JOURNAL OF BACTERIOLOGY, Sept. 2001, p. 5213–5222 Vol. 183, No.

18
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.18.5213–5222.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Overexpression of the MexEF-OprN Multidrug Efflux System

Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa
THILO KÖHLER,* CHRISTIAN VAN DELDEN, LASTA KOCJANCIC CURTY,
MEHRI MICHEA HAMZEHPOUR, AND JEAN-CLAUDE PECHERE
Department of Genetics and Microbiology, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland
Received 2 April 2001/Accepted 12 June 2001

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated
mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type

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mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors
than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled
by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the
decreased transcription of the elastase gene lasB and the rhamnosyltransferase genes rhlAB measured in nfxC
type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In
contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by
50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL
levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be
partially restored by the addition of synthetic C4-HSL and Pseudomonas quinolone signal (PQS). It is proposed
that the MexEF-OprN efflux pump affects intracellular PQS levels.

Pseudomonas aeruginosa is an opportunistic pathogen which lowing antibiotic therapy. Recent investigations in several lab-
may cause pneumonia and bacteremia in immunocompro- oratories have demonstrated that both intrinsic and acquired
mised hosts and is responsible for chronic destructive lung resistance is caused mainly by active efflux systems which effi-
disease in patients suffering from cystic fibrosis. The pathoge- ciently expel antimicrobial compounds without any apparent
nicity of P. aeruginosa is attributable to an arsenal of virulence structural similarity. So far, four genetically distinct efflux sys-
factors, some of which are cell associated (pili, nonpilus ad- tems have been characterized for P. aeruginosa. They are sim-
hesins, lipopolysaccharide, and alginate) while others are se- ilar in genetic and structural organization but differ in sub-
creted (proteases, rhamnolipids, exotoxin A, exoenzyme S, and strate specificity and regulation. The MexAB-OprM system
pyocyanin). The production of many of these extracellular vir- (22, 42) has the broadest substrate spectrum of all bacterial
ulence factors is controlled by two cell-to-cell signaling sys- efflux pumps described so far, including quinolones, tetracy-
tems, called las and rhl, which are both composed of a tran- cline, chloramphenicol (20), trimethoprim (17), ␤-lactam an-
scriptional regulator (LasR and RhlR, respectively) and an tibiotics (21), ␤-lactamase inhibitors (24), and detergents and
autoinducer synthase (LasI and RhlI, respectively). LasI and solvent molecules (23). The transcriptional repressor MexR
RhlI catalyze the last step in the synthesis of the cell-to-cell (43) keeps expression of the mexAB-oprM operon at a low
signaling molecules 3-oxo-C12-homoserine lactone (3-oxo- constitutive level, but one sufficient to contribute significantly
C12-HSL) and C4-HSL, respectively; each of these molecules to the elevated intrinsic antibiotic resistance of this organism.
binds to, and activates, its corresponding transcriptional regu- A second efflux system, MexCD-OprJ (41), is responsible for
lator. The systems are connected via a hierarchical cascade
efflux of quinolones, erythromycin (29), and cephalosporins
(19) and allow coordinated production of extracellular viru-
(12, 27). Its expression is totally repressed by the transcrip-
lence factors, which occurs only when the bacterial cell density
tional regulator NfxB (35, 49). The third efflux pump, MexEF-
has reached a threshold (quorum). Recently, a novel signaling
OprN, transports chloramphenicol as well as quinolones, is
molecule, called PQS, for Pseudomonas quinolone signal (39),
overexpressed in nfxC type mutants (18), and is positively reg-
has been identified. Furthermore, the published genome se-
ulated by the transcriptional activator MexT (16). Recently, a
quence of PAO1 (53) has revealed a new modulator of cell-
fourth efflux system of P. aeruginosa, called MexXY, has been
to-cell signaling, termed QscR (4). This protein is homologous
cloned into Escherichia coli, on which it conferred resistance to
to both LasR and RhlR and seems to prevent premature tran-
scription of quorum-sensing regulated genes. quinolones and erythromycin (31). This efflux system was sub-
Besides its pathogenic capabilities, P. aeruginosa is well sequently shown to be involved in the intrinsic resistance of P.
known for its intrinsic resistance to a wide range of antimicro- aeruginosa to aminoglycosides and erythromycin (45).
bial agents and its ability to develop multidrug resistance fol- nfxC type mutants were originally isolated from P. aerugi-
nosa strain PAO4009 after exposure to the quinolone norfloxa-
cin (9). These mutants displayed cross-resistance to other quin-
olones but also to nonquinolone antibiotics such as imipenem
* Corresponding author. Mailing address: Department of Genetics
and chloramphenicol. The nfxC locus was mapped to 46 min on
and Microbiology, CMU, 9, av. de Champel, CH-1211 Geneva 4,
Switzerland. Phone: 41-22-7025655. Fax: 41-22-7025702. E-mail: Thilo the PAO1 chromosome (9), near the catA gene, which is lo-
[email protected]. cated within 15 kb of the mexEF-oprN operon (18). Mutations

5213
5214 KÖHLER ET AL. J. BACTERIOL.

TABLE 1. Bacterial strains, plasmids, and bacteriophage used in this study

Strain, plasmid, or bacteriophage Relevant characteristicsa Source or reference

Strains
E. coli
MC1061 F⫺ araD139 ⌬(ara-leu)7696 galE15 galK16 ⌬(lac)X74 rpsL thi Laboratory collection
S17-1 thi pro hsdR recA chr::RP4-2 51
DH5␣ endA1 hsdR17(rk⫺mk⫹) glnV44 thi-1 recA1 gyrA relA1 ⌬(lacIZYA-argF)U169 Laboratory collection
deoR [␾80dlac⌬(lacZ)M15]
MG4␭I14 ␭ lysogen carrying a chromosomal lasI::lacZ fusion 48

P. aeruginosa
PAO-BI PAO1 wild type 10
PAO-R1 PAO-BI⌬lasR Tcr 10
JP2 PDO100⌬lasI Tcr Hgr 37
JP3 PDO111⌬lasR Tcr Hgr 37

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PT5 PAO1 wild type Laboratory collection
PT121 PT5mexE::⍀Hg (formerly PAOmexE) Hgr 18
PT149 PT5nfxC (formerly PAO-7H); overproduces MexEF-OprN 18
PT466 PT5⌬lasI Tcr 15
PT498 PT5⌬lasR Tcr 15
PT454 PT5⌬rhlI::Tn501 Hgr 15
PT462 PT5rhlR::Tn501 Hgr 15
PT531 PT5rhlR::Tn501 ⌬lasRI Tcr Hgr This study
PT469 PT149⌬lasI; transduced from PAO-JP1 Tcr This study
PT456 PT149⌬rhlI::Tn501; transduced from PDO100 Hgr This study
PT464 PT149rhlR::Tn501; transduced from PAO-JP3 Hgr This study
PT500 PT149rhlR::Tn501; ⌬lasR Tcr Hgr This study
PT509 PT149⌬lasR; transduced from PAO-JP3 Tcr This study
PT637 PT149mexE::⍀Hg; transduced from PT121 Hgr This study

Plasmids
pTS400 lasB::lacZ on pSW205; Apr 36
pECP60 rhlAB::lacZ on pSW205; Apr 40
pPCS1001 lasR::lacZ on pLP170; Apr 40
pPCS223 lasI::lacZ on pLP170; Apr 56
pPCS1002 rhlR::lacZ on pLP170; Apr 40
pMAL-I rhlI::lacZ on pMP220; Tcr 19
pMP220 Promoterless lacZ fusion vector; Tcr 52
pECP61.5 rhlAB::lacZ, ptac::rhlR; Apr 37
pLP170 Promoterless lacZ fusion vector; Apr 44
pSW205 Promoterless lacZ fusion vector; Apr 11
pEZ5 mexE::lacZ on pSW205; Apr This study

Bacteriophage E79tv2 Transducing phage 32

a
Resistance phenotypes: Hg, mercury; Tc, tetracycline; Ap, ampicillin.

which lead to overexpression of the MexEF-OprN pump have ticularly those controlled by the rhl system. Evidence is pre-
recently been shown to result from variations in the transcrip- sented that the PQS (39) is involved in this response.
tional activator gene mexT (26). NfxC is therefore to be con-
sidered a phenotype, since overexpression of the MexEF- MATERIALS AND METHODS
OprN pump might result from mutations which are not Bacteria, media, and growth conditions. Bacterial strains and plasmids are
necessarily linked to mexT (26) (T. Köhler and J. L. Dumas, listed in Table 1. E. coli and P. aeruginosa were routinely grown in Luria-Bertani
(LB) broth supplemented when necessary with antibiotics at the following con-
unpublished data). centrations, in milligrams per liter: gentamicin, 15; ampicillin (for E. coli), 100;
We previously showed that an nfxC type mutant which over- carbenicillin, 250 (for P. aeruginosa); tetracycline, 10 (for E. coli) or 50 (for P.
expressed the MexEF-OprN efflux operon produces about 20 aeruginosa); mercury chloride, 12.5. For analysis of exoproducts and autoinduc-
ers in culture supernatants and for lacZ fusion experiments, P. aeruginosa strains
times less pyocyanin than the isogenic wild-type strain (18).
were grown as follows. Strains to be tested were streaked from ⫺80°C glycerol
Since pyocyanin is a typical secondary metabolite whose pro- stocks on selective LB agar plates. Single colonies were inoculated into 5 ml of
duction is controlled by the rhl cell-to-cell signaling system (2), PB (2% Bacto Peptone [select peptone 140; Gibco-BRL], 1.4 g of MgCl2/liter,
we decided to investigate the production of other virulence 10 g of K2SO4/liter) (6) supplemented with antibiotics where appropriate. Cul-
tures were grown overnight at 37°C with agitation in 50-ml flasks. One milliliter
factors in nfxC type mutants. Our results show that overexpres-
of this overnight culture was centrifuged and resuspended in 1 ml of fresh PB.
sion of the MexEF-OprN efflux pump is correlated with a From this suspension, 25 ml of prewarmed PB without antibiotics was inoculated
decrease in production of extracellular virulence factors, par- 1:100 and grown in 250-ml flasks with agitation. Defined media were based on
VOL. 183, 2001 EFFLUX SYSTEMS AND VIRULENCE IN PSEUDOMONAS AERUGINOSA 5215

TABLE 2. Exoproduct assays and resistance profiles in P. aeruginosa multidrug efflux and quorum-sensing mutants
Exoproduct activitya MIC of:
Strain Genotype
Pyocyanin Elastase Ciprofloxacin Chloramphenicol Imipenem
b
PT5 wt 100 100 0.125 32 1
PT149 nfxC 5 42 1 1,024 4
PT454 rhlI 3 ND 0.125 32 1
PT462 rhlR ⬍1 4 0.125 32 1
PT466 lasI 80 ND 0.125 64 1
PT498 lasR 45 6 0.125 32 1
PT531 rhlR lasR ⬍1 4 0.125 32 1
PT637 nfxC mexE ND 88 0.125 32 4
PAO-BI nfxC ND 45 1 1,024 4
PAO-RI nfxC lasR ND ⬍1 1 1,024 4

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a
Expressed as a percentage of the activity in the wild-type strain PT5. Pyocyanin activities were determined in supernatants of cultures grown for 18 h in PB medium.
ECR assays were performed on supernatants of cultures grown for 10 h in PB medium. At this point the cultures were in stationary phase, and the OD600s of the cultures
were 5 ⫾ 0.5. ND, not determined.
b
wt, wild type.

M9 salts (25) supplemented with 2 mM MgSO4 and 0.4% glucose. For phage (ECR) method (56). Five milligrams of ECR (Elastin Products Company,
transductions, donor strains were grown in LB broth and recipient strains were Owensville, Mo.) was used per assay. Triplicate samples were analyzed for each
resuspended in TNM medium (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM time point. Pyocyanin was determined in culture supernatants as described pre-
MgSO4). viously (6).
Strain and plasmid constructions. Mutations in the cell-to-cell signaling reg- ␤-Galactosidase assays. Cultures were grown at 37°C with agitation as de-
ulator genes were transferred into the PAO1 wild-type strain PT5 and the nfxC scribed above. Triplicate 100-␮l samples were taken to determine the optical
mutant PT149 using the transducing phage E79tv2 (32). The genotype of the density at 600 nm (OD600) and ␤-galactosidase activity (30). For complementa-
strains was verified by Southern hybridization as described previously (15). The tion assays with autoinducers, strains were grown overnight in PB medium. Cells
mexE gene was inactivated in strain PT149 by using bacteriophage E79tv2 (32) to were resuspended in M9-based medium supplemented with 0.2% glucose (vol/
transduce the mexE::⍀Hg mutation from strain PT121 (called PAOmexE in vol), 2 mM MgSO4, trace elements, and 0.05% (vol/vol) tryptophan instead of
reference 18). In all of the 12 transductants analyzed, wild-type antibiotic sus- NH4Cl as an N source. C4-HSL was added from a dimethyl sulfoxide stock
ceptibility to the efflux pump substrates was restored. However, 4 of the 12 solution. PQS was synthesized as described previously (39)and dissolved in di-
transductants remained imipenem resistant. One of these strains, called PT637, methyl sulfoxide.
was shown by sequencing to contain a full-length mexT open reading frame Autoinducer bioassays. Aliquots (3 ml) were taken at different time points
(ORF) (see Results). The mexE-lacZ fusion pEZ5 was constructed by ligating a during growth in PB medium and centrifuged, and the supernatants were filtered
1.8-kbp BglII-EcoRV fragment from plasmid pNFZ4 (16) into BamHI-EcoRV- (pore size, 0.22 ␮m). Aliquots (2 ml) were extracted twice with 2 ml of ethyl
cleaved pSW205 (11). acetate (containing 0.01% acetic acid). The extracts were kept at ⫺20°C. Ali-
DNA-manipulations. Plasmids were introduced into P. aeruginosa by electro- quots of the ethyl acetate extract were evaporated, and the dried residue was
poration or by triparental mating using pRK2013 as a helper plasmid (8). resuspended directly in 1 ml of the bioassay strain culture. The E. coli bioassay
Genomic DNA was isolated as described previously (1). PCR amplification was strain was grown in M9 glucose medium supplemented with 0.001% thiamine,
performed by using 100 ng of genomic DNA as a template. PCR mixtures 1% LB medium, 50 ␮g of ampicillin/ml, and 1 mM isopropyl-␤-D-thiogalactopy-
contained primers at 0.1 ␮mol, 2.5 mM deoxynucleoside triphosphates, and 2 U ranoside (IPTG) when required. The P. aeruginosa bioassay strain JP2
of Taq polymerase (Appligene, Illkirch, France) in a total volume of 50 ␮l. (pECP61.5) was grown in LB medium. Incubation and ␤-galactosidase determi-
Reaction mixtures were subjected to an initial 1-min denaturation step at 95°C, nations were performed as described above.
followed by 25 cycles of 30 s at 95°C, 30 s at 55°C, and 2 min at 72°C, with a final
5-min elongation at 72°C. Amplification of rhlI with primers RhlI-P30 (5⬘-CCA
TCATCCTGAGCATCTCCAGAGAGC-3⬘) and RhlI-M6 (5⬘-GGAATGACTT RESULTS
CGGCATGGCGACTCC-3⬘) yielded a 1,074-bp fragment, and amplification of
rhlR with primers RhlR-P4 (5⬘-CGGCGTTTCAATGGAATTGTCACAACC- Production of extracellular virulence factors is affected in
3⬘) and RhlR-M5 (5⬘-GGCGGCATCCCTACCCTGATACTCCC-3⬘) yielded a nfxC type mutants. We previously observed that the nfxC type
1,109-bp fragment. PCR products were run on Tris-acetate-EDTA gels (1.2%
mutant PT149 produced 20-fold less pyocyanin than the wild-
agarose) and then purified using a Qiagen gel extraction kit. The mexT DNA
region was amplified using primers nfxC-P1 (5⬘-TCTCGCACGCAAGGCTTG type strain PT5 (18). We therefore tested the production of
ACG-3⬘) and nfxC-M2 (5⬘-TCCCACTCGTTCAGCGGTTGTTC-3⬘). PCR con- other extracellular virulence factors in these strains and com-
ditions were as follows: 1 min at 95°C, followed by 25 cycles of 30 s at 95°C, 20 s pared their levels to those of isogenic lasR, lasI, rhlR, and rhlI
at 52°C, and 2 min at 72°C, with a final 5-min elongation at 72°C. DNA sequences mutants. As expected, the nfxC type mutant and the two rhl
were determined from double-stranded templates according to the dideoxy chain
termination method (47) using an automatic sequencer (model 377A; Applied
mutants showed drastically reduced production of pyocyanin,
Biosystems). while the lasR and lasI mutants still secreted substantial
Qualitative plate assays. Rhamnolipid production was estimated by inoculat- amounts of pyocyanin (Table 2). Elastase activity was reduced
ing strains on M9-based agar plates supplemented with 0.2% glucose (vol/vol), 2 by more than 50% in the nfxC mutant, while the rhlR and lasR
mM MgSO4, trace elements, 0.05% (vol/vol) glutamate (unless otherwise stated)
mutants showed only marginal activity in culture supernatants
instead of NH4Cl as an N source, 0.0005% (vol/vol) methylene blue, and 0.02%
(vol/vol) cetyltrimethylammonium bromide (50). Plates were incubated first at after 10 h of growth in PB medium (Table 2).
37°C for 24 h and then for at least 48 h at room temperature until a blue halo We then compared the production of rhamnolipids using a
appeared around the colony. Swarm plates were prepared and inoculated as standard plate assay. As a reference, we included the PAO1
described previously (15). Incubation was carried out for 18 h at 37°C. wild-type strain (PAO-BI) (10) and its lasR derivative,
Quantitative exoproduct assays on culture supernatants. Samples of 0.5 ml
were taken at various time points during growth in PB, centrifuged (at 8,000 ⫻
PAO-R1 (10). The nfxC mutant PT149 showed strongly re-
g for 5 min), and filtered (pore size, 0.22 ␮m). Filtrates were immediately frozen duced rhamnolipid production (Fig. 1). Surprisingly the wild-
and kept at ⫺80°C. Elastolytic activity was determined by the elastin Congo red type strain PAO-BI also showed reduced rhamnolipid produc-
5216 KÖHLER ET AL. J. BACTERIOL.

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FIG. 2. Swarming was tested on M8-based minimal medium (M9
FIG. 1. Rhamnolipid plate assay. Strains to be tested were grown medium without NH4Cl), supplemented with 0.2% glucose and 0.05%
for 8 h in LB medium, and 2 ␮l was spotted on the plate. Tyrosine at glutamate as the sole nitrogen source and solidified with agar to a final
a final concentration of 0.05% was used as the nitrogen source. Incu- concentration of 0.6%. Strains were inoculated by using a toothpick,
bation was carried out for 24 h at 37°C and then for 72 h at room and plates were incubated at 37°C for 18 h.
temperature. The presence of a dark halo around the colony indicates
production of rhamnolipids.

nfxC mutants are deficient in swarming. We (15) and others

(46) recently demonstrated the swarming motility of P. aerugi-
tion. Unlike PAO-R1, the lasR mutant PT498 constructed in nosa on semisolid agar plates. Swarming was shown to depend
the PT5 background was still able to produce rhamnolipids. As on rhamnolipids as biosurfactants (15). We therefore tested
the nfxC mutant PT149 on swarm plates. While the wild type
expected, the rhlR mutant PT462 (Fig. 1) was completely de-
showed normal swarming behavior, both the nfxC mutant and
ficient in rhamnolipid production as shown previously in other
strain PAO-BI were unable to swarm (Fig. 2). This is in agree-
wild-type backgrounds (2, 33). We therefore verified the resis-
ment with the finding that rhamnolipid production was de-
tance profiles of PAO-BI and PAO-R1. Indeed, both strains
creased in these strains.
were resistant to chloramphenicol, ciprofloxacin, and imi-
Efflux pump overexpression is responsible for decreased
penem, a phenotype reminiscent of our nfxC mutant PT149
virulence factor production. MexEF-OprN is positively regu-
(Table 2). Furthermore ␤-galactosidase levels expressed from
lated by the transcriptional activator MexT (16), located up-
a mexE-lacZ fusion were similar in PAO-BI and the nfxC
stream of the efflux operon. In agreement with the findings of
mutant PT149 (data available upon request). This strongly Maseda et al. (26), we recently found that the nfxC mutant
suggests that strains PAO-BI and PAO-R1 are nfxC mutants PT149 contains a full-length mexT ORF, while in the wild-type
(see also below). strain PT5, the mexT ORF is interrupted by an 8-bp insert
As expected, the rhlR lasR double mutant PT531 was com- (CGGCCAGC), resulting in a truncated MexT protein (Fig.
pletely deficient in production of all exoproducts tested (Table 3). This means that wild-type strains which do not express the
2). MexEF-OprN efflux pump may encode an inactive mexT gene,
To rule out the possibility that a particular mutation in the while nfxC type mutants express a functional mexT gene. As
nfxC mutant strain PT149, which had been selected previously expected from the phenotype, we found that strain PAO-BI
on ciprofloxacin (strain PAO-7H in reference 18), was respon- encodes a functional mexT gene, whose complete sequence is
sible for the decrease in virulence factor production, new nfxC identical to that of the mexT gene in our nfxC mutant PT149
type mutants were selected by plating the wild-type strain PT5 (data available on request).
on LB agar plates containing chloramphenicol at 600 ␮g/ml, a To determine whether possible pleiotropic effects of the
condition which exclusively selects nfxC type mutants (T. functional MexT protein or overexpression of the MexEF-
Köhler, unpublished data). Fifty spontaneous independent OprN efflux pump per se was responsible for the decrease in
Cmr colonies were analyzed. All of them were cross-resistant virulence factor production, the MexEF-OprN pump was in-
to quinolones and imipenem, as expected for nfxC type mu- activated in the nfxC type mutant PT149 by insertion of an
tants. All 50 colonies showed drastically decreased production ⍀Hg cassette into the mexE gene. The resulting strain, called
of rhamnolipids in the plate assay, demonstrating the link PT637, expressed a full-length mexT ORF and was susceptible
between the nfxC phenotype and exoproduct synthesis. to the pump substrates chloramphenicol and ciprofloxacin but
VOL. 183, 2001 EFFLUX SYSTEMS AND VIRULENCE IN PSEUDOMONAS AERUGINOSA 5217

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FIG. 3. Alignment of a partial DNA sequence of mexT from the wild-type strain PT5, the nfxC mutant PT149, and strain PAO-BI. The 8-bp
insert inactivating the mexT ORF is boldfaced. The entire mexT gene was sequenced for all strains, and no other mutation was found.

remained resistant to imipenem. This is in agreement with

previous observations demonstrating that imipenem resistance
is independent of MexEF-OprN overexpression (see the last
row of Table 1 in reference 18) but results from decreased
expression of the porin OprD. Strain PT637 was used in sub-
sequent experiments as a means of distinguishing between
phenotypes related to MexEF-OprN pump overexpression and
those related to other MexT-mediated effects. Indeed, LasB
activities, as measured by elastase production (Fig. 4A), were
restored to wild-type levels in strain PT637, and both rhamno-
lipid production and swarming ability were comparable to
those of the wild type (Fig. 1 and 2). We therefore concluded
that overexpression of the MexEF-OprN efflux pump is solely
responsible for the decrease in virulence factor production in
the nfxC type mutant PT149.
Mutations in the cell-to-cell signaling regulators do not
affect expression of the MexEF-OprN efflux system in the nfxC
type mutant. Since virulence factor production is affected in
nfxC mutants, we asked whether the cell-to-cell signaling reg-
ulators were required for expression of the MexEF-OprN ef-
flux system in the nfxC mutant PT149. For this purpose lasI,
lasR, rhlI, and rhlR knockout mutations were transduced into
PT149. The resistance profiles of the resultant mutants PT469
(nfxC lasI), PT509 (nfxC lasR), PT456 (nfxC rhlI), PT464 (nfxC
rhlR), and PT500 (nfxC rhlR lasR) were compared with those of
PT5 and PT149 on antibiotic gradient plates. All of the cell-
to-cell signaling mutants showed the same susceptibilities to
pefloxacin, chloramphenicol, and imipenem as the parental
strain PT149 (data not shown). We further introduced plasmid
pEZ5, carrying a mexE-lacZ fusion, into the nfxC type mutants
PT149, PT464, PT500, and PT509 and measured ␤-galactosi-
dase activities during exponential growth. In all four strains
similar ␤-galactosidase activities (170 ⫾ 25 Miller units) were
obtained. This clearly establishes the quorum-sensing-indepen-
dent regulation of the mexEF-oprN operon.
Expression of the elastase (lasB) and rhamnosyltransferase
(rhlAB) genes is affected in nfxC type mutants. To further FIG. 4. (A) ECR assay. Elastase production was determined on
analyze the mechanism of extracellular virulence factor pro- filtered culture supernatants of strains grown in PB. Determinations
were performed on three different occasions. Results from one typical
duction of the nfxC type mutant PT149, LasB elastase activity experiment are shown. (B) Expression of the lasB gene was monitored
was recorded over time and compared to the expression of a during growth in PB using a lasB::lacZ fusion carried on plasmid
plasmid-encoded lasB-lacZ fusion. In both the wild type and pTS400. Growth, expressed as theOD600, was monitored (inset).
5218 KÖHLER ET AL. J. BACTERIOL.

the nfxC type mutant, LasB activity and lasB expression started
to appear at an OD600 of 2. However, LasB activity and lasB
expression continued to increase at a lower rate in the nfxC
type mutant, although growth was comparable to that of the
wild type (Fig. 4). Hence, the reduced elastase production in
strain PT149 results from decreased expression of the lasB
gene.
Expression of the rhlAB operon encoding rhamnosyltrans-
ferase was assayed using the translational rhlAB-lacZ fusion
carried by plasmid pECP60. rhlAB expression was determined
after 18 h of incubation in M8 medium supplemented with
0.2% glucose and 0.05% glutamate as the sole nitrogen source.
While the wild-type PT5 yielded 1,976 ⫾ 10 Miller units, the

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nfxC type mutant PT149 and the rhlR mutant PT462 yielded
269 ⫾ 11 and 59 ⫾ 2 Miller units, respectively. These results
suggest that the drastically reduced rhamnolipid production in
strain PT149 (Fig. 1) is caused by a strong reduction in rhlAB
transcription.
Expression of cell-to-cell signaling regulator genes in the
nfxC mutant. Both lasB and rhlAB are controlled by the las and
rhl cell-to-cell signaling systems. We therefore introduced lacZ
fusions carried on plasmids to the lasR, lasI, rhlR, and rhlI
genes into PT5 and PT149 in order to determine whether their
expression was altered, which could account for the decreased
elastase and rhamnolipid production in the nfxC mutant. Both
lasR(pPCS1001) and lasI(pPCS223) expression reached similar
levels in the wild type and the nfxC mutant (Fig. 5). The
expression of lasR increased in both strains during early sta-
tionary phase, as previously reported with the pPCS1001
lasR::lacZ fusion in strain PAO1 (40). In contrast, the expres-
sion of lasI was constant and even decreased slightly in both
strains when stationary phase was reached (Fig. 5B). This sur-
prising expression profile is very likely due to the absence on
pPCS223 of the rsaL gene, encoding the recently described FIG. 5. ␤-Galactosidase activities expressed from lasR::lacZ (A)
and lasI::lacZ (B) fusions were determined during growth in PB.
inhibitor of lasI expression (5). In the absence of multiple Growth, expressed as the OD600, was monitored (inset). Experiments
copies of the RsaL repressor, expression of lasI is already at a were repeated on three different occasions. Error bars represent stan-
maximum during the exponential-growth phase and therefore dard deviations of triplicate LacZ determinations for one typical ex-
does not display an induction profile typical of other genes periment. Where error bars are not shown, the standard deviation was
regulated by the cell-to-cell signaling system. Importantly, the within the size of the symbol. Arrows indicate the end of exponential
growth. The antibiotic phenotypes of the strains at the end of the
nfxC mutation did not affect the expression of lasI compared to experiment were determined on pefloxacin-containing gradient plates.
that in the wild type strain. The LacZ activities of control plasmids pSW205 and pLP170 were
Expression of the regulator gene rhlR(pPCS1002) also in- approximately 3 and 400 Miller units, respectively, and remained fairly
creased after the exponential-growth phase in both the wild constant during growth.
type and the nfxC mutant and remained comparable even
during stationary phase (Fig. 6A). Surprisingly, a significant
difference was found when expression of rhlI(pMAL-I) was
determined. Indeed, in the nfxC type mutant PT149, rhlI tran- concentration of 10 ⫾ 0.6 ␮M, the PT149 supernatants con-
scription was drastically decreased and reached only 35% of tained 3.5 ⫾ 0.7 ␮M C4-HSL (Fig. 7A). As expected, no
wild-type levels in stationary phase (Fig. 6B). We subsequently C4-HSL was detectable in the rhlI mutant PT454 (data not
sequenced the rhlR-rhlI region in strains PT5 and PT149. How- shown). C4-HSL levels determined in supernatants of strain
ever, no differences were found between the two strains, sug- PT637 (nfxC mexE) were comparable to those for the wild type
gesting that the observed effect on rhlI expression in the nfxC (data available on request), suggesting again that MexEF-
type mutant PT149 does not result from mutations in the OprN overexpression is solely responsible for the decreased
rhlR-rhlI regulatory region. amounts of C4-HSL in the nfxC type mutants. When the con-
Autoinducer production in culture supernatants. Since the centrations of the 3-oxo-C12-HSL autoinducer were deter-
transcription of the autoinducer synthase gene rhlI was affected mined, we found increased concentrations in supernatants of
by the mexEF-oprN expression level, production of the C4- strain PT149 at ODs above 4 (Fig. 7B). This suggests that
HSL autoinducer was determined in culture supernatants of MexEF-OprN may contribute to the secretion of the hydro-
PT5 and PT149 and compared to that in the rhlI mutant phobic 3-oxo-C12-HSL molecule, as was previously shown for
PT454. While the wild-type supernatants reached a C4-HSL the MexAB-OprM pump (7, 38).
VOL. 183, 2001 EFFLUX SYSTEMS AND VIRULENCE IN PSEUDOMONAS AERUGINOSA 5219

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FIG. 6. ␤-Galactosidase activities expressed from rhlR::lacZ (A)
and rhlI::lacZ (B) fusions were determined during growth in PB.
Growth, expressed as the OD600, was monitored (inset). Error bars FIG. 7. Autoinducer concentrations determined in culture super-
represent standard deviations of triplicate LacZ determinations for natants. C4-HSL (A) and 3-oxo-C12-HSL (B) were determined using
one typical experiment. Where error bars are not shown, the standard the bioassay strains JP2(pECP61.5) and MG4␭I14, respectively.
deviation was within the size of the symbol. Arrows indicate the end of
exponential growth.
of 50 ␮M had no significant effect on the transcription of the
rhlAB fusion in any of the three strains (Table 3). In contrast,
Complementation of the nfxC mutant with exogenous auto- the presence of 10 ␮M C4-HSL alone increased the transcrip-
inducers. The results described above show an effect of tion of rhlAB about sixfold, but only in the nfxC mutant. Sur-
MexEF-OprN overexpression on rhlI transcription and hence
on the levels of C4-HSL produced. For full induction, the rhl
system requires the 3-oxo-C12-HSL autoinducer and a recently TABLE 3. Complementation of the rhlAB::lacZ fusion
identified novel regulator molecule, PQS (39). We therefore with C4-HSL and PQS
tested the effects of 3-oxo-C12-HSL, C4-HSL, and PQS (kindly
␤-Gal activitiesa (Miller units)
synthesized by the group of U. Burger, Faculty of Chemistry,
Signal added PT5 PT149 PT464
University of Geneva, Geneva, Switzerland) on rhamnolipid
(pECP60) (pECP60) (pECP60)
production using the plate assay. Addition of 3-oxo-C12-HSL
alone or in combination with either PQS or C4-HSL did not None 23,298 ⫾ 4,318 268 ⫾ 10 168 ⫾ 45
affect rhamnolipid production. In contrast, addition of either C4-HSL (10 ␮M) 21,512 ⫾ 274 1,716 ⫾ 484 133 ⫾ 24
PQS (50 ␮M) 16,258 ⫾ 3,453 249 ⫾ 4 103 ⫾ 11
C4-HSL alone or C4-HSL and PQS increased rhamnolipid C4-HSL (10 ␮M) ⫹ PQS
production (data not shown). Hence, we tested the effects of (50 ␮M) 21,058 ⫾ 4,674 4,073 ⫾ 146 129 ⫾ 24
the three signaling molecules on expression of the rhlAB-lacZ a
Cultures were grown for 3 h in the absence of signal; after signal was added,
fusion in strain PT5, in the nfxC mutant PT149, and in the rhl cultures were incubated for 18 h and ␤-galactosidase (␤-Gal) activity was deter-
nfxC double mutant PT464. PQS alone at a final concentration mined. Values are means from two independent experiments done in triplicate.
5220 KÖHLER ET AL. J. BACTERIOL.

prisingly, when both molecules were present at these concen- secretion of 3-oxo-C12-HSL, while deletion of mexAB-oprM
trations, the transcription increased 15-fold in the nfxC mutant. resulted in decreased release of this autoinducer, suggesting
When 3-oxo-C12-HSL (final concentration, 5 ␮M) was added that this hydrophobic molecule is actively secreted by the
to the two other signaling molecules, the expression of rhlAB MexAB-OprM efflux pump.
did not increase further. These results strongly suggest that the Our finding that strain PAO-BI is an nfxC type mutant also
levels of both C4-HSL and PQS are affected by mexEF-oprN allows us to explain discrepancies between several laboratories
overexpression. Since rhlAB transcription in the rhlR nfxC mu- working in the field of quorum sensing. While it was observed
tant was not influenced by the addition of autoinducers, it can that the lasR mutant PAO-R1 was deficient in rhamnolipid
be concluded that the partial complementation observed in the production (37), several investigators showed substantial rh-
nfxC mutant requires the presence of the RhlR regulator. amnolipid production in lasR mutants constructed in other
In conclusion, our work provides evidence that overexpres- strain backgrounds (2, 33). Our findings suggest that the las
sion of the MexEF-OprN multidrug efflux pump reduces the system has only a marginal effect on rhamnolipid production.
production of virulence factors controlled mainly by the rhl Therefore, the strongly reduced rhamnolipid production in

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cell-to-cell signaling system (pyocyanin and rhamnolipids) and strain PAO-R1 is mainly due to its NfxC phenotype.
that this reduction results from decreased rhlI transcription How can we explain the effect of MexEF-OprN overexpres-
and decreased C4-HSL autoinducer production. sion on the rhl quorum system? One possibility is that autoin-
ducers are substrates of the MexEF-OprN efflux pump, in
DISCUSSION which case the overexpression of this pump could lead to
decreased intracellular autoinducer concentrations and hence
P. aeruginosa is known for its ability to develop resistance to diminished production of virulence factors. In the nfxC type
a number of structurally unrelated antibiotics, a phenomenon mutant, we observed increased amounts of 3-oxo-C12-HSL at
which can now be attributed predominantly to chromosomal OD600 values above 4, suggesting that 3-oxo-C12-HSL could
mutations leading to overexpression of multidrug efflux sys- also be a substrate for the MexEF-OprN efflux pump, as sug-
tems. P. aeruginosa also produces a series of exoproducts, sev- gested for MexAB-OprM (7, 38). On the other hand, super-
eral of which, such as elastase, alkaline protease, exotoxins, natants of the nfxC type mutant contained about 60% less of
and pyocyanin, have been shown to be virulence factors (3, 54, the second autoinducer, C4-HSL, than those of the wild-type
55). In this study, we show a link between the active efflux strain. Short-chain autoinducers like 3-oxo-C6-HSL of Photo-
system MexEF-OprN and the production of virulence factors bacterium fischeri (14) and C4-HSL of P. aeruginosa apparently
regulated by the las (10, 11, 19) and rhl (2, 33) cell-to-cell (38) diffuse freely across the bacterial cell membrane. It is
signaling systems. This important finding suggests that P. therefore unlikely that an efflux system, such as the MexEF-
aeruginosa strains becoming resistant to multiple antibiotics by OprN pump, is involved in active export of C4-HSL. Our data
overexpression of MexEF-OprN are likely to be less virulent. support the conclusion that the reduced amounts of C4-HSL
Indeed, we recently found that nfxC mutants exhibit signifi- produced by the nfxC type mutant are the result of altered rhlI
cantly reduced virulence both in a nonmammalian system and expression. Indeed, rhlI transcription levels in PT149 were
in a rat model of acute pneumonia (P. Cosson et al., submitted reduced to 50% of those in the wild type, while rhlR transcrip-
for publication). tion levels were unaffected. Since sequencing of the rhlR-rhlI
The connection between multidrug resistance and virulence DNA region obtained from the nfxC type mutant did not
factor production was previously suggested in a study compar- reveal any mutation, it seems likely that altered expression or
ing 18 multidrug-resistant P. aeruginosa clinical samples col- activity of another regulatory element required for rhlI expres-
lected in a Japanese hospital. All multidrug-resistant strains sion might be involved in the nfxC type mutant. The existence
were deficient in production of pyoverdine, pyocyanin, elas- of such a regulator of the rhl operon has already been sug-
tase, hemolysin, and casein protease, while at least 8 out of 13 gested (19). Furthermore, a novel signaling molecule, called
antibiotic-susceptible strains from the same ward were positive PQS (39), has been identified and shown to positively regulate
for these virulence factors (34). the transcription of lasB and also of rhlI (28). We propose that
Furthermore, two recent reports established a link between the MexEF-OprN pump decreases intracellular PQS levels,
the expression of efflux pumps and the quorum-sensing system which could result either from the transport of PQS by the
in P. aeruginosa. Evans et al. (7) showed that strains overex- pump or from efflux of a precursor required for PQS biosyn-
pressing the MexAB-OprM system secrete less 3-oxo-C12- thesis, like, for example, tryptophan (13).This would explain
HSL. Furthermore, these investigators found reduced produc- the observed decrease in rhlI transcription and the concomi-
tion of pyocyanin, elastase, and casein protease compared to tant decrease in C4-HSL levels. The combined decrease in
that in the wild type. However, several strains used by Evans et PQS and C4-HSL levels could therefore be responsible for the
al. were derived from PAO-BI, which we show here to be an diminished exoproduct synthesis in the nfxC type mutant. In
nfxC mutant. We sequenced the mexT gene in these strains and agreement with this hypothesis is the observation that PQS in
confirmed that all express a functional mexT gene and are combination with C4-HSL is able to partially restore rhlAB
therefore nfxC mutants. Thus, it is not clear whether the effects transcription in the nfxC type mutant. PQS has a quinolone
on virulence factor production observed by Evans et al. are due structure to which a 7-carbon-atom acyl side chain is attached.
to indirect effects of MexAB-OprM overexpression on the ex- This confers a hydrophobic character on the molecule which
pression of MexEF-OprN. In obvious contradiction of these probably prevents diffusion through the membrane, as in the
results, Pearson et al. (38) found that in a non-nfxC back- case of 3-oxo-C12-HSL. The results presented here, together
ground, overexpression of the MexAB-OprM pump increased with our observation on the reduced virulence of the nfxC
VOL. 183, 2001 EFFLUX SYSTEMS AND VIRULENCE IN PSEUDOMONAS AERUGINOSA 5221

mutant PT149 (Cosson et al., submitted), demonstrate that 19. Latifi, A., M. Foglino, K. Tanaka, P. Williams, and A. Lazdunski. 1996. A
hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the
antibiotic resistance can have dramatic effects on the virulence transcriptional activators LasR and RhIR (VsmR) to expression of the
properties of a strain without necessarily affecting its overall stationary-phase sigma factor RpoS. Mol. Microbiol. 21:1137–1146.
fitness. 20. Li, X.-Z., D. M. Livermore, and H. Nikaido. 1994. Role of efflux pump(s) in
intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline,
chloramphenicol, and norfloxacin. Antimicrob. Agents Chemother. 38:1732–
ACKNOWLEDGMENTS 1741.
21. Li, X.-Z., D. Ma, D. M. Livermore, and H. Nikaido. 1994. Role of efflux
We are grateful to J. P. Pearson, B. Iglewski, D. Haas, and M. pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a
Foglino for providing strains, phages, and plasmids. We thank R. contributing factor to ␤-lactam resistance. Antimicrob. Agents Chemother.
Comte for excellent technical assistance and C. Rossier for performing 38:1742–1752.
the sequencing. Many thanks to the members of the group of U. 22. Li, X.-Z., H. Nikaido, and K. Poole. 1995. Role of MexA-MexB-OprM in
Burger, Faculty of Chemistry, University of Geneva, for the synthesis antibiotic efflux in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
of C4-HSL, 3-oxo-C12-HSL, and PQS. 39:1948–1953.
This work was supported by grants 31-55961.98 (to T.K.) and 3231- 23. Li, X. Z., L. Zhang, and K. Poole. 1998. Role of the multidrug efflux systems
of Pseudomonas aeruginosa in organic solvent tolerance. J. Bacteriol. 180:
051940.97 and 3200-052189.97 (to C.V.D.) from the Swiss National
2987–2991.

Downloaded from http://jb.asm.org/ on September 28, 2020 by guest

Science Foundation. 24. Li, X. Z., L. Zhang, R. Srikumar, and K. Poole. 1998. Beta-lactamase inhib-
itors are substrates for the multidrug efflux pumps of Pseudomonas aerugi-
REFERENCES nosa. Antimicrob. Agents Chemother. 42:399–403.
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. 25. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a
Seidman, and K. Struhl. 1987. Current protocols in molecular biology. John laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
Wiley & Sons, Inc., New York, N. Y. N.Y.
2. Brint, J. M., and D. E. Ohman. 1995. Synthesis of multiple exoproducts in 26. Maseda, H., K. Saito, A. Nakajima, and T. Nakae. 2000. Variation of the
Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of mexT gene, a regulator of the MexEF-oprN efflux pump expression in wild-
regulators in strain PAO1 with homology to the autoinducer-responsive type strains of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 192:107–112.
LuxR-LuxI family. J. Bacteriol. 177:7155–7163. 27. Masuda, N., N. Gotoh, S. Ohya, and T. Nishino. 1996. Quantitative corre-
3. Britigan, B. E., T. L. Roeder, G. T. Rasmussen, D. M. Shasby, M. L. Mc- lation between susceptibility and OprJ production in NfxB mutants of
Cormick, and C. D. Cox. 1992. Interaction of the Pseudomonas aeruginosa Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 40:909–913.
secretory products pyocyanin and pyochelin generates hydroxyl radical and 28. McKnight, S. L., B. H. Iglewski, and E. C. Pesci. 2000. The Pseudomonas
causes synergistic damage to endothelial cells. Implications for Pseudomo- quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. J.
nas-associated tissue injury. J. Clin. Investig. 90:2187–2196. Bacteriol. 182:2702–2708.
4. Chugani, S. A., M. Whiteley, K. M. Lee, D. D’Argenio, C. Manoil, and E. P. 29. Michéa-Hamzehpour, M., J.-C. Pechère, P. Plésiat, and T. Köhler. 1995.
Greenberg. 2001. QscR, a modulator of quorum-sensing signal synthesis and OprK and OprM define two genetically distinct multidrug efflux systems in
virulence in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 98:2752– Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:2392–2396.
2757. 30. Miller, J. H. 1972. Experiments in molecular genetics, p. 352–355. Cold
5. de Kievit, T., P. C. Seed, J. Nezezon, L. Passador, and B. H. Iglewski. 1999. Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
RsaL: a novel repressor of virulence gene expression in Pseudomonas aerugi- 31. Mine, T., Y. Morita, A. Kataoka, T. Mizushima, and T. Tsuchiya. 1999.
nosa. J. Bacteriol. 181:2175–2184. Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from
6. Essar, D. W., L. Eberly, A. Hadero, and I. P. Crawford. 1990. Identification Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43:415–417.
and characterization of genes for a second anthranilate synthetase in Pseudo- 32. Morgan, A. F. 1979. Transduction of Pseudomonas aeruginosa with a mutant
monas aeruginosa: interchangeability of the two anthranilate synthases and of bacteriophage E79. J. Bacteriol. 139:137–140.
evolutionary implications. J. Bacteriol. 172:884–900. 33. Ochsner, U. A., A. K. Koch, A. Fiechter, and J. Reiser. 1994. Isolation and
7. Evans, K., L. Passador, R. Srikumar, E. Tsang, J. Nezezon, and K. Poole. characterization of a regulatory gene affecting rhamnolipid biosurfactant
1998. Influence of the MexAB-OprM multidrug efflux system on quorum synthesis in Pseudomonas aeruginosa. J. Bacteriol. 176:2044–2054.
sensing in Pseudomonas aeruginosa. J. Bacteriol. 180:5443–5447. 34. Okazaki, M., T. Onogawa, K. Araki, T. Egami, N. Furuya, N. Endo, and H.
8. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing Uchimura. 1997. Isolation frequency and biological characteristics of the
derivative of plasmid RK2 dependent on a plasmid function provided in multiple-antibiotic resistant Pseudomonas aeruginosa isolated from clinical
trans. Proc. Natl. Acad. Sci. USA 76:1648–1652. specimens. Kansenshogaku Zasshi 71:1181–1186.
9. Fukuda, H., M. Hosaka, K. Hirai, and S. Iyobe. 1990. New norfloxacin 35. Okazaki, T., and K. Hirai. 1992. Cloning and nucleotide sequence of the
resistance gene in Pseudomonas aeruginosa PAO. Antimicrob. Agents Che- Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones.
mother. 34:1757–1761. FEMS Microbiol. Lett. 97:197–202.
10. Gambello, M. J., and B. H. Iglewski. 1991. Cloning and characterization of 36. Passador, L., J. M. Cook, M. J. Gambello, L. Rust, and B. H. Iglewski. 1993.
the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell
expression. J. Bacteriol. 173:3000–3009. communication. Science 260:1127–1130.
11. Gambello, M. J., S. Kaye, and B. H. Iglewski. 1993. LasR of Pseudomonas 37. Pearson, J. P., E. C. Pesci, and B. H. Iglewski. 1997. Roles of Pseudomonas
aeruginosa is a transcriptional activator of the alkaline protease gene (apr) aeruginosa las and rhl quorum-sensing systems in control of elastase and
and an enhancer of exotoxin A expression. Infect. Immun. 61:1180–1184. rhamnolipid biosynthesis genes. J. Bacteriol. 179:5756–5767.
12. Gotoh, N., H. Tsujimoto, M. Tsuda, K. Okamoto, A. Nomura, T. Wada, M. 38. Pearson, J. P., C. van Delden, and B. H. Iglewski. 1999. Active efflux and
Nakahashi, and T. Nishino. 1998. Characterization of the MexC-MexD- diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell
OprJ multidrug efflux system in ⌬mexA-mexB-oprM mutants of Pseudomonas signals. J. Bacteriol. 181:1203–1210.
aeruginosa. Antimicrob. Agents Chemother. 42:1938–1943. 39. Pesci, E. C., J. B. Milbank, J. P. Pearson, S. McKnight, A. S. Kende, E. P.
13. Holden, I., I. Swift, and I. Williams. 2000. New signal molecules on the Greenberg, and B. H. Iglewski. 1999. Quinolone signaling in the cell-to-cell
quorum-sensing block. Trends Microbiol. 8:101–104. communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci.
14. Kaplan, H. B., and E. P. Greenberg. 1985. Diffusion of autoinducer is USA 96:11229–11234.
involved in regulation of the Vibrio fischeri luminescence system. J. Bacteriol. 40. Pesci, E. C., J. P. Pearson, P. C. Seed, and B. H. Iglewski. 1997. Regulation
163:1210–1214. of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol.
15. Köhler, T., L. K. Curty, F. Barja, C. van Delden, and J. C. Pechère. 2000. 179:3127–3132.
Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling 41. Poole, K., N. Gotoh, H. Tsujimoto, Q. Zhao, A. Wada, T. Yamasaki, S.
and requires flagella and pili. J. Bacteriol. 182:5990–5996. Neshat, J. Yamagishi, X. Z. Li, and T. Nishino. 1996. Overexpression of the
16. Köhler, T., S. F. Epp, L. Kocjancic-Curty, and J. C. Pechère. 1999. Charac- mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of
terization of MexT, the transcriptional activator of the MexEF-OprN mul- Pseudomonas aeruginosa. Mol. Microbiol. 21:713–724.
tidrug efflux system of Pseudomonas aeruginosa. J. Bacteriol. 181:6300–6305. 42. Poole, K., K. Krebes, C. McNally, and S. Neshat. 1993. Multiple antibiotic
17. Köhler, T., M. Kok, M. Michea-Hamzehpour, P. Plesiat, N. Gotoh, T. resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux
Nishino, L. K. Curty, and J. C. Pechère. 1996. Multidrug efflux in intrinsic operon. J. Bacteriol. 175:7363–7372.
resistance to trimethoprim and sulfamethoxazole in Pseudomonas aerugi- 43. Poole, K., K. Tetro, Q. Zhao, S. Neshat, D. E. Heinrichs, and N. Bianco.
nosa. Antimicrob. Agents Chemother. 40:2288–2290. 1996. Expression of the multidrug resistance operon mexA-mexB-oprM in
18. Köhler, T., M. Michea-Hamzehpour, U. Henze, N. Gotoh, L. K. Curty, and Pseudomonas aeruginosa: mexR encodes a regulator of operon expression.
J. C. Pechère. 1997. Characterization of MexE-MexF-OprN, a positively Antimicrob. Agents Chemother. 40:2021–2028.
regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Micro- 44. Preston, M. J., P. C. Seed, D. S. Toder, B. H. Iglewski, D. E. Ohman, J. K.
biol. 23:345–354. Gustin, J. B. Goldberg, and G. B. Pier. 1997. Contribution of proteases and
5222 KÖHLER ET AL. J. BACTERIOL.

LasR to the virulence of Pseudomonas aeruginosa during corneal infections. negative bacteria. Bio/Technology 1:784–790.
Infect. Immun. 65:3086–3090. 52. Spaink, H. P., R. J. H. Okker, C. A. Wijffelman, E. Pees, and B. J. J.
45. Ramos-Aires, J., T. Köhler, H. Nikaido, and P. Plésiat. 1999. Involvement of Lugtenberg. 1987. Promoters in the nodulation region of the Rhizobium
an active efflux system in the natural resistance of Pseudomonas aeruginosa to leguminosarum Sym plasmid pRL1JI. Plant Mol. Biol. 9:27–39.
aminoglycosides. Antimicrob. Agents Chemother. 43:2624–2628. 53. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J.
46. Rashid, M. H., and A. Kornberg. 2000. Inorganic polyphosphate is needed Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L.
for swimming, swarming, and twitching motilities of Pseudomonas aerugi- Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L.
nosa. Proc. Natl. Acad. Sci. USA 97:4885–4890. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D.
47. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with Spencer, G. K. Wong, Z. Wu, and I. T. Paulsen. 2000. Complete genome
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467. sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.
48. Seed, P. C., L. Passador, and B. H. Iglewski. 1995. Activation of the Pseudo- Nature 406:959–964.
monas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: 54. Tamura, Y., S. Suzuki, and T. Sawada. 1992. Role of elastase as a virulence
an autoinduction regulatory hierarchy. J. Bacteriol. 177:654–659. factor in experimental Pseudomonas aeruginosa infection in mice. Microb.
49. Shiba, T., K. Ishiguro, N. Takemoto, H. Koibuchi, and K. Sugimoto. 1995. Pathog. 12:237–244.
Purification and characterization of the Pseudomonas aeruginosa NfxB pro- 55. Twining, S. S., S. E. Kirschner, L. A. Mahnke, and D. W. Frank. 1993. Effect
tein, the negative regulator of the nfxB gene. J. Bacteriol. 177:5872–5877. of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on
50. Siegmund, I., and F. Wagner. 1991. New method for detecting rhamnolipids corneal proteinases and proteins. Investig. Ophthalmol. Vis. Sci. 34:2699–
excreted by Pseudomonas species during growth in mineral agar. BioTech- 2712.

Downloaded from http://jb.asm.org/ on September 28, 2020 by guest

niques 5:265–268. 56. van Delden, C., E. C. Pesci, J. P. Pearson, and B. H. Iglewski. 1998. Star-
51. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization vation selection restores elastase and rhamnolipid production in a Pseudo-
system for in vivo genetic engineering: transposon mutagenesis in Gram- monas aeruginosa quorum-sensing mutant. Infect. Immun. 66:4499–4502.