SummaryMexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa , the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC . PA3721 ( nalC ) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.

Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple classes of antimicrobials (11), attributable in part to a family of broadly specific, so-called multidrug efflux systems (23, 24) that work synergistically with low outer membrane permeability (9, 19) to limit antimicrobial accumulation in this organism. Several multidrug efflux systems in P. aeruginosa have been described to date (23), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (10, 18, 26). MexAB-OprM exports a wide variety of antimicrobials, including most classes of antibiotics, biocides, dyes, detergents, organic solvents (i.e., aromatic hydrocarbons; reviewed in reference 23]), and homoserine lactones associated with quorum sensing (8,22). The last play a role in cell densitydependent expression of a number of virulence factors in P. aeruginosa, and thus, the activity of this efflux system can influence virulence (30). Indeed, a recent study suggests that the MexAB-OprM efflux system of P. aeruginosa promotes the release of a molecule(s) ultimately important for the virulence of this organism (12). The observation that MexAB-OprM hyperexpression in nalB strains impairs fitness and virulence (30) also suggests that this efflux system has a physiological role in P. aeruginosa independent of antimicrobial efflux and resistance. Consistent with this, mutants hyperexpressing MexABOprM were readily selected in vivo in a rat model of acute P. aeruginosa pneumonia in the absence of any antibiotic selection (16). The specific nature of the selective in vivo growth advantage provided by this efflux system is, however, unknown.Hyperproduction of MexAB-OprM has been documented in lab and clinical multidrug-resistant isolates carrying lesions in the mexR gene (4,15,20,28,35,37) (so-called nalB mutants [21]), encoding a repressor of mexAB-oprM expression (27, 35).MexAB-OprM hyperexpression also occurs independently of mutations in mexR or the mexR and mexAB-oprM promoter regions (35, 37). These so-called nalC mutants (5, 20, 35) carry a mutation in a recently identified gene (PA3721, also known as nalC) that encodes a TetR family repressor of an adjacent two-gene operon, PA3720-PA3719 (5). It is, in fact, the increased express...

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.Pseudomonas aeruginosa is an opportunistic human pathogen that demonstrates an innate resistance to multiple classes of antimicrobials (5), a property explained in part by the operation of multidrug efflux systems of the resistance-nodulation division (RND) family (19,20). Several RND-type multidrug efflux systems have been described in P. aeruginosa (20), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (4, 10, 21). MexAB-OprM exports a variety of clinically used antimicrobials (including several classes of antibiotics and biocides) (19,20) but, as well, several additional agents with antimicrobial activity (e.g., dyes [9], detergents [34], and organic solvents such as aromatic hydrocarbons [11]) and acylhomoserine lactones (AHLs) associated with quorum sensing (3, 18). AHLs play a role in cell density-dependent expression of a number of virulence factors in P. aeruginosa and, thus, the activity of this efflux system can influence virulence (6). The observation that MexAB-OprM overproduction can compromise bacterial "fitness" (29) and that pump-overproducing mutants of P. aeruginosa can be selected in animal models of infection in the absence of antimicrobial selection (7) also point to in vivo functions for this pump independent of antimicrobial efflux and resistance.Hyperproduction of MexAB-OprM has been documented in so-called nalB mutants (13) carrying lesions in the mexR gene (13,26,35,38) encoding a repressor of mexAB-oprM expression (22,35). MexR is a member of the MarR family of regulators (16) and binds as a dimer (12) to two sites in the mexRmexA intragenic region, near mexR and overlapping promoters for mexR and mexAB-oprM (2). MexAB-OprM hyperexpression also occurs independently of mutations in mexR; these so-called nalC mutants (1, 13, 35) carry a mutation in a gene (PA3721, also known as nalC) that encodes a TetR family repressor of an adjacent two-gene operon, PA3720-PA3719 (1). It is, in fact, the increased expression of PA3719 that results from disruption of the nalC repressor gene that promotes mexAB-oprM hyperexpression (1), apparently as a result of PA3719 modulation of MexR repressor activity (L. Cao, S.

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...