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, 187 (4), 1246-53

Mutations in PA2491 (mexS) Promote MexT-dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas Aeruginosa

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Mutations in PA2491 (mexS) Promote MexT-dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas Aeruginosa

Mara L Sobel et al. J Bacteriol.

Abstract

Disruption of the PA2491 gene in a mini-Tn5-tet insertion mutant of a clinical isolate of Pseudomonas aeruginosa increased expression of the mexEF-oprN multidrug efflux genes and decreased production of outer membrane protein OprD, concomitant with enhanced resistance to chloramphenicol, quinolones, and imipenem, which was reminiscent of previously described nfxC mutants. PA2491 encodes a probable oxidoreductase previously shown to be positively regulated by the MexT positive regulator of mexEF-oprN expression (T. Kohler, S. F. Epp, L. K. Curty, and J. C. Pechere, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrug-resistant mutants of the P. aeruginosa clinical isolate hyperexpressing mexEF-oprN and showing reduced production of OprD were readily selected in vitro, and all of them were shown to carry mutations in PA2491, highlighting the probable significance of such mutations as determinants of MexEF-OprN-mediated multidrug resistance in vivo.

Figures

FIG. 1.
FIG. 1.
Expression of mexE (A), mexT (B), and rpsL (C) in P. aeruginosa as assessed by RT-PCR. Lane 1, strain K2153 (PA2491+); lane 2, K2313 (PA2491); lane 3, K2313/pUCP18 (PA2491); lane 4, K2313/pSN2491 (PA2491+); lane 5, K2316 (K2313 ΔmexT); lane 6, K2153 (PA2491+); lane 7, K2317 (PA2491, Tyr127Stop); lane 8, K2318 (PA2491, Val333Gly); lane 9, K2319 (PA2491, 91-bp deletion); lane 10, K2320 (PA2491, Ser124Arg) (genotypes are indicated in parentheses). The PCR portions of the reactions were carried out for 28 cycles (top panel) and 29 cycles (bottom panel) for mexE (A), for 29 cycles (top panel) and 30 cycles (bottom panel) for mexT (B), and for 18 cycles (top panel) and 19 cycles (bottom panel) for rpsL (C). The rpsL reaction served as an internal control that ensured that equal amounts of RNA were employed in all of the RT-PCRs.
FIG. 2.
FIG. 2.
Immunoblot detection of OprD. Lane 1, clinical isolate K2153 (PA2491+); lane 2, K2313 (PA2491); lane 3, K2313/pUCP18 (PA2491); lane 4, K2313/pSN2491 (PA2491+); lane 5, K2316 (K2313 ΔmexT); lane 6, K2317 (PA2491, Tyr127Stop); lane 7, K2318 (PA2491, Val333Gly); lane 8, K2319 (PA2491, 91-bp deletion); lane 9, K2320 (PA2491, Ser124Arg) (genotypes are indicated in parentheses).

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