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-...