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Antimicrobial Agents and Chemotherapy, May 2005, p. 1844-1851, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1844-1851.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523,¹ Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand²

Received 12 October 2004/ Returned for modification 8 December 2004/ Accepted 19 January 2005

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.

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