Transcriptional regulation of mexR, the repressor of Pseudomonas aeruginosa mexAB-oprM multidrug efflux pump

FEMS Microbiology Letters 207 (2002) 63^68

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Transcriptional regulation of mexR, the repressor of Pseudomonas aeruginosa mexAB-oprM multidrug e¥ux pump Patricia Sa¨nchez, Fernando Rojo, Jose¨ L. Mart|¨nez * Departamento de Biotecnolog|¨a Microbiana, Centro Nacional de Biotecnolog|¨a (CSIC), Campus Universidad Auto¨noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 9 September 2001; received in revised form 19 November 2001 ; accepted 29 November 2001 First published online 10 January 2002

Abstract The transcription start site of mexR, encoding the repressor of the Pseudomonas aeruginosa mexAB-oprM multidrug efflux pump, has been determined by S1 mapping. One signal corresponding to a single promoter has been found, whereas three major signals were observed for the mexA messenger. Further analysis demonstrated that mexA has just one promoter that overlaps with the mexR promoter, with the other two signals observed by S1 probably being the consequence of RNA processing. Transcription of mexR and mexA from the aforementioned promoters is regulated by MexR. We show that bacterial growth phase affects expression of these promoters as well. mexR expression was higher at the exponential growth phase and declined afterwards, whereas mexA expression was triggered at the onset of the stationary growth phase. A model for the regulation of mexR and mexA expression, which includes an analysis of the interplay between both promoters, is proposed. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Multidrug resistance; E¥ux pump; Antibiotic resistance; Gene expression

1. Introduction Antibiotic-resistant bacteria constitute one of the most dangerous problems in current medical practice [1], namely Pseudomonas aeruginosa, being a conspicuous example of antibiotic-resistant nosocomial pathogen [2]. This bacterial species is intrinsically resistant to a wide range of drugs, and mutants with increasing levels of resistance are easily selectable both in vitro [3,4] and in vivo [5,6]. Intrinsic antibiotic resistance in Gram-negative bacteria is due to an interplay between low outer membrane permeability and the expression of multidrug resistance (MDR) e¥ux pumps. Three proteins compose MDR systems in Gram-negative bacteria: an inner-membrane protein, a periplasmic protein, and an outer-membrane protein [7].

* Corresponding author. Tel : +34 (91) 5854571; Fax: +34 (91) 5854506. E-mail address : [email protected] (J.L. Mart|¨nez).

It has been described that the MexAB-OprM e¥ux pump plays an important role for the intrinsic antibiotic resistance of P. aeruginosa [8]. The genes encoding the synthesis of such pump constitute an operon [9], the expression of which is usually down-regulated. The negative regulator of mexAB-oprM expression is MexR [10]. The mexR gene is located upstream of mexA, and is transcribed divergently to the operon. Mutations in mexR (nalB mutants) lead to overproduction of MexAB-OprM, and to increased levels of antibiotic resistance in clinical P. aeruginosa isolates [6,11]. It has been recently demonstrated that puri¢ed histidine-tagged MexR binds the mexA^mexR intergenic region [12] at two sites that contain each one an inverted repeat of the pentamer GTTGA [13]. The inverted repeats are localised in regions that have been suggested to contain the promoters for mexR and mexAB-oprM [13]. It has been suggested that mexA may have one inducible promoter [12] or two promoters, one constitutive and one inducible [13]. In the present work, we analyse the regulation of mexR transcription, and present data supporting that mexA has just one inducible promoter. The growth-phase regulation of mexR expression as well as the interplay between mexR and mexA expression were also explored.

0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 6 2 - 6

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2. Materials and methods 2.1. Bacterial strains and growth conditions The P. aeruginosa wild-type strain ML5087 and its isogenic nalB mutant strain K1112 [14] were obtained from Dr. K. Poole. The Escherichia coli strain CC118Vpir [15] was obtained form Dr. V. de Lorenzo. The E. coli strains HB101 and TG1 [16] were from our laboratory collection. Bacteria were routinely grown in Luria broth [16] at 37³C. 2.2. RNA analysis Total RNA from P. aeruginosa ML5087 and K1112 was obtained using the guanidine thiocyanate-based Tri Reagent-LS (Molecular Research Center Inc.), according to manufacturer's instructions. Residual DNA was removed by treatment with RNase-free DNaseI (Boehringer Mannheim), followed by phenol extraction. RNA concentration and purity was estimated by measuring UV optical density at 260 and 280 nm [16] For Northern analysis, 25 Wg of total RNA was electrophoresed on 1% agarose under denaturing conditions (formaldehyde/formamide procedure [16]) and transferred to Hybond-N‡ (Amersham) membranes. RNA molecular mass markers were from Boehringer Mannheim. Membranes were stained with 0.02% methylene blue in 0.3 M sodium acetate pH 5.2 to verify that RNA amounts in each lane were comparable. Membranes were subjected to overnight hybridisation and subsequent washings under stringent conditions [16] at 55³C with a mexR probe. To generate such probe, the mexR gene was ampli¢ed from genomic P. aeruginosa DNA (obtained as described [17]) with primers mexR1 (5P-CGCCATGGCCCATATTGAG3P) and mexR2 (5P-GGCATTCGCCAGTAAGCGG-3P). The PCR reaction mixture (0.2 mM of each deoxynucleotide triphosphate, 0.5 WM of each primer, 1.5 mM MgCl2 , 10 mM Tris^HCl,, pH 8.3, 50 mM KCl, 1 U Taq DNA polymerase and 100 ng of chromosomal DNA) was heated for 3 min at 94³C, followed by 32 cycles of 60 s at 94³C, 60 s at 58³C, 90 s at 72³C, and ¢nally one 10-min extension step at 72³C. The PCR product obtained was puri¢ed with Micro Bio-Spin chromatography columns (Bio-Rad) and labelled with [K-32 P]dCTP using the Ready-To-GoDNA Labelling Kit-dCTP (Pharmacia Biotech), according to manufacturer's instructions. S1 nuclease reactions were performed as described [16] using 40 Wg of total RNA and an excess of 32 P-labelled single-stranded DNA (ssDNA) probe hybridising to the 5Pregion of the mRNA to be analysed. The DNA template used the for generation of ssDNA probes was obtained by PCR, under the same conditions as described above but with an annealing temperature of 55³C, and using the primers mexA1 (5P-CAATACATGGACGTCGGG3P) and mexA2 (5P-CGCCTCGCTTTTTCCGCCAC-3P),

which amplify the ¢rst 489 nucleotides of the L11616 sequence containing the intergenic region between mexA and mexR. ssDNA probes were thus generated by linear PCR, using the same conditions described above, but including only one primer (10 pmol) in each reaction and 500 ng of puri¢ed mexA^mexR intergenic DNA template. The primers were 5P-32 P end-labelled with 16 U of polynucleotide kinase (New England Biolabs) and 100 WCi of [Q-32 P]ATP (Amersham) as described. The S1 probe for mapping the transcription start of mexR was obtained using the mexA1 primer. The annealing temperature in the ssDNA PCR reaction was 55³C. The probe for mapping the transcription start of mexA was obtained using the mexA14 primer (5P-GCATAGCGTTGTCCTCATGAGCG-3P). The annealing temperature in the ssDNA PCR reaction was 58³C. The probes were puri¢ed from 6% polyacrylamide^urea denaturing gels by the crush-and-soak method [16]. 5U104 cpm of the probe were used for each S1 reaction. The S1 reactions were run in 6% polyacrylamide^urea denaturing gels. A G+A ladder, obtained by chemical degradation of the ssDNA probes [16], was included as molecular size marker. RT-PCR analysis of mexA expression was carried out by means of a two-step reaction. First, 5 Wg of RNA was incubated for 1 h at 37³C with primer mexA10 (5P-CCAGCAGCTTGTAGCGCTGG-3P) and avian myeloblastosis virus (AMV) reverse transcriptase (USB) to obtain cDNA. cDNA was then PCR-ampli¢ed using primers mexA7 (5P-CCTGCTGGTGCGGATTTCGG-3P) and mexA8 (5P-GGCGCTCTGGTAGTCGGCCT-3P) as described above with an annealing temperature of 55³C. Amplicons were analysed on 1.5% agarose gels, and stained with ethidium bromide. To detect any spurious DNA contamination in the RNA preparation, the same reaction protocol as performed with RT-PCR was carried out for each sample, but without AMV reverse transcriptase. 2.3. Cloning of mexR and mexA promoters The mexA^mexR full-length intergenic region was obtained by PCR using mexA1 and mexA2 primers as described above. The putative distal mexA promoter was obtained by PCR as described above, using the primers mexA1 and mexA12 (5P-TCGCGTGAAAACACCTGA3P). The putative proximal mexA promoter was obtained by PCR as described above, using the primers mexA2 and mexA9 (5P-TCAGGTGTTTTCACGCGA-3P). In both cases, PCR was performed at the annealing temperature of 50³C. PCR products were puri¢ed as described above, cloned in the pGEMT-Easy vector (Promega) and recovered as EcoRI fragments. These fragments were cloned in opposite orientations in the promoter-reporter plasmid pUJ8 [18]. Sequencing of pUJ8-derived plasmids by the method of Sanger [16] with primer puj8 (5P-TTGTACT-

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GAGAGTGCACC-3P) con¢rmed the orientations of PmexR: :lacZ and the PmexA: :lacZ fusions.

Table 1 L-Galactosidase activity of di¡erent regions of the mexR^mexA intergenic region fused to a reporter lacZ gene

2.4. L-Galactosidase activity

Promoter region

L-Galactosidase activity (%)a

mexA P1+P2 mexA P1 mexA P2 mexA P2b mexR mexRb

32.9 þ 0.9 2.3 þ 1.7 39.9 þ 0.9 0.0 þ 0.8 100 þ 1.9 134 þ 4.1

L-Galactosidase activity was measured as described [19], and expressed as the percentage of activity referred to the promoter that showed higher levels. Determinations were made in E. coli TG1, harbouring the pUJ8-derived plasmids, after overnight growth in M9 minimal medium [16] with 0.2% glucose as the carbon source and containing ampicillin 100 Wg ml31 . Since pUJ8 presents by itself a certain residual L-galactosidase activity [15], this background was subtracted in all determinations. 3. Results and discussion 3.1. The mexR promoter S1 mapping of mexR mRNA indicates that it has just one transcriptional start site lying close to the 5P-end of the mexR gene (Fig. 1). The position of this transcription start site agrees with the predicted position of mexR promoter [13]. The mexR transcription start site is clearly detectable in the nalB mutant, and is also detectable,

P1: Proximal mexA region; P2: distal mexA region. a The activity is referred to the mexR promoter. b Mutant promoter, see text.

although at lower level, in the wild-type strain ML5087. This indicates that a single promoter drives expression of the mexR gene, and the promoter is de-repressed in a MexR-de¢cient strain. Considering that the MexR binding sites [13] overlap the detected mexR promoter, our results clearly show that mexR expression is autoregulated at a transcriptional level from a single promoter. To further analyse the proposed mexR promoter, a transcriptional fusion of the intergenic region to the reporter lacZ gene was obtained in the appropriate orientation. As shown in Table 1, the activity of this fusion was even higher than that of the mexA promoter (see below). This con¢rms the results obtained by S1 nuclease protection assays showing that the mexR promoter lies close to the 5P-end of the mexR gene. 3.2. The mexA promoter

Fig. 1. Localisation of the transcription start site of mexR. The position of the transcription start site of mexR was determined by S1 mapping in the wild-type P. aeruginosa strain ML5087 and the nalB mutant strain K1112. One signal (more intense in the nalB mutant) was observed in both strains. M: A G+A ladder of the ssDNA probe was used as the molecular size marker. The transcription start site is shown in grey in the ¢gure.

Recently published works suggested that mexA could have either one [12] or two (one inducible an one constitutive) promoters [13]. Thus, before going further in the analysis of mexR transcription, we tried to clarify whether mexA has one or two promoters. Three putative transcription starts were detected by S1 mapping for the mexA RNA (Fig. 2). The signals were detectable both in the wild-type ML5087 strain and in the nalB mutant K1112, being much more intense in the latter one. The relative intensity of the three signals was similar in the wild-type strain and in the nalB mutant. This indicates either that they correspond to di¡erent MexR-repressed promoters or that they are processed forms from a single transcript originated from a single MexR-responsible promoter. It has been suggested [13] that mexA has two promoters. One might correspond to the upper signal we detect by S1 mapping, and believed to be only active in the MexRde¢cient strains [13]. The second, closer to mexA [10,13], has been suggested to be a constitutive promoter, responsible for mexA expression in wild-type P. aeruginosa strains [13]. We have detected that the larger transcript is observed not only in the nalB mutant, but also in the wild-type strain, which indicates that it contributes to the expression of mexA in both strains. Fine S1 mapping of mexA transcripts showed that the mexA signal corre-

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Fig. 3. Structure of the mexR and mexA promoters. The positions of the transcription start sites are shown in white letters with a dark grey background. The MexR-binding sites [13] are highlighted in bold and with grey arrows. The position of the 335 and 310 regions are highlighted in italics and with black arrows. Note that the 335 regions of the mexR and mexA promoters overlap with the putative MexR-binding inverted repeats [13]. The C^T mutation that abolished the mexABoprM activity is outlined. The position of the earlier suggested mexA transcription start [13] is indicated with an asterisk.

Fig. 2. Localisation of the transcription start sites of mexA. The position of the putative start sites of mexA was determined by S1 mapping in the wild-type P. aeruginosa strain ML5087 and the nalB mutant strain K1112. Three main signals were observed in both strains, the three being more intense in the nalB mutant. M: A G+A ladder of the ssDNA probe was used as the molecular size marker. The positions of the origin of the transcripts was determined by ¢ne S1 mapping and is shown in grey in the ¢gure.

sponding to the larger transcript was composed by two bands, suggesting either RNA degradation, or the presence of two transcription start sites separated by ¢ve nucleotides (Fig. 2). These sites are very close to one of the previously suggested starts [13] of mexA transcription (the C labelled with an asterisk in Fig. 3). The discrepancies in the position of the transcription starts relative to those reported earlier [13] might probably be due to the di¡erent methodologies used: S1 protection assays in our case, and rapid ampli¢cation of cDNA ends (RACE) in reference [13], since it has recently been reported that mapping of bacterial mRNA transcripts by RACE might produce inaccurate results because of premature termination of the cDNA synthesis [20]. Expression of the smaller transcript is not constitutive (Fig. 2). Furthermore, the origin of the smaller transcript maps inside the 310 region of the previously proposed constitutive mexA promoter [13]. Although this region lacks a MexR binding site [13], the S1 signals seem to be regulated by MexR (they are more intense in the nalB mutant). We think that these smaller transcripts are probably the consequence of the processing of the larger one, and that they do not come from more proximal bona ¢de promoters.

To ascertain if only the distal region had promoter activity, or if both regions had a role in driving transcription of mexA, we made transcriptional fusions of di¡erent fragments of the intergenic region to the reporter gene lacZ. The fusions were made in a multicopy plasmid and introduced in the heterologous host E. coli TG1, so that even low-level promoter activities could be detectable. E. coli does not have a mexR gene, so the repressor activity of MexR would not interfere with the analysis. The results of these experiments are shown in Table 1. The distal region drove the expression of the reporter lacZ gene to the same level than the full intergenic region, whereas the proximal region did not show any relevant activity. These results agree with previously published data [12], and support the notion that mexA has just one promoter, which overlaps with the MexR binding sites close to the 5P-end of mexR [13]. The other bands detected by S1 mapping (Fig. 1) and RACE assays [13] must be the result of mexA RNA processing.

Fig. 4. Growth-phase regulation of mexR and mexA expression in P. aeruginosa. The expression of mexR at di¡erent stages of the growth phase was determined by Northern blot (panel A). A hybridisation signal with a size of ca. 550 bp was observed both in the wild-type strain and the nalB mutant. The intensity of the signal was higher at mid-exponential phase in both strains. In panel B, the expression of mexA in the wild-type strain ML5087 was analysed by RT-PCR. The intensity of the signal increased at late exponential phase. In both cases, ee : early exponential (OD600 = 0.2), me : mid-exponential (OD600 = 0.4), le: late exponential (OD600 = 0.8), st: stationary (overnight).

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3.3. Growth-phase regulation of mexR expression

4. Conclusions

The expression of MDR pumps is usually down-regulated. However, they can be de-repressed in response to environmental signals or to the bacterial physiological state. To gain some insight on the physiological regulation of mexR expression, the e¡ect of growth phase on its transcriptional regulation was studied. Northern blot analysis showed that the amount of mexR RNA was higher at midexponential phase and declined during stationary growth phase, both in the wild-type strain P. aeruginosa ML5087 and in the nalB mutant K1112, although the amount of mRNA was much higher in the MexR-de¢cient mutant (Fig. 4A). The size of the RNA detected in the blot agrees with the position of the mexR transcriptional start previously determined by S1 mapping (Fig. 1). Using a fusion of the mexA promoter region to the phoA reporter gene, it had previously been described that mexA transcription increased in stationary growth phase [21]. RT-PCR analysis of mexA RNA (Fig. 4B) con¢rmed that mexA transcription is triggered at the end of exponential phase, a moment in which mexR RNA levels start to decrease. It could be therefore suggested that MexR might have a role on mexR and mexA growth-phase regulation. However, mexR growth-phase regulation was observed as well in a mexR-null mutant (Fig. 4A). This indicates that MexR is not involved in mexR growth-phase regulation, a situation previously described for mexA [21].

Data presented in this article allow to get a clearer picture of the regulation of mexR and mexA expression. Our results indicate mexA has most probably one single promoter responsible for the low-expression of the operon observed in the wild-type strains, and which is de-repressed in the MexR-de¢cient mutant. Expression of mexR messenger RNA is driven by a single promoter that overlaps with the mexA promoter and with the MexR binding boxes. Then, mexR and mexA transcription can be regulated at di¡erent levels. First, the RNA polymerase should compete with the MexR repressor for binding, since both mexR and mexA 335 regions overlap with the putative MexR binding sequences (Fig. 3). Second, mexR and mexA promoters should compete for the binding of the RNA polymerase, so that mutations that increase the activity of one of the promoters will reduce the activity of the other one (Table 1). Third, expression of mexR and mexA is triggered at di¡erent stages of growth phase. This growth-phase regulation appears to be MexR-independent, indicating that additional factor(s) must be involved in controlling mexR and mexA expression.

3.4. Interplay between mexR and mexA promoters Our results show that mexR and mexA promoters overlap, and both overlap as well with the previously de¢ned MexR binding sites [13] (see Fig. 3). In the course of the cloning of the mexA promoter, we obtained a mutant promoter that was unable to drive the expression of the reporter lacZ gene in E. coli. Sequencing of this mutant promoter demonstrated that the mutation that abolished the promoter activity was a T^C change seven nucleotides downstream from the ¢rst mexA transcription start (Fig. 3). The ¢rst 20 nucleotides of transcribing RNAs might contribute to the strength of some promoters, however the dramatic e¡ect we observed is di¤cult to explain, unless some other factors are involved. The T^C mutation in the mexA strand leads to an A^G change in the mexR strand (Fig. 3). This change lies within the 335 mexR box. Analysis of lacZ expression driven by the mexR mutant promoter indicated that it is stronger than the wild-type (Table 1). These results suggest that this mutation might favour RNA polymerase binding to the 335 mexR box in the mexR strand. Such binding might compete with the progression of the transcription of the mexA operon in the mexA strand, so that the mutant mexA promoter will be inactive as the consequence of a higher activity of the mexR promoter.

Acknowledgements Thanks are given to Dr. Keith Poole for the gift of the strains P. aeruginosa ML5087 and K1112 used in the present work. This work has been supported by Grants BIO98-0808 and BIO2000-0939. P.S. is a recipient of a fellowship from Ministerio de Educacio¨n y Cultura.

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