title: 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.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
authors.forename: Rungtip
authors.initials: R
authors.lastname: Chuanchuen
authors.forename: Jared B
authors.initials: JB
authors.lastname: Gaynor
authors.forename: RoxAnn
authors.initials: R
authors.lastname: Karkhoff-Schweizer
authors.forename: Herbert P
authors.initials: HP
authors.lastname: Schweizer
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006706:Homeostasis; D007700:Kinetics; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D009876:Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins; D003850:Deoxyribonuclease I