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.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (15), a resistance that is increasingly attributable to the operation of broadly specific, tripartite multidrug efflux systems of the resistance-nodulation-division (RND) family (39,40). Several RND family multidrug efflux systems have been described for P. aeruginosa, although the major systems contributing to intrinsic and/or acquired multidrug resistance include MexAB-OprM, MexXY-OprM, 40). Unlike MexAB-OprM and MexXYOprM, which contribute to intrinsic resistance (1,7,26,32), the MexEF-OprN and MexCD-OprJ systems are typically quiescent in wild-type cells (under usual laboratory growth conditions) (19,28,50), with expression and, therefore, a contribution to antimicrobial resistance following a mutational upregulation of the efflux genes (13,20,30,41,48). In the case of MexEF-OprN, this involves the reversion of mutations present in the mexT gene of a number of so-called wild-type strains (29, 30) or a mutation of the mexS gene (also known as qrh [25]), encoding an oxidoreductase of unknown function (48). mexT, which occurs upstream of mexEF-oprN and downstream of mexS, encodes a LysR family positive regulator that promotes mexEF-oprN (25,28,36) and mexS (25) expression. mexEF-oprN expression and modest multidrug resistance have also been reported for a mutant disrupted in the mvaT gene (53), encoding a global regulator of virulence gene expression (8).Originally identified as a determinant of fluoroquinolone resistance (12, 13), MexEF-OprN accommodates a variety of antimicrobials, including trimethoprim and chloramphenicol (28), with chloramphenicol readily selecting multidrug-...

nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K D ], 158.0 ؎ 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR I104F , was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K D , 853.2 ؎ 151.1 nM). Consistent with this, MexR I104F repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal ␣-helix of the protein that alone is sufficient for interaction with MexR.Multidrug efflux systems of the resistance-nodulation-division (RND) family are broadly distributed among gram-negative bacteria, where they are, in many instances, important determinants of intrinsic and/or acquired antimicrobial resistance (48). Comprised of an inner membrane drug-proton antiporter (the RND component), an outer membrane channel-forming component, and a periplasmic membrane fusion protein (40), these pumps function to capture periplasmic and possibly cytosolic substrates and deliver them to the cell exterior (2, 42, 49). In Pseudomonas aeruginosa, seven RND-type pumps have been described to date (36, 46), although the major efflux determinant of intrinsic multidrug resistance and the best studied of these pumps in P. aeruginosa is MexABOprM (45,46). In addition to numerous medically relevant antimicrobials (47), MexAB-OprM also exports a variety of dyes and detergents (25,62,64), inhibitors of fatty acid biosynthesis (58), biocides (10), organic solvents (26, 27), homoserine lact...

Meropenem potentiates AG activity against laboratory and CF strains of P. aeruginosa, both dependent on and independent of MexXY, highlighting the complexity of AG resistance in this organism.