Laboratory of Microbial Interactions, Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute of Biotechnology, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium1
Author for correspondence: Pierre Cornelis. Tel: +32 2 3590221. Fax: +32 2 3590399. e-mail: pcornel{at}vub.ac.be
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ABSTRACT |
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Keywords: Mex-type efflux pump, quorum sensing, N-acyl-homoserine lactone production
Abbreviations: AHL, N-acyl-homoserine lactone; BHL, N-butanoyl-L-homoserine lactone; OdDHL, N-(3-oxododecanoyl)-L-homoserine lactone; Cm, chloramphenicol; Gm, gentamicin; Tc, tetracycline
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INTRODUCTION |
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In this study, we describe one P. aeruginosa mutant with an increased susceptibility to V, with an insertion upstream from a previously undescribed efflux pump, with similarity to Mex efflux systems from P. aeruginosa known to contribute to antibiotic resistance. We also show that this new MexGHI-OpmD pump plays a role in the quorum sensing network of P. aeruginosa.
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METHODS |
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DNA sequencing was performed by Eurogentec, using universal primers. Sequences obtained were compared to sequences present in the database of the Pseudomonas genome (http://www.pseudomonas.com) using the BLASTX algorithm.
Mutagenesis by allelic exchange.
From P. aeruginosa strain PAO1 (Stover et al., 2000 ) a 2·6 kb sequence between genes PA4204 and PA4205 was amplified by PCR using primers 4204 and 4205 (Table 2
). This fragment was ligated to pCR2.1 using the TA cloning kit (Invitrogen). The recombinant plasmid was further restricted with SacII and the ends polished by treatment with Klenow polymerase. The Gm cassette from the pBBR-1-MCS vector (Kovach et al., 1994
) was amplified with Taq polymerase (Gibco-BRL) using the primers gent1 and gent2 (Table 2
), and cloned into the pCR2.1 vector, resulting in plasmid pGM. The Gm cassette was removed from plasmid pGM by NotI restriction, the fragment was blunt-ended and then ligated to the SacII-digested plasmid. After transformation, the resulting plasmid containing the Gm-interrupted DNA was isolated from a recombinant cloned and digested by HindIII and BclI to liberate the DNA fragment containing the Gm cassette. This fragment was in turn ligated to HindIII/BamHI-restricted pBR325 DNA (Bolivar, 1978
) before transformation of E. coli DH5
or S17-1. Transformed E. coli S17-1 was used to mobilize the suicide plasmid to P. aeruginosa PAO1 (Simon et al., 1983
). Selection for double recombination events was done by plating the recombinants on CAA plates containing Gm (100 µg ml-1), and Gm plus chloramphenicol (Cm) (200 µg ml-1), and selecting the clones that were sensitive to Cm. Confirmation of the double recombination event in these clones was done by PCR amplification using primers 4205upf and 4205upr (Table 2
), since double recombination should result in the amplification of a unique band of 1·3 kb.
The P. aeruginosa PAO1 opmD gene (PA4208) was amplified by PCR using primers opmD1 and opmD2 (Table 2). The resulting fragment was ligated into vector pCR2.1 (Invitrogen).
After restriction with EcoN1, the Gm cassette was ligated into the opmD gene. The complete DNA fragment with the inserted Gm cassette was liberated by restriction with EcoRI and ligated to EcoRI-restricted pBR325. Similarly, the mexI gene (PA4207) was amplified using primers mexI1 and mexI2 (Table 2). After StuI restriction, the Gm cassette was ligated into mexI. The complete fragment was liberated from pCR2.1 by restriction with HindIII and BclI, and ligated to HindIII/BamHI-restricted pBR325. After transformation of E. coli GJ23, the plasmids were transferred by conjugation to P. aeruginosa PAO1. Transconjugants showing the loss of Cm resistance (200 µg ml-1) were selected and the double recombination confirmed by PCR using the above described primers.
RT-PCR analysis.
RNA was extracted (High Pure RNA Isolation Kit; Roche Diagnostics) from P. aeruginosa PA 59.20 wild-type, PA 59.20 vs1, PAO1 wild-type and PAO1 ncr mutants grown in LB and harvested in the middle of the exponential phase. cDNA, synthesized using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia) was done using two sets of primers: mexI3 and mexI1, and opmD3 and opmD4 (Table 2). The relative position of the primers is shown in Fig. 1(A)
. As a control for RNA contamination by DNA, the PCR reaction was done on the same samples, without first strand cDNA synthesis.
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Vanadyl oxydo sulfate (VOSO4.5H2O) was prepared as a 100 mM stock solution and kept at 4 °C. Growth curves were also followed in the presence of increasing concentrations (0·5, 1, 1·5, 2 and 2·5 mM) of different metal salts in CAA medium at 37 °C: CoCl2, ZnSO4, NiCl2, CuSO4, CrCl3 and Bi(NO3)3. CdCl2 was used at 0·2 mM, final concentration.
Antibiotic susceptibility.
Antimicrobial susceptibility tests were performed by the disk diffusion method on MuellerHinton agar plates. The antibiotics tested (Sanofi Diagnostic Pasteur) included amikacin (30 µg), ticarcillin-clavulanate (75/10 µg), tobramycin (10 µg), tetracycline (Tc; 30 UI), imipenem (10 µg), netilmicin (5 µg), ciprofloxacin (5 µg) and colistin (10 µg). Zones of inhibition were measured after incubation at 37 °C for 24 h.
Production of elastase.
Overnight LB-grown cultures were centrifuged and the supernatants collected. One-hundred microlitres of culture supernatant were added to glass test tubes containing 10 mg elastin Congo Red ml-1 in 0·9 ml 0·1 M Tris/HCl. After 6 h incubation at 37 °C, the tubes were centrifuged and the OD495 of the supernatants were measured (Rust et al., 1994 ). All measurements were repeated three times.
Production of rhamnolipids.
M9-glutamate minimal medium agar plates containing 0·2 g cetyltrimethylammoniumbromide and 5 mg methylene blue l-1 were inoculated with 10 µl of an overnight LB culture of P. aeruginosa strains. After an overnight incubation at 37 °C, the diameter of the clearing zone around the bacterial spots was measured as evidence of rhamnolipid production (Siegmund & Wagner, 1991 ).
Swarming motility.
Two microlitres from overnight agitated cultures in LB (5 ml) were spotted on LB plates containing 1·3 % agarose. The plates were incubated overnight at 37 °C and photographed. Spreading bacteria indicated swarming from the inoculation spot.
Measurement of siderophore and pyocyanine production.
The pyoverdine content in the supernatant was determined by measuring A400 or by spectrofluorimetry (excitation at 405 nm, emission at 460 nm, using a Shimadzu spectrofluorimeter) and normalized by a biomass unit expressed as OD600 of the culture (Höfte et al., 1993 ). Measurements are means of three independent experiments.
Pyocyanine production was visualized by plating the bacteria on Pseudomonas P agar (Difco), followed by 48 h incubation. Pyocyanine production caused the medium to be coloured deep blue. Pyocyanine was measured in culture supernatants according to Mavrodi et al. (2001) .
Bio-assay for acyl-homoserine lactone (AHL) production.
As indicator strain, a Chromobacter violaceum CV026 AHL-deficient mutant was used as described by McClean et al. (1997) . TLC was used to separate the AHLs and overlaid with the CV026 indicator strain as described by McClean et al. (1997)
. Total culture supernatants from overnight-agitated cultures grown at 37 °C in LB were extracted with dichloromethane as described by McClean et al. (1997)
to extract the AHLs.
Complementation in trans with wild-type DNA.
A genomic bank of PAO1 DNA was constructed as described previously (Lim et al., 1997 ) in the wide-host range cosmid pRG930Cm (van den Eede et al., 1992
), using PstI to partially digest the genomic DNA. The bank was screened by colony blotting using the 2·6 kb PCR fragment amplified using primers 4204 and 4205 as probe. The Roche Diagnostics non-radioactive Dig detection system was used, in combination with the chemiluminescent substrate CPD-star (Roche Diagnostics). Positive clones were confirmed by PCR analysis using the following primers: opmD3 and opmD4, 4207f and 4207r, 4206f and 4206r, and 4205upf and 4205upr (Table 2
).
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RESULTS |
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Localization of the place of transposon insertion in PA 59.20 vs1
DNA from P. aeruginosa PA 59.20 vs1 was isolated and the sequence of the DNA flanking the transposon insertion was determined as described in Methods. After the nested PCR reaction, a fragment of about 600 bp was obtained, which was cloned in pCR2.1 and sequenced. The sequence analysis revealed 94% identity for a 171 bp sequence with a PAO1 sequence (http://www.pseudomonas.com) found between genes PA4204 and PA4205 (Fig. 1A, B
). According to the PAO1 genome annotation, gene PA4205 encodes a hypothetical integral membrane protein with four predicted transmembrane helices. The predicted ATG start codon for this protein is at position 4705955, but is not preceded by a convincing ribosome-binding site (AAGGA, Fig. 1B
). An alternative ATG start codon is present, 128 nt downstream, in the same frame, just after a ribosome-binding site closer to the consensus (GGAGG, Fig. 1B
). From this new start codon, an ORF extends that encodes a protein of 105 aa containing three transmembrane domains (1028, 3555, 6485) (Zhai & Saier, 2001
). Gene PA4206 encodes a putative RND efflux protein of 370 residues (Tseng et al., 1999
), while gene PA4207 encodes a protein with highest similarity to P. aeruginosa MexF, a member of the family of membrane fusion proteins (Zgurskaya & Nikaido, 2000
). The last protein, encoded by gene PA4208, is most similar to the P. aeruginosa efflux porin OprN (Köhler et al., 1997
). After this gene cluster, and transcribed in the opposite orientation, is gene PA4209 that has recently been shown to correspond to the phzM gene encoding an O-methyltransferase necessary for the biosynthesis of pyocyanine (Mavrodi et al., 2001
; Pattery et al., 2001
). This gene cluster, with the exception of gene PA4205, is very similar to the already described complete tripartite gene clusters that encode the MexABOprM, MexCDOprJ and MexEFOprN systems, which function in drug efflux in P. aeruginosa (Poole, 2001a
, b
).
Generation of new mutants by allelic exchange
Since the transposon was inserted in a non-coding region where its presence could eventually have positively influenced the transcription of downstream genes, it was decided to create a mutant in P. aeruginosa PAO1 with a Gm cassette insertion in the same locus, but downstream of the original Tn5 mutation. This was done by insertion of a Gm cassette into the unique SacII site (Fig. 1B). The double recombinant PAO1 ncr mutant had exactly the same phenotype as the original vs1 mutant in strain PA 59.20. This mutant could not grow in CAA or in LB medium in the presence of 1·5 mM VOSO4, and showed reduced growth in the presence of 1 mM VOSO4 (Fig. 2
).
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Complementation of the PAO1 ncr, mexI and opmD mutants
A PAO1 DNA genomic bank was constructed by cloning partially PstI-digested DNA into the wide host-range cosmid vector pRG930Cm (van den Eede et al., 1992 ; S. Matthijs, unpublished results). About 3500 colonies were screened by colony hybridization using as probe the 2·5 kb PCR fragment amplified with primers 4204 and 4205. One positive clone was selected and was confirmed by PCR amplifications with primers designed to amplify the mexG, mexH, mexI and opmD genes. Based on the results of these amplifications, we confirmed that the insert contained the genes PA42004209 (Table 1
and Fig. 1A
). Subclones were obtained that contained only mexG, or mexG-mexH, or mexG-mexH-mexI (Table 1
and Fig. 1A
). These subclones, as well as the original cosmid, were transferred by conjugation to PAO1 ncr, mexI or opmD and the transconjugants were tested for growth in the presence of VOSO4. From the results, it was clear that only recombinants containing mexG-mexH-mexI-opmD could grow in the presence of 1 mM V, indicating that the complete pump is needed to confer resistance to the metal (results shown only for opmD complemented with mexG-mexH-mexI-opmD in Fig. 2
).
RT-PCR analysis of the mexGHI-opmD transcript
To confirm the operon organization of the cluster and to evaluate the effect of the two mutations in the intergenic region (the original transposon insertion in strain PA 59.20 and the PAO1 ncr mutation) on transcription efficiency, we did an RT-PCR analysis on mRNAs extracted from wild-type PA 59.20, PA 59.20 vs1, PAO1 and PAO1 ncr. Different primer combinations were used (Fig. 1A). Transcripts were clearly less abundant in the PA 59.20 vs1 transposon mutant and in the PAO1 ncr mutant compared to the wild-type (see Fig. 3a
for mexI primers, Fig. 3b
for opmD primers). When amplifications were performed using the opmD primer combinations, almost no transcript could be amplified from the two mutant mRNA preparations.
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DISCUSSION |
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One of the first obvious phenotypes of our V-sensitive mutants was the clearly reduced production of the phenazine pigment pyocyanine. One possibility is that pyocyanine excretion is directly mediated by this pump. In this regard, it is worth noting that phzM, a gene recently demonstrated to be necessary for the biosynthesis of pyocyanine, is transcribed just downstream of opmD, in the opposite orientation (Mavrodi et al., 2001 ; Pattery et al., 2001
). Another more likely explanation for the decreased pyocyanine production is that decreased extracellular release of AHLs by the pump mutants results in decreased transcription of the phz phenazine biosynthesis cluster. Indeed, we observed that different, well known, quorum-sensing-regulated traits were affected in our different ncr, mexI and opmD mutants, while the presence of the pump in trans restored the wild-type phenotype in all instances. P. aeruginosa is known to produce two major AHL signal molecules, one long-chain, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and one short-chain, N-butanoyl-L-homoserine lactone (BHL). The C. violaceum assay is known to detect short-chains AHLs and, conversely, to be inhibited by long-chain homoserine lactones, including OdDHL (McClean et al., 1997
). The fact that total AHL fractions from the mexI and opmD mutants failed to stimulate the production of violacein by C. violaceum may be due to an imbalance in the BHL/OdDHL ratio. If the proportion of long-chain AHLs is higher in these mutant extracts, an inhibition of the biosensor is likely to occur (McClean et al., 1997
). Supporting this hypothesis is the fact that one stimulating AHL can be detected in the extracts of these mutants when separated first by TLC. This AHL spot is the same as one that is detected by TLC separation of wild-type PAO1 extracts and is likely to be BHL. However, only a more thorough analysis of AHL production in these mutants by HPLC will definitively answer this question. It is interesting to note that the opmD mutant is also affected in its growth in CAA medium. Its morphology is also different on LB plates since it does not form isolated colonies but grows in patches (results not shown). Also, this mutant aggregates in liquid cultures (results not shown). These observations indicate that OpmD is an important outer-membrane porin for P. aeruginosa and that it cannot be replaced by another member of the OprM porin family (http://www.cmdr.ubc.ca/bobh/OprMfamily.html). Recently, Köhler et al. (2001)
showed that overproduction of the MexEF-OprN efflux pump results in decreased production of virulence factors dependent on RhlI-RhlR, including pyocyanine and rhamnolipids. Two groups, Evans et al. (1998)
and Pearson et al. (1999)
, reached opposite conclusions concerning the influence of the MexAB-OprM pump in regard to quorum sensing. The first group found that strains overexpressing mexAB-oprM excrete less OdDHL while the opposite was observed by Pearson and co-workers. The difference between the two observations could be explained by the fact that Evans and co-workers worked with a strain that is a nalB mutant overexpressing mexAB-oprM, while Pearson and co-workers worked with a PAO1 strain that was not mutated for nalB. Köhler et al. (2001)
propose that another quorum sensing molecule, 2-heptyl-3-hydroxy-4-quinolone, termed PQS (Pesci et al., 1999
; Holden et al., 2000
), could be the substrate of MexEF-OprN. PQS is indeed known to positively regulate the rhl system (McKnight et al., 2000
). Interestingly, Whiteley et al. (1999)
previously identified the mexGHI-opmD gene cluster (called qsc133) as being regulated by quorum sensing, in response to both BHL and OdDHL. With this in mind, we looked for putative lux boxes in the intergenic region between genes PA4204 and PA4205. Interestingly, two sequences that match the consensus sequence NNCT-(N12)-AGNN, as defined by Whiteley & Greenberg (2001)
, were found. Although no conclusions can be taken at this stage concerning their localization compared to the transcription start point, it is tempting to assume that one would be a site for binding of LasR while the other would be a sequence where RhlR would bind. In their analysis, Whiteley et al. (1999)
did not find a putative lux box upstream of qsc133, but they did not, at that time, take the PA4205 gene into account.
Curiously, the V-sensitive mutants did not display a decreased resistance to any of the antibiotics that we tested. Conversely, we observed an increased resistance towards Tc, ticarcillin plus clavulanic acid, and netilmicin. It has been shown that loss of mexAB-oprM expression correlates with an increased expression of mexEF-oprN and mexCD-oprJ (Li et al., 2000 ). It is therefore possible that decreased expression of mexGHI-opmD could be compensated by increased expression of other pumps, such as mexCD-oprJ that is known to confer resistance to Tc, Cm and some cephems (Poole et al., 1996
). The presence of the full mexGHI-opmD in trans results in increased resistance to ticarcillin and clavulanic acid. One possible explanation for this phenomenon is that clavulanic acid (itself a lactone) is excreted by the pump. This, however, will need experimental confirmation. It will therefore be necessary in the future to study the expression of mexGHI-opmD and its interplay with other efflux systems in P. aeruginosa.
While this paper was being reviewed, an article was published (Diggle et al., 2002 ) where a Tn5 insertion in mexI is described as causing a reduction of a lecA::lacZ fusion expression. The lecA gene encodes a lectin that is regulated positively by the RhlIRhlR system (Winzer et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 6 December 2001;
revised 25 April 2002;
accepted 2 May 2002.