Abstract

ABSTRACT MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of themtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand howMtrA activatesmtrCDE expression, we successfully purifiedMtrA and found that it could bind specifically to themtrCDE promoter region. The affinity of MtrA for themtrCDE promoter increased 2-fold in the presence of a known effector and sub-strate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition withMtrR, the tran-scriptional repressor ofmtrCDE, MtrA was found to bind with apparent lower affinity thanMtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increasedMtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important inMtrA binding) defined theMtrA-binding site 20–30 bp upstream of the knownMtrR-binding site. Collectively, these results suggest that the MtrA andMtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for themtrCDE promoter such that MtrR binding is negatively impacted. Our results pro-vide a mechanism for transcriptional activation ofmtrCDE byMtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci. IMPORTANCE Antibiotic resistance inNeisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for