The mexCD-oprJ multidrug efflux operon in Pseudomonas aeruginosa: regulation by the NfxB-like novel regulator PA4596 and envelope stress

Abstract

Expression of the mexCD-oprJ multidrug efflux operon is enhanced by the presence of membrane damaging agents [e.g., the biocide chlorhexidine (Chx)] or mutations in the nfxB gene encoding a repressor of efflux gene expression, both dependent on the AlgU envelope stress response sigma factor. Details of mexCD-oprJ regulation are, however, lacking. In examining the mexCD-oprJ locus, a gene, PA4596, encoding a homologue of NfxB (61% identity) was identified downstream of oprJ, a location conserved in all sequenced Pseudomonas aeruginosa isolates and in Pseudomonas putida. Opposite to mexCD-oprJ, PA4596 expression was reduced by Chx exposure, as assessed using RT-PCR; although like mexCD-oprJ, this was AlgU-dependent (i.e., lost in a ΔalgU strain). Deletion of PA4596 had no impact on Chx resistance indicating that it is not required for Chx-inducible mexCD-oprJ expression/ MexCD-OprJ-dependent Chx resistance. In contrast, mexCD-oprJ expression and the attendant multidrug resistance of nfxB deletion mutants were compromised upon deletion of PA4596, indicating that PA4596 plays a positive role in mexCD-oprJ expression in such mutants. Consistent with this, PA4596 expression increased in nfxB deletion and missense mutants in parallel with mexCD-oprJ. Intriguingly, mexCD-oprJ expression and multidrug resistance were observed in a mutant lacking an nfxB mutation (demonstrating an NfxB-like phenotype) and in an nfxB missense mutant and these were not compromised upon deletion of PA4596. Thus, mexCD-oprJ hyperexpression can be both PA4596-dependent and -independent. A bacterial 2-hybrid assay revealed a PA4596-PA4596 interaction, consistent with the protein forming dimers as NfxB has been shown to do. Two-hybrid assays also demonstrated that NfxB and PA4596 interact. While the functional significance of this remains to be elucidated, it is consistent with their common role in regulating mexCD-oprJ expression and is suggestive of a complex and possibly novel regulatory mechanism. These data highlight the complexity of mexCD-oprJ regulation and the apparently multiple pathways to efflux gene expression, suggestive of multiple roles for this efflux system in P. aeruginosa independent of antimicrobial efflux.