JAC Advance Access originally published online on May 10, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):31-36; doi:10.1093/jac/dkl172
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Multidrug resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in Escherichia coli
Departamento de Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional de Tucumán) and Instituto de Química Biológica ‘Dr Bernabé Bloj’ Chacabuco 461, 4000 San Miguel de Tucumán, Tucumán, Argentina
*Correspondence address. Departamento de Bioquímica de la Nutrición, INSIBIO, Chacabuco 461, 4000 San Miguel de Tucumán, Argentina. Tel/Fax: +54-381-4248921; E-mail: salomon{at}fbqf.unt.edu.ar
Received 23 November 2005; returned 19 January 2006; revised 23 February 2006; accepted 7 April 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Objectives: Starting from the observation that Escherichia coli tolC mutations severely reduced the high-level resistance to tetracycline afforded by Tn10- and plasmid-encoded Tet(A) pumps, we studied the mechanism of this susceptibility.
Methods: The MIC of tetracycline for MC4100 tolC::Tn10 and several tolC mutants carrying the Tn10 in other sites on the chromosome (thr::Tn10) was determined. The effect of a tolC mutation on the level of expression of Tn10 tet(A) was examined by using a tet(A)::lacZ gene fusion. Influence of tolC mutations on tetracycline efflux and accumulation was quantified by spectrofluorometric assays. The contribution of the AcrAB multidrug efflux system to high-level tetracycline resistance was measured in a Tn10-carrying acrAB null mutant strain.
Results: Tn10- and plasmid-encoded Tet(A) conferred 5- to 6-fold lower levels of tetracycline resistance in tolC mutants, as compared with control strain tolC+. Spectrofluorometric analyses showed that this resulted from a decrease in drug efflux in tolC mutants. Chlortetracycline resistance was also compromised by loss of TolC. Mutational loss of the AcrAB multidrug efflux transporter had the same effect as tolC mutations on tetracycline resistance. This indicated that tolC mutations act through inactivation of the AcrAB system.
Conclusions: Our results are compatible with the hypothesis that the AcrAB pump is an important component in the development of high levels of resistance to tetracycline in E. coli, perhaps by working in combination with Tet(A).
Keywords: tolC mutation , Tet(A)-AcrAB interplay , tetracycline susceptibility , E. coli
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Bacterial resistance to many classes of antibiotics is provided mainly by membrane transporter proteins called drug efflux pumps.1 These pumps may occur as either single-component or multi-component systems. In Gram-negative bacteria, single-component efflux pumps expel their substrates into the periplasmic space. An example of such single-component efflux pumps is the transposon-encoded, tetracycline-specific Tet(A).2 Multi-component efflux pumps (which are found exclusively in Gram-negative bacteria) traverse both inner and outer membranes. The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC.3–5 This complex consists of the inner-membrane component AcrB, belonging to the resistance-nodulation-division (RND) family of proteins,6 the outer membrane channel TolC and a periplasmic linker protein, AcrA, which is a member of the membrane fusion protein (MFP) superfamily.7 The latter was thought to bring into contact the membrane-associated efflux components. However, Tamura et al.8 have recently proposed that AcrB and TolC first directly dock with each other and then the complex is stabilized by AcrA. This structural organization allows extrusion of substrates from the cell into the external medium, bypassing the periplasm and the outer membrane.9
By using tolC mutants of strains carrying the transposon Tn10 we demonstrate in this work that the outer membrane protein TolC is required for high-level resistance to tetracycline (>40 mg/L) afforded by the Tn10- and plasmid-encoded Tet(A) pumps. Our results support the conclusion that tolC mutations act through the inactivation of the AcrAB efflux system in determining increased susceptibility of Tn10-carrying E. coli strains to tetracycline. We propose that high-level resistance conferred by Tet(A) results from its cooperation with AcrAB, which would capture tetracycline from the periplasm, where it has been accumulated by the action of Tet(A), and then would extrude the drug directly into the external medium.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
Strains, plasmids and media
Strains and plasmids used in this work are listed in Table 1. Growth medium was Luria– Bertani (LB) broth. Antibiotics were used at the following concentrations: kanamycin, 30 mg/L; ampicillin, 50 mg/L; and tetracycline, as indicated. The tolC::Tn10, tolC::Tn5 and acrAB derivatives of MC4100 were constructed by phage P1 transduction,10 using strains CAG12184, SC44 and AG100A as donors, respectively. The MC4100 TolC– and AcrAB– transductants had the expected deoxycholate-susceptible phenotype. Batch cultures were grown at 37°C with aeration by shaking.
|
Chemicals
Tetracycline was obtained from ICN Biomedicals, Inc. (Irvine, CA, USA) and chlortetracycline (7-chlorotetracycline) was purchased from Sigma Chemical Co. (St Louis, MO, USA).
Determination of the MIC of tetracycline
MICs of tetracycline were determined by a colony forming assay. LB plates were prepared with increasing concentrations of tetracycline (ranging from 10 to 200 mg/L, with 10 mg/L increments). They were inoculated (0.1 mL) with a 10–4 dilution of stationary-phase cultures grown in LB supplemented with 10 mg/L tetracycline, such that about 105 bacteria were spread per plate, and incubated at 37°C for 14–18 h. The MIC was the lowest concentration of tetracycline required to completely inhibit colony formation.
Tetracycline efflux and accumulation assays
Tetracycline release and accumulation were measured using spectrofluorometry, as described previously.11 For the efflux assay, bacteria (about 108 cells/mL) grown in LB were centrifuged and resuspended in 2 mL aliquots of Mg2+ buffer (50% methanol, 10 mM Tris–HCl, pH 8, 0.1 mM MgCl2, 0.2% glucose). At time zero, tetracycline (100 mg/L) was added and the fluorescence (excitation at 400 nm and emission at 520 nm) recorded for at least 10 min with a spectrofluorometer (Gilson). Tetracycline accumulation was determined by using bacterial suspensions in Mg2+ buffer, prepared as described above. Tetracycline was added at 100 mg/L, and after 15 min of incubation bacterial suspensions were centrifuged, the pellets were resuspended in 2 mL of Mg2+ buffer and the released fluorescence (excitation at 400 nm and emission at 520 nm) was immediately recorded with a spectrofluorometer (Gilson). In other experiments, the absolute amount of tetracycline accumulated was determined as indicated by Ball et al.12 LB overnight cultures of the strains to be tested were appropriately diluted with LB so as to get an optical density at 600 nm of 0.8. Samples (1 mL) were centrifuged, washed with 100 mM Tris/HCl buffer, pH 8, and resuspended in 1 mL of 10 mM Tris/HCl buffer, pH 8. Tetracycline was added at 100 mg/L, and the mixture was incubated for 15 min. Bacteria were harvested and the pellet was disrupted with 5 M HCl (1 mL), which, after boiling for 10 min, quantitatively converts tetracycline into anhydrotetracycline.13 Cooled samples were centrifuged to remove cell debris. The absorbance at 440 nm of the anhydrotetracycline contained in the supernatants was measured. The amount of anhydrotetracycline contained in these samples was determined with a standard curve (0–100 mg tetracycline/L). The experiment was repeated six times, and the results were expressed in terms of µg of tetracycline/mg of cell protein. To correct for the amount of external tetracycline trapped within the bacterial pellet after centrifugation, one of the assays was performed at 0–4°C.14 Low temperature inhibits diffusion15 and active transport through lipid bilayers, thus preventing uptake of tetracycline through the inner membrane. Under these conditions, the amount of drug trapped in the pellet was 1.5 µg/mg of protein. This value was used for correction.
Analytical methods
Bacterial cell protein was estimated by the Lowry method.16 Prior to assay, cells were heated at 90°C in 1 M NaOH for 10 min to obtain complete solubilization. ß-Galactosidase activity was determined and expressed according to Miller.10
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
High-level tetracycline resistance afforded by Tn10-encoded Tet(A) is affected by tolC mutations
This study arose from the observation that when tolC::Tn10 strains are selected with tetracycline at a concentration conventionally used for this antibiotic (10 mg/L) they form colonies smaller than those growing in the absence of tetracycline. We thought it likely that even in the presence of Tn10 the strains were being partially inhibited by the antibiotic. We determined the MIC of tetracycline for MC4100 tolC::Tn10 and several tolC mutants carrying the Tn10 in other sites on the chromosome (thr::Tn10). It can be seen in Table 2 that while the chromosomal copy of Tn10 provides the expected high-level tetracycline resistance in the control strain MC4100 thr::Tn10 (200 mg/L), it confers 5-fold lower levels (40 mg/L) of tetracycline resistance in the tolC derivatives. The experiments with the tolC thr::Tn10 strains seemed to discard a polar effect of the tolC::Tn10 insertion on a downstream gene. To confirm the role of the TolC protein itself in tetracycline resistance, plasmid pAX629, which carries a 1.9 kb chromosomal DNA fragment encoding the TolC protein only,17 was introduced into the MC4100 tolC::Tn10 strain. The transformants fully recovered the high tetracycline resistance levels (Table 2). Taken together, these results led us to conclude that tolC mutations somehow compromise Tet(A)-mediated resistance.
|
High-level resistance of constitutive tet(A) genes encoded by plasmids is also affected by tolC mutations
As described above, in TolC– cells expressing the single-component Tet(A) efflux pump of Tn10, high-level resistance to tetracycline is severely compromised. We wished to know whether this also occurred with another class of Tet(A) protein, that expressed constitutively from plasmid pBR322. MC4100 and MC4100 tolC::Tn5/10 cells were transformed with this plasmid and their resistance to tetracycline was measured. As shown in Table 2, in a tolC background the highest level of tetracycline resistance conferred by the plasmid was 30 mg/L, as compared with 180 mg/L for the control. Thus, the efficiency of the pBR322-encoded Tet(A) pump was also reduced by the tolC mutation. Similar results were obtained with plasmid pACYC184, which carries the same tetracycline resistance gene as pBR322 (results not shown).
tolC::Tn10 mutants are also hypersusceptible to chlortetracycline
During the course of our study we attempted to generate a tolC deletion mutant from strain MC4100 tolC::Tn10. To achieve this we used the technique developed by Bochner et al.,18 which is based on the finding that fusaric acid kills tetracycline-resistant cells in which the tetracycline resistance gene of the transposon Tn10 has been induced by autoclaved chlortetracycline (50 mg/L). Note that when chlortetracycline is autoclaved in broth it is denatured so as to lose its toxicity towards tetracycline-susceptible cells while retaining its inducing ability for tetracycline-resistant cells.18 Despite repeated attempts, we did not succeed in obtaining tetracycline-susceptible clones. We reasoned that the failure could be due to either fusaric acid or autoclaved chlortetracycline being toxic to the tetracycline-susceptible tolC deletion mutants we were seeking (the cells that had lost the transposon would probably still be tolC, since this leaves at high frequency a genetic lesion at the site where the Tn10 was inserted). We therefore tested well-characterized tolC mutants in which the gene has been inactivated by Tn5 insertion (MC4100 tolC::Tn5), deletion (PB3) or a point mutation (A586), and all of them proved susceptible to heated chlortetracycline at 50 mg/L. We concluded that the heated chlortetracycline in the selective medium was responsible for the toxicity to the tolC mutants that otherwise would be generated. This came as a surprise, since heated chlortetracycline has been reported to be non-toxic to E. coli cells. In fact, the parent MC4100 was unaffected by the heat-detoxified antibiotic at 50 mg/L. Although TolC– strains were susceptible to heated chlortetracycline at 50 mg/L, they grew well at a concentration of 10 mg/L of the autoclaved antibiotic. A possible explanation is that autoclaving does not abolish chlortetracycline activity, and that the antibiotic accumulates within tolC cells until it reaches a toxic level. MC4100 tolC::Tn10 did not grow in the presence of 10 mg/L of native chlortetracycline. In this regard, Traub and Beck19 found that the level of resistance to chlortetracycline of an E. coli K-12 strain carrying Tn10 was 42 mg/L (uninduced cells) or 65 mg/L (preinduced). Thus, chlortetracycline resistance is also compromised by loss of tolC.
tet(A) gene expression is not altered in tolC mutants
Mutations in tolC elevate the transcription of micF antisense RNA, resulting in the concomitant reduction of OmpF.20 Likewise, the decreased resistance to tetracycline in tolC mutants could be the result of an effect on the level of expression of Tn10 tet(A). Therefore, the influence of a tolC mutation on the expression of a tet(A)::lacZ gene fusion was examined. For these experiments, the medium-copy-number plasmid pRKH40,21 harbouring a tet(A)::lacZ fusion whose transcription is under the control of the repressor TetR, was transformed into strains MC4100 and MC4100 tolC::Tn5. Expression of the fusion in the TolC– strain (1618 and 1629 Miller units in log and stationary phase, respectively) was similar to that in the TolC+ parent strain (1283 and 1134 Miller units). This suggests that the effect of tolC mutations on tetracycline resistance was not due to a down-regulation of the tet(A) gene.
Tetracycline efflux and accumulation assays
Given the involvement of TolC with known efflux systems, the increased tetracycline and chlortetracycline susceptibilities probably result from a decrease in drug efflux. To test this possibility we examined the kinetics of tetracycline uptake and release by using a method which relies on the fact that internalized tetracycline becomes fluorescent, and once released from the cells it can be quantified with a spectrofluorometer.11,22 As shown in Figure 1, there was a slower efflux from the tolC::Tn10 mutant than in the MC4100 thr::Tn10 strain. Values for the tolC strain approached those for the parental, tetracycline-susceptible strain MC4100.
|
Since an impairment of tetracycline efflux would lead to more drug being retained within cells, we next investigated tetracycline accumulation levels in the same strains used for the efflux experiments. To this end, cells were preloaded with tetracycline (100 mg/L) during a 15 min incubation, pelleted and resuspended in tetracycline-free buffer. Under these conditions, intracellular fluorescent tetracycline is immediately released into the buffer and could be detected by a spectrofluorometer, providing a quantitative estimate of the amount of tetracycline accumulated during the 15 min loading phase. As can be seen in Figure 2(a), fully tetracycline-resistant MC4100 thr::Tn10 cells accumulate the smallest amount of tetracycline, whereas the tolC::Tn10 mutation resulted in significantly higher intracellular tetracycline levels, which were comparable to that observed in the susceptible MC4100 strain. When the tolC::Tn10 strain was transformed with pAX629, which complements the tolC mutation, the accumulated antibiotic became closer to that of the thr::Tn10 strain. In other experiments, the absolute amount of tetracycline accumulated was measured by its conversion into anhydrotetracycline, as described in the Materials and methods section. As shown in Figure 2(b), there is a precise correlation between the results obtained with this method and those of the fluorescence assay. In particular, the tolC mutant accumulated a higher level of tetracycline than did strain MC4100 thr::Tn10. Taken together, the above results are consistent with a defect in drug efflux in the tolC::Tn10 mutant.
|
Contribution of the AcrAB multidrug efflux system to high-level tetracycline resistance
That the operation of TolC involves drug efflux strongly supported the involvement of additional efflux components, since outer membrane proteins are unable to function alone in energy-dependent transport processes. We considered the possibility that TolC could be associated with the Tet protein, and probably with a linker protein connecting them. However, Thanassi et al.23 found convincing evidence showing that the Tet protein pumps out tetracycline into the periplasm and not directly into the medium.
Mutations in tolC would lead to a non-functional AcrAB-TolC system. However, the AcrAB efflux mechanism confers a low level of resistance as compared with Tet(A). For example, the tetracycline MIC for a wild-type E. coli K-12 strain, such as W3110, is typically around 1.25 mg/L.24 Thus, we thought it unlikely that the large decrease in MIC of tetracycline, from 200 to 40 mg/L, seen in tolC mutants carrying a Tn10 could be accounted for by the loss of the AcrAB efflux activity caused by the mutation. We decided to assess the contribution of the AcrAB pump to high-level tetracycline resistance. The 
acrAB::kan mutation from strain AG100A25 was transduced into MC4100 thr::Tn10 with phage P1vir, selecting for kanamycin resistance. Interestingly, the tetracycline MIC for the resultant transductant, defective in the AcrAB pump, dropped from 200 mg/L in the parent strain to 40 mg/L, the same value observed for the MC4100 thr::Tn10 tolC mutant. The converse experiment was also performed. The thr::Tn10 mutation was transferred to strains AG100 and AG100A, and the tetracycline resistance of the transductants was tested. As shown in Table 2, a similar reduction in tetracycline resistance was observed. That the acrAB and tolC mutations, in otherwise isogenic strains, reduced tetracycline resistance to the same extent indicates that the mutations act through a mutual mechanism. By searching in the literature, we noticed a paper by Lee et al.26 which could provide a possible explanation to our results. These authors showed that when a single-component efflux pump and a multi-component efflux pump with shared substrates are co-expressed in the same cell, the observed antibiotic resistance is much higher than that conferred by each of the pumps expressed singly. Moreover, recent functional27–29 and structural30–32 data favour the view that for multi-component efflux pumps, exemplified by the E. coli AcrAB system, the substrate capture may occur in the periplasm. Altogether, our results suggested that the single-component Tet(A) transports tetracycline from the cytosol to the periplasmic space and the AcrAB multi-component pump promotes efflux from this space to the external medium, resulting in a multiplicative enhancement of the level of drug resistance. At tetracycline concentrations of 40 mg/L or lower, in the absence of a functional AcrAB-TolC pump, the action of Tet(A) and the rapid exit of tetracycline extruded into the periplasm through the OmpF porin would be sufficient to balance the re-entry of tetracycline back into the cytoplasm. However, at higher external tetracycline concentrations this mechanism could be overrided and Tet(A) should have to work in combination with AcrAB to ensure high levels of resistance.
Lee et al.26 have observed a multiplicative level of tetracycline resistance in Pseudomonas aeruginosa by combining Tet(A) and the multidrug efflux system MexAB–OprM (MexAB is an AcrAB homologue). In the present work, starting by a chance observation that a strain with a Tn10 insertional inactivation of tolC gave smaller colonies on tetracycline plates, we experimentally validated for the first time that a similar mechanism may be operational in E. coli.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank Drs Mary Berlin (at the E. coli Genetic Stock Center), Hiroshi Nikaido, Cécile Wandersman, Carol Gross, Laura McMurry and Stuart Levy for generosity in providing bacterial strains and plasmids. This work was supported by a grant from Agencia Nacional de Promoción Científica y Tecnológica (PICT 01-17819). R. E. de C. was the recipient of a CONICET fellowship. R. A. S. and P. A. V. are career investigators of CONICET.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionTransparency declarationsReferences |
|---|
1 Nikaido H. (1998) Multiple antibiotic resistance and efflux. Curr Opin Microbiol 1:516–23.[CrossRef][ISI][Medline]
2
Chopra I and Roberts M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65:232–60.
3 Ma D, Cook DN, Alberti M, et al. (1995) Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol 16:45–55.[CrossRef][ISI][Medline]
4
Nikaido H. (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382–8.
5 Nikaido H and Zgurskaya HI. (2001) AcrAB and related multidrug efflux pumps of Escherichia coli. J Mol Microbiol Biotechnol 3:215–18.[ISI][Medline]
6 Saier MH Jr, Tam R, Reizer A, et al. (1994) Two novel families of bacterial membrane proteins concerned with nodulation, cell division, and transport. Mol Microbiol 11:841–7.[CrossRef][ISI][Medline]
7
Dinh T, Paulsen IT, Saier MH Jr. (1994) A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J Bacteriol 176:3825–31.
8 Tamura N, Murakami S, Oyama Y, et al. (2005) Direct interaction of multidrug efflux transporter AcrAB and outer membrane channel TolC detected via site-directed disulfide cross-linking. Biochemistry 44:11115–21.[CrossRef][Medline]
9
Nikaido H. (1996) Multidrug efflux pumps of gram-negative bacteria. J Bacteriol 178:5853–9.
10 Miller JH. (1992) A Short Course in Bacterial Genetics (Cold Spring Harbor Laboratory, New York).
11
Roccaro AS, Blanco AR, Giuliano F., et al. (2004) Epigallocatechin-gallate enhances the activity of tetracycline in staphylococci by inhibiting its efflux from bacterial cells. Antimicrob Agents Chemother 48:1968–73.
12 Ball PR, Shales SW, Chopra I. (1980) Plasmid-mediated tetracycline resistance in Escherichia coli involves increased efflux of the antibiotic. Biochem Biophys Res Commun 93:74–81.[CrossRef][ISI][Medline]
13 Mitscher LA. (1978) The Chemistry of the Tetracycline Antibiotics (Marcel Dekker Inc., New York).
14
Smith MCM and Chopra I. (1983) Limitations of a fluorescence assay for studies on tetracycline transport into Escherichia coli. Antimicrob Agents Chemother 23:175–8.
15 Nikaido H. (1976) Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some hydrophobic substances. Biochim Biophys Acta 433:118–32.[Medline]
16
Lowry OH, Rosebrough NJ, Farr AL, et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–75.
17
Hiraga S, Niki H, Ogura T, et al. (1989) Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells. J Bacteriol 171:1496–505.
18
Bochner BR, Huang H-C, Schieven GL, et al. (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–33.
19
Traub B and Beck CF. (1985) Resistance to various tetracyclines mediated by transposon Tn10 in Escherichia coli K-12. Antimicrob Agents Chemother 27:879–81.
20
Misra R and Reeves P. (1987) Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12. J Bacteriol 169:4722–30.
21 Hickman RK, McMurry LM, Levy SB. (1990) Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to ß-galactosidase. Mol Microbiol 4:1241–51.[CrossRef][ISI][Medline]
22 Samra Z, Krausz-Steinmetz J, Sompolinsky D. (1978) Transport of tetracyclines through the bacterial cell membrane assayed by fluorescence: a study with susceptible and resistant strains of Staphylococcus aureus and Escherichia coli. Microbios 21:7–21.[ISI][Medline]
23
Thanassi DG, Suh GSB, Nikaido H. (1995) Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli. J Bacteriol 177:998–1007.
24
Sulavik MC, Houseweart C, Cramer C, et al. (2001) Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother 45:1126–36.
25
Okusu H, Ma D, 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 178:306–8.
26
Lee A, Mao W, Warren MS, et al. (2000) Interplay between efflux pumps may provide either additive or multiplicative effects on drug resistance. J Bacteriol 182:3142–50.
27 Elkins CA and Nikaido H. (2003) 3D structure of AcrB: the archetypal multidrug efflux transporter of Escherichia coli likely captures substrates from periplasm. Drug Resist Updat 6:9–13.[CrossRef][ISI][Medline]
28 Mao W, Warren MS, Black DS, et al. (2002) On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol 46:889–901.[CrossRef][ISI][Medline]
29
Tikhonova EB, Wang Q, Zgurskaya HI. (2002) Chimeric analysis of the multicomponent multidrug efflux transporters from Gram-negative bacteria. J Bacteriol 184:6499–507.
30 Lomovskaya O, Zgurskaya HI, Nikaido H. (2002) It takes three to tango. Nat Biotechnol 20:1210–12.[CrossRef][ISI][Medline]
31 Murakami S, Nakashima R, Yamashita E, et al. (2002) Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419:587–93.[CrossRef][Medline]
32
Yu EW, McDermott G, Zgurskaya HI, et al. (2003) Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump. Science 300:976–80.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. E. de Cristobal, P. A. Vincent, and R. A. Salomon A Combination of sbmA and tolC Mutations in Escherichia coli K-12 Tn10-Carrying Strains Results in Hypersusceptibility to Tetracycline J. Bacteriol., February 15, 2008; 190(4): 1491 - 1494. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||