Characterization of NfxB and PA4596, Two Repressors of the mexCD-oprJ Operon Encoding an RND-Type Multidrug Efflux Pump in Pseudomonas aeruginosa
Gene Regulation , Pseudomonas aeruginosa , Multidrug Efflux
MexCD-OprJ is an RND-type multidrug efflux pump present in P. aeruginosa and is capable of exporting, and as such providing resistance to, several clinically important antimicrobials including fluoroquinolones, cephems, macrolides, and several biocides including chlorhexidine (CHX). Expression of mexCD-oprJ is negatively regulated by NfxB, a LacI-type repressor. The promoter region of mexCD-oprJ was identified and included two inverted repeat operator sites, B1 and B2, both of which are required in order for NfxB to bind, thereby repressing mexCD-oprJ. NfxB oligomerizes into a tetramer in solution and likely functions as a dimer of NfxB homodimers. In addition to being derepressed by loss of NfxB, MexCD-OprJ is inducible by a variety of non-antibiotic membrane-damaging agents (MDAs) such as CHX. A homologue of NfxB, PA4596, was found to be induced in response to CHX-promoted envelope stress in an AlgU-dependent manner and is directly repressed by NfxB. Loss of PA4596 resulted in increased resistance to the biocide CHX, shown to be a result of increased CHX-dependent expression of mexCD-oprJ. Susceptibility to CHX was restored upon expression of PA4596 from the plasmid pAK1900 as was decreased expression of mexCD-oprJ in the presence of CHX, indicating that PA4596 contributes to mexCD-oprJ repression in the presence of CHX. PA4596 was found to form oligomers in solution, likely dimers and tetramers. In the absence of NfxB, PA4596 is unable to contribute to repression of mexCD-oprJ. However, NfxB and PA4596 interact and together form a repressor capable of regulating mexCD-oprJ expression. Screening of transposon mutants for increased resistance to erythromycin potentially indicative of increased mexCD-oprJ expression lead to the identification of several novel genes including PA0479, cupA3, faoA, PA3259, mucD, and clpA whose loss generated a multidrug resistance profile consistent with increased production of MexCD-OprJ. However, further studies are required to determine how each of these genes may be affecting expression of mexCD-oprJ.