For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

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.The MexJK efflux system is not expressed at significant levels in wild-type cells but is constitutively expressed in mexL mutants (3, 4). The Pseudomonas aeruginosa mexL mutant PAO238-1 was previously isolated by exposure of the susceptible ⌬(mexAB-oprM) ⌬(mexCD-oprJ) mutant PAO238 to the broad-spectrum biocide triclosan (3). PAO238-1 overexpresses MexJK, presumably because it encodes an inactive MexL repressor due to a single-nucleotide change in mexL that causes a change from alanine 47 to aspartate (A47D) in the putative MexL helix-turn-helix DNA binding motif. Our initial studies showed that mexL is located 94 bp upstream of and transcribed divergently from mexJK. MexL belongs to the TetR repressor family that includes MexZ, a regulator of mexXY in P. aeruginosa (1), AmrR, a regulator of amrAB-oprA in Burkholderia pseudomallei (19), QacR, a repressor of qacA/qacB in Staphylococcus aureus (10), AcrR, a repressor of acrAB in Escherichia coli (17, 30), MtrR, a repressor of mtrCDE in Neisseria gonorrhoeae (11), and SmeT, a repressor of smeDEF in Stenotrophomonas maltophilia (25). The MexJK substrate spectrum is relatively narrow, and to date, only triclosan and erythromycin were shown to be effluxed by this pump. However, neither of these substrates induces mexJK operon expression. To gain a better understanding of mexJK regulation and possible effectors, we purified and characterized MexL. Our data support the notion that MexL is a specific repressor of mexJK transcription and autoregulates its own ...