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, 55 (2), 508-14

mexEF-oprN Multidrug Efflux Operon of Pseudomonas Aeruginosa: Regulation by the MexT Activator in Response to Nitrosative Stress and Chloramphenicol

Affiliations

mexEF-oprN Multidrug Efflux Operon of Pseudomonas Aeruginosa: Regulation by the MexT Activator in Response to Nitrosative Stress and Chloramphenicol

Hossam Fetar et al. Antimicrob Agents Chemother.

Abstract

A null mutation in the mexS gene of Pseudomonas aeruginosa yielded an increased level of expression of a 3-gene operon containing a gene, xenB, whose product is highly homologous to a xenobiotic reductase in Pseudomonas fluorescens shown previously to remove nitro groups from trinitrotoluene and nitroglycerin (D. S. Blehert, B. G. Fox, and G. H. Chambliss, J. Bacteriol. 181:6254, 1999). This expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, dependent on the MexT LysR family positive regulator previously implicated in mexEF-oprN expression. As nitration is a well-known result of nitrosative stress, a role for xenB (and the coregulated mexEF-oprN) in a nitrosative stress response was hypothesized and tested. Using s-nitrosoglutathione (GSNO) as a source of nitrosative stress, the expression of xenB and mexEF-oprN was shown to be GSNO inducible, although in the case of xenB, this was seen only for a mutant lacking MexEF-OprN. In both instances, this GSNO-inducible expression was dependent upon MexT. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN, was shown to induce mexEF-oprN but not xenB, again dependent upon the MexT regulator, possibly because it resembles a nitrosated nitrosative stress product accommodated by MexEF-OprN.

Figures

FIG. 1.
FIG. 1.
Influence of mexS and mexT mutations on mexEF-oprN and xenB expression. Expression levels of mexEF-oprN, xenB, and/or rpsL in P. aeruginosa strains K2153 (wild type) (lane 1), K2376 (MexS) (lane 2), K2492 (MexS MexT) (lane 3), K2492 carrying plasmid pRK415 (MexS MexT) (lane 4), and K2492 carrying mexT plasmid pHF001 (MexS MexT+) (lane 5) (A to C) as well as strains K2153 (lane 1), K2376 (lane 2), and K2946 (MexS MexF) (lane 3) (D and E) were assessed by using RT-PCR. The rpsL reaction served as an internal control that ensured that equal amounts of RNA were employed in all of the RT-PCRs shown. The PCR portion of the reactions was carried out for 31 cycles (top) or 34 cycles (bottom) in all instances except in C, where 21 cycles (top) and 23 cycles (bottom) were used, and E, where 24 cycles (top) and 26 cycles (bottom) were used. Data are representative of at least 2 independent experiments.
FIG. 2.
FIG. 2.
Influence of nitrosative stress on xenB expression. Expression levels of xenB and rpsL in P. aeruginosa strains K2153 (wild type) (lanes 1 and 2), K2892 (MexF) (lanes 3 and 4), K2945 (MexF MexT) (lanes 5 and 6), K2960 (MexF MexT+ [as a result of a chromosomal insertion of mexT at the attB site]) (lanes 7 and 8), and K2892 (MexF) (lanes 9 and 10) without or with GSNO or DETA (5 mM for 30 min) exposure were assessed by using RT-PCR. The PCR portions of the xenB reactions were carried out for 26 cycles (top) or 29 cycles (bottom) (lanes 1 and 2) or for 23 cycles (top) or 26 cycles (bottom) (lanes 3 to 6). The PCR portions of the rpsL reactions was carried out for 18 cycles (top) or 20 cycles (bottom) (lanes 1 and 2) or for 17 cycles (top) or 19 cycles (bottom) (lanes 3 to 6). Data are representative of at least 2 independent experiments.
FIG. 3.
FIG. 3.
Influence of nitrosative stress on mexEF-oprN expression. (A) Expression levels of mexE and rpsL in P. aeruginosa strains K2153 (wild type) (lanes 1 and 2), K2520 (MexT) (lanes 3 and 4), K2959 (MexT+, as a result of a mexT insertion at the chromosomal attB site) (lanes 5 and 6), and K2153 (wild type) (lanes 7 and 8) without or with GSNO or DETA (5 mM for 30 min) exposure were assessed by using RT-PCR. The PCR portion of the reactions was carried out for 29 cycles (top) or 32 cycles (bottom) for mexE and for 18 cycles (top) or 21 cycles (bottom) for rpsL. Data are representative of at least 2 independent experiments. (B) Expression of mexE and rpsL in P. aeruginosa strain K2153 without (lane 1) or with (lanes 2 to 4) GSNO (5 mM) exposure for 5 min (lane 2), 15 min (lane 3), or 30 min (lane 4). The PCR portion of the reactions was carried out for 27 cycles (top) or 30 cycles (bottom) for mexE and for 20 cycles (top) or 23 cycles (bottom) for rpsL. Data are representative of at least 2 independent experiments.
FIG. 4.
FIG. 4.
Influence of nitrosative stress on expression of PA2214 (left), PA3229 (middle), and PA4881 (right). (Left) Expression levels of PA2214 and rpsL in P. aeruginosa strain K2892 (MexF) without (lane 1) or with (lane 2) GSNO (5 mM for 30 min) exposure were assessed by using RT-PCR. The PCR portion of the reactions was carried out for 28 cycles (top) or 31 cycles (bottom) for PA2214 and for 17 cycles (top) or 20 cycles (bottom) for rpsL. (Middle and right) Expression levels of PA3229 (middle), PA4881 (right), and rpsL (bottom middle and bottom right) in P. aeruginosa strain K2153 (wild type) without (lane 1) or with (lane 2) GSNO (5 mM for 30 min) exposure were assessed by using RT-PCR. The PCR portion of the reactions was carried out for 28 cycles (top) or 31 cycles (bottom) for PA3229 and PA4881 and for 20 cycles (top) or 23 cycles (bottom) for rpsL. PA2214 was not induced by GSNO in wild-type strain K2153. Data are representative of at least 2 independent experiments.
FIG. 5.
FIG. 5.
Influence of chloramphenicol on mexEF-oprN expression. Expression levels of mexE and rpsL in P. aeruginosa strains K2153 (lanes 1 and 2) and K2520 (MexT) (lanes 3 and 4) without (odd-numbered lanes) or with (even-numbered lanes) chloramphenicol (32 μg/ml for 90 min) exposure were assessed by using RT-PCR. The PCR portion of the reactions was carried out for 30 cycles (top) or 32 cycles (bottom) for mexE and for 21 cycles (top) or 23 cycles (bottom) for rpsL. Data are representative of at least 2 independent experiments.
FIG. 6.
FIG. 6.
Influence of antimicrobials on mexEF-oprN expression. The β-galactosidase activity of P. aeruginosa K2153 (wild type [WT]) carrying mexE-lacZ fusion vector pHF003 was assessed following exposure to chloramphenicol (CAM) (16 μg/ml), ciprofloxacin (CIP) (0.625 μg/ml), or gentamicin (GEN) (0.5 μg/ml) for 30 min. −, no antimicrobial exposure. The β-galactosidase activity of P. aeruginosa K2376 (MexS) (mexS) harboring pHF003 is included for comparison purposes. The data have been corrected for the background activity of strains carrying the pMP220 lacZ fusion vector without mexE and are reported as the means ± standard deviations of data from at least 2 independent experiments. Individual unexposed or wild-type controls are included for each antibiotic-exposed culture of K2153 or K2376.

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