanism of multidrug transporters of the MFS, RND, and SMR families. The conserved motifs may be directly involved in drug or proton translocation. The charged residue in motif B (Arg) and in motif D1 (Asp) in members of the MFS, in motif A (Glu) in SMR-type multidrug transporters, and in motif C (two Arg) in drug efflux systems of the RND family are predicted to be located in TMS. The presence of positively or negatively charged amino acid residues in the membrane is energetically unfavorable, suggesting that these motifs play a functional role in the binding and transport of charged drugs and/or translocation of protons. Alternatively, they may play a structural role in the interaction between the transporters of the RND family (motif A) and proteins of the MFP family, or in the formation of homo- and heterooligomers of SMR-type multidrug transporters. The conserved sequences are characteristic of proteins of the MFS, SMR, and RND families and can be used to identify new members of these families in bacterial genomes.

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Table 4.

Consensus sequences of conserved motifs in transporters of the MFS, SMR, and RND families

RésuméThe secondary multidrug transporters identified in bacteria (Tables 1 to 3) belong to one of four distinct families of proteins, MFS, SMR, RND, and MATE. Whereas multidrug transporters of the MFS and SMR families have been found in both gram-positive and gram-negative bacteria, RND- and MATE-type multidrug transporters have so far only been identified in gram-negative organisms. Table5 indicates that bacteria can contain several multidrug transporters from different families. The presence of a variety of multidrug transporters with an overlapping substrate spectrum would seem redundant. However, the availability of an array of multidrug transporters, which are controlled by both global and specific regulatory proteins, enables the bacterium to fine tune the response to different environmental signals.

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Table 5.

Number of sequenced and functionally characterized multidrug transporters of various families in selected bacteria

The most striking difference between multidrug transporters of the SMR family and drug efflux systems from the other families is their size. Whereas members of the MFS, RND, and MATE families consist of 12 or 14 TMS, SMR-type multidrug transporters only contain 4 TMS. These small multidrug efflux pumps are thought to function as homooligomers, presumably composed of three monomers. However, two new SMR-type multidrug transporters were recently identified in B. subtilis which require two homologous but dissimilar subunits for drug efflux. This observation raises the question of what molecular determinants dictate whether an SMR transporter functions as a homo- or heterooligomer.

Secondary multidrug transporters extrude drugs in exchange with protons (or sodium ions). Both components of the PMF can drive the extrusion of drugs by multidrug transporters from the MFS, SMR, and RND families, indicating that an electrogenic antiport mechanism is a general feature of secondary multidrug transporters.

Multiple sequence alignments of transporters within one family revealed the presence of three or more conserved motifs in multidrug transporters of the MFS, SMR, and RND families (Table 4). Hydropathy analysis, the recognition of family-specific conserved motifs, and the availability of the complete genomic sequences of 28 bacteria and 8 archaea (http://www.tigr.org/tdb/mdb) enable the identification of putative multidrug transporters. However, as multidrug transporters are frequently more homologous to substrate-specific transporters than to other multidrug transporters, the activity of newly identified proteins needs to be characterized.

ATP-DEPENDENT MULTIDRUG TRANSPORTERS

Although most bacterial multidrug drug transporters utilize the PMF (or sodium) for the extrusion of cytotoxic compounds, some drug efflux systems are driven by the free energy of ATP hydrolysis (Fig.7). All ATP-dependent drug efflux proteins known to date are members of the ABC superfamily (87), also referred to as traffic ATPases (8). In general, ABC transporters require four distinct domains: two highly hydrophobic membrane domains, which usually consist of six putative transmembrane α-helices each, and two hydrophilic nucleotide-binding domains (NBDs), containing the Walker A and B motifs (263) and the ABC signature (96). The individual domains can be expressed as separate proteins or may be fused into multidomain polypeptides in a variety of ways (55, 87, 96).

Fig. 7.
Fig. 7.

Structural model for multidrug transporters of the ABC superfamily. Shown is the multidrug transporter LmrA, with six transmembrane helices and an NBD, containing the Walker A and B motifs and the ABC signature sequence. In view of the general four-domain organization, it may function as a homodimer. The Walker A and B motifs and the ABC signature sequence are shaded.

Most bacterial ABC drug transporters mediate the export of specific antibiotics (17, 55, 76, 130, 183, 197, 210, 212). The first true bacterial ABC multidrug transporter was found in Lactococcus lactis (20). The gene encoding this transporter, designated lmrA, encodes a 590-amino-acid membrane protein with an N-terminal hydrophobic domain with six putative transmembrane helices and a C-terminal hydrophilic domain, containing the ATP-binding cassette (256). LmrA is homologous to each of the two halves of the human multidrug transporter P-glycoprotein (256), suggesting that LmrA is functional as a homodimer. This suggestion was confirmed by studies on covalently linked dimers composed of wild-type LmrA units (designated KK), mutant LmrA units containing an inactivated NBD (designated MM), and combinations of wild-type and mutant units (designated KM and MK) (258). The ethidium transport activity and substrate-stimulated ATPase activity of the KK dimeric form of LmrA was comparable to that of wild-type LmrA. However, the KM, MK, and MM mutant forms of dimeric LmrA had lost all LmrA-associated ATPase activity and the ability to transport ethidium from the cell. These results are supported by reconstitution experiments which demonstrate the negative dominance of M units over K units in proteoliposomes and suggest that homodimeric LmrA is the minimal functional unit. Hence, functional crosstalk between two LmrA monomers is essential for activity (258).

When expressed in human lung fibroblast cells, LmrA is targeted to the membrane and confers multidrug resistance on these human cells (257). The pharmacological characteristics of LmrA and the human multidrug transporter P-glycoprotein (67) and the affinities of both proteins for vinblastine and Mg2+-ATP are very similar. Blockers of P-glycoprotein also inhibited LmrA-mediated resistance. These results demonstrate that LmrA and the human multidrug transporter P-glycoprotein are functionally interchangeable, indicating that this type of multidrug transporter is conserved from bacteria to humans (258).

Kinetic analysis of vinblastine dissociation from LmrA and characterization of transport of Hoechst 33342 or vinblastine in the presence of low concentrations of a second substrate revealed that LmrA contains at least two allosterically linked drug-binding sites with overlapping substrate specificities (see below) (257, 258). Purified LmrA reconstituted in proteoliposomes mediates ATP-dependent transport of Hoechst 33342 (147). Certain ABC transporters, including the LmrA homolog MsbA of E. coli (285), are able to translocate lipids. The specificity of reconstituted LmrA for C6-NBD-labeled phosphatidylethanolamine and phosphatidylcholine was investigated. Interestingly, LmrA catalyzes the transport of C6-NBD-labeled phosphatidylethanolamine but not of C6-NBD-labeled phosphatidylcholine. These results indicate that LmrA exhibits specificity for phospholipid headgroups and that LmrA may be involved in the translocation of specific lipid or lipid-linked precursors in L. lactis (147).

Hop-resistant lactobacilli can cause serious spoilage of beer. Sami et al. (218, 219) demonstrated that plasmid pRH45 ofLactobacillus brevis is involved in the high resistance to hop compounds (iso-α-acids). pRH45 contains the horA gene, which encodes a protein of the ABC superfamily. HorA is a structural homolog of LmrA, containing six putative TMS and a C-terminal ATP-binding domain. In addition to conferring hop resistance, HorA confers resistance to the structurally unrelated drugs novobiocin and ethidium (219). HorA has been heterologously expressed inLactococcus lactis, purified, and functionally reconstituted in proteoliposomes. HorA-mediated Hoechst 33342 transport in proteoliposomes is inhibited by hop compounds (K. Sakamoto et al., submitted for publication).

Kaidoh et al. (105) described an ATP-dependent drug efflux system in the archaeon Haloferax volcanii. This efflux system mediates the transport of doxorubicin, vinblastine, vincristine, ethidium, and monensin. Expression of the transporter is induced in media containing a large excess of essential amino acids, glucose, or fructose. These observations suggest that this efflux system plays a role in the active extrusion of cytotoxic compounds or toxic metabolites generated in the cell. The gene encoding this ABC-type multidrug transporters remains to be cloned and sequenced (105). Genome sequencing revealed the presence of putative LmrA homologs in E. coli, B. subtilis, and the pathogens Helicobacter pylori, Mycobacterium genitalium, H. influenzae, and S. aureus(259). Functional characterization of these proteins is required to demonstrate their involvement in the ATP-dependent transport of structurally unrelated drugs.

Mutant analysis and photoaffinity labeling experiments have been used to identify essential residues and putative substrate-binding sites in the human multidrug transporter P-glycoprotein. The results demonstrated that, although some mutations leading to changes in specificity are located in other regions of the protein, TMS 5, 6, 11, and 12 and the loop between TMS 2 and 3 and TMS 11 and 12 are most important in drug binding and/or transport (39, 43, 68, 69, 82,106, 137, 139, 164, 282). Interestingly, sequence conservation between the lactococcal multidrug transporter LmrA and P-glycoprotein includes parts of TMS 5 and 6 and the loop between TMS 2 and 3 (256), indicating that these particular regions may be important for substrate binding in both human and bacterial ABC-type multidrug transporters.

REGULATION

Several studies demonstrated that the expression levels of multidrug transporters such as Bmr, NorA, QacA, and P-glycoprotein can be enhanced by the addition of structurally diverse compounds which are exported by these pumps (3, 74, 104, 107). These observations indicate that the expression of multidrug transporters is controlled by regulatory proteins which, like the transporters, are capable of recognizing structurally diverse compounds. The activator and repressor proteins involved in the transcriptional regulation of multidrug transporter genes are summarized in Table6.

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Table 6.

Reported transcriptional regulators that control the expression of multidrug transporters in bacteria

The best-studied regulator of multidrug transport is BmrR, the transcriptional activator of the B. subtilis multidrug transporter Bmr. BmrR, which is encoded by a gene immediately downstream of bmr, belongs to the MerR family of regulators and has strong homology with other members of the family only in the N-terminal helix-turn-helix DNA-binding domain. Binding of one rhodamine 6G molecule to a dimer of BmrR increases the affinity of BmrR for the bmr promoter, resulting in enhanced transcription of the bmr gene (3). The individually expressed C-terminal region of BmrR forms dimers and binds a broad range of drugs with the same affinity as full-length BmrR (148, 150). High-resolution crystal structures of the BmrR C-terminal region and its complex with TPP+ revealed drug-induced unfolding and relocation of an α-helix, which exposes an internal drug-binding pocket. Residue Glu-134, which is completely buried in the unliganded structure, is the key to cation selectivity, while a number of nonpolar and aromatic side chains impose specific requirements on the size and shape of the substrate (284). The transcription of theblt gene is regulated by another member of the MerR family, BltR. The C-terminal domains of BmrR and BltR have no sequence similarity, which is consistent with the observation that Bmr and Blt are expressed in response to different inducers. This difference in inducer specificity may explain why Bmr and not Blt is normally expressed in wild-type B. subtilis (4).

The expression of the S. aureus multidrug efflux geneqacA is regulated by the divergently transcribed repressor protein QacR. QacR binds specifically to the qacA promoter, preventing transcription of the gene. Substrates of QacA, such as ethidium and rhodamine 6G, interact with QacR directly and inhibit the binding of QacR to the qacA promoter, resulting in derepression of qacA expression (74).

The EmrAB multidrug pump of E. coli is induced in the presence of CCCP, the weak acid salicylate, and a number of other structurally unrelated drugs. The derepression is controlled by the EmrR, a MarR type of repressor protein (136). EmrR was found to bind putative substrates of the EmrAB pump, such as 2,4-dinitrophenol, CCCP, and carbonyl cyanidep-(trifluoro-methoxy)-phenylhydrazone (FCCP), with micromolar affinity. Equilibrium dialysis experiments suggested one bound ligand per EmrR dimer (27).

Analysis of the sequence upstream of the mtrCDE multidrug efflux system of N. gonorrhoeae revealed the presence of the divergently transcribed repressor gene mtrR. MtrR binds to the 13-bp inverted repeat sequence between the mtrR andmtrC promoters, thereby inhibiting expression of themtrRCDE gene complex (80, 185). Studies by Hagman and Shafer (79), however, showed that the MtrCDE efflux system is subject to both MtrR-dependent and MtrR-independent regulation. Recently, the transcriptional activator MtrA was shown to be involved in the modulation of mtrCDE gene expression (214).

The transcription of the acrAB operon in E. coliis regulated by the repressor AcrR (144). Gel mobility shift assays and lacZ transcriptional fusions suggested that the general-stress-enhanced transcription ofacrAB (144) is primarily mediated by global regulatory pathways such as the mar regulon (see below) and that a major function of AcrR is that of a specific secondary modulator, which fine tunes the level of acrAB transcription (144).

The transcriptional repressor AmrR of Burkholderia pseudomallei shows strong similarity to the regulator MtrR. The expression of the AmrAB-OprA efflux system was not affected by the substrate streptomycin or by stress conditions (4% ethanol or 0.5% NaCl), and further studies are required to identify the inducers of this system (161).

The expression of the P. aeruginosa multidrug transporter MexAB-OprM is regulated by MexR, a MarR-type regulator (53,201). The transcriptional repressor NfxB regulates a second MDR in P. aeruginosa, MexCD-OprJ, as well as its own expression (200). The mexEF-oprN efflux operon differs from the other P. aeruginosa efflux systems in that it is positively regulated by MexT, a protein belonging to the LysR family of transcriptional activators (115, 116). A third MDR inP. aeruginosa, MexXY, may be regulated by MexZ, which is homologous to the repressor proteins MtrR and AmrR (6).

In addition to the specific regulatory mechanisms that affect the expression of single multidrug transporters, induction of multidrug efflux systems can also result from global regulatory mechanisms. Multiple-antibiotic resistance (Mar) mutants of E. coliexpress elevated levels of resistance to a wide range of antibiotics. The alleles that affect the Mar phenotype are located in themarRAB operon (34). Expression of this operon is normally repressed by the transcriptional repressor MarR (9,225) but can be derepressed by various compounds, such as tetracycline, chloramphenicol, and salicylates (35, 78). ThemarA gene encodes an AraC-type activator that is structurally and functionally similar to two other global regulators ofE. coli, SoxS and Rob (60, 235, 270). The MarA, SoxS, and Rob proteins are transcriptional activators of at least a dozen promoters, the mar and soxRS regulons, that confer resistance to a variety of antibiotics and superoxide-generating agents (10, 62, 99, 100, 226). The observed resistance is partly due to increased micF transcription and the consequent decrease in expression of the outer membrane porin OmpF (33, 60). The RND-type multidrug transporter AcrAB-TolC plays a major role in the antibiotic and organic solvent resistance induced by MarA, SoxS, or Rob (182, 266). The maroperon is widespread among enteric bacteria, including the generaSalmonella, Shigella, Klebsiella,Citrobacter, Hafnia, and Enterobacter(36). Expression of E. coli MarA inMycobacterium smegmatis resulted in increased resistance to multiple antimicrobial agents, suggesting that MarA functions inM. smegmatis and that a mar-like regulatory system exists in mycobacteria (156). Recently, Baranova et al. (16) identified a global MerR-type regulator in B. subtilis, termed Mta. The individually expressed N-terminal DNA-binding domain of Mta mimics the inducer-bound form and interacts directly with the promoters of bmr and blt, thereby inducing transcription of these genes. Additionally, this domain stimulates the expression of the mta gene and at least one more gene, ydfK, which encodes a hypothetical membrane protein (16).

RECONSTITUTION

Multidrug transporters have predominantly been studied in whole cells and membrane vesicles, in which other proteins and cellular components or processes may complicate the interpretation of the data. In order to elucidate the structure, function, and mechanism of multidrug transporters at the molecular level, methods have been developed for overexpression, purification, and functional reconstitution into liposomes (72, 73, 147, 203, 227, 275,280). Although the overexpression of membrane proteins is often deleterious for the cells (48, 119, 203, 243), drug transporters could be overexpressed up to 5 to 30% of total membrane protein using homologous expression systems and inducible promoters, such as the T7 polymerase promoter (275), the lacpromoter (236), and the nisA promoter (147,203). The first step in the purification of membrane proteins involves extraction of the protein from the membrane. The E. coli multidrug transporter EmrE can be quantitatively and functionally extracted with a mixture of organic solvents, such as chloroform-methanol (275). However, solubilization of most other membrane proteins requires the use of detergents (115). The choice of detergent is crucial, since some detergents can irreversibly inactivate the protein (227; J. Knol, R. H. E. Friesen, and B. Poolman, submitted for publication). In addition, detergents are potential substrates for multidrug efflux proteins and can affect the transport activity (46, 203, 268). Retention of detergent during the reconstitution procedure is a serious problem which further restricts the choice of detergent for the solubilization and reconstitution of multidrug transporters (115). Affinity chromatography, a highly specific and efficient technique, allows the purification of several multidrug transporters to a high degree of purity (>90%) in a single step (72, 147, 203, 227, 280). After reconstitution in liposomes, Smr, P-glycoprotein, EmrE, NorA, LmrA, LmrP, AcrB, and HorA mediated the transport of multiple drugs in response to the imposition of an artificial proton gradient or the addition of ATP (72, 73, 147, 203, 227, 275, 280; K. Sakamoto, H. W. van Veen, and W. N. Konings, unpublished data). Taken together, these results demonstrate that multidrug transporters of the MFS, SMR, RND, and ABC families are capable of mediating drug transport independent of accessory proteins or cytosolic components.

SUBSTRATE BINDING

Both secondary and ATP-dependent multidrug transporters extrude a wide variety of toxic compounds. Although the substrates have very different structures, they share physical characteristics, such as high hydrophobicity, an amphiphilic nature, and a positive or neutral charge. In the absence of three-dimensional structural information, the identification of structure-function relationships in multidrug transporters has relied on amino acid sequence alignments and site-directed mutagenesis. The localization of a charged residue in the membrane-embedded part of the protein is normally considered energetically unfavorable, yet many multidrug transporters of the MFS and SMR families, such as MdfA, QacA, EmrE, and Smr, contain an acidic amino acid residue in the middle of TMS 1 (50). Furthermore, Guan et al. (75) identified three highly conserved negatively charged amino acid residues in transmembrane domains of efflux proteins of the RND family. Since the majority of the MDR substrates are positively charged, the negative charge of an intramembranous acidic residue may be involved in substrate binding. The role of the conserved Glu in TMS 1 of Smr has been investigated using site-directed mutagenesis. Even conservative substitutions, such as the replacement of Glu with Asp, effectively abolished the activity of the efflux system, indicating that this residue may be directly involved in the multidrug efflux process, potentially in substrate binding and/or the exchange of drug molecules for protons (72). Edgar and Bibi (50) demonstrated that a single membrane-embedded negative charge is critical for the recognition of positively charged drugs by the multidrug transporter MdfA of E. coli but not for transport activity. Replacement of the Glu residue in TMS 1 of EmrE abolished binding activity, but replacement with a negatively charged Asp created a mutant protein that retained its ability to bind substrate (165). Whereas QacA transports both monovalent and divalent cations, the closely related multidrug transporter QacB only transports monovalent cations. Sequence analysis and mutagenesis revealed that the presence of an acidic residue in TMS 10 (position 322 or 323) of these transporters is crucial for the recognition of divalent cations (192). Altogether, these results suggest that charge-charge interactions play an important role in the recognition and export of lipophilic cations by multidrug transporters.

Based on the observation that aromatic amino acid residues in transmembrane regions of the human multidrug transporter P-glycoprotein are highly conserved, Pawagi et al. (195) hypothesized that the side chains of these residues can participate in the initial binding and subsequent transport of a variety of ring-containing compounds. Indeed, Dougherty (47) indicated that cation-π interactions between cations, such as quarternary ammonium compounds, and aromatic side chains from the amino acids Phe, Tyr, and Trp are important in a variety of proteins that bind cationic substrates. The potential role of the conserved aromatic residues Tyr59 and Trp62 in the staphylococcal multidrug transporter Smr has been investigated by site-directed mutagenesis (189). Substitutions at these positions abolished the multidrug efflux capacities of the protein, indicating that these residues play a critical structural or functional role. Mutations of residues Phe143, Val286, and Phe306 of Bmr altered the sensitivity to reserpine and changed the substrate specificity, indicating that these residues are likely involved in substrate recognition (2, 111). Furthermore, mutations of Phe, Trp, and Tyr residues in TMS 6, 11, and 12 drastically altered the drug resistance profile of P-glycoprotein (82, 137). These results illustrate the importance of aromatic and nonpolar amino acid residues in drug binding.

The three-dimensional structure of the multidrug-binding domain of BmrR, the transcriptional activator of Bmr, supports a model in which a negatively charged amino acid residue as well as aromatic and nonpolar residues is involved in drug binding (284). The binding of TPP+ to Bmr induces the displacement of a short amphipatic α-helix to allow the drug to enter the drug-binding pocket. Inside the binding pocket, TPP+ forms a number of van der Waals' forces and stacking interactions with the surrounding hydrophobic and aromatic residues. The bottom of the binding pocket contains a buried glutamate residue, which makes an electrostatic interaction with the positively charged substrate (284). The substrate-binding site(s) of a multidrug transporter may have a similar organization. Most secondary multidrug transporters contain an essential membrane-embedded negatively charged amino acid residue. This residue could act like a magnet to attract positively charged substrates from the inner leaflet of the membrane. The surrounding hydrophobic residues would impose requirements on the size and shape of the substrate. In contrast to secondary multidrug transporters, the ABC multidrug transporters LmrA, P-glycoprotein, and others do not contain negatively charged residues in predicted TMS. It should also be noted that the binding of neutral or negatively charged substrates cannot be explained by the model described above.

There is increasing evidence for the presence of multiple drug-binding sites in both secondary and ATP-dependent multidrug transporters, rather than a single binding site, as suggested previously (126,195). Tamai and Safa (245) indicated for the first time that the human multidrug transporter P-glycoprotein may contain at least two kinetically distinguishable drug-binding sites. Kinetic analysis of the drug-stimulated ATPase activity of P-glycoprotein showed that cyclosporin A competitively inhibits the verapamil-stimulated ATPase activity, whereas daunorubicin, gramicidin D, vinblastine, and colchicine give an allosteric inhibition. Interestingly, cooperative stimulation of the verapamil-induced ATPase activity was found with progesterone (131). Several other studies demonstrated a similar complex effect of pairs of substrates or modulators on the drug-induced ATPase activity of P-glycoprotein (30, 61, 153, 186). Noncompetitive inhibition of drug binding (44, 56, 153, 264) and drug transport by P-glycoprotein (13, 196, 230, 238) further support a model with multiple cooperating drug interaction sites.

Noncompetitive inhibition of rhodamine 6G transport by drugs such as quinidine, puromycin, and colchicine also suggested multiple cooperating drug interaction sites in the yeast multidrug transporter Pdr5p (117). Van Veen et al. (257) demonstrated that nicardipin interacts with the lactococcal multidrug transporter LmrA at a binding site distinct from but allosterically linked to the vinca alkaloid-binding site. Competition studies showed that QacA-mediated transport of ethidium is inhibited competitively by the monovalent cations benzalkonium and tetraphenylarsonium and noncompetitively by the divalent cations chlorhexidine and propamide. These observations also suggest that the S. aureus multidrug transporter QacA contains distinct binding sites for monovalent and divalent cations (159). Furthermore, kinetic analysis of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles revealed a competitive inhibition by verapamil and quinine, a noncompetitive inhibition by nicardipin and vinblastine, and a noncompetitive inhibition by TPP+, implying that LmrP must contain at least two drug interaction sites (204). These results indicate that the presence of two or more interacting substrate-binding sites may be a general feature of multidrug efflux systems, including those of the SMR and RND families.

MECHANISM OF TRANSPORT

The common ability of MDR substrates to intercalate in the membrane led to the suggestion that multidrug transporters transport their substrates from the lipid bilayer (88, 207, 252) rather than from the cytoplasmic aqueous phase (7). Drug recognition within the membrane is suggested by a number of observations: (i) site-directed mutagenesis has identified specific residues implicated in substrate specificity, which are predominantly located within predicted TMS (2, 50, 65, 103, 184, 192), (ii) photoactive substrates label the human multidrug transporter P-glycoprotein in or near transmembrane helices (69, 207), and (iii) nonfluorescent acetoxymethyl esters of various fluorescent indicators, such as 2′,7′-bis-(2-carboxymethyl)-5-(and 6)-carboxy-fluorescein (BCECF) and calcein, are extruded from the membrane before they can be converted into fluorescent carboxylates by nonspecific esterases in the cytoplasm (23, 89, 91).

The most convincing evidence for drug efflux from the membrane to the aqueous phase, however, is provided by transport studies using the fluorescent compounds TMA-DPH, Hoechst 33342, and 2-{4-[4-(dimethylamino)phenyl]-1,3-butadienyl}-3-ethylbenzo-thiazolium perchlorate (LDS-751) which are strongly fluorescent in a hydrophobic environment but essentially nonfluorescent in an aqueous environment. Transport of these compounds from the membrane to the aqueous phase can be monitored as a decrease in fluorescence over time (22, 23, 180, 228, 229, 231). Shapiro et al. (229) demonstrated that the initial rate of P-glycoprotein-mediated Hoechst 33342 transport is directly proportional to the amount of Hoechst 33342 in the membrane, demonstrating that P-glycoprotein removes the drug from the lipid membrane. The mechanism of transport of LmrA (23), LmrP (22), MexAB-OprM (180), and QacA (159) was studied with TMA-DPH. TMA-DPH displays a biphasic interaction with bacterial membranes; a fast partitioning of the probe in the outer leaflet of the phospholipid bilayer, followed by a slower transbilayer movement of the probe from the outer to the inner leaflet (23,159). The initial rate of TMA-DPH extrusion by the lactococcal multidrug transporter LmrA increases with the concentration of TMA-DPH in the inner leaflet, demonstrating that LmrA transports drugs from the inner leaflet of the membrane (23). A similar relationship between the initial rate of transport and the concentration of substrate in the inner leaflet of the membrane was observed for LmrP (22) and P-glycoprotein (229, 231). Also, the MDR transporters MexAB-OprM and QacA are capable of strongly reducing the accumulation of TMA-DPH in the inner leaflet of the bilayer (159,180). Altogether, these results demonstrate that the multidrug transporters of the MFS, RND, and ABC families extrude toxic compounds from the inner leaflet of the cytoplasmic membrane to the aqueous extracellular medium (Fig. 8). Thus, despite differences in energy coupling, ATP- and PMF-dependent multidrug efflux pumps display a general mechanism of transport for hydrophobic drugs.

Fig. 8.
Fig. 8.

Extrusion of hydrophobic drugs by multidrug transporters. Drugs are expelled from the cytoplasmic leaflet of the membrane to the external medium.

Drug transport from the inner leaflet of the cytoplasmic membrane to the extracellular aqueous medium requires a high-affinity substrate-binding site that is accessible from the inner leaflet of the phospholipid bilayer. This high-affinity site is converted to a low-affinity outward-facing drug-binding site at the expense of ATP hydrolysis or proton translocation. Recently, drug transport cycles were described for the lactococcal ABC-type multidrug transporter LmrA (258) and the E. coli SMR-type multidrug transporter EmrE (165). Equilibrium binding experiments, photoaffinity labeling studies, and drug transport assays support the notion that homodimeric LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug release site on the outer membrane surface. The interconversion of these two sites is driven by the hydrolysis of ATP. Indeed, a combination of totalreflection Fourier transform infrared spectroscopy,2H/H exchange, and fluorescence-quenching experiments indicated that LmrA undergoes a secondary-structure change and passes through three different conformational states during its drug transport cycle (262). The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters (258). For P-glycoprotein, evidence has been obtained which supports a model of alternating catalytic sites (223). Both NBDs are able to bind and hydrolyze ATP (92, 138). The two nucleotide-binding sites interact strongly and cannot hydrolyze Mg2+-ATP simultaneously, suggesting that the two NBDs may alternate in catalysis (93, 224, 253, 254).

EmrE, which contains a membrane-embedded glutamate residue that is required for ligand binding and is proposed to function as a homotrimer, binds 1 mol of TPP+ per ∼3 mol of ErmE. From the observation that both binding and release of TPP+ from EmrE are strongly influenced by pH, Muth and Schuldiner (165) proposed a mechanism of transport by EmrE. As the substrate approaches the hydrophobic binding pocket, two protons are released from the negatively charged glutamate triplet. The positively charged substrate is bound through electrostatic interactions with the negatively charged carboxylate groups of the glutamates. Following a currently unknown conformational transition, the opening to the substrate-binding pocket becomes accessible to the external face of the membrane while being closed off from the internal face. The subsequent movement of two protons towards the binding pocket catalyzes the release of the bound substrate. EmrE then undergoes a conformational transition that converts the binding pocket accessibility back to the original membrane face. As most secondary multidrug transporters contain one or more negatively charged amino acid residues in TMS, a similar mechanism may be valid for other transporters. The available techniques for purification and functional reconstitution of the secondary multidrug transporters Smr, EmrE, NorA, LmrP, and AcrB enable structural studies that can reveal conformational changes during the catalytic cycle.

ANTIBIOTIC RESISTANCE

Bacterial multidrug efflux systems are a serious problem in the pharmacological treatment of patients with infectious diseases, since the substrate spectra of many multidrug transporters include clinically relevant antibiotics. Multidrug transporters are associated with both intrinsic and acquired resistance to antibiotics. Disruption of the genes encoding AcrAB, MexAB-OprM, HI0894/HI0895, NorA, and others causes increased sensitivity to several antibiotics, demonstrating that these multidrug transporters are expressed in wild-type strains and contribute to the intrinsic antibiotic resistance of E. coli, P. aeruginosa, H. influenzae, andS. aureus, respectively (143, 199, 220, 271). In addition, it has been shown that the expression of several genes encoding multidrug efflux pumps is inducible by the drugs to which they offer protection and that the expression of some multidrug transporters is modulated in response to environmental conditions. Acquired MDR can arise via three mechanisms: (i) amplification and mutations of genes encoding multidrug transporters, which change the expression level (174) or activity (113), (ii) mutations in specific or global regulatory genes which lead to the increased expression of multidrug transporters, exemplified by themexR mutation in the OCR1 strain of P. aeruginosa (201), and (iii) intercellular transfer of resistance genes on transposons or plasmids, such as qacE(108).

The clinical importance of multidrug transporters strongly depends on the antibiotic spectrum to which they confer resistance. The antibiotic resistance profiles of the multidrug transporters known to date are summarized in Table 7. The data in Table7 clearly show that the drug resistance profiles of the multidrug transporters vary strongly and family-specific profiles are not found. Resistance is observed against all important classes of antibiotics. MexAB-OprM of P. aeruginosa and the ABC-type multidrug transporter LmrA of L. lactis display very broad antibiotic specificity, demonstrating that both secondary and ATP-dependent multidrug transporters can seriously affect the efficacy of many antibiotics. LmrA and the human multidrug transporter P-glycoprotein are functionally interchangeable (257), suggesting that P-glycoprotein may also be involved in export of antibiotics. Indeed, several Steptomyces macrolide antibiotics have been reported to inhibit P-glycoprotein function (140). The plasmid-encoded multidrug transporter HorA confers resistance to a smaller range of antibiotics than the chromosomally encoded LmrA. However, since few antibiotic resistance data are available for other plasmid-encoded multidrug transporters, such as QacA, QacB, and Smr, it is not known whether a more restricted antibiotic resistance profile is a general feature of plasmid-encoded multidrug transporters. NorA and Bmr confer higher levels of resistance to hydrophilic quinolones, such as norfloxacin and ciprofloxacin, than to hydrophobic quinolones, such as sparfloxacin, ofloxacin, and nalidixic acid (104, 171, 271,278). Similarly, norfloxacin transport by LfrA is inhibited by hydrophilic and not by hydrophobic quinolones (134). This specificity for hydrophilic quinolones was also observed for PmrA and NorM (63, 163). However, EmrB and VceB confer resistance only to hydrophobic quinolones, whereas LmrA and the multidrug transporters of the RND family transport both hydrophilic and hydrophobic quinolones (37, 66, 135, 158, 200, 205). In addition to conferring resistance, the overexpression of multidrug transporters can also cause increased drug sensitivity. The E. coli multidrug transporter MdfA confers resistance to the aminoglycosides neomycin and kanamycin but causes hypersensitivity to spectinomycin (19, 49). Overexpression of MexCD-OprJ results in hypersusceptibility to gentamicin and carbenicillin but not to ampicillin and penicillin (200, 240, 241). The mechanism of this increased drug susceptibility remains to be studied.

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Table 7.

Reported drug resistance profiles of PMF- and ATP-dependent multidrug transportersa

PHYSIOLOGICAL FUNCTION

The efflux of a broad range of structurally unrelated toxic compounds can be the primary physiological function of multidrug transporters or, alternatively, can be merely a fortuitous side effect of the transport of an unidentified specific physiological substrate (173). The latter role of MDRs would be consistent with the observation that multidrug transporters belong to four distinct families which are frequently more homologous to substrate-specific transporters than to each other (126). More convincing evidence for a role of multidrug transporters in the efflux of a specific substrate comes from the B. subtilis multidrug transporter Blt. Woolridge et al. (269) demonstrated that overexpression of Blt results in an increased efflux of spermidine in the medium. The blt gene is cotranscribed with a downstream gene, btlD, which encodes a spermidine acetyltransferase (4, 269). This genetic arrangement suggests that the efflux of spermidine is the natural function of the Blt transporter, whereas multiple drugs may be recognized merely opportunistically. The ability of some MDR transporters to mediate lipids or fluorescent lipid derivatives may indicate that the physiological function of these multidrug transporters involves phospholipid translocation or transport of lipid-linked precursors of peptidoglycan (24, 90, 147, 254a,280).

Alternatively, multidrug transporters may have evolved to protect cells from diverse environmental toxins. Schinkel et al. (222) demonstrated that disruption of the mouse mdr1aP-glycoprotein gene leads to increased sensitivity to drugs such as ivermectin and vinblastine. Similarly, increased antibiotic susceptibility was found in norA knockout mutants (94,271). P-glycoprotein and the E. coli multidrug transporter AcrAB are induced in response to cellular damage (31) and stress conditions (143), respectively. Furthermore, the substrate spectra of several multidrug transporters include detergents (46, 203, 268), bile salts (80, 143,249), organic solvents (266), and ionophores (54,218, 233), suggesting that multidrug transporters play a role in protection of the membrane integrity or energy state of the cell.

CONCLUDING REMARKS

Multidrug transporters are a serious problem in the treatment of patients with hospital- or community-acquired infectious diseases, as they confer resistance to various of the antibiotics employed. A major force in the overexpression of endogenous multidrug transporters and spread of plasmid-encoded multidrug transporters is the huge consumption of antibiotics in human therapy, animal husbandry, and agriculture (12, 123). To restrain the evolution of antibiotic resistance, several strategies have been proposed to reduce the selective pressure of the presence of antibiotics (14, 123,124, 267). The inappropriate use of antibiotics in household products, for the treatment of viral diseases, and as growth promoters in farm animals should be reduced. In addition, the development of new therapeutic agents is essential to fight the increasing number of multidrug-resistant microorganisms. One approach is to search for antibiotics with novel modes of action, which will not face previously selected resistance determinants. Alternatively, resistance-blocking agents can be identified and developed, allowing existing antibiotics to remain effective (125, 149). Several inhibitors of the multidrug transporters NorA and PmrA have been shown to increase the activity of fluoroquinolones against S. aureus and S. pneumoniae, respectively. Furthermore, these inhibitors dramatically suppress the in vitro emergence of resistant strains (1, 151, 152). However, since the known inhibitors, such as reserpine, are toxic to humans, the development of nontoxic inhibitors is required. Detailed information about the structure-function relationships that determine drug binding and transport by multidrug transporters should provide us with a rationale for these strategies.

Considerable progress has been made towards the identification of amino acid residues implicated in drug binding. Mutants of both ATP-dependent and secondary multidrug transporters demonstrated that aromatic, nonpolar, and negatively charged amino acid residues in predicted TMS are involved in substrate binding by multidrug transporters. Increasing evidence suggests the presence of multiple drug-binding sites, but the exact number and their three-dimensional organization are not known. Understanding the molecular mechanism of multidrug transport will ultimately require high-resolution structures of MDR proteins. As integral membrane proteins are generally very difficult to crystalize, structures of MDR regulator proteins which can bind a similar broad range of drugs may provide useful information on the structure-function relationships that dictate drug recognition. The low-resolution structure of P-glycoprotein (211) and the high-resolution (2.7 Å) three-dimensional structure of the drug-binding region of the regulator protein BmrR (284) are the first steps towards resolving the mystery of the broad substrate specificity of multidrug transporters.

FOOTNOTES

  • ↵* Corresponding author. Mailing address: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands. Phone: 31 50 3632150. Fax: 31 50 3632154.

  • ↵† Present address: Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom.

REFERENCES

  1. 1.↵
    1. Aeschlimann J. R.,
    2. Dresser L. D.,
    3. Kaatz G. W.,
    4. Rybak M. J.
    (1999) Effects of NorA inhibitors on in vitro antibacterial activities and postantibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 43:335–340.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Ahmed M.,
    2. Borsch C. M.,
    3. Neyfakh A. A.,
    4. Schuldiner S.
    (1993) Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensitive alkaloid reserpine. J. Biol. Chem. 268:11086–11089.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Ahmed M.,
    2. Borsch C. M.,
    3. Taylor S. S.,
    4. Vásquez-Laslop N.,
    5. Neyfakh A. A.
    (1994) A protein that activates expression of a multidrug transporter upon binding the transporter substrates. J. Biol. Chem. 269:28506–25813.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Ahmed M.,
    2. Lyass L.,
    3. Markham P. N.,
    4. Taylor S. S.,
    5. Vásquez-Laslop N.,
    6. Neyfakh A. A.
    (1995) Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated. J. Bacteriol. 177:3904–3910.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Aı́nsa J. A.,
    2. Blokpoel M. C. J.,
    3. Otal I.,
    4. Young D. B.,
    5. De Smet K. A. L.,
    6. Martı́n C.
    (1998) Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis. J. Bacteriol. 180:5836–5843.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Aires J. R.,
    2. Köhler T.,
    3. Nikaido H.,
    4. Plésiat P.
    (1999) Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother. 43:2624–2628.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Altenberg G. A.,
    2. Vanoye C. G.,
    3. Horton J. K.,
    4. Reuss L.
    (1994) Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane. Proc. Natl. Acad. Sci. USA 91:4654–4657.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Ames G. F.-L.,
    2. Mimura C. S.,
    3. Shyamala V.
    (1990) Bacterial periplasmic permeases belong to a family of transport proteins operating from E. coli to human: traffic ATPases. FEMS Microbiol. Rev. 75:429–446.
    OpenUrlCrossRefWeb of Science
  9. 9.↵
    1. Ariza R. R.,
    2. Cohen S. P.,
    3. Bachhawat N.,
    4. Levy S. B.,
    5. Demple B.
    (1994) Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli. J. Bacteriol. 176:143–148.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Ariza R. R.,
    2. Li Z.,
    3. Ringstad N.,
    4. Demple B.
    (1995) Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein. J. Bacteriol. 177:1655–1661.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Arkin I. T.,
    2. Russ W. P.,
    3. Lebendiker M.,
    4. Schuldiner S.
    (1996) Determining the secondary structure and orientation of EmrE, a multi-drug transporter, indicates a transmembrane four-helix bundle. Biochemistry 35:7233–7238.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Austin D. J.,
    2. Kristinsson K. G.,
    3. Anderson R. M.
    (1999) The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance. Proc. Natl. Acad. Sci. USA 96:1152–1156.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Ayesh S.,
    2. Shao Y.-M.,
    3. Stein W. D.
    (1996) Co-operative, competitive and non-competitive interactions between modulators of P-glycoprotein. Biochim. Biophys. Acta 1316:8–18.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Baquero F.,
    2. Blázquez J.
    (1997) Evolution of antibiotic resistance. TREE 12:482–487.
    OpenUrl
  15. 15.↵
    1. Baranova N. N.,
    2. Neyfakh A. A.
    (1997) Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 41:1396–1398.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Baranova N. N.,
    2. Danchin A.,
    3. Neyfakh A. A.
    (1999) Mta, a global MerR-type regulator of the Bacillus subtilis multidrug-efflux transporters. Mol. Microbiol. 31:1549–1559.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. Barrasa M. I.,
    2. Tercero J. A.,
    3. Lacalle R. A.,
    4. Jimenez A.
    (1995) The ard1 gene from Streptomyces capreolus encodes a polypeptide of the ABC-transporters superfamily which confers resistance to the aminonucleoside antibiotic A201A. Eur. J. Biochem. 228:562–569.
    OpenUrlPubMedWeb of Science
  18. 18.↵
    1. Bentley J.,
    2. Hyatt L. S.,
    3. Ainley K.,
    4. Parish J. H.,
    5. Herbert R. B.,
    6. White G. R.
    (1993) Cloning and sequence analysis of an Escherichia coli gene conferring bicyclomycin resistance. Gene 127:117–120.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Bohn C.,
    2. Bouloc P.
    (1998) The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl-β-d-thiogalactopyranoside exclusion and spectinomycin sensitivity. J. Bacteriol. 190:6072–6075.
    OpenUrl
  20. 20.↵
    1. Bolhuis H.,
    2. Molenaar D.,
    3. Poelarends G.,
    4. van Veen H. W.,
    5. Poolman B.,
    6. Driessen A. J. M.,
    7. Konings W. N.
    (1994) Proton motive force-driven and ATP-dependent drug extrusion systems in multidrug-resistant Lactococcus lactis. J. Bacteriol. 176:6957–6964.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Bolhuis H.,
    2. Poelarends G.,
    3. van Veen H. W.,
    4. Poolman B.,
    5. Driessen A. J. M.,
    6. Konings W. N.
    (1995) The lactococcal lmrP gene encodes a proton motive force-dependent drug transporter. J. Biol. Chem. 270:26092–26098.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Bolhuis H.,
    2. van Veen H. W.,
    3. Brands J. R.,
    4. Putman M.,
    5. Poolman B.,
    6. Driessen A. J. M.,
    7. Konings W. N.
    (1996) Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP. J. Biol. Chem. 271:24123–24128.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Bolhuis H.,
    2. van Veen H. W.,
    3. Molenaar D.,
    4. Poolman B.,
    5. Driessen A. J. M.,
    6. Konings W. N.
    (1996) Multidrug resistance in Lactococcus lactis: Evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane. EMBO J. 15:4239–4245.
    OpenUrlPubMedWeb of Science
  24. 24.↵
    1. Bosch I.,
    2. Dunussi-Joannopoulos K.,
    3. Wu R.-L.,
    4. Furlong S.,
    5. Croop J.
    (1997) Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. Biochemistry 36:5685–5694.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Brenwald N. P.,
    2. Gill M. J.,
    3. Wise R.
    (1997) The effect of reserpine, an inhibitor of multi-drug efflux pumps, on the in-vitro susceptibilities of fluoroquinolone-resistant strains of Streptococcus pneumoniae to norfloxacin. J. Antimicrob. Chemother. 40:458–460.
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    1. Brenwald N. P.,
    2. Gill M. J.,
    3. Wise R.
    (1998) Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42:2032–2035.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Brooun A.,
    2. Tomashek J. J.,
    3. Lewis K.
    (1999) Purification and ligand binding of EmrE, a regulator of a multidrug transporter. J. Bacteriol. 181:5131–5133.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Brown M. H.,
    2. Paulsen I. T.,
    3. Skurray R. A.
    (1999) The multidrug efflux protein NorM is a prototype of a new family of transporters. Mol. Microbiol. 31:394–395.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    1. Bush K.,
    2. Jacoby G. A.,
    3. Medeiros A. A.
    (1995) A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211–1233.
    OpenUrlFREE Full Text
  30. 30.↵
    1. Buxbaum E.
    (1999) Co-operative binding sites for transported substrates in the multiple drug resistance transporter Mdr1. Eur. J. Biochem. 265:64–70.
    OpenUrlPubMedWeb of Science
  31. 31.↵
    1. Chaudhary P. M.,
    2. Roninson I. B.
    (1993) Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs. J. Natl. Cancer Inst. 85:632–639.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    1. Cohen M. L.
    (1992) Epidemiology of drug resistance: implications for a postantimicrobial era. Science 257:1050–1055.
  33. 33.↵
    1. Cohen S. P.,
    2. McMurry L. M.,
    3. Levy S. B.
    (1988) marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli. J. Bacteriol. 170:5416–5422.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Cohen S. P.,
    2. Hächler H.,
    3. Levy S. B.
    (1993) Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J. Bacteriol. 175:1484–1492.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Cohen S. P.,
    2. Levy S. B.,
    3. Foulds J.,
    4. Rosner J. L.
    (1993) Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway. J. Bacteriol. 175:7856–7862.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Cohen S. P.,
    2. Yan W.,
    3. Levy S. B.
    (1993) A multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria. J. Infect. Dis. 168:484–488.
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    1. Colmer J. A.,
    2. Fralick J. A.,
    3. Hamood A. N.
    (1998) Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae. Mol. Microbiol. 27:63–72.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    1. Culliton B. J.
    (1992) Drug-resistant TB may bring epidemic. Nature 356:473.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    1. Currier S. J.,
    2. Kane S. E.,
    3. Willingham M. C.,
    4. Cardarelli C. O.,
    5. Pastan I.,
    6. Gottesman M. M.
    (1992) Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters. J. Biol. Chem. 267:25153–25159.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Davies J.
    (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264:375–382.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Delahay R. M.,
    2. Robertson B. D.,
    3. Balthazar J. T.,
    4. Shafer W. M.,
    5. Ison C. A.
    (1997) Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents. Microbiology 143:2127–2133.
    OpenUrlCrossRefPubMedWeb of Science
  42. 42.↵
    1. De Rossi E.,
    2. Branzoni M.,
    3. Cantoni R.,
    4. Milano A.,
    5. Riccardi G.,
    6. Ciferri O.
    (1998) mmr, a Mycobacterium tuberculosis gene conferring resistance to small cationic dyes and inhibitors. J. Bacteriol. 180:6068–6071.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Devine S. E.,
    2. Ling V.,
    3. Melera P. W.
    (1992) Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance. Proc. Natl. Acad. Sci. USA 89:4564–4568.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Dey S.,
    2. Ramachandra M.,
    3. Pastan I.,
    4. Gottesman M. M.,
    5. Ambudkar S. V.
    (1997) Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc. Natl. Acad. Sci. USA 94:10594–10599.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Dinh T.,
    2. Paulsen I. T.,
    3. Saier M. H.
    (1994) A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J. Bacteriol. 176:3825–3831.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    1. Doige C. A.,
    2. Yu X.,
    3. Sharom F. J.
    (1993) The effect of lipids and detergents on ATPase-active P-glycoprotein. Biochim. Biophys. Acta 1146:65–72.
    OpenUrlCrossRefPubMedWeb of Science
  47. 47.↵
    1. Dougherty D. A.
    (1996) Cation-π interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271:163–168.
    OpenUrlAbstract
  48. 48.↵
    1. Eckert B.,
    2. Beck C. F.
    (1989) Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential. J. Bacteriol. 171:3557–3559.
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Edgar R.,
    2. Bibi E.
    (1997) MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J. Bacteriol. 179:2274–2280.
    OpenUrlAbstract/FREE Full Text
  50. 50.↵
    1. Edgar R.,
    2. Bibi E.
    (1999) A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA. EMBO J. 18:822–832.
    OpenUrlAbstract
  51. 51.↵
    1. Edwards R. A.,
    2. Turner R. J.
    (1998) Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters. Biochem. Cell Biol. 76:791–797.
    OpenUrlCrossRefPubMed
  52. 52.↵
    1. Evans K.,
    2. Passador I.,
    3. Srikumar R.,
    4. Tsang E.,
    5. Nezezon J.,
    6. Poole K.
    (1998) Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 180:5443–5447.
    OpenUrlAbstract/FREE Full Text
  53. 53.↵
    1. Evans K.,
    2. Poole K.
    (1999) The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated. FEMS Microbiol. Lett. 173:35–39.
    OpenUrlCrossRefPubMedWeb of Science
  54. 54.↵
    1. Eytan G. D.,
    2. Borgnia M. J.,
    3. Regev R.,
    4. Assaraf Y. G.
    (1994) Transport of polypeptide ionophores into proteoliposomes reconstituted with rat liver P-glycoprotein. J. Biol. Chem. 269:26058–26065.
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    1. Fath M. J.,
    2. Kolter R.
    (1993) ABC transporters: bacterial exporters. Microbiol. Rev. 57:995–1017.
    OpenUrlAbstract/FREE Full Text
  56. 56.↵
    1. Ferry D. R.,
    2. Russell M. A.,
    3. Cullen M. H.
    (1992) P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is allosterically coupled to a vinca-alkaloid-selective binding site. Biochem. Biophys. Res. Commun. 188:440–445.
    OpenUrlCrossRefPubMedWeb of Science
  57. 57.↵
    1. Fleischmann R. D.,
    2. Adams M. D.,
    3. White O.,
    4. Clayton R. A.,
    5. Kirkness E. F.,
    6. Kerlavage A. R.,
    7. Bult C. J.,
    8. Tomb J. F.,
    9. Dougherty B. A.,
    10. Merrick J. M.,
    11. McKenney K.,
    12. Sutton G.,
    13. FitzHugh W.,
    14. Field C.,
    15. Gocayne J. D.,
    16. Scott J.,
    17. Shirley R.,
    18. Liu L.-I.,
    19. Glodek A.,
    20. Kelly J. M.,
    21. Weidman J. F.,
    22. Phillips C. A.,
    23. Spriggs T.,
    24. Hedblom E.,
    25. Cotton M. D.,
    26. Utterback T. R.,
    27. Hanna M. C.,
    28. Nguyen D. T.,
    29. Saudek D. M.,
    30. Brandon R. C.,
    31. Fine L. D.,
    32. Fritchman J. L.,
    33. Fuhrman J. L.,
    34. Geohagen N. S. M.,
    35. Gnehm C. L.,
    36. McDonald L. A.,
    37. Small K. V.,
    38. Fraser C. M.,
    39. Smith H. O.,
    40. Venter J. C.
    (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512.
    OpenUrlAbstract/FREE Full Text
  58. 58.↵
    1. Fralick J. A.
    (1996) Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J. Bacteriol. 178:5803–5805.
    OpenUrlAbstract/FREE Full Text
  59. 59.↵
    1. Furukawa H.,
    2. Tsay J.-T.,
    3. Jackowski S.,
    4. Takamura Y.,
    5. Rock C. O.
    (1993) Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB. J. Bacteriol. 175:3723–3729.
    OpenUrlAbstract/FREE Full Text
  60. 60.↵
    1. Gambino L.,
    2. Gracheck S. J.,
    3. Miller P. F.
    (1993) Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli. J. Bacteriol. 175:2888–2894.
    OpenUrlAbstract/FREE Full Text
  61. 61.↵
    1. Garrigos M.,
    2. Mir L. M.,
    3. Orlowski S.
    (1997) Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase; further experimental evidence for a multisite model. Eur. J. Biochem. 244:664–673.
    OpenUrlCrossRefPubMedWeb of Science
  62. 62.↵
    1. Gaudu P.,
    2. Weiss B.
    (1996) SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form. Proc. Natl. Acad. Sci. USA 93:10094–10098.
    OpenUrlAbstract/FREE Full Text
  63. 63.↵
    1. Gill M. J.,
    2. Brenwald N. P.,
    3. Wise R.
    (1999) Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 43:187–189.
    OpenUrlAbstract/FREE Full Text
  64. 64.↵
    1. Gotoh N.,
    2. Tsujimoto H.,
    3. Poole K.,
    4. Yamagishi J.-I.,
    5. Nishino T.
    (1995) The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by oprK of the mexA-mexB-oprK multidrug resistance operon. Antimicrob. Agents Chemother. 39:2567–2569.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    1. Gotoh N.,
    2. Tsujimoto H.,
    3. Tsuda M.,
    4. Okamoto K.,
    5. Nomura A.,
    6. Wada A.,
    7. Nakahashi M.,
    8. Nishino T.
    (1998) Characterization of the MexC-MexD-OprJ efflux system in ΔmexA-mexB-oprM mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 42:1938–1943.
    OpenUrlAbstract/FREE Full Text
  66. 66.↵
    1. Gotoh N.,
    2. Tsujimoto H.,
    3. Nomura A.,
    4. Okamoto K.,
    5. Tsuda M.,
    6. Nishino T.
    (1998) Functional replacement of OprJ by OprM in the MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 165:21–27.
    OpenUrlCrossRefPubMed
  67. 67.↵
    1. Gottesman M. M.,
    2. Pastan I.
    (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 62:385–427.
    OpenUrlCrossRefPubMedWeb of Science
  68. 68.↵
    1. Gottesman M. M.,
    2. Hrycyna C. A.,
    3. Schoenlein P. V.,
    4. Germann U. A.,
    5. Pastan I.
    (1995) Genetic analysis of the multidrug transporter. Annu. Rev. Genet. 29:607–649.
    OpenUrlCrossRefPubMedWeb of Science
  69. 69.↵
    1. Greenberger L. M.
    (1993) Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P-glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12. J. Biol. Chem. 268:11417–11425.
    OpenUrlAbstract/FREE Full Text
  70. 70.↵
    1. Griffith J. K.,
    2. Baker M. E.,
    3. Rouch D. A.,
    4. Page M. P. G.,
    5. Skurray R. A.,
    6. Paulsen I. T.,
    7. Chater K. F.,
    8. Baldwin S. A.,
    9. Henderson P. J. F.
    (1992) Membrane proteins: implications of sequence comparisons. Curr. Opin. Cell Biol. 4:684–695.
    OpenUrlCrossRefPubMed
  71. 71.↵
    1. Grinius L.,
    2. Dreguniene G.,
    3. Goldberg E. B.,
    4. Liao C.-H.,
    5. Projan S. J.
    (1992) A staphylococcal multidrug resistance gene product is a member of a new protein family. Gene 27:119–129.
  72. 72.↵
    1. Grinius L.,
    2. Goldberg E. B.
    (1994) Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump. J. Biol. Chem. 269:29998–30004.
    OpenUrlAbstract/FREE Full Text
  73. 73.↵
    1. Grinius L. L.,
    2. Siehnel R. J.,
    3. Morris C. M.
    (1997) Quinolone efflux protein NorA is a proton-driven drug pump. FASEB J. 11:A958.
    OpenUrl
  74. 74.↵
    1. Grkovic S.,
    2. Brown M. H.,
    3. Roberts N. J.,
    4. Paulsen I. T.,
    5. Skurray R. A.
    (1998) QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA. J. Biol. Chem. 273:18665–18673.
    OpenUrlAbstract/FREE Full Text
  75. 75.↵
    1. Guan L.,
    2. Ehrmann M.,
    3. Yoneyama H.,
    4. Nakae T.
    (1999) Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM Extrusion pump in Pseudomonas aeruginosa. J. Biol. Chem. 274:10517–10522.
    OpenUrlAbstract/FREE Full Text
  76. 76.↵
    1. Guilfoile P. G.,
    2. Hutchinson C. R.
    (1991) A bacteriol analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc. Natl. Acad. Sci. USA 88:8553–8557.
    OpenUrlAbstract/FREE Full Text
  77. 77.↵
    1. Gutmann L.,
    2. Williamson R.,
    3. Moreau N.,
    4. Kitzis M.-D.,
    5. Collatz E.,
    6. Acar J. F.,
    7. Goldstein F. W.
    (1985) Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella, Enterobacter, and Serratia. J. Infect. Dis. 151:501–507.
    OpenUrlCrossRefPubMed
  78. 78.↵
    1. Hächler H.,
    2. Cohen S. P.,
    3. Levy S. B.
    (1991) marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. J. Bacteriol. 173:5532–5538.
    OpenUrlAbstract/FREE Full Text
  79. 79.↵
    1. Hagman K. E.,
    2. Shafer W. M.
    (1995) Transcriptional control of the mtr efflux system of Neisseria gonorrhoeae. J. Bacteriol. 177:4162–4165.
    OpenUrlAbstract/FREE Full Text
  80. 80.↵
    1. Hagman K. E.,
    2. Pan W.,
    3. Spratt B. G.,
    4. Balthazar J. T.,
    5. Judd R. C.,
    6. Shafer W. M.
    (1995) Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141:611–622.
    OpenUrlCrossRefPubMedWeb of Science
  81. 81.
    1. Hagman K. E.,
    2. Lucas C. E.,
    3. Balthazar J. T.,
    4. Snyder L.,
    5. Nilles M.,
    6. Judd R. C.,
    7. Shafer W. M.
    (1997) The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system. Microbiology 143:2117–2125.
    OpenUrlCrossRefPubMedWeb of Science
  82. 82.↵
    1. Hanna M.,
    2. Brault M.,
    3. Kwan T.,
    4. Kast C.,
    5. Gros P.
    (1996) Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning. Biochemistry 35:3625–3635.
    OpenUrlCrossRefPubMed
  83. 83.↵
    1. Hayes J. D.,
    2. Wolf C. R.
    (1990) Molecular mechanisms of drug resistance. Biochem. J. 272:281–295.
    OpenUrlFREE Full Text
  84. 84.↵
    1. Heir E.,
    2. Sundheim G.,
    3. Holck A. L.
    (1998) The Staphylococcus aureus qacH gene product: a new member of the SMR family encoding multidrug resistance. FEMS Microbiol. Lett. 163:49–56.
    OpenUrlCrossRefPubMed
  85. 85.↵
    1. Heir E.,
    2. Sundheim G.,
    3. Holck A. L.
    (1999) The qacG gene on plasmid pST94 confers resistance to quarternary ammonium compounds in staphylococci isolated from the food industry. J. Appl. Microbiol. 86:378–388.
    OpenUrlCrossRefPubMed
  86. 86.
    Reference deleted.
  87. 87.↵
    1. Higgins C. F.
    (1992) ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8:67–113.
    OpenUrlCrossRefPubMedWeb of Science
  88. 88.↵
    1. Higgins C. F.,
    2. Gottesman M. M.
    (1992) Is the multidrug transporter a flippase? Trends Biochem. Sci. 17:18–21.
    OpenUrlCrossRefPubMedWeb of Science
  89. 89.↵
    1. Holló Z.,
    2. Homolya L.,
    3. Davis C. W.,
    4. Sarkadi B.
    (1994) Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim. Biophys. Acta 1191:384–388.
    OpenUrlPubMed
  90. 90.↵
    1. Höltje J.-V.
    (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62:181–203.
    OpenUrlAbstract/FREE Full Text
  91. 91.↵
    1. Homolya L.,
    2. Holló Z.,
    3. Germann U. A.,
    4. Pastan I.,
    5. Gottesman M. M.,
    6. Sarkadi B.
    (1993) Fluorescent cellular indicators are extruded by the multidrug resistance protein. J. Biol. Chem. 268:21493–21496.
    OpenUrlAbstract/FREE Full Text
  92. 92.↵
    1. Horio M.,
    2. Gottesman M. M.,
    3. Pastan I.
    (1988) ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc. Natl. Acad. Sci. USA 85:3580–3584.
    OpenUrlAbstract/FREE Full Text
  93. 93.↵
    1. Hrycyna C. A.,
    2. Ramachandra M.,
    3. Ambudkar S. V.,
    4. Hee Ko Y.,
    5. Pedersen P. L.,
    6. Pastan I.,
    7. Gottesman M. M.
    (1998) Mechanism of action of human P-glycoprotein ATPase activity; photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites. J. Biol. Chem. 273:16631–16634.
    OpenUrlAbstract/FREE Full Text
  94. 94.↵
    1. Hsieh P.-C.,
    2. Siegel S. A.,
    3. Rogers B.,
    4. Davis D.,
    5. Lewis K.
    (1998) Bacteria lacking a multidrug pump: a sensitive tool for drug discovery. Proc. Natl. Acad. Sci. USA 95:6602–6606.
    OpenUrlAbstract/FREE Full Text
  95. 95.↵
    1. Huovinen P.,
    2. Sundström L.,
    3. Swedberg G.,
    4. Sköld O.
    (1995) Trimethoprim and sulfonamide resistance. Antimicrob. Agents Chemother. 39:279–289.
    OpenUrlFREE Full Text
  96. 96.↵
    1. Hyde S. C.,
    2. Emsley P.,
    3. Hartshorn M. J.,
    4. Mimmack M. M.,
    5. Gileady U.,
    6. Pearce S. R.,
    7. Gallagher M. P.,
    8. Gill D. R.,
    9. Hubbard R. E.,
    10. Higgins C. F.
    (1990) Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature 346:362–365.
    OpenUrlCrossRefPubMedWeb of Science
  97. 97.↵
    1. Jack D. L.,
    2. Storms M. L.,
    3. Tchieu J. H.,
    4. Paulsen I. T.,
    5. Saier M. H. Jr.
    (2000) A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins. J. Bacteriol. 182:2311–2313.
    OpenUrlAbstract/FREE Full Text
  98. 98.↵
    1. Jäger W.,
    2. Kalinowski J.,
    3. Pühler A.
    (1997) A Corynebacterium glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli. J. Bacteriol. 179:2449–2451.
    OpenUrlAbstract/FREE Full Text
  99. 99.↵
    1. Jair K.-W.,
    2. Martin R. G.,
    3. Rosner J. L.,
    4. Fujita N.,
    5. Ishihama A.,
    6. Wolf R. E. Jr.
    (1995) Purification and regulatory properties of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters. J. Bacteriol. 177:7100–7104.
    OpenUrlAbstract/FREE Full Text
  100. 100.↵
    1. Jair K.-W.,
    2. Yu X.,
    3. Skarstad K.,
    4. Thöny B.,
    5. Fujita N.,
    6. Ishihama A.,
    7. Wolf R. E. Jr.
    (1996) Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. J. Bacteriol. 178:2507–2513.
    OpenUrlAbstract/FREE Full Text
  101. 101.↵
    1. Jarlier V.,
    2. Nikaido H.
    (1994) Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol. Lett. 123:11–18.
    OpenUrlCrossRefPubMedWeb of Science
  102. 102.↵
    1. Jessen-Marshall A. E.,
    2. Paul N. J.,
    3. Brooker R. J.
    (1995) The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease. J. Biol. Chem. 27:16251–16257.
  103. 103.↵
    1. Kaatz G. W.,
    2. Seo S. M.,
    3. Ruble C. A.
    (1993) Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 37:1086–1094.
    OpenUrlAbstract/FREE Full Text
  104. 104.↵
    1. Kaatz G. W.,
    2. Seo S. M.
    (1995) Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 39:2650–2655.
    OpenUrlAbstract/FREE Full Text
  105. 105.↵
    1. Kaidoh K.,
    2. Miyauchi S.,
    3. Abe A.,
    4. Tanabu S.,
    5. Nara T.,
    6. Kamo N.
    (1996) Rhodamine 123 efflux transporter in Haloferax volcanii is induced when cultured under ‘metabolic stress’ by amino acids: the efflux system resembles that in a doxorubicin-resistant mutant. Biochem. J. 314:355–359.
  106. 106.↵
    1. Kajiji S.,
    2. Talbot F.,
    3. Grizzuti K.,
    4. Van Dyke-Phillips V.,
    5. Agresti M.,
    6. Safa A. R.,
    7. Gros P.
    (1993) Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry 32:4185–4194.
    OpenUrlCrossRefPubMed
  107. 107.↵
    1. Kato S.,
    2. Nishimura J.,
    3. Yufu Y.,
    4. Ideguchi H.,
    5. Umemura T.,
    6. Nawata H.
    (1992) Modulation of expression of multidrug resistance gene (mdr-1) by adriamycin. FEBS Lett. 308:175–178.
    OpenUrlCrossRefPubMed
  108. 108.↵
    1. Kazama H.,
    2. Hamashima H.,
    3. Sasatsu M.,
    4. Arai T.
    (1998) Distribution of the antiseptic-resistance genes qacE and qacEΔ1 in Gram-negative bacteria. FEMS Microbiol. Lett. 159:173–178.
    OpenUrlPubMedWeb of Science
  109. 109.↵
    1. Kazama H.,
    2. Hamashima H.,
    3. Sasatsu M.,
    4. Arai T.
    (1998) Distribution of the antiseptic-resistance gene qacEΔ1 in Gram-positive bacteria. FEMS Microbiol. Lett. 165:295–299.
    OpenUrlPubMedWeb of Science
  110. 110.↵
    1. Klein J. R.,
    2. Henrich B.,
    3. Plapp R.
    (1991) Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. Mol. Gen. Genet. 230:230–240.
    OpenUrlCrossRefPubMed
  111. 111.↵
    1. Klyachko K. A.,
    2. Schuldiner S.,
    3. Neyfakh A. A.
    (1997) Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr. J. Bacteriol. 179:2189–2193.
    OpenUrlAbstract/FREE Full Text
  112. 112.
    1. Klyachko K. A.,
    2. Neyfakh A. A.
    (1998) Paradoxical enhancement of the activity of a bacterial multidrug transporter caused by substitutions of a conserved residue. J. Bacteriol. 180:2817–2821.
    OpenUrlAbstract/FREE Full Text
  113. 113.↵
    1. Knol J.,
    2. Veenhoff L.,
    3. Liang W.-J.,
    4. Henderson P. J. F.,
    5. Leblanc G.,
    6. Poolman B.
    (1996) Unidirectional reconstitution into detergent destabilized liposomes of the purified lactose transport systems of Streptococcus thermophilus. J. Biol. Chem. 271:15358–15366.
    OpenUrlAbstract/FREE Full Text
  114. 114.
    Reference deleted.
  115. 115.↵
    1. Köhler T.,
    2. Michéa-Hamzehpour M.,
    3. Henze U.,
    4. Gotoh N.,
    5. Kocjancic Curty L.,
    6. Pechère J.-C.
    (1997) Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 23:345–354.
    OpenUrlCrossRefPubMedWeb of Science
  116. 116.↵
    1. Köhler T.,
    2. Epp S. F.,
    3. Kocjancic Curty L.,
    4. Pechère J.-C.
    (1999) Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. J. Bacteriol. 181:6300–6305.
    OpenUrlAbstract/FREE Full Text
  117. 117.↵
    1. Kolaczkowski M.,
    2. van der Rest M.,
    3. Cybularz-Kolaczkowska A.,
    4. Soumillion J.-P.,
    5. Konings W. N.,
    6. Goffeau A.
    (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J. Biol. Chem. 271:31543–31548.
    OpenUrlAbstract/FREE Full Text
  118. 118.↵
    1. Kronakis V.,
    2. Sharff A.,
    3. Koronakis E.,
    4. Luisi B.,
    5. Hughes C.
    (2000) Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914–919.
    OpenUrlCrossRefPubMedWeb of Science
  119. 119.↵
    1. Kurland C. G.,
    2. Dong H.
    (1996) Bacterial growth inhibition by overproduction of protein. Mol. Microbiol. 21:1–4.
    OpenUrlCrossRefPubMedWeb of Science
  120. 120.↵
    1. Leelaporn A.,
    2. Paulsen I. T.,
    3. Tennent J. M.,
    4. Littlejohn T. G.,
    5. Skurray R. A.
    (1994) Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci. J. Med. Microbiol. 40:214–220.
    OpenUrlCrossRefPubMedWeb of Science
  121. 121.↵
    1. Leelaporn A.,
    2. Firth N.,
    3. Paulsen I. T.,
    4. Hettiaratchi A.,
    5. Skurray R. A.
    (1995) Multidrug resistance plasmid pSK108 from coagulase-negative staphylococci: relationships to Staphylococcus aureus qacC plasmids. Plasmid 34:62–67.
    OpenUrlCrossRefPubMedWeb of Science
  122. 122.↵
    1. Levy S.
    (1992) Active efflux mechanisms for antibiotic resistance. Antimicrob. Agents Chemother. 36:695–703.
    OpenUrlFREE Full Text
  123. 123.↵
    1. Levy S.
    (1998) The challenge of antibiotic resistance. Sci. Am. 278:32–39.
    OpenUrl
  124. 124.↵
    1. Levy S.
    (1998) Multidrug resistance—a sign of the times. N. Engl. J. Med. 338:1376–1378.
    OpenUrlCrossRefPubMedWeb of Science
  125. 125.↵
    1. Levy S.,
    2. Nelson M.
    (1998) Reversing tetracycline resistance: a renaissance for the tetracycline family of antibiotics. in Resolving the antibiotic paradox. eds Rosen B. P., Mobashery S. (Kluwer Academic/Plenum Publishers, New York, N.Y), pp 17–25.
  126. 126.↵
    1. Lewis K.
    (1994) Multidrug resistance pumps in bacteria: variations on a theme. Trends Biochem. Sci. 19:119–123.
    OpenUrlCrossRefPubMedWeb of Science
  127. 127.↵
    1. Lewis K.,
    2. Hooper D. C.,
    3. Ouellette M.
    (1997) Multidrug resistance pumps provide broad defense. ASM News 63:605–610.
    OpenUrlWeb of Science
  128. 128.↵
    1. Li X.-Z.,
    2. Livermore D. M.,
    3. Nikaido H.
    (1994) Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob. Agents Chemother. 38:1732–1741.
    OpenUrlAbstract/FREE Full Text
  129. 129.↵
    1. Li X.-Z.,
    2. Nikaido H.,
    3. Poole K.
    (1995) Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:1948–1953.
    OpenUrlAbstract/FREE Full Text
  130. 130.↵
    1. Linton K. J.,
    2. Cooper H. N.,
    3. Hunter I. S.,
    4. Leadleay P. F.
    (1994) An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin. Mol. Microbiol. 11:777–785.
    OpenUrlCrossRefPubMed
  131. 131.↵
    1. Litman T.,
    2. Zeuthen T.,
    3. Skovsgaard T.,
    4. Stein W. D.
    (1997) Competitive, non-competitive and cooperative ineractions between substrates of P-glycoprotein as measured by its ATPase activity. Biochim. Biophys. Acta 1361:169–176.
    OpenUrlCrossRefPubMed
  132. 132.↵
    1. Littlejohn T. G.,
    2. DiBerardino D.,
    3. Messerotti L. J.,
    4. Spiers S. J.,
    5. Skurray R. A.
    (1991) Structure and evolution of a family of genes encoding antiseptic and disinfectant resistance in Staphylococcus aureus. Gene 101:59–66.
    OpenUrlCrossRefPubMedWeb of Science
  133. 133.↵
    1. Littlejohn T. G.,
    2. Paulsen I. T.,
    3. Gillespie M. T.,
    4. Tennent J. A.,
    5. Midgley M.,
    6. Jones I. G.,
    7. Purewal A. S.,
    8. Skurray R. A.
    (1992) Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus. FEMS Microbiol. Lett. 95:259–266.
    OpenUrlCrossRef
  134. 134.↵
    1. Liu J.,
    2. Takiff H. E.,
    3. Nikaido H.
    (1996) Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump. J. Bacteriol. 178:3791–3795.
    OpenUrlAbstract/FREE Full Text
  135. 135.↵
    1. Lomovskaya O.,
    2. Lewis K.
    (1992) emr, an Escherichia coli locus for multidrug resistance. Proc. Natl. Acad. Sci. USA 89:8938–8942.
    OpenUrlAbstract/FREE Full Text
  136. 136.↵
    1. Lomovskaya O.,
    2. Lewis K.,
    3. Matin A.
    (1995) EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB. J. Bacteriol. 177:2328–2334.
    OpenUrlAbstract/FREE Full Text
  137. 137.↵
    1. Loo T. W.,
    2. Clarke D. M.
    (1993) Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein. J. Biol. Chem. 268:19965–19972.
    OpenUrlAbstract/FREE Full Text
  138. 138.↵
    1. Loo T. W.,
    2. Clarke D. M.
    (1994) Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J. Biol. Chem. 269:7750–7755.
    OpenUrlAbstract/FREE Full Text
  139. 139.↵
    1. Loo T. W.,
    2. Clarke D. M.
    (1994) Mutation of amino acids located in predicted transmembrane domain segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein. Biochemistry 33:14049–14057.
    OpenUrlCrossRefPubMed
  140. 140.↵
    1. Loor F.
    (1996) Cyclosporins and related fungal products in the reversal of P-glycoprotein-mediated multidrug resistance. in Multidrug resistance in cancer cells: cellular, biochemical and biological aspects. eds Gupta S., Tsuruo T. (John Wiley & Sons Ltd, Chichester, U.K), pp 385–412.
  141. 141.↵
    1. Ma D.,
    2. Cook D. N.,
    3. Alberti M.,
    4. Pon N. G.,
    5. Nikaido H.,
    6. Hearst J. E.
    (1993) Molecular cloning and characterization of acrA and acrE genes Escherichia coli. J. Bacteriol. 175:6299–6313.
    OpenUrlAbstract/FREE Full Text
  142. 142.↵
    1. Ma D.,
    2. Cook D. N.,
    3. Hearst J. E.,
    4. Nikaido H.
    (1994) Efflux pumps and drug resistance in Gram-negative bacteria. Trends Microbiol. 2:489–493.
    OpenUrlCrossRefPubMed
  143. 143.↵
    1. Ma D.,
    2. Cook D. N.,
    3. Alberti M.,
    4. Pon N. G.,
    5. Nikaido H.,
    6. Hearst J. E.
    (1995) Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol. Microbiol. 16:45–55.
    OpenUrlCrossRefPubMedWeb of Science
  144. 144.↵
    1. Ma D.,
    2. Alberti M.,
    3. Lynch C.,
    4. Nikaido H.,
    5. Hearst J. E.
    (1996) The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol. Microbiol. 19:101–112.
    OpenUrlCrossRefPubMedWeb of Science
  145. 145.↵
    1. Maiden M. C. J.,
    2. Davis E. O.,
    3. Baldwin S. A.,
    4. Moore D. C. M.,
    5. Henderson P. J. F.
    (1987) Mammalian and bacterial sugar transport proteins are homologous. Nature 325:41–643.
    OpenUrlCrossRef
  146. 146.↵
    1. Marger M.,
    2. Saier M. H. Jr.
    (1993) A major superfamily of transmembrane facilitators that can catalyze uniport, symport and antiport. Trends Biochem. Sci. 18:13–20.
    OpenUrlCrossRefPubMedWeb of Science
  147. 147.↵
    1. Margolles A.,
    2. Putman M.,
    3. van Veen H. W.,
    4. Konings W. N.
    (1999) The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids. Biochemistry 38:16298–16306.
    OpenUrlCrossRefPubMed
  148. 148.↵
    1. Markham P. N.,
    2. Ahmed M.,
    3. Neyfakh A. A.
    (1996) The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain. J. Bacteriol. 178:1473–1475.
    OpenUrlAbstract/FREE Full Text
  149. 149.↵
    1. Markham P. N.,
    2. Neyfakh A. A.
    (1996) Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:2673–2674.
    OpenUrlFREE Full Text
  150. 150.↵
    1. Markham P. N.,
    2. LoGuidice J.,
    3. Neyfakh A. A.
    (1997) Broad ligand specificity of the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. Biochem. Biophys. Res. Commun. 239:269–272.
    OpenUrlCrossRefPubMed
  151. 151.↵
    1. Markham P. N.
    (1999) Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug inhibitor reserpine. Antimicrob. Agents Chemother. 43:988–989.
    OpenUrlAbstract/FREE Full Text
  152. 152.↵
    1. Markham P. N.,
    2. Westhaus E.,
    3. Klyachko K.,
    4. Johnson M. E.,
    5. Neyfakh A. A.
    (1999) Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Antimicrob. Agents Chemother. 43:2404–2408.
    OpenUrlAbstract/FREE Full Text
  153. 153.↵
    1. Martin C.,
    2. Berridge G.,
    3. Higgins C. F.,
    4. Callaghan R.
    (1997) The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction. Br. J. Pharmacol. 122:765–771.
    OpenUrlCrossRefPubMedWeb of Science
  154. 154.↵
    1. Masaoka Y.,
    2. Ueno Y.,
    3. Morita Y.,
    4. Kuroda T.,
    5. Mizushima T.,
    6. Tsuchiya T.
    (2000) A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis. J. Bacteriol. 182:2307–2310.
    OpenUrlAbstract/FREE Full Text
  155. 155.↵
    1. Maseda H.,
    2. Yoneyama H.,
    3. Nakae T.
    (2000) Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:658–664.
    OpenUrlAbstract/FREE Full Text
  156. 156.↵
    1. McDermott P. F.,
    2. White D. G.,
    3. Podglajen I.,
    4. Aleksun M. N.,
    5. Levy S. B.
    (1998) Multidrug resistance following expression of the Escherichia coli marA gene in Mycobacterium smegmatis. J. Bacteriol. 180:2992–2998.
    OpenUrlAbstract/FREE Full Text
  157. 157.↵
    1. Medeiros A. A.,
    2. O'Brien T. F.,
    3. Rosenberg E. Y.,
    4. Nikaido H.
    (1987) Loss of OmpC porin in a strain of Salmonella typhimurium causes increased resistance to cephalosporins during therapy. J. Infect. Dis. 156:751–757.
    OpenUrlCrossRefPubMed
  158. 158.↵
    1. Mine T.,
    2. Morita Y.,
    3. Kataoka A.,
    4. Mizushima T.,
    5. Tsuchiya T.
    (1999) Expression in Escherichia coli of a new efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43:415–417.
    OpenUrlAbstract/FREE Full Text
  159. 159.↵
    1. Mitchell B. A.,
    2. Paulsen I. T.,
    3. Brown M. H.,
    4. Skurray R. A.
    (1999) Bioenergetics of the staphylococcal multidrug export protein QacA: identification of distinct binding sites for monovalent and divalent cations. J. Biol. Chem. 274:3541–3548.
    OpenUrlAbstract/FREE Full Text
  160. 160.↵
    1. Moellering R. C. Jr.
    (1998) Introduction: problems with antimicrobial resistance in Gram-positive cocci. Clin. Infect. Dis. 16:1177–1178.
    OpenUrl
  161. 161.↵
    1. Moore R. A.,
    2. DeShazer D.,
    3. Reckseidler S.,
    4. Weissman A.,
    5. Woods D. E.
    (1999) Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrob. Agents Chemother. 43:465–470.
    OpenUrlAbstract/FREE Full Text
  162. 162.↵
    1. Morimyo M.,
    2. Hongo E.,
    3. Hama-Inaba H.,
    4. Machida I.
    (1992) Cloning and characterization of the mvrC gene of Escherichia coli K-12 which confers resistance against methyl viologen toxicity. Nucleic Acids Res. 20:3159–3165.
    OpenUrlCrossRefPubMedWeb of Science
  163. 163.↵
    1. Morita Y.,
    2. Kodama K.,
    3. Shiota S.,
    4. Mine T.,
    5. Kataoka A.,
    6. Mizushima T.,
    7. Tsuchiya T.
    (1998) NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob. Agents Chemother. 42:1778–1782.
    OpenUrlAbstract/FREE Full Text
  164. 164.↵
    1. Morris D. I.,
    2. Greenberger L. M.,
    3. Bruggemann E. P.,
    4. Cardarelli C.,
    5. Gottesman M. M.,
    6. Pastan I.,
    7. Seamon K. B.
    (1994) Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin. Mol. Pharmacol. 46:329–337.
    OpenUrlAbstract
  165. 165.↵
    1. Muth T. R.,
    2. Schuldiner S.
    (2000) A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE. EMBO J. 19:232–240.
    OpenUrl
  166. 166.↵
    1. Nakamura H.
    (1965) Gene-controlled resistance to acriflavine and other basic dyes in Escherichia coli. J. Bacteriol. 90:8–14.
    OpenUrlAbstract/FREE Full Text
  167. 167.↵
    1. Nakamura H.
    (1968) Genetic determination of resistance to acriflavine, phenethyl alcohol, and sodium dodecyl sulfate in Escherichia coli. J. Bacteriol. 96:987–996.
    OpenUrlAbstract/FREE Full Text
  168. 168.
    1. Naroditskaya V.,
    2. Schlosser M. J.,
    3. Fang N. Y.,
    4. Lewis K.
    (1993) An E. coli gene emrD is involved in adaptation to low energy shock. Biochem. Biophys. Res. Commun. 196:803–809.
    OpenUrlCrossRefPubMedWeb of Science
  169. 169.↵
    1. Neu H. C.
    (1992) The crisis in antibiotic resistance. Science 257:1064–1073.
  170. 170.↵
    1. Neyfakh A. A.,
    2. Bidnenko V. E.,
    3. Chen L. B.
    (1991) Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc. Natl. Acad. Sci. USA 88:4781–4785.
    OpenUrlAbstract/FREE Full Text
  171. 171.↵
    1. Neyfakh A. A.
    (1992) The multidrug efflux transporter of Bacillus subtilis is a structural and functional homolog of the Staphylococcus NorA protein. Antimicrob. Agents Chemother. 36:484–485.
    OpenUrlAbstract/FREE Full Text
  172. 172.↵
    1. Neyfakh A. A.,
    2. Borsch C. M.,
    3. Kaatz G. W.
    (1993) Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux protein. Antimicrob. Agents Chemother. 37:128–129.
    OpenUrlAbstract/FREE Full Text
  173. 173.↵
    1. Neyfakh A. A.
    (1997) Natural functions of bacterial multidrug transporters. Trends Microbiol. 8:309–313.
  174. 174.↵
    1. Ng E. Y. W.,
    2. Trucksis M.,
    3. Hooper D. C.
    (1994) Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob. Agents Chemother. 38:1345–1355.
    OpenUrlAbstract/FREE Full Text
  175. 175.↵
    1. Nikaido H.,
    2. Vaara M.
    (1985) Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49:1–32.
    OpenUrlFREE Full Text
  176. 176.↵
    1. Nikaido H.
    (1989) Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob. Agents Chemother. 33:1831–1836.
    OpenUrlFREE Full Text
  177. 177.↵
    1. Nikaido H.
    (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382–388.
    OpenUrlAbstract/FREE Full Text
  178. 178.↵
    1. Nikaido H.
    (1996) Multidrug efflux pumps of gram-negative bacteria. J. Bacteriol. 178:5853–5859.
    OpenUrlFREE Full Text
  179. 179.↵
    1. Nilsen I. W.,
    2. Bakke I.,
    3. Vader A.,
    4. Olsvik Ø.,
    5. El-Gewely M. R.
    (1996) Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump. J. Bacteriol. 178:3188–3193.
    OpenUrlAbstract/FREE Full Text
  180. 180.↵
    1. Ocaktan A.,
    2. Yoneyama H.,
    3. Nakae T.
    (1997) Use of fluorescent probes to monitor the subunit proteins of the MexA-MexB-OprM drug extrusion machinery in Pseudomonas aeruginosa. J. Biol. Chem. 272:21964–21969.
    OpenUrlAbstract/FREE Full Text
  181. 181.↵
    1. Ohki R.,
    2. Murata M.
    (1997) bmr3, a third multidrug transporter gene of Bacillus subtilis. J. Bacteriol. 179:1423–1427.
    OpenUrlAbstract/FREE Full Text
  182. 182.↵
    1. Okusu H.,
    2. Ma D.,
    3. Nikaido H.
    (1996) AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple antibiotic resistance (Mar) mutants. J. Bacteriol. 178:306–308.
    OpenUrlAbstract/FREE Full Text
  183. 183.↵
    1. Olano C.,
    2. Rodrı́guez A. M.,
    3. Méndez C.,
    4. Salas J. A.
    (1995) A second ABC transporter is involved in oleandomycin resistance and its secretion by Streptomyces antibioticus. Mol. Microbiol. 16:333–343.
    OpenUrlCrossRefPubMed
  184. 184.↵
    1. Oshita Y.,
    2. Hiramatsu K.,
    3. Yokota T.
    (1990) A point mutation in the norA gene is responsible for quinolone resistance in Staphylococcus aureus. Biochem. Biophys. Res. Commun. 172:1028–1034.
    OpenUrlCrossRefPubMed
  185. 185.↵
    1. Pan W.,
    2. Spratt B. G.
    (1994) Regulation of the permeability of the gonococcal cell envelope by the mtr system. Mol. Microbiol. 11:769–775.
    OpenUrlCrossRefPubMedWeb of Science
  186. 186.↵
    1. Pascaud C.,
    2. Garrigos M.,
    3. Orlowski S.
    (1998) Multidrug resistance transporter P-glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents. Biochem. J. 333:351–358.
  187. 187.↵
    1. Paulsen I. T.,
    2. Littlejohn T. G.,
    3. Rådström P.,
    4. Sundström L.,
    5. Sköld O.,
    6. Swedberg G.,
    7. Skurray R. A.
    (1993) The 3′ conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob. Agents Chemother. 37:761–768.
    OpenUrlAbstract/FREE Full Text
  188. 188.↵
    1. Paulsen I. T.,
    2. Skurray R. A.
    (1993) Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes—an analysis. Gene 124:1–11.
    OpenUrlCrossRefPubMedWeb of Science
  189. 189.↵
    1. Paulsen I. T.,
    2. Brown M. H.,
    3. Dunstan S. J.,
    4. Skurray R. A.
    (1995) Molecular characterization of the staphylococcal multidrug resistance export protein QacC. J. Bacteriol. 177:2827–2833.
    OpenUrlAbstract/FREE Full Text
  190. 190.↵
    1. Paulsen I. T.,
    2. Brown M. H.,
    3. Skurray R. A.
    (1996) Proton-dependent multidrug efflux systems. Microbiol. Rev. 60:575–608.
    OpenUrlAbstract/FREE Full Text
  191. 191.↵
    1. Paulsen I. T.,
    2. Skurray R. A.,
    3. Tam R.,
    4. Saier M. H. Jr.,
    5. Turner R. J.,
    6. Weiner J. H.,
    7. Goldberg E. B.,
    8. Grinius L. L.
    (1996) The SMR family: A novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs. Mol. Microbiol. 19:1167–1175.
    OpenUrlCrossRefPubMedWeb of Science
  192. 192.↵
    1. Paulsen I. T.,
    2. Brown M. H.,
    3. Littlejohn T. G.,
    4. Mitchell B. A.,
    5. Skurray R. A.
    (1996c) Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity. Proc. Natl. Acad. Sci. USA 93:3630–3635.
    OpenUrlAbstract/FREE Full Text
  193. 193.↵
    1. Paulsen I. T.,
    2. Brown M. H.,
    3. Skurray R. A.
    (1998) Characterization of the earliest known Staphylococcus aureus plasmid encoding a multidrug efflux system. J. Bacteriol. 180:3477–3479.
    OpenUrlAbstract/FREE Full Text
  194. 194.↵
    1. Paulsen I. T.,
    2. Sliwinski M. K.,
    3. Saier M. H. Jr.
    (1998) Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities. J. Mol. Biol. 277:573–592.
    OpenUrlCrossRefPubMedWeb of Science
  195. 195.↵
    1. Pawagi A. B.,
    2. Wang J.,
    3. Silverman M.,
    4. Reithmeier R. A. F.,
    5. Deber C. M.
    (1994) Transmembrane aromatic amino acid distribution in P-glycoprotein: a functional role in broad substrate specificity. J. Mol. Biol. 235:554–564.
    OpenUrlCrossRefPubMedWeb of Science
  196. 196.↵
    1. Pereira E.,
    2. Borrel M. N.,
    3. Fiallo M.,
    4. Garnier-Suillerot A.
    (1994) Noncompetitive inhibition of P-glycoprotein-associated efflux of THP-adriamycin by verapamil in living K562 leukemia cells. Biochim. Biophys. Acta 1225:209–216.
    OpenUrlPubMed
  197. 197.↵
    1. Podlesek Z.,
    2. Comino A.,
    3. Herzog-Velikonja B.,
    4. Žgur-Bertok D.,
    5. Komel R.,
    6. Grabnar M.
    (1995) Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance. Mol. Microbiol. 16:969–976.
    OpenUrlCrossRefPubMedWeb of Science
  198. 198.↵
    1. Poole K.,
    2. Heinrichs D. E.,
    3. Neshat S.
    (1993) Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine. Mol. Microbiol. 10:529–544.
    OpenUrlCrossRefPubMedWeb of Science
  199. 199.↵
    1. Poole K.,
    2. Krebes K.,
    3. McNally C.,
    4. Neshat S.
    (1993) Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J. Bacteriol. 175:7363–7372.
    OpenUrlAbstract/FREE Full Text
  200. 200.↵
    1. Poole K.,
    2. Gotoh N.,
    3. Tsujimoto H.,
    4. Zhao Q.,
    5. Wada A.,
    6. Yamasaki T.,
    7. Neshat S.,
    8. Yamagishi J.-I.,
    9. Nishino T.
    (1996) Overexpression of the mexC-mexD-oprJ efflux operon in nfx-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol. Microbiol. 21:713–724.
    OpenUrlCrossRefPubMedWeb of Science
  201. 201.↵
    1. Poole K.,
    2. Tetro K.,
    3. Zhao Q.,
    4. Neshat S.,
    5. Heinrichs D. E.,
    6. Bianco N.
    (1996) Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrob. Agents Chemother. 40:2021–2028.
    OpenUrlAbstract/FREE Full Text
  202. 202.↵
    1. Purewal A. S.
    (1991) Nucleotide sequence of the ethidium efflux gene from Escherichia coli. FEMS Microbiol. Lett. 82:229–232.
    OpenUrlCrossRef
  203. 203.↵
    1. Putman M.,
    2. van Veen H. W.,
    3. Poolman B.,
    4. Konings W. N.
    (1999) Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP. Biochemistry 38:1002–1008.
    OpenUrlCrossRefPubMed
  204. 204.↵
    1. Putman M.,
    2. Koole L. A.,
    3. van Veen H. W.,
    4. Konings W. N.
    (1999) The secondary multidrug transporter LmrP contains multiple drug interaction sites. Biochemistry 38:13900–13905.
    OpenUrlCrossRefPubMed
  205. 205.↵
    1. Putman M.,
    2. van Veen H. W.,
    3. Degener J. E.,
    4. Konings W. N.
    (2000) Antibiotic resistance: era of the multidrug pump. Mol. Microbiol. 36:772–774.
    OpenUrlCrossRefPubMed
  206. 206.↵
    1. Rasmussen B. A.,
    2. Bush K.
    (1997) Carbapenem-hydrolyzing β-lactamases. Antimicrob. Agents Chemother. 41:223–232.
    OpenUrlFREE Full Text
  207. 207.↵
    1. Raviv Y.,
    2. Pollard H. B.,
    3. Bruggemann E. P.,
    4. Pastan I.,
    5. Gottesman M. M.
    (1990) Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. J. Biol. Chem. 265:3975–3980.
    OpenUrlAbstract/FREE Full Text
  208. 208.↵
    1. Reeve E. C.,
    2. Suttie D. R.
    (1968) Chromosomal location of a mutation causing chloramphenicol resistance in Escherichia coli K 12. Genet. Res. 11:97–104.
    OpenUrlPubMed
  209. 209.↵
    1. Roberts M. C.
    (1996) Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Rev. 19:1–24.
    OpenUrlCrossRefPubMed
  210. 210.↵
    1. Rodrı́guez A. M.,
    2. Olano C.,
    3. Vilches C.,
    4. Méndez C.,
    5. Salas J. A.
    (1993) Streptomyces antibioticus contains at least three oleandomycin-resistance determinants, one of which shows similarity with proteins of the ABC-transporter superfamily. Mol. Microbiol. 8:571–582.
    OpenUrlCrossRefPubMed
  211. 211.↵
    1. Rosenberg M. F.,
    2. Callaghan R.,
    3. Ford R. C.,
    4. Higgins C. F.
    (1997) Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J. Biol. Chem. 272:10685–10694.
    OpenUrlAbstract/FREE Full Text
  212. 212.↵
    1. Ross J. I.,
    2. Eady E. A.,
    3. Cove J. H.,
    4. Cunliffe W. J.,
    5. Baumberg S.,
    6. Wootton J. C.
    (1990) Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Mol. Microbiol. 4:1207–1214.
    OpenUrlCrossRefPubMedWeb of Science
  213. 213.↵
    1. Rouch D. A.,
    2. Cram D. S.,
    3. DiBerardino D.,
    4. Littlejohn T. G.,
    5. Skurray R. A.
    (1990) Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol. Microbiol. 4:2051–2062.
    OpenUrlCrossRefPubMedWeb of Science
  214. 214.↵
    1. Rouquette C.,
    2. Harmon J. B.,
    3. Shafer W. F.
    (1999) Induction of the mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an AraC-like protein. Mol. Microbiol. 33:651–658.
    OpenUrlCrossRefPubMedWeb of Science
  215. 215.↵
    1. Rubin R. A.,
    2. Levy S. B.,
    3. Heinrikson R. L.,
    4. Kétzy F. J.
    (1990) Gene duplication in the evolution of the two complementing domains of Gram-negative bacterial tetracyclin efflux proteins. Gene 87:7–13.
    OpenUrlCrossRefPubMed
  216. 216.↵
    1. Saier M. H. Jr.,
    2. Tam R.,
    3. Reizer A.,
    4. Reizer J.
    (1994) Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol. Microbiol. 11:841–847.
    OpenUrlCrossRefPubMedWeb of Science
  217. 217.↵
    1. Saier M. H. Jr.,
    2. Paulsen I. T.,
    3. Sliwinski M. K.,
    4. Pao S. S.,
    5. Skurray R. A.,
    6. Nikaido H.
    (1998) Evolutionary origins of multidrug and drug-specific efflux pumps in bacteria. FASEB J. 12:265–274.
    OpenUrlPubMedWeb of Science
  218. 218.↵
    1. Sami M.,
    2. Yamahita H.,
    3. Hirono T.,
    4. Kadokura H.,
    5. Kitamoto K.,
    6. Yoda K.,
    7. Yamasaki M.
    (1997) Hop-resistant Lactobacillus brevis contains a novel plasmid harboring a multidrug resistance-like gene. J. Ferment. Bioeng. 84:1–6.
  219. 219.↵
    1. Sami M.,
    2. Suzuki K.,
    3. Sakamoto K.,
    4. Kadokura H.,
    5. Kitamoto K.,
    6. Yoda K.
    (1998) A plasmid pRH45 of Lactobacillus brevis confers hop resistance. J. Gen. Appl. Microbiol. 44:361–363.
  220. 220.↵
    1. Sánchez L.,
    2. Pan W.,
    3. Viñas M.,
    4. Nikaido H.
    (1997) The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump. J. Bacteriol. 179:6855–6857.
    OpenUrlAbstract/FREE Full Text
  221. 221.↵
    1. Sasatsu M.,
    2. Shima K.,
    3. Shibata Y.,
    4. Kono M.
    (1989) Nucleotide sequence of a gene that encodes resistance to ethidium bromide from a transferable plasmid in Staphylococcus aureus. Nucleic Acids Res. 17:10103.
    OpenUrlCrossRefPubMedWeb of Science
  222. 222.↵
    1. Schinkel A. H.,
    2. Smit J. J. M.,
    3. van Trellingen O.,
    4. Beijnen J. H.,
    5. Wagenaar E.,
    6. van Deemter L.,
    7. Mol C. A. A. M.,
    8. van der Valk M. A.,
    9. Robanus-Maandag E. C.,
    10. te Riele H. P. J.,
    11. Berns A. J. M.,
    12. Borst P.
    (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77:491–502.
    OpenUrlCrossRefPubMedWeb of Science
  223. 223.↵
    1. Senior A. E.,
    2. Al-Shawi M.,
    3. Urbatsch I. L.
    (1995) The catalytic cycle of P-glycoprotein. FEBS Lett. 377:285–289.
    OpenUrlCrossRefPubMedWeb of Science
  224. 224.↵
    1. Senior A. E.,
    2. Bhagat S.
    (1998) P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites. Biochemistry 37:831–836.
    OpenUrlCrossRefPubMed
  225. 225.↵
    1. Seoane A. S.,
    2. Levy S. B.
    (1995) Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli. J. Bacteriol. 177:3414–3419.
    OpenUrlAbstract/FREE Full Text
  226. 226.↵
    1. Seoane A. S.,
    2. Levy S. B.
    (1995) Identification of new genes regulated by the marRAB operon in Escherichia coli. J. Bacteriol. 177:530–535.
    OpenUrlAbstract/FREE Full Text
  227. 227.↵
    1. Shapiro A. B.,
    2. Ling V.
    (1995) Reconstitution of drug transport by purified P-glycoprotein. J. Biol. Chem. 270:16167–16175.
    OpenUrlAbstract/FREE Full Text
  228. 228.↵
    1. Shapiro A. B.,
    2. Corder A. B.,
    3. Ling V.
    (1997) P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer. Eur. J. Biochem. 250:115–121.
    OpenUrlPubMedWeb of Science
  229. 229.↵
    1. Shapiro A. B.,
    2. Ling V.
    (1997) Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein. Eur. J. Biochem. 250:122–129.
    OpenUrlCrossRefPubMedWeb of Science
  230. 230.↵
    1. Shapiro A. B.,
    2. Ling V.
    (1997) Positively cooperative sites for drug transport by P-glycoprotein with distinct drug specificities. Eur. J. Biochem. 250:130–137.
    OpenUrlCrossRefPubMedWeb of Science
  231. 231.↵
    1. Shapiro A. B.,
    2. Ling V.
    (1998) Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein. Eur. J. Biochem. 254:181–188.
    OpenUrlPubMed
  232. 232.
    1. Shapiro A. B.,
    2. Fox K.,
    3. Lam P.,
    4. Ling V.
    (1999) Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone: evidence for a third drug-binding site. Eur. J. Biochem. 259:841–850.
    OpenUrlPubMedWeb of Science
  233. 233.↵
    1. Sharom F. J.,
    2. DiDiodato G.,
    3. Yu X.,
    4. Ashbourne K. J. D.
    (1995) Interaction of the P-glycoprotein multidrug transporter with peptides and ionophores. J. Biol. Chem. 270:10334–10341.
    OpenUrlAbstract/FREE Full Text
  234. 234.↵
    1. Shaw K. J.,
    2. Rather P. N.,
    3. Hare R. S.,
    4. Miller G. H.
    (1993) Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138–163.
    OpenUrlAbstract/FREE Full Text
  235. 235.↵
    1. Skarstad K.,
    2. Thöny B.,
    3. Hwang D.,
    4. Kornberg A.
    (1993) A novel binding protein of the origin of the Escherichia coli chromosome. J. Biol. Chem. 268:5365–5370.
    OpenUrlAbstract/FREE Full Text
  236. 236.↵
    1. Someya Y.,
    2. Moriyama Y.,
    3. Futai M.,
    4. Sawai T.,
    5. Yamaguchi A.
    (1995) Reconstitution of the metal-tetracyclin/H+ antiporter of Escherichia coli in proteoliposomes including F0F1-ATPase. FEBS Lett. 374:72–76.
    OpenUrlCrossRefPubMedWeb of Science
  237. 237.↵
    1. Speer B. S.,
    2. Shoemaker N. B.,
    3. Salyers A. A.
    (1992) Bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. Clin. Microbiol. Rev. 5:387–399.
    OpenUrlAbstract/FREE Full Text
  238. 238.↵
    1. Spoelstra E. C.,
    2. Westerhoff H. V.,
    3. Pinedo H. M.,
    4. Dekker H.,
    5. Lankelma J.
    (1994) The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunomycin as a non-competitive substrate. Eur. J. Biochem. 221:363–373.
    OpenUrlPubMed
  239. 239.↵
    1. Spratt B. G.
    (1994) Resistance to antibiotics mediated by target alterations. Science 264:388–393.
    OpenUrlAbstract/FREE Full Text
  240. 240.↵
    1. Srikumar R.,
    2. Li X.-Z.,
    3. Poole K.
    (1997) Inner membrane efflux components are responsible for β-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa. J. Bacteriol. 179:7875–7881.
    OpenUrlAbstract/FREE Full Text
  241. 241.↵
    1. Srikumar R.,
    2. Kon T.,
    3. Gotoh N.,
    4. Poole K.
    (1998) Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain. Antimicrob. Agents Chemother. 42:65–71.
    OpenUrlAbstract/FREE Full Text
  242. 242.↵
    1. Steiner Mordoch S.,
    2. Granot D.,
    3. Lebendiker M.,
    4. Schuldiner S.
    (1999) Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes. J. Biol. Chem. 274:19480–19486.
    OpenUrlAbstract/FREE Full Text
  243. 243.↵
    1. Sun L.,
    2. Sreedharan S.,
    3. Plummer K.,
    4. Fisher L. M.
    (1996) NorA plasmid resistance to fluoroquinolones: role of copy number and norA frameshift mutations. Antimicrob. Agents Chemother. 40:1665–1669.
    OpenUrlAbstract/FREE Full Text
  244. 244.↵
    1. Takiff H. E.,
    2. Cimino M.,
    3. Musso M. C.,
    4. Weisbrod T.,
    5. Martinez R.,
    6. Delgado M. B.,
    7. Salazar L.,
    8. Bloom B. R.,
    9. Jacobs W. R. Jr.
    (1996) Efflux pump of the proton antiporter family confers low level fluoroquinolone resistance in Mycobacterium smegmatis. Proc. Natl. Acad. Sci. USA 93:362–366.
    OpenUrlAbstract/FREE Full Text
  245. 245.↵
    1. Tamai I.,
    2. Safa A. R.
    (1991) Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug-resistant cells. J. Biol. Chem. 266:16796–16800.
    OpenUrlAbstract/FREE Full Text
  246. 246.↵
    1. Tanabe H.,
    2. Yamasaki K.,
    3. Furue M.,
    4. Yamamoto K.,
    5. Katoh A.,
    6. Yamamoto M.,
    7. Yoshioka S.,
    8. Tagami H.,
    9. Aiba H.,
    10. Utsumi R.
    (1997) Growth phase-dependent transcription of emrKY, a homolog of multidrug efflux emrAB genes of Escherichia coli, is induced by tetracycline. J. Gen. Appl. Microbiol. 43:257–263.
  247. 247.↵
    1. Taylor D. E.,
    2. Hou Y.,
    3. Turner R. J.,
    4. Weiner J. H.
    (1994) Location of a potassium tellurite resistance operon (tehA tehB) within the terminus of Escherichia coli K-12. J. Bacteriol. 166:2740–2742.
    OpenUrl
  248. 248.↵
    1. Tennent J. M.,
    2. Lyon B. R.,
    3. Midgley M.,
    4. Jones G.,
    5. Purewal A. S.,
    6. Skurray R. A.
    (1989) Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus. J. Gen. Microbiol. 135:1–10.
    OpenUrlCrossRefPubMedWeb of Science
  249. 249.↵
    1. Thanassi D. G.,
    2. Cheng L. W.,
    3. Nikaido H.
    (1997) Active efflux of bile salts by Escherichia coli. J. Bacteriol. 179:2512–2518.
    OpenUrlAbstract/FREE Full Text
  250. 250.↵
    1. Turner R. J.,
    2. Taylor D. E.,
    3. Weiner J. H.
    (1997) Expression of Escherichia coli TehA gives resistance to antiseptics and disinfectants similar to that conferred by multidrug efflux pumps. Antimicrob. Agents Chemother. 41:440–444.
    OpenUrlAbstract/FREE Full Text
  251. 251.↵
    1. Ubukata K.,
    2. Itoh N.-Y.,
    3. Konno M.
    (1989) Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 33:1535–1539.
    OpenUrlAbstract/FREE Full Text
  252. 252.↵
    1. Ueda K.,
    2. Taguchi Y.,
    3. Morishima M.
    (1997) How does P-glycoprotein recognize its substrates? Semin. Cancer Biol. 8:151–159.
    OpenUrlCrossRefPubMedWeb of Science
  253. 253.↵
    1. Urbatsch I. L.,
    2. Sankaran B.,
    3. Weber J.,
    4. Senior A. E.
    (1995) P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270:19383–19390.
    OpenUrlAbstract/FREE Full Text
  254. 254.↵
    1. Urbatsch I. L.,
    2. Sankaran B.,
    3. Bhagat S.,
    4. Senior A. E.
    (1995) Both P-glycoprotein nucleotide-binding sites are catalytically active. J. Biol. Chem. 270:26956–26961.
    OpenUrlAbstract/FREE Full Text
  255. 254a.↵
    1. van Helvoort A.,
    2. Smith A. J.,
    3. Sprong H.,
    4. Fritzsche I.,
    5. Schinkel A. H.,
    6. H. A.,
    7. Borst P.,
    8. Vermeer G.
    (1996) MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 87:507–517.
    OpenUrlCrossRefPubMedWeb of Science
  256. 255.
    1. van Veen H. W.,
    2. Konings W. N.
    (1997) Multidrug transporters from bacteria to man: similarities in structure and function. Semin. Cancer Biol. 8:183–191.
    OpenUrlCrossRefPubMedWeb of Science
  257. 256.↵
    1. van Veen H. W.,
    2. Venema K.,
    3. Bolhuis H.,
    4. Oussenko I.,
    5. Kok J.,
    6. Poolman B.,
    7. Driessen A. J. M.,
    8. Konings W. N.
    (1996) Multidrug resistance mediated by a bacterial homolog of the human drug transporter MDR1. Proc. Natl. Acad. Sci. USA 93:10668–10672.
    OpenUrlAbstract/FREE Full Text
  258. 257.↵
    1. van Veen H. W.,
    2. Callaghan R.,
    3. Soceneantu L.,
    4. Sardini A.,
    5. Konings W. N.,
    6. Higgins C. F.
    (1998) A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene. Nature 391:291–295.
    OpenUrlCrossRefPubMedWeb of Science
  259. 258.↵
    1. van Veen H. W.,
    2. Margolles A.,
    3. Müller M.,
    4. Higgins C. F.,
    5. Konings W. N.
    (2000) The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism. EMBO J. 19:2503–2514.
    OpenUrlAbstract
  260. 259.↵
    1. van Veen H. W.,
    2. Konings W. N.
    (1998) The ABC family of multidrug transporters in microorganisms. Biochim. Biophys. Acta 1365:31–36.
    OpenUrlPubMedWeb of Science
  261. 260.
    1. Varela M. F.,
    2. Sansom C. E.,
    3. Griffith J. K.
    (1995) Mutational analysis and molecular modelling of an amino acid sequence motif conserved in antiporters but not symporters in a transporter superfamily. Mol. Membr. Biol. 12:313151–159.
    OpenUrl
  262. 261.↵
    1. Vedantam G.,
    2. Guay G. G.,
    3. Austria N. A.,
    4. Doktor S. Z.,
    5. Nichols B. P.
    (1998) Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli. Antimicrob. Agents Chemother. 42:88–93.
    OpenUrlAbstract/FREE Full Text
  263. 262.↵
    1. Vigano C.,
    2. Margolles A.,
    3. van Veen H. W.,
    4. Konings W. N.,
    5. Ruysschaert J.-M.
    (2000) Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis; a Fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis. J. Biol. Chem. 275:10962–10967.
    OpenUrlAbstract/FREE Full Text
  264. 263.↵
    1. Walker J. E.,
    2. Saraste M.,
    3. Runswick M. J.,
    4. Gay N. J.
    (1982) Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP requiring enzymes and a common nucleotide binding fold. EMBO J. 1:945–951.
    OpenUrlPubMedWeb of Science
  265. 264.↵
    1. Wang G.,
    2. Pincheira R.,
    3. Zhang J.-T.
    (1998) Dissection of drug-binding-induced conformational changes in P-glycoprotein. Eur. J. Biochem. 255:383–390.
    OpenUrlCrossRefPubMed
  266. 265.↵
    1. Weisblum B.
    (1995) Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother. 39:577–585.
    OpenUrlFREE Full Text
  267. 266.↵
    1. White D. G.,
    2. Goldman J. D.,
    3. Demple B.,
    4. Levy S. B.
    (1997) Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J. Bacteriol. 179:6122–6126.
    OpenUrlAbstract/FREE Full Text
  268. 267.↵
    1. Williams R. J.,
    2. Heymann D. L.
    (1998) Containment of antibiotic resistance. Science 279:1153–1154.
    OpenUrlAbstract/FREE Full Text
  269. 268.↵
    1. Woodcock D. M.,
    2. Linsenmeyer M. E.,
    3. Chojnowski G.,
    4. Kriegler A. B.,
    5. Nink V.,
    6. Webster L. K.,
    7. Sawyer W. H.
    (1992) Reversal of multidrug resistance by surfactants. Br. J. Cancer 66:62–68.
    OpenUrlCrossRefPubMedWeb of Science
  270. 269.↵
    1. Woolridge D. P.,
    2. Vasquez-Laslop N.,
    3. Markham P. N.,
    4. Chevalier