Molecular Characterization of MexL, the Transcriptional Repressor of the mexJK Multidrug Efflux Operon in Pseudomonas aeruginosa

Abstract

The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexL(A47D) mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexL(A47D) oligomerization was confirmed using a genetic approach, and the MexL(A47D) mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexL(A47D), binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions -84 and -20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted -10 and -35 promoter sequences for both mexL and mexJ, with partially overlapping -10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.