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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

Rungtip Chuanchuen,2 Jared B. Gaynor,1 RoxAnn Karkhoff-Schweizer,1 and Herbert P. Schweizer1*

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


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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 MexLA47D 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 MexLA47D oligomerization was confirmed using a genetic approach, and the MexLA47D mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexLA47D, 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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INTRODUCTION
 
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 {Delta}(mexAB-oprM) {Delta}(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 expression.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are described in Table 1. Luria-Bertani (LB) medium from Difco (Detroit, MI) was used as a growth medium for all bacterial strains, unless otherwise stated. The minimal medium used was M9 medium (23) supplemented with 0.2% Casamino Acids (Difco) or 0.2% glucose (Sigma, St. Louis, MO). Cultures were routinely incubated at 37°C with shaking (250 rpm). Where necessary, antibiotics were used in growth media as follows: for Escherichia coli, ampicillin (100 to 150 µg/ml) and tetracycline (15 µg/ml); and for Pseudomonas aeruginosa, tetracycline (10 to 15 µg/ml) and carbenicillin (100 to 200 µg/ml). Antibiotics were purchased from Sigma, with the exception of carbenicillin, which was purchased from Fisher Scientific (Pittsburgh, PA).


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  		TABLE 1. Bacterial strains and plasmids used in this study

General DNA methodology. Routine DNA manipulations, including PCR amplifications from genomic DNA templates, were performed as previously described (12, 24). Preparations of chromosomal and plasmid DNAs were performed using the ISOQUICK nucleic acid extraction kit (ORCA Research, Bothell, WA) and QIAprep Mini-spin kit (QIAGEN, Valencia, CA), respectively, and these kits were used according to the manufacturer's instructions. The lacZ fusion plasmid pTZ120 was derived from pTZ110 (28) by inserting rehybridized NcoI/NsiI linkers 1 and 2 (oligonucleotides used in this study are listed in Table 2) into the BamHI site of pTZ110 using previously described methods (26, 28). The mexL'-lacZ fusion plasmid pPS1237 was created by inserting the 632-bp XhoI-EcoRI fragment from pPS1176 between the same sites of pTZ110. For complementation experiments, pPS1245 carrying the mexL coding region in the same orientation as Plac was constructed as follows. Plasmid pPS1153 was digested with EcoRI, blunt ended, and then digested with SalI. The mexL fragment was cloned into pRK415 obtained by digesting with HindIII, blunt ending, and then digesting with SalI.


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  		TABLE 2. Oligonucleotides used in this study

Construction of mexJ'-lacZ transcriptional fusions for mexJK promoter mapping. Various portions of the mexL-mexJ intergenic regions were PCR amplified from genomic DNA templates by using different pairs of primers as follows: LJ1U-LJ2D, LJ4U-LJ5D, LJ6U-LJ5D, LJ7U-LJ5D, LJ8U-LJ5D, and mexLJ-up-mexLJ-down. Each primer contained base mismatch(es) that introduced a restriction site suitable for directional cloning (Table 2). PCR mixtures (50 µl) contained 1 U of Taq DNA polymerase (Invitrogen), 30 pmol of each primer, 5% (vol/vol) dimethyl sulfoxide, 0.2 µM of each deoxynucleotide, 1.5 mM MgCl2, 0.5 µg chromosomal DNA, and 1x PCR buffer (Invitrogen). General cycle conditions were 95°C for 5 min, followed by 30 cycles (1 cycle consisting of 95°C for 45 s, 65°C for 45 s, and 72°C for 1 min) per kb of DNA fragment to be amplified, and a final extension at 72°C for 10 min. Following the manufacturer's instructions, the PCR fragments were cloned into pCR2.1 to yield pPS1188, pPS1190, pPS1191, pPS1232, pPS1233, and pPS1173, respectively. An additional fragment was obtained by hybridizing two oligonucleotides, LJ9Ulinker and LJ9Dlinker, encompassing positions –36 to –87 relative to the first nucleotide of the mexJ start codon.

Next, the various portions of the mexL-mexJ intergenic region were fused to lacZ in pTZ120. To do this, pPS1188, pPS1190, pPS1173, pPS1232, and pPS1233 were digested with EcoRI-PstI, SalI-PstI, XhoI-EcoRV, EcoRI-PstI, and EcoRI-PstI, respectively. The digested DNA fragments were purified from an agarose gel and then ligated to pTZ120 digested with EcoRI-NsiI, SalI-NsiI, XhoI-SmaI, EcoRI-NsiI, and EcoRI-NsiI to produce pPS1201, pPS1202, pPS1204, pPS1209, and pPS1210, respectively. For construction of pPS1236, the LJ9U and LJ9D oligonucleotide linkers with EcoRI and HindIII overhangs was ligated between the same sites of pTZ120. The inserts were PCR amplified from each plasmid and their nucleotide sequences determined. The fusion plasmids were then transformed into {Delta}(mexLJK) strain PAO314 by electroporation (6). ß-Galactosidase (ß-Gal) activity was measured, and activity units were determined by the method of Miller (18). For complementation experiments, pPS1245 (mexL+) was transformed into strain PAO314 carrying the respective fusion plasmids.

Construction of LexA-DBD-MexL fusions. Wild-type mexL was PCR amplified from pPS1153 by using primer MexLF (introducing a SacI site and engineered to allow in-frame fusion of the MexL start codon to LexA) and primer MexLR (introducing a KpnI site downstream of mexL). PCR conditions were as described above except that annealing temperatures of 83°C and 1-min extension times were used. The 728-bp PCR product was cloned into pCR2.1 to form pPS1284. This plasmid was then digested with SacI and KpnI, and the 718-bp fragment was cloned between the same sites of pSR658 to form pPS1286. This plasmid encodes a MexL-LexA-DBD (DNA-binding domain) fusion protein of 312 amino acids (102 amino acids from LexA-DBD and 210 from MexL; note that methionine and serine, the first two amino acids of the 212-amino-acid MexL protein were changed to glutamic acid and leucine). A mutant MexLA47D-LexA-DBD fusion was constructed in a similar fashion. PCR was performed by using pPS1175 as the template and using the MexLF and MexLR primers. The 728-bp PCR product was cloned into pCR2.1 to form pPS1285. Subcloning of the pPS1285 insert into pSR658 then formed pPS1287.

Purification of MexL protein. To produce hexahistidine-tagged MexL (MexL-His6), the mexL gene was amplified from P. aeruginosa PAO1 genomic DNA using primers mexL-pET21b-up, which anneals at the 5' end of mexL and incorporates a NdeI restriction site, and mexL-pET21b-down, which anneals at the 3' end of mexL and incorporates a NotI restriction site while removing the mexL stop codon. PCR conditions were as described above, except that the elongation step was for 45 s at 72°C. The PCR fragment was gel purified and cloned into the pCR2.1 TA cloning vector to generate pPS1216. The 630-bp NdeI-NotI fragment from pPS1216 was cloned into NdeI- NotI-restricted pET-21b (Novagen) to produce pPS1217. To generate a clone expressing a His6-tagged PAO238-1 mutant MexLA47D protein, the mutant mexL gene was amplified from pPS1150 and cloned into pCR2.1 to yield pPS1166. Then, a NdeI-NotI fragment containing mutant mexL was cloned between the same sites of pET-21b to yield pPS1167.

To overexpress and purify MexL-His6, E. coli BL21(DE3) carrying plasmid pPS1217 was grown overnight at 37°C in LB broth containing 100 µg/ml ampicillin. The culture was diluted 1:10 into 250 ml of LB broth with 100 µg/ml ampicillin and incubated at 37°C until it reached an optical density at 600 nm of 0.3 to 0.5 (approximately 1 to 2 h). Then, isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and incubation was continued for 3 to 4 h. Bacterial cells were harvested, and MexL-H6 was purified by Ni2+ affinity chromatography utilizing previously described procedures (13). The purity of eluted fractions was assessed by electrophoresis on a 0.1% sodium dodecyl sulfate (SDS)-10% polyacrylamide gel, followed by Coomassie blue staining. Three 0.5-ml eluate fractions containing the majority of MexL-His6 were combined and dialyzed overnight at 4°C against 1.5 liters of dialysis buffer (20 mM Tris-HCl [pH 7.5], 10% glycerol) which was changed once during the dialysis period. The purified MexL-His6 protein was stored in dialysis buffer at –70°C. MexLA47D-His6 protein from P. aeruginosa PAO238-1 was prepared from pPS1312-containing E. coli BL21(DE3) cells by using the same procedures. MexL-His6 protein concentrations were determined by using the Bradford assay (Bio-Rad, Hercules, CA) and bovine serum albumin (BSA) (Sigma) as the protein standard. Total yields were ~1 mg of purified MexL-His6 and MexLA47D-His6 from 250 ml of culture.

Nondenaturing gradient PAGE. The native molecular size of MexL-His6 was estimated by nondenaturing gradient polyacrylamide gel electrophoresis (PAGE) (8). The gel contained a 5 to 20% linear gradient of polyacrylamide stabilized with a 0 to 20% linear glycerol gradient. Ten micrograms of each sample was mixed with an equal volume of loading buffer (100 mM Tris-HCl [pH. 7.5], 40% glycerol, 2 mg/ml bromophenol blue) and loaded on the gel. The electrophoresis was carried out at 80 V for 16 to 20 h at room temperature. The native molecular size of MexL-His6 was estimated by comparison of its migration distance to those of standard proteins (Sigma).

Size exclusion chromatography. Gel filtration chromatography of MexL-His6 was performed at 4°C as previously described (16) with some modifications. Sephadex G200 (Bio-Rad) was packed into a column (1.5 cm by 50 cm) (Bio-Rad) at a flow rate of 10.5 ml/h using an ECONO gradient pump (Bio-Rad). The packed column was equilibrated with 0.02 M Tris-HCl [pH 7.5], 1 mM EDTA, and 0.1 mM dithiothreitol (DTT) at the packing flow rate. Aliquots (500 µl) containing 1 mg/ml of protein, 25 µg/ml of pUCP20T DNA (29), and 1 mM ATP were loaded and chromatographed with 0.02 M Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM DTT, and 15% glycerol using a flow rate of 7 ml/h. The absorbance (280 nm) of the effluent was monitored using an ECONO UV monitor (Bio-Rad) and recorded utilizing a Bio-Rad chart recorder. The standard proteins included ß-amylase, BSA, chicken ovalbumin, alcohol dehydrogenase, and carbonic anhydrase (Sigma). The elution volume (Ve) of each protein was determined using the time at which the sample peak (absorbance at 280 nm) was detected and the operating flow rate. The voided volume (V0) and total volume (Vt) were determined using plasmid DNA and ATP, respectively. The average partition coefficient (Kav) was determined as follows: Kav = (Ve – V0)/(Vt – V0).

DNA binding assay and measurement of binding affinity. To obtain templates for MexL binding assays, pPS1173 and pPS1232 were digested with EcoRI-XhoI and EcoRI-PstI, respectively. The EcoRI-XhoI fragment of pPS1173 is a 205-bp DNA fragment containing the 94-bp mexL-mexJ intergenic region. The fragments were gel purified, end labeled with [{alpha}-35S]dATP (3,000 Ci mmol–1; Perkin-Elmer Life and Analytical Sciences, Boston, MA) using Klenow fragment (Invitrogen), and purified by using the QIAquick nucleotide removal kit (QIAGEN) (23). The end-labeled DNA fragments (~10,000 cpm) were incubated with purified MexL-His6 (~4.2 µM) in a binding reaction mixture containing 1x binding buffer (10 mM Tris-HCl [pH 7.5], 150 mM KCl, 20% glycerol, 2 mM EDTA, 2 mM DTT) and BSA (66 ng/µl) for 15 min at room temperature (21). The mixtures were immediately loaded on a 4% nondenaturing polyacrylamide gel in 1x TG buffer (25 mM Tris-HCl [pH 8.3], 192 mM glycine) and electrophoresed at 160 to 165 V for 1.5 to 2 h using the same buffer. The gels were dried under vacuum, and labeled fragments were visualized by autoradiography. To determine the specificity of MexL, an excess amount of the same unlabeled 205-bp fragment from pPS1173 was included as competitor DNA.

To estimate MexL binding affinity, the labeled 205-bp DNA fragment from pPS1173 and the same experimental conditions described above were used except that the binding reactions were performed with increasing concentrations of MexL (0.5, 1.0, 2.0, 3.1, 4.1, 5.2, 6.2, 7.2, and 8.3 µM). After autoradiography, the amounts of the remaining free DNA fragments were estimated by scanning the autoradiograph and analyzing band intensities with Scion Image software (NIH Image 1.6). The values of free DNA fractions were plotted against the log [MexL] to generate a Bjerrum plot (31). The MexL concentration at which 50% of the MexL binding sites were occupied (Kd) was estimated from the plot. The relative constant (Kb) for MexL binding was determined from the fraction of DNA bound (fbound) at a given protein concentration (P) in the linear range of the graph by calculation of Kb = fbound/[P (1 – fbound)] as previously described (22). For all calculations, it was assumed that the MexL used in these assays was 100% active.

DNase I footprinting assay. For DNase I footprinting assays of the mexJ coding strand, target DNA was the XhoI-NotI fragment (mexL-mexJ intergenic region) from pPS1173 which was end labeled with [{alpha}-35S]dATP at the XhoI end. For the mexL coding strand, target DNA was the XbaI fragment from pPS1173 that was end labeled before digestion with BamHI. DNase I digestion was performed as previously described (9). Briefly, the labeled DNA fragments (~100,000 cpm) were incubated with purified MexL-His6 at concentrations of 5, 7.5, and 10 µM in 50-µl reaction mixtures containing gel mobility shift assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM KCl, 20% glycerol, 2 mM EDTA, 2 mM DTT) and BSA (66 ng/µl). The mixtures were incubated for 15 min at room temperature and then digested with 0.03 U of DNase I (Invitrogen) for 2 min at room temperature. After extraction and ethanol precipitation, DNA fragments were separated by electrophoresis on a 5% (wt/vol) polyacrylamide-8 M urea sequencing gel. Labeled DNA fragments were visualized by autoradiography. Chain termination sequencing reactions using the same labeled templates were performed by using the Sequenase version 2.0 DNA sequencing kit (Amersham Bioscience, Piscataway, NJ) as described by the manufacturer. Primers LJ14U and LJ16D were used for sequencing of the mexL and mexJ coding strands, respectively.

RNase protection assay. RNase protection analysis on total RNA isolated from P. aeruginosa PAO318 by the hot phenol method was performed as previously described (2, 20). To generate a riboprobe, a DNA fragment containing the mexL promoter was amplified from PAO1 genomic DNA by using primer LJ11U annealing 147 bp downstream of the mexL start codon and primer LJ11D annealing 150 bp downstream of the mexJ start codon. The purified DNA fragment was cloned into pCR2.1 in such a way that MexL transcription is opposite that of the T7 promoter. The resulting plasmid, pPS1463, was digested with BamHI and used for the synthesis of a 32P-labeled mexL riboprobe by using the Riboprobe System-T7 (Promega, Madison, WI) following the manufacturer's instructions. The integrity of total RNA was confirmed by RNase protection analysis using a riboprobe specific for the constitutively expressed omlA gene (20).


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RESULTS AND DISCUSSION
 
Purified MexL is a tetrameric protein. To study repressor action at the molecular level, MexL-His6 was overexpressed in E. coli and purified to near homogeneity by Ni2+ affinity chromatography (Fig. 1A). The apparent molecular mass of purified MexL-His6 was ~25,000 Da, which is close to the 24,242 Da calculated for the 220-amino-acid fusion protein monomer. The molecular mass of MexL-His6 in solution was determined by native PAGE and size exclusion chromatography (Fig. 1B and C). By native PAGE, the mass of MexL-His6 was estimated at 97,600 Da, and by gel filtration, its mass was indicated as 83,500 Da. Both of these values are consistent with native MexL-His6 being a tetrameric protein, with a calculated molecular mass of 96,968 Da.



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  		FIG. 1. Purification and characterization of MexL-His6. A) MexL was overexpressed with a carboxy-terminal hexahistidine tag in E. coli BL21(DE3). The expressed MexL-His6 fusion protein was purified by Ni2+ nitrilotriacetic acid affinity chromatography and analyzed by SDS-PAGE. The gel was stained with Coomassie blue. Lanes: M, marker proteins (from top to bottom, myosin, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor); 1, uninduced cells containing pET-21b; 2, induced cells containing pET-21b; 3, uninduced cells containing pPS1216 expressing MexL-His6; 4, induced cells containing pPS1216; 5, purified MexL-His6. B) Native polyacrylamide gradient gel electrophoresis. The standard proteins and their molecular masses (in kilodaltons) were as follows: 11, urease dimer (545 kDa); 12, urease monomer (272 kDa); 21, bovine serum albumin dimer (132 kDa); 22, bovine serum albumin monomer (66 kDa); and 3, chicken ovalbumin (45 kDa). The relative migration of MexL is indicated by the black diamond. C) Size exclusion chromatography on Sephadex G-200. The numbers and symbols refer to some of the same proteins employed in panel B. The additional standard proteins were as follows: 4, ß-amylase (202 kDa); 5, alcohol dehydrogenase (167 kDa); and 6, carbonic anhydrase (29 kDa). The black diamond marks the Kav of MexL-His6.

MexL multimerization was genetically verified using the LexA-based system devised for this purpose (5, 7). To this end, the entire MexL protein-coding sequence was fused in frame to the LexA-DBD. The resulting plasmid construct, pPS1286, along with the vector control encoding only LexA-DBD, was transformed into E. coli strain SU101, which contains a chromosomally integrated sulA'-lacZ fusion, whose expression is under LexA control. As a positive control, a LexA-DBD-NeuD construct was used, since NeuD, a protein involved in polysialic acid synthesis in E. coli, is known to multimerize in this system (5, 7). IPTG-induced cells expressing only LexA-DBD (pSR658) exhibited high levels of ß-Gal activity (Table 3), since LexA-DBD cannot multimerize and thus does not bind to the sulA promoter region and repress sulA'-lacZ transcription. In contrast, cells containing the positive-control pSR658neuD expressing LexA-DBD-NeuD expressed lower (repressed) levels of ß-Gal activity. Levels of ß-Gal in IPTG-induced SU101 cells containing either pPS1286 (LexA-DBD-MexL) or pPS1287 (LexA-DBD-MexLA47D) were both greatly reduced, indicating that both forms of MexL can assist in LexA-DBD multimerization. Thus, the inability of MexLA47D to repress mexJK transcription is not due to lack of oligomerization of the mutant protein. ß-Gal activity levels in uninduced cells containing the various plasmids were similar to those of uninduced cells containing pSR658 (data not shown).


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  		TABLE 3. Genetic evidence for multimerization of MexLa

MexL binds specifically to the mexL-mexJ intergenic region. To demonstrate that MexL binds to the mexL-mexJ intergenic region in a specific manner, gel mobility shift assays were performed. Purified MexL-His6 band shifted an end-labeled 205-bp DNA fragment containing the 94-bp mexL-mexJ intergenic region (Fig. 2A). MexL-His6 binding to this fragment was specific, since the band shift was not observed in the presence of excess competitor DNA (the same unlabeled 205-bp fragment). When the same labeled DNA fragment was incubated with MexLA47D-His6 mutant protein, no band shift was observed (Fig. 2B), demonstrating that the single A47D change in the putative helix-turn-region of the mutant protein affects its ability to bind to target DNA, although the protein was stable after purification and multimerized in solution. A DNA fragment containing the region from positions –97 to +113 (relative to the first nucleotide of the mexJ ATG start codon) showed the same band shift pattern as that observed with the pPS1173 insert.



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  		FIG. 2. Gel mobility shift assays. A) An end-labeled 205-bp fragment from pPS1173 containing the entire 94-bp mexL-mexJ intergenic region was incubated with MexL-His6 in the absence (–) or presence (+) of unlabeled competitor DNA (the same unlabeled 205-bp fragment). B) Approximate localization of the MexL binding sites in the mexL-mexJ intergenic region. Plasmid pPS1173 contains DNA from positions –165 to +36, and pPS1232 contains DNA from –90 to +113. A synthetic oligonucleotide (Oligo) contains nucleotides located between –86 and –37 of the intergenic region (sequences are numbered relative to the first nucleotide of the mexJ ATG codon). All fragments were incubated with MexL-His6, except for the fragment in the leftmost lane which was incubated with MexLA47D-His6. Asterisks mark weak band-shifted fragments. C) Estimation of affinity by Bjerrum plot analysis. Band shift assays were performed by incubating the 205-bp fragment employed in panel A with increasing concentrations of MexL. The autoradiograph was scanned, band intensities were quantitated, and the fraction of free DNA was plotted versus log MexL concentration. The MexL concentration at which 50% of the MexL binding sites were occupied (Kd) was estimated from the plot.

To roughly localize the MexL binding (operator) sites, PCR fragments containing various portions of the mexL-mexJ intergenic regions were generated and used as templates in band shift assays. These experiments (data not shown) localized the MexL operator(s) to a region located between positions –90 and –41 upstream of mexJ. This was verified by the finding that MexL band shifted a synthetic 51-bp oligonucleotide encompassing the –86 and –37 region from the mexJ start codon (Fig. 2B). The observed shift was not as complete as those observed with the pPS1173 and pPS1232 inserts, probably due to incomplete labeling of the oligonucleotide leaving unlabeled "competitor" DNA.

Using the 205-bp end-labeled fragment, band shift assays and Bjerrum plot analyses (Fig. 2C), the relative binding constant (Kb) was estimated to be 10–7 M–1 and the dissociation constant (Kd) was 9 x 10–7 M. Although acceptable for a DNA binding protein, the affinity of MexL for its target is not extraordinarily high, since Kds in the 10–8 to 10–9 M range have been observed for many DNA binding proteins (21). This may mean the following. (i) Binding conditions still have to be optimized. (ii) The techniques used for determining Kd and Kb were not optimal. (iii) An "assisting factor" was missing from the in vitro binding reactions, which is less likely.

Identification of the MexL operator sites. A closer analysis of the mexL-mexJ intergenic region (Fig. 3) revealed two inverted GTATTT repeats that are separated by 16 nucleotides. To more precisely map the MexL operator sites with respect to these inverted repeats, the mexL-mexJ intergenic region was end labeled on either the mexL or mexJ coding strand and subjected to DNase I footprinting. The footprinting profiles revealed a single protected region on either strand (Fig. 4A and B), and these two protected regions overlapped almost perfectly. Whereas the protected region on the mexJ coding strand was located between positions –22 and –84 upstream of the mexJ start codon, that of the mexL coding strand was situated between –20 and –81. The protected regions encompassed the two inverted repeats, as well as the predicted –35 and –10 regions of the mexJ and mexL promoters.



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  		FIG. 3. Regulatory elements within the mexL-mexJ intergenic region. The mexL and mexJ coding regions are shown on the respective strands and indicated by light grey shading. The regions containing the inverted GTATTT repeat are boxed and shaded. The putative –10 and –35 regions of the mexJ and mexL promoters, as predicted by the promoter prediction program www.fruitfly.org/seq_tools/promoter.html, are boxed. The mexL transcription start site is marked with an asterisk. The black bar indicates the –84 to –22 region on the mexJ coding strand that is protected by MexL, and the white bar indicates the –81 to –20 region on the mexL coding strand that is protected by MexL. All coordinates are relative to +1, which was arbitrarily chosen as the first nucleotide in the mexJ start codon. The boundaries of a double-stranded synthetic oligonucleotide used for band shift assays are indicated by large brackets.



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  		FIG. 4. DNase I footprinting analysis of MexL-DNA interactions in the mexL-mexJ intergenic region. DNase I footprinting was performed A) with the XhoI-NotI fragment of pPS1173, which was labeled on the mexJ coding strand and B) with the XbaI-BamHI fragment from pPS1173, which was labeled on the mexL coding strand. Labeled DNA fragments were treated with DNase I in the absence of MexL-His6 (–MexL) or in the presence of increasing amounts (5 µM, 7.5 µM, and 10 µM) of MexL-His6 (+MexL). The nucleotide sequence of the protected area is indicated to the right of the autoradiographs. Inverted repeats are marked with arrows, and the predicted –10 and –35 regions of the mexJ and mexL promoters are boxed. Sequencing ladders of the same DNA templates are also shown.

Several attempts were made to map the mexJ transcription start sites using either primer extension, rapid amplification of cDNA ends, or RNase protection, but all attempts failed. However, the mexL transcription start was successfully mapped using RNase protection (data not shown). The labeled riboprobe covered the mexL-mexJ intergenic region and started 147 bp downstream of the mexL initiation codon. The protected area was 176 bp in length, which corresponded to a transcriptional start at a T residue located 29 bp upstream of the mexL start codon. This transcription start site was verified by reverse transcription-PCR on P. aeruginosa PAO238 total RNA using a common primer annealing within the expected mexL transcript and a second primer either annealing inside (3') or outside (5') of the start of the expected mexL transcript. The common primer yielded a PCR product only in combination with the inside primer (data not shown).

Autoregulation of mexL was ascertained by gene fusion analysis (Table 4). To do this, pPS1237 carrying a mexL'-lacZ transcriptional fusion and pPS1245 containing the wild-type mexL gene under Plac control were electroporated either alone or in combination into {Delta}(mexLJK) strain PAO314. ß-Gal activity measurements revealed that mexL'-lacZ transcription was reduced approximately sixfold in the presence of MexL. Similar results were obtained when the same plasmids were analyzed in E. coli HPS1, but the repression by MexL was only approximately twofold (data not shown). These data verified that MexL negatively autoregulates its own expression.


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  		TABLE 4. Autoregulation of mexL expressiona

The predicted mexJ promoter contains a –35 TTGAAA region, which is close to the TTGACA consensus sequence, but its –10 region TGTATT shares only 3 of 6 bases of the TATAAT consensus. The –10 and –35 regions of the mexL promoter are far from those of the consensus sequence, but this promoter location was confirmed by mapping the mexL transcript start site and regulators are often weakly expressed. Although the involvement of the GTATTT hexanucleotide sequences in MexL binding has not yet been directly proven, their central localizations within the protected regions and overlap with the mexL and mexJ promoter regions strongly supports their involvement in MexL binding and thus regulation of mexJ and mexL transcription.

Mapping of the mexJ promoter. Since we failed to obtain the mexJ transcriptional start site(s) using conventional mapping technologies, transcriptional lacZ gene fusions carrying various portions of the mexL-mexJ intergenic regions were constructed and used to approximate the location of the mexJ promoter. The various portions of the mexL-mexJ intergenic region contained in the mexJ'-lacZ fusion plasmids are shown in Fig. 5. The fusion plasmids were electroporated into {Delta}(mexLJK) strain PAO314, and ß-Gal activities were measured. Cells containing either vector pTZ120 or pPS1201, which contains the –244 to –71 region devoid of obvious promoter sequences, did not show any detectable ß-Gal activity. In contrast, cells containing pPS1204, which carries the entire intergenic region from positions –165 to +36, showed the highest activity, followed by cells containing pPS1236 (–86 to –37) and pPS1209 (–90 to +113), which exhibited significant levels of ß-Gal activities. MexL, coexpressed from a compatible plasmid, repressed mexJ'-lacZ transcription from pPS1204, pPS1209, and pPS1236. Plasmids pPS1202 (–41 to +133) and pPS1210 (–71 to +113) directed expression of lower levels of ß-galactosidase activity; however, expression of this activity was not repressed by MexL. In summary, these experiments narrowed the mexJ promoter and its associated regulatory sequences to a region located between –86 and –37 nucleotides upstream of mexJ, and the results are consistent with the results of footprinting experiments.



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  		FIG. 5. Localization of the mexJ promoter within the mexL-mexJ intergenic region using lacZ transcriptional fusions. PCR fragments containing the portions of mexL-mexJ intergenic region indicated by the boxes were cloned in front of a promoterless lacZ on pTZ120 to form the respective mexJ'-lacZ fusion plasmids pPS1201 to pPS1210; pPS1236 was obtained by ligating a synthetic oligonucleotide encompassing the sequences between positions –86 and –37 into pTZ120. The plasmids were transformed into {Delta}(mexLJK) strain PAO314, and ß-Gal activities were measured in triplicate samples of cells grown on M9 medium plus glucose. For expression of MexL, cells were cotransformed with pPS1245. Sequences are arbitrarily numbered relative to the first nucleotide of the mexJ start codon. The averages ± standard deviations (error bars) from a representative experiment are shown.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant AI051588.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682. Phone: (970) 491-3536. Fax: (970) 491-1815. E-mail: Herbert.Schweizer{at}colostate.edu. Back


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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.



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