FEMS Microbiology Letters 253 (2005) 125–131
www.fems-microbiology.org
Osmotic stress and phosphate limitation alter production
of cell-to-cell signal molecules and rhamnolipid biosurfactant
by Pseudomonas aeruginosa
Alexis Bazire a, Alexandra Dheilly a, Farès Diab b, Danièle Morin a, Mohamed Jebbar b,
Dominique Haras a, Alain Dufour a,*
b
a
Laboratoire de Biotechnologie et Chimie Marines, EA 3884, Université de Bretagne Sud, BP 92116, 56321 Lorient, France
Département Osmorégulation chez les Bactéries, UMR-CNRS 6026, Université de Rennes I, Campus de Beaulieu, 35042 Rennes, France
Received 13 July 2005; received in revised form 19 September 2005; accepted 19 September 2005
First published online 5 October 2005
Edited by M. Schembri
Abstract
In Pseudomonas aeruginosa, rhamnolipid production is controlled by the quorum-sensing system RhlRI, which itself depends on
LasRI. These systems use cell-to-cell signal molecules: N-butyryl-L-homoserine lactone (C4–HSL) and N-(3-oxododecanoyl)-Lhomoserine lactone (3OC12–HSL), respectively. Whereas both HSLs were produced in M63 medium, rhamnolipid synthesis was
not achieved. Phosphate limitation reduced the HSL concentrations, while allowing rhamnolipid production. Hyperosmotic shock
applied during the exponential growth phase stopped the accumulation of 3OC12–HSL, and prevented C4–HSL and rhamnolipid
production. These defects result from lower expression of genes involved in C4–HSL and rhamnolipid syntheses. The osmoprotectant glycine betaine partially restored C4–HSL and rhamnolipid production.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Quorum sensing; Homoserine lactone; Rhamnolipid; Biosurfactant; Pseudomonas aeruginosa; Osmotic stress
1. Introduction
Pseudomonas aeruginosa is an opportunistic pathogen, which is in particular responsible for chronic lung
infection of cystic fibrosis patients. P. aeruginosa produces a number of extra-cellular virulence factors,
including rhamnolipids [1]. The latter are biosurfactants,
i.e., amphipathic molecules reducing surface tension at
oil/water or air/water interfaces. Rhamnolipids are involved in multiple activities, including the maintenance
*
Corresponding author. Tel.: +33 2 97 87 45 93; fax: +33 2 97 87 45
00.
E-mail address: [email protected] (A. Dufour).
of biofilm architecture (biofilm is a lifestyle displayed
during chronic infection [1]), cell detachment from biofilms, and enhancing the bioactivity of the Pseudomonas
quinolone signal (PQS), which is itself a virulence factor
[2,3, and references therein]. In addition, rhamnolipids
offer various applications by enhancing biodegradation
and/or removal of low solubility pollutants such as
hydrocarbons [4].
Mono- and di-rhamnolipids consist of a fatty acid
moiety and of one and two rhamnosyls, respectively
[4]. Their syntheses require the rhamnosyltransferase
RhlAB to link a rhamnosyl to a fatty acid chain of variable length [4]. Di-rhamnolipids result from a subsequent reaction catalyzed by RhlC [4,5]. Study of the
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.09.029
126
A. Bazire et al. / FEMS Microbiology Letters 253 (2005) 125–131
rhlAB genes led to the discovery of the RhlRI quorumsensing (QS) system [6]. RhlI synthesizes the cell-to-cell
signal molecule N-butyryl-L-homoserine lactone (C4–
HSL, also known as autoinducer PAI-2 or BHL) [7].
The C4–HSL binding to the regulator RhlR leads to
transcriptional activation of target genes. The RhlRI
system is controlled by the similar LasRI system, the signal molecule of which is N-(3-oxododecanoyl)-L-homoserine lactone (3OC12–HSL, or PAI-1, or OdDHL)
[7,8]. These two hierarchical QS systems regulate the
expression of more than 300 P. aeruginosa genes [7,9],
including virulence genes [1]. Furthermore, 3OC12–
HSL directly acts as a virulence factor [1]. The rhlAB
promoter contains a predicted las–rhl box which would
specifically bind RhlR–C4–HSL [10]. Nevertheless, full
rhlAB transcription requires both HSLs [9,11], probably
because of the interplay between the two systems. Similarly, the PA1131–rhlC operon contains a las–rhl box [5]
and requires both HSLs for full expression [9]. The QS
network is further complicated by the involvement of
the LasR homologue QscR, the PQS, the sigma factors
RpoN and RpoS, and numerous transcriptional regulators [7]. The regulatory pathway remains unclear since
the signals to which these regulators respond remain
generally unknown. The identification of these signals
is a prerequisite to the understanding of this regulatory
network.
Since high NaCl concentrations are encountered in
respiratory tract fluid from cystic fibrosis patients [12],
we examined the osmotic stress effect on HSL and
rhamnolipid production by P. aeruginosa. This led us
to show that phosphate limitation and high NaCl concentration are two signals negatively affecting HSL production with various intensities, while having opposite
effects on a QS-dependent phenotype, rhamnolipid
production.
2. Materials and methods
2.1. Bacterial strains and culture conditions
The strains were P. aeruginosa PAO1 (a kind gift
from M. Foglino, Marseille, France) and Escherichia
coli JM109. The culture media were LB, M63 (KH2PO4,
13.6 g l1; (NH4)2SO4, 2 g l1; MgSO4 Æ 7H2O, 0.2 g l1;
FeEDTA and thiamine, 0.5 mg l1 each; benzoic acid,
1.44 g l1), or phosphate-limited M63 (PLM63), in
which KH2PO4 concentration was reduced to 0.01 and
14.9 g l1 of KCl were added. In all media, the final
pH was adjusted to 7.2. Gentamycin was used at 35
and 50 lg ml1 for E. coli and P. aeruginosa, respectively, containing plasmid pBBR1MCS-5 [13] or derivatives. Cultures were performed at 37 C with shaking
and growth was followed by measuring optical density
at 600 nm (OD600). Osmotic shock (0.5 M NaCl) was
applied by adding one volume of 1 M NaCl-containing
medium (preheated at 37 C) to NaCl-free cultures.
When osmotic shock was performed in the presence of
glycine betaine (GB), 2 mM GB was included in the
NaCl-containing medium. The final GB concentration
was thus 1 mM.
2.2. Extraction and analysis of HSLs and rhamnolipids
HSLs were extracted twice from 50 ml of culture
supernatant with a total of 30 ml of dichloromethane.
The extracts were dehydrated with anhydrous magnesium sulfate, filtered and evaporated to dryness at
35 C using a rotovapor apparatus (Buchi, Flawil, Switzerland). Residues were dissolved in 0.5 ml of water–
acetonitrile (50:50), filtered through 0.45 lm pore-size
filters, and stored frozen until analysis by liquid chromatography–mass spectrometry (LC–MS–MS) as described [14]. For rhamnolipids, 1 ml of culture
supernatant was filtered through a 0.2 lm pore-size filter, fivefold diluted in water–acetonitrile (65:35) ammonium acetate (4 mM), and analyzed by LC–MS as
described [15]. The detection of pseudomolecular
[M H] ions and fragment ions allowed a selective
identification. The proportion of each rhamnolipid was
obtained from the corresponding m/z [M H] chromatograms by measure of peak areas. For cell content
analyses, bacteria were washed twice and suspended in
250 ll of 1% H2SO4. After 3 h at room temperature,
250 ll of acetonitrile were added, the supernatant was
recovered after centrifugation at 10,000g for 5 min,
and treated like a supernatant sample.
2.3. Nucleic acid procedures
Restriction enzymes, T4 DNA ligase, and alkaline
phosphatase were purchased from Invitrogen (Carlsbad,
Ca.), and used according to the manufacturers instructions. PCR reactions were performed using the
GC-RICH PCR System (Roche Diagnostics, Basel,
Switzerland). Plasmids were purified using the QIAprep
Spin Miniprep Kit (Qiagen, Hilden, Germany).
2.4. Plasmid construction and bioluminescence assays
To construct a phzA-luxCDABE fusion, a 6-kb DNA
fragment containing the luxCDABE genes was subcloned from pSB406 [16] into the EcoRI restriction site
of pBBR1MCS-5 [13], leading to pAB133. The phzA
(qsc131) promoter [11] was PCR-amplified with the
primers qsc131F (5 0 -TTAATGAGCTCGGCACCTACCAGATCTTG-3 0 ) and qsc131R (5 0 -TAATAAACTAGTGGGCTCTCCAGGTATGC-3 0 ). The SacI–
SpeI-digested PCR product was inserted into pAB133,
yielding pDA224. The insert was verified by DNA
sequencing (Sequentia, Evry, France). Bioluminescence
A. Bazire et al. / FEMS Microbiology Letters 253 (2005) 125–131
3. Results
3.1. Time-course of HSL production in M63 medium
Hyperosmotic stress studies requiring the use of a
minimum medium to avoid the uncontrolled presence
of osmoprotectants, we first examined the ability of P.
aeruginosa PAO1 to produce HSLs in M63 medium.
M63
PLM63
1.0
0.5
OD600
Bacteria were grown during 20 h and two volumes of
RNAprotect bacteria reagent (Qiagen, Hilden, Germany)
were added to a culture containing about 5 · 109 cells.
RNAs were extracted with the RNeasy Midi Kit and
RNase-Free DNase Set (Qiagen). Residual DNAs were
eliminated by acid phenol treatment. The absence of
DNA was confirmed by verifying that PCR reactions
failed without prior cDNA synthesis. RNAs were nonspecifically converted to single-stranded cDNAs using
the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Ca.). rhlI, rhlAB, and rhlC mRNAs
were quantified by real-time PCR amplification of their
cDNAs, using primers RHLI33 (5 0 -CATCAGGTCTTCATCGAGAAGCT-3 0 ), RHLI34 (5 0 -CGACGATGTAGCGGGTTTG-3 0 ), RHLA33 (5 0 -GATCGAGCTGGACGACAAGTC-3 0 ), RHLA34 (5 0 -GCTGATGGTTGCTGGCTTTC-3 0 ), RHLC33 (5 0 -ACCGGATAGACATGGGCGT-3 0 ), and RHLC34 (5 0 -GATCGCTGTGCGGTGAGTT-3 0 ). PCR reactions were performed in
triplicate with the 7300 Real-Time PCR System apparatus
(Applied Biosystems). The 25-ll reactions contained
12.5 ll of SYBR Green PCR Master Mix (including
AmpliTaq Gold DNA Polymerase [Applied Biosystems]), 900 lM of each primer, and cDNAs generated
from 0.01 ng of total RNA. The conditions were 95 C
for 10 min for polymerase activation, and 40 cycles at 95
and 60 C for 60 and 30 s, respectively. ROX dye was used
as passive reference to normalize for non-PCR related
fluorescence variations. The relative quantification of
the mRNAs of interest was obtained by the comparative
CT (2DDCT) method [17], using 16S rRNA as endogenous
control [18]. Each primer pair was validated by verifying
that the PCR efficiency E was above 0.95, and that a single
PCR product with the expected Tm was obtained.
a
[3OC12-HSL]/OD600
2.5. RNA quantification by real-time reverse
transcription-PCR
Extra-cellular 3OC12- and C4–HSLs were quantified
[14] at different time points during bacterial growth.
To take into account the bacterial population increase,
the HSL concentrations were divided by the corresponding OD600 values (Fig. 1). The highest HSL concentrations are given in Table 1. 3OC12–HSL concentration
per OD600 unit increased rapidly at the beginning of
the exponential phase, reached a peak at OD600 0.4,
and decreased after mid-exponential phase (Fig. 1(b)).
3OC12–HSL had almost disappeared from culture
supernatants after 12 h in stationary phase. C4–HSL
concentration per OD600 unit increased more slowly
0.1
0
10
20
30
0
10
20
30
0
10
20
30
1.0
0.8
0.6
0.4
0.2
0
b
[C4-HSL]/OD600
was measured in 96-well optiplates using a LumiCount
apparatus (PerkinElmer, Boston, Ma.): 100 ll of bacterial suspensions were adjusted at the same OD600, and
bioluminescence values of the negative control strain
P. aeruginosa PAO1 (pAB133) were subtracted from
values of pDA224-containing PAO1. Bioluminescence
was expressed in relative light units per 0.5 s (RLU
0.5 s1). Each set of experiment was performed twice.
127
5
4
3
2
1
0
c
Time (h)
Fig. 1. P. aeruginosa PAO1 growth curves (a), and time-course of
extra-cellular production of 3OC12–HSL (b) and C4–HSL (c). The
HSL concentrations (in lM) were divided by the OD600 values of the
cultures at the sampling time. The media were M63 and the phosphatelimited PLM63. The graphs are representative of at least three
experiments.
Table 1
Highest extra-cellular concentrations of 3OC12- and C4–HSL
Medium
3OC12–HSL (nM)b
C4–HSL (nM)b
M63
PLM63
PLM63 NaCla
PLM63 + NaCla
PLM63 + NaCl + GBa
590 ± 80
185 ± 12
210 ± 30
28 ± 1
22 ± 3
3914 ± 120
1369 ± 180
1630 ± 160
17 ± 4
585 ± 83
a
At OD600 0.3, one volume of preheated PLM63 medium was
added. The latter contained no NaCl (NaCl), 1 M NaCl (+NaCl), or
1 M NaCl and 2 mM glycine betaine (+NaCl + GB).
b
The values are averages of at least two experiments.
128
A. Bazire et al. / FEMS Microbiology Letters 253 (2005) 125–131
M63
PLM63
103 Area
8
6
4
2
0
0
20 40
10 3 Area/OD600
b
60
80 100 120
Time (h)
a
12
8
4
0
m/z
649
675
677
Fig. 2. Rhamnolipid production by P. aeruginosa PAO1: (a) timecourse of production of the major rhamnolipid species, di-rhamnolipid
Rha–Rha–C10–C10 (ion m/z 649), in M63 and PLM63 media;
(b) quantification of the three more abundant rhamnolipid species in
PLM63 medium at the peak of production (23 h cultures). Ions m/z
649, 675, and 677 are due to di-rhamnolipids Rha–Rha–C10–C10,
Rha–Rha–C10–C12:1, and Rha–Rha–C10–C12, respectively. The
values are averages from three experiments, with the standard
deviations indicated by the error bars.
throughout the exponential phase, reaching its highest
level at the stationary phase entry (Fig. 1(c)). It decreased then slowly and was still at about 45% of its
highest value after 18 h in stationary phase. Despite
both HSLs were produced in M63 medium, rhamnolipids were barely detected in culture supernatants
(Fig. 2(a)). Since no or very few rhamnolipids were detected from cell extracts (not shown), the defect was in
rhamnolipid synthesis rather than in export.
3.2. Phosphate limitation reduces HSL levels but allows
rhamnolipid production
Based on previous results [19] and on the rich PPGAS
medium, which is limited in phosphate and favors
rhamnolipid production [5], we modified the M63 medium by decreasing its KH2PO4 concentration. The latter
was set to 75 lM (0.01 g l1) after searching for the lowest concentration allowing a reasonable P. aeruginosa
growth. In the final phosphate-limited (PLM63) medium, the potassium concentration was maintained by
adding KCl, since K+ accumulation is involved in
P. aeruginosa adaptation to osmotic stress [20]. In
PLM63 medium, P. aeruginosa PAO1 had a similar
growth rate as in M63, but it entered stationary phase
at a lower cell density (Fig. 1(a)). The time-courses of
HSL production per OD600 unit were unchanged
(Fig. 1(b) and (c)), but the highest extra-cellular concentrations of 3OC12- and C4–HSL were 3.2- and 2.9-fold
lower than in M63 (Table 1). Whereas C4–HSL freely
diffuses through membranes, 3OC12–HSL is subject to
active efflux via the MexAB–OprM pump [7]. A reduction in efflux pump activity would thus increase
3OC12–HSL intra-cellular concentration, while preventing its extra-cellular accumulation. We examined this
possibility by assaying the activity of the phzA
(qsc131) promoter, which depends on both HSLs [11].
We constructed pAB133, a vector carrying the promoter-less reporter genes luxCDABE, upstream of
which the phzA promoter was inserted, creating
pDA224. P. aeruginosa PAO1(pDA224) displayed a 3fold lower bioluminescence during the first 10 h of
growth in PLM63 medium compared to M63 (not
shown). The lower 3OC12–HSL extra-cellular level observed in PLM63 medium resulted thus from a lower
synthesis rather than an alteration of its efflux pump
activity.
Despite the negative effect of phosphate limitation on
HSL production, rhamnolipids were produced in
PLM63 medium during the first 20 h of incubation
(Fig. 2(a)). In PLM63, the main rhamnolipids were,
from the more to the less abundant, the di-rhamnolipids
Rha–Rha–C10–C10, Rha–Rha–C10–C12 and Rha–
Rha–C10–C12:1, represented by ions m/z 649, 677 and
675, respectively (Fig. 2(b)). We also detected low
amounts of the mono-rhamnolipid Rha–C10–C10 and
the di-rhamnolipid Rha–Rha–C8–C10 (not shown).
3.3. Hyperosmotic shock prevents C4–HSL and
rhamnolipid production
The addition of 0.5 M NaCl into PLM63 medium
prior to inoculate P. aeruginosa prevented bacterial
growth. NaCl was thus added during the exponential
growth phase, at OD600 0.3 (arrows in Fig. 3), by diluting the cultures with one volume of preheated medium
containing 1 M NaCl (shocked cultures) or no NaCl
(control). NaCl was not added in crystalline form to
avoid formation of high local salt concentration during
its dissolution. The dilution explains the OD600 drops
(Fig. 3(a)) and the delays in the peaks of HSL concentration per OD600 unit (Fig. 3(b) and (c) vs. Fig. 1(b)
and (c)). After osmotic shock, P. aeruginosa was able
to resume its growth (Fig. 3(a)), but 3OC12–HSL production was interrupted and its extra-cellular concentration per OD600 unit dropped (Fig. 3(b)). Consequently,
the highest 3OC12–HSL concentration was 7.5-fold
lower in osmotically shocked cultures than in the
un-stressed control (Table 1). C4–HSL production was
totally inhibited after NaCl addition (Fig. 3(c)), leading
to a 96-fold lower C4–HSL concentration compared to
the un-stressed control (Table 1). In agreement with
the negative effect of hyperosmotic shock on HSL accumulation, the activity of the phzA promoter was strongly
inhibited by the stress and remained low thereafter
(Fig. 3(d)). Another construction showed that this stress
A. Bazire et al. / FEMS Microbiology Letters 253 (2005) 125–131
NaCl+GB
NaCl
OD600
1.0
0.5
0.1
[3OC12-HSL]/OD600
a
0
[C4-HSL]/OD600
10
20
30
0.6
0.5
0.4
0.3
0.2
0.1
0
b
18
16
14
12
10
8
6
4
2
0
NaCl
GB
Time (h)
103 Area/OD600
No NaCl
129
0
10
20
30
0
10
20
30
– + +
– – +
24
– ++ – ++
– –+ – – +
48
72
Fig. 4. Quantification of di-rhamnolipid Rha–Rha–C10–C10 (ion m/z
649) in culture supernatants of P. aeruginosa PAO1 grown in PLM63
medium for the indicated time. Where indicated, NaCl and glycine
betaine (GB) were added at the final concentrations of 0.5 M and
1 mM, respectively, at OD600 0.3. The liquid chromatography–mass
spectrometry peak surface areas were divided by the OD600 values of
the cultures. The values are averages of three experiments, with the
standard deviations indicated by the error bars.
3
2
1
0
c
103 RLU/0.5 s
18
14
10
6
2
0
d
0
10
20
30
Time (h)
Fig. 3. Effect of hyperosmotic shock (0.5 M NaCl) on HSL production
by P. aeruginosa PAO1, and promoter phzA activity. Bacteria were
grown in PLM63 medium and one volume of medium containing NaCl
or no NaCl was added (vertical arrows) when OD600 reached 0.3. The
growth was followed (a) and 3OC12–HSL (b) and C4–HSL (c) were
assayed in culture supernatants. The HSL concentrations (in lM) were
divided by the OD600 values of the cultures. (d) Promoter phzA activity
was followed by measuring the luminescence from P. aeruginosa PAO1
harbouring pDA224, which contains a phzA-luxCDABE transcriptional fusion. The bacteria were un-stressed (no NaCl, squares),
submitted to osmotic shock (NaCl, triangles), or submitted to osmotic
shock in the presence of 1 mM glycine betaine (NaCl + GB, circles).
The graphs are representative of two complete experiments and of
several additional experiments with fewer time points.
impairs neither the luxCDABE reporter system nor the
vector replication (not shown). Osmotic stress also abolished rhamnolipid production (Fig. 4) and cell content
analyses revealed that this resulted from a defect in synthesis rather than in export (not shown). Real-time PCR
showed that the rhlI mRNA level was reduced 14-fold
(Fig. 5), indicating that the C4–HSL production defect
had a genetic cause. As expected, the rhlAB and rhlC
mRNA amounts were in turn reduced, although to a lesser extent: 4 and 7 fold, respectively (Fig. 5).
Fig. 5. Expression levels of rhlI, rhlAB and rhlC in P. aeruginosa
PAO1 submitted to 0.5 M osmotic shock without glycine betaine (GB)
or in its presence. The expression levels were determined by quantitative reverse transcription-PCR, relatively to the expression in PLM63
medium without stress. Values lower than 1 indicate reduction of gene
expression upon stress. 16S rRNA was used as endogenous control to
normalize the RNA input and reverse transcription efficiency. PCR
reactions were performed in triplicate and the standard deviations were
lower than 0.15 CT.
3.4. Glycine betaine restores C4–HSL and rhamnolipid
production in hyperosmotic condition
Since GB provides osmoprotection to P. aeruginosa
[20], we added a final concentration of 1 mM GB simultaneously to NaCl. In our conditions, GB accumulated
in the cells without being metabolized (not shown).
GB did not improve the production of 3OC12–HSL
(Fig. 3(b) and Table 1), but restored 60% of C4–HSL
production per OD600 unit (Fig. 3(c)), leading to a highest C4–HSL concentration corresponding to 36% of that
seen in un-stressed cultures (Table 1). C4–HSL production was thus partially re-established independently of
3OC12–HSL. GB also restored a strong but delayed
phzA promoter activity (Fig. 3(d)), and a poor rhamnolipid production (Fig. 4). At the genetic level, GB partially restored the rhlI and rhlC expression levels,
which were then only 2.5- and 2-fold lower, respectively,
than in un-stressed cells (Fig. 5). Surprisingly, the partial
restorations of rhlI expression and C4–HSL production
130
A. Bazire et al. / FEMS Microbiology Letters 253 (2005) 125–131
had only a marginal positive effect on the rhlAB expression (Fig. 5), which might explain why the rhamnolipid
production was not more strongly re-established in the
presence of GB (Fig. 4).
4. Discussion
Although QS is heavily studied in P. aeruginosa [7],
only few reports previously described time-course studies of HSL production [21]. The use of a LC–MS–MS
procedure to quantify HSLs [14] allowed us to do so.
Chen et al. [21] recently reported that the C4–HSL concentration peaked at the transition from exponential to
stationary phases. In M63 medium, we obtained a similar result, and observed that the peak of 3OC12–HSL
concentration per OD600 unit preceded that of C4–HSL.
This chronology is consistent with the QS hierarchy in
which LasR–3OC12–HSL activates the transcription of
the rhlI gene encoding the C4–HSL synthase [8]. C4–
HSL degradation was associated to the presence of P.
aeruginosa cells [21]. The decrease in 3OC12–HSL concentration per OD600 unit during late exponential phase
could also result from degradation by P. aeruginosa [22].
Alternatively, HSL inactivation by alkaline pH has been
reported [23]. During P. aeruginosa growth in M63, the
pH of the medium remained at 7.2, which is high enough
to explain at least in part the C4–HSL decrease [23]. The
absence of rhamnolipid synthesis in this medium indicated that C4–HSL production is not a sufficient condition to induce rhamnolipid production, as previously
reported [24]. Phosphate limitation had opposite effects:
it led to rhamnolipid production while reducing HSL
synthesis and/or stability. Its effect on 3OC12–HSL occurred too early to result from a growth rate difference
or a pH increase, since the pH of the medium remained
at 7.2 during the first 16 h of growth in PLM63 medium.
Phosphate limitation thus probably exerts a negative effect on 3OC12–HSL synthesis, suggesting that at least
one of the regulators controlling the expression of lasI
(encoding the 3OC12–HSL synthase) responds to phosphate concentration. The effect of phosphate limitation
on C4–HSL could be a consequence of the entry into stationary phase at a lower cell density. It could also result
from the reduced level of 3OC12–HSL, leading to a
lower expression of rhlI [8].
Hyperosmotic stress applied to exponentially growing P. aeruginosa cells in PLM63 medium interrupted
3OC12–HSL production and prevented C4–HSL and
rhamnolipid syntheses. Our gene expression analyses
and osmoprotection experiments suggested that osmotic
stress prevents rhamnolipid production by acting at least
at two distinct levels: it impairs (i) rhlI expression, leading to a lack of C4–HSL production, which in turns has
a negative effect on rhlAB and rhlC expression and (ii)
rhlAB expression independently from C4–HSL. Whereas
osmoprotection by GB was able to partially re-establish
the first level, it had no effect on the second.
The QS impairment by osmotic stress suggests that
the latter likely affects other QS – dependent phenotypes. We indeed observed that pyocianin production
was abolished after osmotic stress and partially restored
by GB (not shown). A number of studies showed that
HSLs are produced in infected lungs of cystic fibrosis
patients [1], whereas this environment is hyperosmotic
[12]. This could be explained by the presence of phosphatidylcholine, which can be converted to precursors
of the osmoprotectants choline and GB by phospholipase C [25]. This hypothesis is strengthened by the
observation that 1 mM choline or a mixture of choline
and GB (0.5 mM each) had the same positive effect as
1 mM GB on C4–HSL and rhamnolipid production during osmotic stress (not shown).
In conclusion, we identified two conditions, phosphate limitation and osmotic stress, which had mild
and strong negative effects on HSL production. The P.
aeruginosa QS systems are not only cell-density dependent, but are also controlled by a growing list of regulators [7], which respond presumably to a variety of still
unknown signals. Therefore, identifying such signals
constitutes a step towards the unraveling of the regulatory circuit affecting the QS network.
Acknowledgements
This work was supported by the Region Bretagne,
FEDER funds, and the Ministère de la Recherche et
de la Technologie, France (RITMER grant and doctoral
fellowships to A.B., A.Dh., and F.D.). We are grateful
to M. Foglino, K.M. Peterson, and S. Swift for the gifts
of strain and plasmids. We thank C. Blanco for discussions and J.-P. Le Pennec for critically reading the
manuscript.
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