Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains and plasmids used in this study are listed in Table I.5,1519 LuriaBertani (LB) broth [1% (w/v) Difco tryptone, 0.5% (w/v) Difco yeast extract and 0.5% (w/v) sodium chloride] was used throughout the study and bacteria were cultivated at 37°C. Introduction of plasmids pVLT31 and pXZL34 from E. coli DH5
to the OprM-deficient P. aeruginosa strains was performed by conjugation (i.e. triparental mating) as described previously17 and the P. aeruginosa conjugants were obtained on LB agar containing tetracycline (10 mg/L) and chloramphenicol (25 mg/L), or tetracycline (10 mg/L) and imipenem (0.5 mg/L). Both chloramphenicol and imipenem were used to counterselect E. coli cells. Successful conjugation was confirmed by preparation of plasmids from the tetracycline-resistant P. aeruginosa strains.
|
The mexAB-oprM deletion mutants were constructed using gene replacement vector pRSP21 (for PAO4098- and K1034-derived strains) or pELCT04 (for PAO4098E-derived strains) as described previously17,19 with some modifications. Briefly, pRSP21 or pELCT04 was mobilized from E. coli S17-1 into P. aeruginosa strains by conjugation (i.e. biparental mating).17 pRSP21- or pELCT04-containing P. aeruginosa were selected on LB agar supplemented with kanamycin (1500 mg/L) or mercuric chloride (15 mg/L), respectively, and tetracycline (10 mg/L, to counterselect E. coli S17-1). Kanamycin- or mercuric chloride-resistant transconjugants were recovered and re-streaked on LB agar containing 10% (w/v) sucrose. Sucrose-resistant colonies were screened for loss of kanamycin and/or mercuric chloride resistance, and those carrying a mexAB-oprM deletion were identified by their drug susceptibility phenotype and loss of MexA, MexB and OprM as assayed by Western immunoblotting (see below) with antibodies specific for MexA (X.-Z. Li and K. Poole, unpublished data), MexB20 and OprM.15
SDSpolyacrylamide gel electrophoresis and Western immunoblotting of membrane proteins
Cell envelopes of P. aeruginosa were prepared from the cells grown to the exponential phase by sonic disruption followed by differential centrifugation.21 Outer membranes were prepared by extraction of cell envelopes with 1.5% (w/v) sodium N-lauroyl sarkosinate (sarkosyl; Sigma, Oakville, Ontario, Canada) as described previously.21 Protein contents of cell envelopes and outer membranes were determined by the method of Lowry et al. using bovine serum albumin as the standard.22 The membrane proteins were subsequently analysed using slab sodium dodecyl sulphatepolyacrylamide (11% w/v) gel electrophoresis (SDSPAGE).23 Each sample containing 50 µg of proteins (cell envelopes) or 30 µg of proteins (outer membranes) was heated at 100°C for 5 min before being subjected to SDSPAGE. Following electrophoresis, proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bradford, MA, USA) at 20 mA for 16 h at 4°C. Membranes were processed as described previously.15 Antibodies (anti-OprM,15 anti-OprJ6 and anti-OprN7) and a horseradish peroxidase-coupled donkey anti-rabbit or anti-mouse immunoglobulin G were used as primary and secondary antibodies, respectively. Blots were developed with the enhanced chemiluminescence system (Amersham, Pharmacia Biotech, Bai d'Urfé, Québec, Canada) according to the manufacturer's instructions.
Antibiotic susceptibility assays
Drug susceptibility testing was carried out in LB broth using the two-fold serial broth dilution method with an inoculum of 5 x 105 cells/mL. Data were reported as MICs, which reflected the lowest concentration of antibiotic inhibiting visible growth after overnight incubation at 37°C. Antibiotics were obtained from the following sources: carbenicillin, cefoperazone, ciprofloxacin, tetracycline, chloramphenicol and novobiocin from SigmaAldrich Canada Ltd (Oakville, Ontario, Canada); cefpirome from Roussel UCLAF (Paris, France); and imipenem from Merck Sharp Dohme Canada (Montreal, Canada).
RTPCR
Total bacterial RNA was isolated from late-log-phase cultures (12 mL) of P. aeruginosa strains using the Qiagen RNeasy Mini Kit (Qiagen Inc., Mississauga, Ontario, Canada), treated with RNase-free DNase (Promega, Madison, WI, USA) (1 U of enzyme/µg RNA for 60 min at 37°C) and re-purified using the same kit. A 0.2 µg sample of DNase-treated RNA was used as template for reverse transcriptionpolymerase chain reaction (RTPCR) with the Qiagen OneStep RTPCR kit (Qiagen Inc.) according to a protocol supplied by the manufacturer. Primer pairs specific for and internal to mexA [jt-18, 5'-ACCTACGAGGCCGACTACCAGA-3' (forward); jt-12, 5'-GTTGGTCACCAGGGCGCCTTC-3' (reverse)], mexC [mexc1xz, 5'-AGCCAGCAGGACTTCGATACC-3' (forward); mexc2xz, 5'-ACGTCG-GCGAACTGCAAC-3' (reverse)] and mexE [mexe1xz, 5'-GTCATCGAACAACCGC-TG-3' (forward); mexe2xz, 5'-GTCGAAGTAGGCGTAGACC-3' (reverse)] were used to amplify and quantitate the corresponding mRNA, as a measure of mexAB-oprM, mexCD-oprJ and mexEF-oprN expression. The mRNA of the constitutively expressed rspL gene was amplified and quantitated by RTPCR using primers rspl1xz [5'-GCAACTATCAACCAGGCTG-3' (forward)] and rspl2xz [5'-GCTGTGCTCTTGCAGGTTGTG-3' (reverse)].10 Thirty picomoles of each primer was used per reaction (final volume of 50 µL), which involved a 30 min incubation at 50°C, followed by 15 min at 95°C, and 40 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, before finishing with 10 min at 72°C. A 15 µL sample of each reaction product was analysed by agarose (1.4% w/v) gel electrophoresis for the expected RTPCR products (rpsL, 220 bp; mexA, 252 bp; mexC, 314 bp; mexE, 516 bp).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains reportedly wild type with respect to the MDR efflux systems of P. aeruginosa failed to express detectable levels of MexCD-OprJ in Western immunoblots (Figure 1b, lanes 1, 5 and 9, and data not shown). Consistent with this, deletion of this operon failed to alter the intrinsic drug susceptibility of wild-type strains.6,17 To assess whether MexCD-OprJ might respond to the loss of the MexAB-OprM efflux system, expression of MexCD-OprJ was examined in mexAB-oprM or oprM deletion derivatives of PAO1 strain K767 (K1119), PAO6609 (K1032), PAO4098 (K1232) and ML5087 (K1110 and K1121). In all instances, loss of MexAB-OprM or OprM (confirmed by immunoblotting with MexA-, MexB- and/or OprM-specific antisera; Figure 1a
, lanes 2, 6 and 10, and data not shown) correlated with an increase in MexCD-OprJ, as assessed using an OprJ-specific antiserum (Figure 1b
, lanes 2 and 6, and Figure 2b
, lanes 2 and 8). These deletion strains were hypersusceptible to multiple antibiotics, including those known to be substrates for MexCD-OprJ (Table II
). As expected, hyperexpression of MexAB-OprM in nalB strains K1034, PAO4098E and K1112 (Figure 1a
, lanes 3 and 7, and Figure 2a
, lane 3) correlated with a lack of MexCD-OprJ expression (Figure 1b
, lanes 3 and 7, and Figure 2b
, lane 3), while mexAB-oprM (or oprM) deletion derivatives of these nalB strains (i.e. K1230, K1234 and K1113) (Figure 1a
, lanes 4 and 8, Figure 2a
, lane 4) did express MexCD-OprJ (Figure 1b
, lanes 4 and 8, and Figure 2b
, lane 4). Again, these deletion derivatives were drug hypersusceptible (Table II
).
|
|
|
Influence of the MexAB-OprM status of P. aeruginosa on MexEF-OprN expression
Western immunoblotting revealed that different strains apparently wild-type with regard to the MDR pumps expressed different levels of OprN (and, thus, probably MexEF-OprN). P. aeruginosa PAO1 strain K767 (data not shown) and PAO4098 (Figure 1c, lane 5) produced undetectable levels of OprN, and strains PAO6609 and ML5087 (Figure 1c
, lanes 1 and 9) produced modest levels of this protein. OprM levels were generally consistent in the aforementioned strains (Figure 1a
and data not shown). Elimination of MexAB-OprM or OprM in those strains producing modest levels of MexEF-OprN (PAO6609 and ML5087) yielded strains K1032, K1110 and K1121, respectively, which exhibited increased expression of MexEF-OprN (Figure 1c
, lanes 2 and 10, and Figure 2c
, lane 8). MexEF-OprN remained undetectable in those mexAB-oprM deletion strains (K1119, data not shown; K1232, Figure 1c
, lane 6) which were derived from strains originally producing undetectable OprN (K767 and PAO4098).
In all instances, where OprN was detectable in the original wild-type strains (PAO6609 and ML5087), nalB derivatives of these exhibited undetectable levels of OprN (Figure 1c, lanes 3 and 11). Again, elimination of mexAB-oprM or oprM in the above nalB strains (efflux system is absent but the nalB mutation remains) restored MexEF-OprN expression in K1230, a mexAB-oprM deletion of the nalB strain K1034 (Figure 1c
, lane 4), and in K1113, the oprM deletion of nalB strain K1112 (Figure 1c
, lane 12). It indicates, therefore, that in these strains there is an inverse correlation between MexAB-OprM and MexEF-OprN, much like that seen for MexAB-OprM and MexCD-OprJ. In spite of this, the OprN level of K1113 was less than that of K1110, suggesting the nalB mutation itself seems to be impacting on MexEF-OprN, independent of any effect on MexAB-OprM expression. The mutation responsible for the nalB phenotype in K1112 (parent of K1113) occurs within the mexR gene encoding a repressor of mexAB-oprM expression.24 Still, introduction of a plasmid-borne wild-type mexR gene (on plasmid pRSP5524) into K1113 failed to restore OprN (i.e. MexEF-OprN) production (data not shown), suggesting that additional mutations are also contributing the nalB phenotype in this strain. In any case, the reduced amount of MexEF-OprN in K1113 was consistent with antibiotic resistance data showing this strain to be markedly more susceptible to ciprofloxacin, chloramphenicol and novobiocin than K1110 (Table II
). Both strains are ML5087 derivatives lacking OprM, although only K1110 hyperexpresses MexEF-OprN (Figure 1c
, lane 10) and this efflux system is known to accommodate both ciprofloxacin and chloramphenicol.7,13
As for MexCD-OprJ (above), it was important to establish that changes in MexEF-OprN (measured as OprN changes) were a response to loss of a functional MexAB-OprM MDR efflux system. Again, introduction of the cloned wild-type oprM gene (pXZL34) into K1110 (ML5087 oprM), completely reversed the increase in OprN (Figure 2c
, lanes 5). In contrast, the oprM plasmid had no effect on OprN levels in the MexAB-OprMdeficient strain K1121, which already produced substantial amounts of OprN (Figure 2c
, lanes 9 and 10).
Efflux status of the multidrug hypersusceptible P. aeruginosa mutant Z61
The drug hypersusceptibility (attributed to increased outer membrane permeability)18,25 and efflux-deficiency of P. aeruginosa Z61 are now well known,12 although the nature of the defect in this strain has yet to be elucidated. Western immunoblotting with an anti-OprM and anti-MexB antisera revealed that, in contrast to its parent strain (Figure 1a, lane 13), this mutant lacks OprM (Figure 1a
, lane 14) but still produces MexB (data not shown). Thus, Z61 was similar to the OprM-deficient strain K1110 in terms of the MexAB-OprM expression. Intriguingly, like K1110, this strain exhibited elevated production of both OprJ (Figure 1b
, lane 10) and OprN (Figure 1c
, lane 14) compared with the parent strain K799 (Figure 1b
, lane 9, and Figure 1c
, lane 13), indicating an enhanced production of the MexCD-OprJ and MexEF-OprN efflux systems in response to loss of OprM (i.e. loss of a functional MexAB-OprM pump) in this strain.
Influence of MexAB-OprM status on mexCD-oprJ and mexEF-oprN expression
To assess if the presence or absence of MexAB-OprM was influencing expression of the mexCD-oprJ and mexEF-oprN genes, and not impacting solely on production of the OprJ and OprN outer membrane proteins, RTPCR was employed using internal primers specific for the first gene of each of these operons. As seen in Figure 3b, RTPCR was an accurate measure of mexA (as a measure of mexAB-oprM) expression, with a mexA-specific RTPCR product absent only in strains lacking the mexAB-oprM genes (Figure 3b
, lanes 3 and 7). Using mexC-specific primers, it was clear that strains wild-type with respect to efflux expressed barely detectable levels of mexC (as a measure of mexCD-oprJ) mRNA (Figure 3c
, lanes 2, 6 and 8) while the nfxB strain K1111, as expected, hyperexpressed this gene (Figure 3c
, lane 5). In contrast to the wild-type strains, substantial expression of mexC was evident in their
mexAB-oprM (Figure 3b
, lanes 3 and 7) and
oprM (Figure 3b
, lane 4) derivatives. Similarly, the Z61 strain, shown above to lack OprM and thus, a functional MexAB-OprM efflux system, showed increased expression of mexC relative to its OprM+ parent strain (Figure 3b
, lane 9, cf. lane 8). Similar results were observed with the mexE-specific RTPCR, with weak mexE (as a measure of mexEF-oprN) expression evident for wild-type strains (Figure 3d
, lanes 2, 6 and 8) and substantially increased expression seen in their MexAB-OprM-/OprM-deficient derivatives (Figure 3b
, lanes 35, 7 and 9). RTPCR using primers for the constitutively expressed rpsL gene served as a control and confirmed that the differences cited above were not due to variability in RNA recovery from the strains being examined (Figure 3a
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Similarly, decreases in MexAB-OprM were paralleled by a compensatory increase in MexEF-OprN (in K1032, K1110 and K1121, where the parent strains produced modest levels of MexEF-OprN), while hyperexpression of MexAB-OprM in strains K1034 and K1112 correlated with a seemingly compensatory decrease in MexEF-OprN. MexEF-OprN remained undetectable in those mexAB-oprM deletion strains (K1119 and K1232) that were derived from strains originally producing undetectable OprN (K767 and PAO4098). In the case of K1119, this may relate to the presence, within the mexT gene of its parent K767, of two mutations (K. Poole, unpublished data) that probably abrogate the function of this mexEF-oprN activator gene.26 Possibly, any increase in MexEF-OprN seen upon deletion of mexAB-oprM requires MexT activation of mexEF-oprN expression. That restoration of MexAB-OprM in the MexAB+-OprM strain (K1110) by adding back OprM (pXZL34) reversed the increase in OprN while lack of a functional MexAB-OprM pump in K1121/ pXZL34 (MexAB-OprM+) did not reverse the increase in OprN indicates that MexEF-OprN was responding solely to the presence or absence of a functional MexAB-OprM efflux system. Moreover, the observed increase in mexE expression in strains lacking MexAB-OprM indicates that this OprN increase is a result of enhanced mexEF-oprN gene expression and, thus, reflective of an increase in MexEF-OprN pump production. The higher expression of MexEF-OprN in K1110 (ML5087 oprM) than that in K1113 (ML5087 nalB
oprM) indeed correlates with the increased resistance to ciprofloxacin, chloramphenicol and novobiocin (four- to 16-fold in MICs; Table II
), indicating a clinical significance for the differential MDR pump expression.
The examination of the well-studied hypersusceptible P. aeruginosa Z61 and its parent strain demonstrated that Z61 lacks a functional MexAB-OprM system since it lacks OprM expression. This result confirms our previous conclusion that the mutant Z61 is deficient in antibiotic efflux12 and, thus, hypersusceptible to multiple antibiotics.12,18,25 Interestingly, the elevated production of both OprJ and OprN in the mutant Z61 is in agreement with the aforementioned compensatory changes seen in MexCD-OprJ and MexEF-OprN expression in response to the loss of a functional MexAB-OprM system. Again, too, this appears to occur at the level of gene expression.
The influence of the MexAB-OprM status on the expression of MexCD-OprJ and MexEF-OprN suggests that the cell can assess the status of its MDR efflux systems and provide for compensatory changes in the levels of one system in response to increases or decreases in another, perhaps to maintain a basal (if not optimal) level of efflux gene expression. Consistent with this, it was noted previously that nfxB strains hyperexpressing MexCD-OprJ produced decreased levels of MexAB-OprM.27 In spite of this, a mexCD-oprJ
mexAB-oprM double mutant of ML5087 was no more susceptible to antibiotics than was a
mexAB-oprM strain of ML5087 (data not shown), indicating that the increased expression of MexCD-OprJ in the absence of MexAB-OprM is insufficient to provide meaningful resistance to antibiotics. It is possible, however, that the elevated expression of MexCD-OprJ and MexEF-OprN complements, to some extent, the lack of MexAB-OprM with respect to its role in the export of some cell-associated compound(s). Given the very similar antibiotic substrate profiles of MexAB-OprM and MexCD-OprJ, it is likely that the latter could effectively replace the former with respect to export of whatever cell-associated compound(s) are the probable natural substrates for these MDR efflux systems.
The compensatory changes in MexCD-OprJ and MexEF-OprN upon the loss of functional MexAB-OprM may serve to maintain net expression of this family of efflux systems at some possibly pre-determined overall level. It is unclear whether this reflects some form of global regulation of MDR efflux transporters in P. aeruginosa. Alternatively, the loss of MexAB-OprM could promote increased accumulation of certain compounds within the cell and that these could then trigger expression of the other MDR systems via their own regulatory circuits. Given the broad and often overlapping substrate specificity of the MDR efflux systems of this organism, it would not be surprising to find these pumps to be somewhat interchangeable within the cell. In any case, it is obvious that the cell possesses mechanisms by which it maintains a certain level of expression of these efflux pumps, suggesting that these pumps play an important role in the cell, one that is probably independent of any contributions to antibiotic resistance.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Nikaido, H. (1996). Multidrug efflux pumps of gram-negative bacteria. Journal of Bacteriology 178, 58539.
3
.
Li, X.-Z., Zhang, L. & Poole, K. (2000). Interplay between the MexA-MexB-OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 45, 4336.
4 . Poole, K., Krebes, K., McNally, C. & Neshat, S. (1993). Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. Journal of Bacteriology 175, 736372.[Abstract]
5 . Li, X.-Z., Nikaido, H. & Poole, K. (1995). Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 194853.[Abstract]
6 . Poole, K., Gotoh, N., Tsujimoto, H., Zhao, Q., Wada, A., Yamasaki, T. et al. (1996). Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Molecular Microbiology 21, 71324.[ISI][Medline]
7 . Köhler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L. K. & Pechere, J. C. (1997). Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Molecular Microbiology 23, 34554.[ISI][Medline]
8
.
Mine, T, Morita, Y., Kataoka, A., Mizushima, T. & Tsuchiya, T. (1999). Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 4157.
9
.
Aires, J. R., Köhler, T., Nikaido, H. & Plésiat, P. (1999). Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrobial Agents and Chemotherapy 43, 26248.
10
.
Westbrock-Wadman, S., Sherman, D. R., Hickey, M. J., Coulter, S. N., Zhu, Y. Q., Warrener, P. et al. (1999). Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrobial Agents and Chemotherapy 43, 297583.
11 . Saier, M. H., Tam, R., Reizer, A. & Reizer, J. (1994). Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Molecular Microbiology 11, 8417.[ISI][Medline]
12 . Li, X.-Z., Livermore, D. M. & Nikaido, H. (1994). Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrobial Agents and Chemotherapy 38, 173241.[Abstract]
13 . Masuda, N., Sakagawa, E. & Ohya, S. (1995). Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 6459.[Abstract]
14 . Masuda, N., Gotoh, N., Ohya, S. & Nishino, T. (1996). Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 40, 90913.[Abstract]
15
.
Zhao, Q., Li, X.-Z., Srikumar, R. & Poole, K. (1998). Contribution of outer membrane efflux protein OprM to antibiotic resistance in Pseudomonas aeruginosa independent of MexAB. Antimicrobial Agents and Chemotherapy 42, 16828.
16
.
Li, X.-Z., Zhang, L., Srikumar, R. & Poole, K. (1998). Betalactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 399403.
17 . Srikumar, R., Li, X.-Z. & Poole, K. (1997). Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa. Journal of Bacteriology 179, 787581.[Abstract]
18 . Zimmermann, W. (1980). Penetration of ß-lactam antibiotics into their target enzymes in Pseudomonas aeruginosa: comparison of a highly sensitive mutant with its parent strain. Antimicrobial Agents and Chemotherapy 18, 94100.[ISI][Medline]
19
.
Evans, K, Passador, L., Srikumar, R., Tsang, E., Nezezon, J. & Poole, K. (1998). Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa. Journal of Bacteriology 180, 54437.
20
.
Srikumar, R., Kon, T., Gotoh, N. & Poole, K. (1998). Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain. Antimicrobial Agents and Chemotherapy 42, 6571.
21
.
Zhang, L., Li, X.-Z. & Poole, K. (2000). Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system. Antimicrobial Agents and Chemotherapy 44, 28793.
22
.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 26575.
23 . Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P. & van Alphen, L. (1975). Electrophoretic resolution of the major outer membrane protein of Escherichia coli K-12 into four bands. FEBS Letters 58, 2548.[ISI][Medline]
24
.
Srikumar, R., Paul, C. J. & Poole, K. (2000). Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa. Journal of Bacteriology 182, 14104.
25 . Angus, B. L., Carey, A. M., Caron, D. A., Kropinski, A. M. B. & Hancock, R. E. (1982). Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrobial Agents and Chemotherapy 21, 299309.[ISI][Medline]
26
.
Köhler, T., Epp, S. F., Curty, L. K. & Pechere, J. C. (1999). Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. Journal of Bacteriology 181, 63005.
27
.
Gotoh, N., Tsujimoto, H., Tsuda, M., Okamoto, K., Nomura, A., Wada, T. et al. (1998). Characterization of the MexC-MexD-OprJ multidrug efflux system in mexA-mexB-oprM mutants of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 193843.
Received 2 August 2000; accepted 4 September 2000