Microbiology (2005), 151, 1127–1138
DOI 10.1099/mic.0.27566-0
Expression of the quorum-sensing regulatory
protein LasR is strongly affected by iron and
oxygen concentrations in cultures of Pseudomonas
aeruginosa irrespective of cell density
Eun-Jin Kim,1 Wei Wang,2 Wolf-Dieter Deckwer2 and An-Ping Zeng1
Division of Molecular Biotechnology1 and Group of TU-BCE2, GBF – German Research Centre
for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany
Correspondence
An-Ping Zeng
[email protected]
Received 18 August 2004
Revised
20 December 2004
Accepted 21 December 2004
The expression of the transcriptional regulatory protein LasR, a main component of the
quorum-sensing (QS) system in Pseudomonas aeruginosa, was recently found to be sensitive
to several environmental factors in addition to its dependency on cell density. However, the
inherent effects of the different factors have seldom been separately demonstrated due to
concurrent changes of culture conditions in typical experimental settings. Furthermore, the
interplays of the different factors are unknown. In this work, the effects and interplay of iron
concentration and dissolved oxygen tension (pO2) on the expression of lasR in P. aeruginosa
were studied in defined growth media with varied iron concentration and pO2 values in
computer-controlled batch and continuous cultures. b-Galactosidase activity in a recombinant
P. aeruginosa PAO1 (NCCB 2452) strain with a lasRp–lacZ fusion was used as a reporter
for lasR expression. In batch culture with a constant pO2<10 % air saturation, a strong
correlation between the exhaustion of iron and the increase of lasR expression was observed.
In continuous culture with nearly constant cell density but varied pO2 values, lasR expression
generally increased with increasing oxidative stress with the exception of growth under O2-limited
conditions (pO2<0 %). Under O2 limitation, the expression of lasR strongly depended on the
concentration of iron. It showed a nearly twofold increase in cells grown under iron deprivation in
comparison with cells grown in iron-replete conditions and reached the expression level seen at
high oxidative stress. A preliminary proteomic analysis was carried out for extracellular proteins
in samples from batch cultures grown under different iron concentrations. Several of the
extracellular proteins (e.g. AprA, LasB, PrpL) which were up-regulated under iron-limited
conditions were found to be QS regulated proteins. Thus, this study clearly shows the links
between QS and genes involved in iron and oxygen regulation in P. aeruginosa.
INTRODUCTION
Pseudomonas aeruginosa, a Gram-negative opportunistic
human pathogen, is often found in nosocomial infections.
In particular, it can cause chronic pulmonary infection
of cystic fibrosis patients, leading to a high mortality rate
due to the formation of virulence factors and the inappropriate host response (Hybiske et al., 2004; Wolfgang
et al., 2004; Lyczak et al., 2002). In P. aeruginosa, the
formation of numerous virulence factors is controlled to a
large extent by a mechanism called quorum sensing (QS)
(Fuqua & Greenberg, 1998; Wagner et al., 2003; Schuster
et al., 2003; Hentzer et al., 2003; Juhas et al., 2004). Two
major genetic components of QS, the las and rhl systems,
have been identified in P. aeruginosa. The las system consists of LasR, a transcriptional regulatory protein, and LasI,
the autoinducer synthase that controls the production of
the signal molecule N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL, PAI-1). The rhl system consists of
RhlR, the regulatory protein, and RhlI, involved in the
production of the autoinducer N-butyrylhomoserine lactone (C4-HSL, PAI-2). The rhl system is under the control
of the las system (Wagner et al., 2003). The regulatory
proteins interact with the autoinducers, thereby activating
the production of a large number of virulence factors such
as proteases, exotoxin A, rhamnolipids, pyocyanin and
siderophores.
Abbreviations: 2-DE, two-dimensional gel electrophoresis; MALDI/TOF
MS, matrix-assisted laser desorption ionization time-of-flight mass
spectrometry; PMF, peptide mass fingerprinting; pO2, dissolved oxygen
tension; QS, quorum sensing.
QS is generally considered as a cell-density-dependent and
globally regulated cell-to-cell signalling process in prokaryotes. In pathogens, it controls virulence and combats
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E.-J. Kim and others
host defences in a cell-density-dependent manner (Smith &
Iglewski, 2003). There is, however, increasing evidence to
suggest that the cell-to-cell signalling mediated by the QS
system can also be strongly affected by environmental
factors other than the cell density. For example, bioluminescence, which is regulated by QS in Vibrio harveyi, was
found to be sensitive to the availability of iron, oxygen and
carbohydrate (Nealson & Hastings, 1991). In P. aeruginosa,
environmental conditions such as nutrient availability have
also been shown to affect the expression of QS genes
(Withers et al., 2001; Albus et al., 1997). Cyanide production, which is related to QS in P. aeruginosa, increased
under low oxygen concentration (Pessi & Haas, 2000).
Furthermore, Bollinger et al. (2001) found an increased
expression of lasI under conditions of iron deprivation
in uncontrolled shake-flask culture. Whiteley et al. (1999)
suggested links between the QS and iron regulons. More
recently, Cornelis & Aendekerk (2004) discussed the relationship between iron and QS based on evaluation of
experimental data, in particular those from transcriptomic
and proteomic analyses of Juhas et al. (2004) and ArevaloFerro et al. (2003). In the work of Juhas et al. (2004), who
studied the regulatory role of VqsR (virulence and QS
regulator) in the QS hierarchy, 25 genes known to be ironregulated were found to be repressed in a vqsR mutant,
among which some were also regulated by QS.
oxygen from the gas phase into the liquid phase under
iron-deficient conditions, thereby causing oxygen limitation in the culture. This may result in decreased formation
of oxidants or increased solubility and availability of iron.
Under these conditions, the generation of a number of
virulence factors, including elastase and siderophores, is
strongly increased, leading to a higher virulence (Kim et al.,
2003). Our studies also demonstrated the importance of
a quantitative and systematic approach in studying cell
physiology. In the typical shake-flask cultures that are widely
used, many environmental parameters may change simultaneously during the time-course, rendering an interpretation of experimental results inconclusive in many situations.
Even in well-controlled culture systems changes of some
important parameters cannot be easily separated, as we
demonstrated in the case of oxygen limitation triggered by
iron deficiency in P. aeruginosa cultures (Kim et al., 2003).
The interplay of multiple parameters is particularly critical
for studying cell-density-dependent phenomena such as
QS. As mentioned above, there is evidence to suggest that
the expression of genes involved in QS is controlled not
only by cell density but also by environmental factors such
as the concentration of iron and oxygen. However, the
inherent effects of these parameters have not been clearly
demonstrated, because they have not been studied separately. In this work, we used a computer-controlled cultivation system to quantitatively and separately study the effects
of iron and oxygen concentrations on the expression of
lasR of the QS system in P. aeruginosa PAO1. To exclude
the effects of cell density, continuous culture was also used
to ensure a relatively constant cell density.
Iron acquisition from the environment is important for the
growth of P. aeruginosa and is related to its pathogenicity
(e.g. the change to mucoid form and biofilm formation)
(Singh et al., 2000; Costerton et al., 1999). Iron availability
for P. aeruginosa is often limited in biofilm, the primary
form of growth of this pathogen in the lung of cystic fibrosis
patients (Haas et al., 1991; Mathee et al., 1999; Frederick
et al., 2001; Singh et al., 2000). Iron availability is also
important for the host in the context of host–pathogen
interaction. As a defence mechanism, the host cell may
sequester iron to limit the growth of the pathogen. Iron
deprivation interferes with one of the important innate
immune responses of host cells, i.e. the formation of
oxidants for oxidative killing of pathogens, and can lead to
an accumulation of superoxide and hydrogen peroxide.
Several mechanisms are proposed for P. aeruginosa to
protect against environmental oxidative stresses, such as
the formation of superoxide dismutases and catalase, which
is controlled by QS, and the formation of the exopolysaccharide alginate on the cell surface (Mathee et al., 1999;
Hassett et al., 1999; Stewart et al., 2000; Valente et al.,
2000). Alginate formation is an efficient strategy for cells to
quench oxidative stress and P. aeruginosa has been shown
to increase its alginate formation under oxidative stress
(Sabra et al., 2002; Mathee et al., 1999; Hassett et al., 1999;
Stewart et al., 2000; Valente et al., 2000).
Bacterial strains and growth conditions. P. aeruginosa PAO1
(deposit NCCB 2452 obtained from the Netherlands Culture
Collection of Bacteria) was used for generating a recombinant strain
in this study. Plasmid pMAM301, which contains the promoter
region of lasR, was kindly donated by Dr B. H. Iglewski (Albus et al.,
1997). Plasmid pMAM301 was based on plasmid pQF50, which has
the promoterless lacZ gene and a pRO1600 replicon for a broad
range of host plasmids in Gram-negative bacteria. Fifteen bases of
the lacZ gene were replaced with the Shine–Dalgarno sequence
(Farinha & Kropinski, 1990) and the lasR promoter region from
nucleotide 2324 to 24 was fused to lacZ. This plasmid was introduced into P. aeruginosa NCCB 2452 by electroporation. The electrical setting for electroporation was as follows: set voltage, 2?5 kV;
discharge capacitor, 25 mF; pulse controller parallel resister, 200 V.
After electroporation, transformed cells were selected on LB agar
plates containing 300 mg carbenicillin ml21. Cells were cultivated in
a modified glucose minimal medium described previously (Sabra
et al., 2002). The medium contained 300 mg carbenicillin ml21 and
varied concentration of FeSO4.7H2O. Seed culture was prepared in
medium A without iron (Mian et al., 1978) and shaken vigorously
at 37 uC.
Recently, we proposed a possible new defence mechanism
of P. aeruginosa against oxidative stress in relation to iron
deficiency (Sabra et al., 2002; Kim et al., 2003). We showed
that P. aeruginosa could strongly reduce the transfer of
The wild-type P. aeruginosa NCCB 2452 strain was used for proteomic study. For shake-flask cultures, medium A without iron was
used for seed culture. Medium A which contained 0?6 mg FeSO4.7H2O
l21 was used for low-iron medium and 7 mg FeSO4.7H2O l21 for ironrich medium for growth cultivation. For batch cultures, conditions
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METHODS
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Microbiology 151
Effects of iron and oxygen on lasR expression
were as described for the recombinant strain batch cultivation. Lateexponential-phase cultures were centrifuged and the supernatant was
harvested for proteomic analysis.
Cultivation system and parameter control. Batch cultivations
with dissolved oxygen tension (pO2) controlled at 10 % of air
saturation were carried out in a computer-controlled and highly
instrumented bioreactor system under replete (7 mg l21) and low
(0?6 mg l21) FeSO4.7H2O concentrations as described previously
(Kim et al., 2003; Sabra et al., 2002) for both the wild-type and
recombinant P. aeruginosa strains. Continuous cultivations of
recombinant P. aeruginosa strains were carried out in a 1?5 l stirredtank bioreactor with a working volume of 1?0 l at a constant dilution rate of 0?2 h21 as described by Sabra et al. (2003). The agitation
speed was constant at 300 r.p.m. pO2 was controlled at various
ranges of 0–220 % of air saturation by mixing nitrogen and pure
oxygen in the inlet gas. Varied FeSO4.7H2O concentrations (0, 1,
2?5, 3?5, 4 and 7 mg l21) in the feed medium were used for the continuous culture under microaerobic conditions (pO2 air saturation<0 %). The total aeration rate was kept at a constant value
(1 litre min21) by a proportional-integral-differential controller
defined through the real-time operation computer-control system
(UBICON, Universal Bioprocess Control System; ESD). State-steady
data are mean values from three samples after at least four reactor
volume exchanges.
Biochemical analysis. The total amount of extracellular protein in
cell-free supernatant was determined by the Lowry method. Elastase
activity was determined in a spectrophotometric assay using elastinCongo red (Sigma) as a substrate as described by Kessler et al.
(1993). Siderophores (pyoverdine and pyochelin) were measured
with a microtitre plate fluorometer (MFX Microtitre Plate Fluorometer, DYNEX). Fluorescence was determined by exciting the supernatant of the culture at 400 nm for pyoverdine and 355 nm for
pyochelin; the emission was measured at 460 nm for pyoverdine and
440 nm for pyochelin (McMorran et al., 2001; Ankenbauer et al.,
1985). Biomass dry weight was determined gravimetrically as
described previously (Sabra et al., 2000). The concentration of iron
ions in the culture supernatant was separately determined as Fe2+
and Fe3+ by spectrophotometric assay using iron test kits (Merck)
as described previously (Kim et al., 2003).
b-Galactosidase activity measurement. Cells were centrifuged
at 13 000 r.p.m. for 5 min and the pellet was resuspended in
Z buffer (per litre of distilled water: 16?1 g Na2HPO4.7H2O;
5?5 g NaH2PO4.H2O; 0?75 g KCl; 0?25 g MgSO4.7H2O; 2?7 ml 2mercaptoethanol) at a ratio of 1 : 2 to 1 : 10 to obtain a final OD420
of 0?2–0?8. Chloroform and 0?1 % SDS were added to this suspension and mixed vigorously. Preheated (28 uC) o-nitrophenyl b-Dgalactoside (0?2 ml, 4 mg ml21) was added to the above mixture
and incubated for 30 min at 37 uC. The reaction was then stopped
by adding 1 M Na2CO3. The mixture was centrifuged to remove cell
debris, and the A420 measured. b-Galactosidase activity was determined in Miller units (Miller, 1972).
Separation of extracellular proteins by two-dimensional gel
electrophoresis (2-DE). Extracellular proteins were extracted by
precipitation of filtered culture supernatant with 20 % (w/v)
trichloroacetic acid after overnight stirring at 4 uC and collected by
centrifugation (18 000 r.p.m. for 30 min at 4 uC) (Nouwens et al.,
2002). The protein pellet was then washed three times with ice-cold
acetone containing 0?1 % (w/v) DTT and frozen at 280 uC until
analysed.
For 2-DE analysis protein pellets were solubilized in a 2-DEcompatible buffer consisting of 7 M urea, 2 M thiourea, 4 % (w/v)
CHAPS, 50 mM DTT, 0?5 % (w/v) carrier ampholytes pH 3–10.
Total protein concentrations were measured using Bradford protein
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assay reagent Roti-Quant (Roth) according to the manufacturer’s
protocol. Isoelectric focusing was performed using the IPGphor
Isoelectric Focusing System and Immobiline DryStrip 18 cm pH 3–
10NL (Amersham Biosciences). One hundred micrograms of each
protein sample was applied to an IPG strip by in-gel rehydration. Each
sample was run in parallel. Isoelectric focusing was carried out as
described by Wang et al. (2003a). Proteins were focused for a total
of 80 kVh. The second-dimension electrophoresis (SDS-PAGE) was
carried out using the vertical slab separation unit Ettan Dalt II
System and pre-cast Ettan Dalt II 12?5 % gels (Amersham Biosciences).
The focused IPG strips were equilibrated as recommended by the
manufacturer, then separated at 20 uC using the constant-power
mode of 2 W per gel for 1 h, followed by 20 mA per gel until the
bromophenol blue dye front reached the bottom of the gel. For
visualization, gels were stained with Brilliant Blue G-colloidal
Concentrate (Sigma) (Wang et al., 2003a). After image scanning, the
2-D gels were evaluated with PHORETIX 2D ADVANCE software, VERSION
6.00 (Phoretix).
Protein identification by mass spectrometric analysis. Protein
spots excised from 2-D gels were digested with trypsin (Promega),
purified with reversed-phased C18 ZipTip pipette tips (Millipore)
and analysed by matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI/TOF MS) as described by Wang
et al. (2003b).
Peptide masses generated from the MALDI/TOF MS analysis were
used for protein identification by peptide mass fingerprinting (PMF).
Using the search program MASCOT (Matrix Science) peptide masses
were compared to the predicted peptide masses in a protein database
of P. aeruginosa installed on a local MASCOT server. Trypsin was given
as the digestion enzyme, one missed cleavage site was allowed, cysteine
was modified by iodoacetamide and methionine was assumed to be
partially oxidized. All peptide mass values are monoisotopic and the
mass tolerance was set at 100 p.p.m.
RESULTS
Response of lasR expression to iron deficiency
and oxygen limitation in controlled batch
culture
To investigate the effects of iron concentration on lasR
expression, we cultivated a recombinant P. aeruginosa PAO1
strain containing the reporter plasmid pMAM301 (lasRp–
lacZ) in computer-controlled batch cultures with different
iron concentrations at a preset dissolved oxygen tension
(pO2) of 10 % air saturation (Fig. 1). As previously observed
for the wild-type strain (Kim et al., 2003), a control of pO2
at 10 % of air saturation was only possible in the culture
with iron-rich medium (Fig. 1a). In the culture with a low
iron concentration, iron was completely consumed after
10–12 h cultivation (Fig. 1b). After that the pO2 dropped
drastically and reached zero although the flow rate of O2
in the inlet gas was strongly increased by the computercontrol system with pure O2, which resulted in oxygen
limitation (microaerobic conditions) in the culture. During
the cultivation period with pO2 controlled at 10 % air
saturation, the cell density in both cultures was similar.
However, in the culture with low iron concentration, the
cell density rapidly increased during the period of oxygen
limitation. The lasR expression, determined by the activity
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Fig. 1. Control of pO2, growth parameters and LasR (b-galactosidase) expression in batch cultures of recombinant P.
aeruginosa PAO1 (plasR–lacZ) grown in (a) iron-rich medium and (b) low-iron medium. w, b-Galactosidase expression level;
$, biomass; –, pO2; &, inlet flow rate of O2; %, Fe2+ concentration; m, Fe3+ concentration. Each point represents the
mean±SD of three separate measurements.
of b-galactosidase, followed similar trends as the cell density
in the two cultures.
The above results seem to fit well with the generally accepted
conjecture that the expression of the QS transcriptionalregulatory gene lasR in P. aeruginosa is primarily regulated
by the cell density. However, as revealed in Fig. 2, a strict
correlation between the expression level of lasR and the
cell density did not exist over the whole range of cell
densities studied. The b-galactosidase activity appeared to
be higher under iron-limiting conditions (i.e. for cell
densities>0?30 g l21). Since an iron limitation simultaneously leads to an oxygen limitation in batch cultures of
P. aeruginosa (Kim et al., 2003), it is not possible to separate
the effects of iron and oxygen limitation on the expression of
lasR with the above experiments in typical batch cultures.
LasR is known to affect the production of a number of
virulence factors related to QS in P. aeruginosa (Wagner
et al., 2003). The secretion of total protein and the formation of two typical virulence factors elastase and pyoverdine
are shown in Fig. 3 as functions of cultivation time and
cell density for cultures with low and high iron concentrations. In general, they did not directly correlate with the
cell density, and higher amounts of secreted proteins (3a),
elastase (3b) and pyoverdine (3c) were found for the lowiron culture.
Effect of iron concentration on lasR expression
in continuous cultures excluding influences of
cell density and oxygen concentration
Fig. 2. b-Galactosidase expression level as a function of biomass for batch cultivation of recombinant P. aeruginosa PAO1
(plasR–lacZ). ,, Iron-rich medium; $, low-iron medium. Each
point represents the mean±SD of three separate measurements.
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To distinguish the inherent effect of iron concentration on
lasR expression from the effects of cell density and pO2,
we investigated the expression of lasR in cells grown in
continuous culture with relatively constant values of cell
density and pO2 but with varied iron concentrations. The
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Effects of iron and oxygen on lasR expression
Fig. 4. b-Galactosidase activity (&), iron concentration (m)
and biomass (#) in glucose- and oxygen-limited chemostat
culture with different iron inlet concentrations. ., outlet iron
concentration; &, b-galactosidase expression level; #, biomass. Each point represents the mean±SD of three separate
measurements.
the batch cultures (Figs 1 and 2). The activity of bgalactosidase significantly decreased with increasing iron
concentration in the cultures. The b-galactosidase activity
was maximal under iron-deficient conditions. Secretion
of proteins showed similar trends to lasR expression (data
not shown).
Effect of oxygen concentration on lasR
expression in continuous cultures
To assess the effect of oxygen concentration on lasR
expression, additional experiments with the recombinant
P. aeruginosa strain were performed in continuous cultures with a constant dilution rate of 0?2 h21 but varied
pO2 in the range of nearly zero to as high as 200 % air
saturation (sparging with pure O2).
Fig. 3. Specific and total (inset graphs) protein secretion (a),
elastase (b) and siderophore production (c) in batch cultures of
P. aeruginosa in iron-rich medium (%) and low-iron medium
(&). Each point represents the mean±SD of three separate
measurements.
continuous culture had a constant dilution rate of 0?2 h21
(i.e. a constant growth rate) and a negligible percentage of
air in the inlet flow gas, resulting in pO2<0 %. Fig. 4 shows
the activity of b-galactosidase (measuring lasR expression)
as a function of inlet iron concentration in different steady
states of the continuous cultures. Also shown is the relatively constant cell density (0?15–0?18 g l21), which is
significantly lower than that (0?33–0?44 g l21) reached in
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In iron-deficient continuous cultures, the biomass concentration varied from steady state to steady state according
to pO2 levels (Fig. 5a). The cell density reached a maximum
in the culture with a pO2 of 5 % air saturation. This is
consistent with the finding that P. aeruginosa grows
optimally under microaerobic conditions (Sabra et al.,
2002). However, it is interesting to note that lasR expression reached a minimum at this condition. The expression
of lasR increased with decreased cell density at pO2>5 %
of air saturation. The highest value of lasR was observed at
pO2<0 %, where the cell density was at a minimum.
Such an increase of lasR expression at pO2<0 % was not
observed in the iron-rich continuous culture (Fig. 5b). In
fact, under iron-rich conditions, the expression of lasR
increased only slightly with increased oxidative stress. The
cell density in the iron-rich culture also showed a different
trend compared with that in the low-iron continuous
culture under similar oxygen-stress conditions. There was
no decrease of the cell density with increased oxygen stress
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and the highest biomass concentration was observed under
the high oxygen stress (pO2<217 %).
We also measured the extracellular protein production
under different oxygen-stress conditions. As found previously with the wild-type strain (Sabra et al., 2002; Kim
et al., 2003), the extracellular protein concentration
increased with increasing oxygen stress in both the lowiron and iron-rich media. It was generally somewhat
higher under low-iron conditions. The effects of iron
and oxygen concentrations on the formation of virulence
factors such as pyoverdine and elastase are also similar,
as reported previously (Sabra et al., 2002; Kim et al., 2003;
data not shown).
Proteomic analysis of extracellular proteins
To investigate the effects of iron and O2 limitation on
the secretion of proteins, proteomic analysis of the extracellular proteins was carried out for samples from ironrich and low-iron batch cultivations of the wild-type
P. aeruginosa strain. Fig. 6 shows the different protein
secretion patterns for cells grown under iron-limiting (a)
and iron-rich (b) conditions. The major extracellular proteins identified by peptide fingerprinting include alkaline
protease (AprA), elastase (LasB), Pvds-regulated endoprotease (PrpL), chitin-binding protein (CbpD), sulfatebinding protein (Sbp), sulfate-binding protein of ABC
transporter (CysP), flagellin typeB (FliC), flagellar cap
protein (FliD), porin protein (OprD), outer-membrane
Fig. 5. b-Galactosidase activity (bars) and biomass (&) in (a)
low-iron and glucose-limited, and (b) iron-rich and glucoselimited chemostat cultures at steady states under different
oxygen stresses. Each point represents the mean±SD of three
separate measurements.
(a)
(b)
3
pH
10
3
10
pH
kDa
97
66
45
30
20.1
14.4
Fig. 6. 2-DE of P. aeruginosa extracellular proteins from batch cultivations with iron deprivation (a) (numbers correspond to
those in Table 1; dotted arrows, down-regulated protein; continuous arrows, up-regulated protein), and iron-rich conditions
(b) on pH 3–10 IPG gels.
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Effects of iron and oxygen on lasR expression
protein OprL precursor (OprL), azurin precursor (Azu),
outer-membrane metalloproteinase precursor (PA4370),
type III export protein PscL and several other hypothetical
proteins. Several membrane or intracellular proteins were
also found in the secreted proteins. This may be partly due
to exocytosis and formation of membrane vesicles on the
outer membrane (Beveridge, 1999; Sabra et al., 2003).
Membrane vesicles contain intracellular and membrane
proteins (mostly virulence factors) and can be released into
the extracellular milieu, where they lyse.
limitation also occurred in the shake-flask culture. In fact,
because of the restricted gas exchange and the low oxygen
transfer coefficient the shake-flask culture should have
a more profound oxygen limitation than the controlled
batch cultures, which had a much high aeration rate (even
with pure oxygen in the late growth phase). The expression
and secretion of several virulence factors and hypothetical
proteins were found to be sensitive to the availability of
iron and O2 in these cultures. For example, AprA, a
toxic protease involved in obtaining iron from host cells
(Shigematsu et al., 2001), significantly increased under
oxygen limitation triggered by iron deprivation in the late
exponential phase of the batch culture. However, in shakeflask cultivation, the increase of AprA secretion was not
as significant as in the batch cultivation. A similar change
was also found for the secretion of PrpL, an endoprotease
which is regulated by PvdS and the QS system. It can
hydrolyse casein, elastin and lactoferrin (Wilderman et al.,
2001; Lamont et al., 2002; Ravel & Cornelis, 2003; ArevaloFerro et al., 2003). It seems that the secretion of both AprA
and PrpL is very sensitive to oxygen concentration. So far,
little is known about the inherent effects of iron and O2 on
the expression of these proteins.
Most of the proteins identified were up-regulated in response to iron limitation. In contrast to the other proteins,
the flagellin type B protein FliC was found to decrease under
iron-limited conditions. Flagellin type B is involved in
flagellar-mediated chemotactic motility. Loss of flagella has
been reported for mucoid P. aeruginosa strains and chronic
infection strains from the lungs of cystic fibrosis patients
(Drake & Montie, 1988; Feldman et al., 1998; Wolfgang
et al., 2004). Our results support the notion that ironlimited conditions induce mucoidy and reduce movement
in P. aeruginosa. In fact, it is known that the mucoid condition is due to alginate production and is related to iron
limitation (Shand et al., 1991). This is also consistent with
our previous finding that alginate overproduction mainly
occurred under iron-limited conditions (Kim et al., 2003).
Our analysis of the secretome (although less comprehensive) agrees well with the results of recent transcriptomic
analysis of iron limitation in P. aeruginosa (Ochsner et al.,
2002) (Table 1). We also compared our results with recent
proteomic and transcriptomic studies of QS (Arevalo-Ferro
et al., 2003; Hentzer et al., 2003; Schuster et al., 2003, 2004;
We also carried out proteomic analysis of shake-flask
cultures grown under iron-rich and low-iron conditions
(Fig. 7). As shown previously (Sabra et al., 2002), oxygen
(a)
3
(b)
pH
3
10
pH
10
kDa
97
66
45
30
20.1
14.4
Fig. 7. 2-DE of P. aeruginosa extracellular proteins from shake-flask cultivations under iron-deprivation (a) and iron-rich (b)
conditions on pH 3–10 IPG gels.
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Results are also compared to proteomic and transcriptomic data published in the literature. References: (1), Nouwens et al. (2003); (2), Arevalo-Ferro et al. (2003); (3), Ochsner et al.
(2002); (4), Hentzer et al. (2003); (5), Schuster et al. (2003); (6), Schuster et al. (2004); (7), Wagner et al. (2003); (8), Juhas et al. (2004). +, up-regulation; 2, down-regulation; ND, not
determined; NC, not changed; DvqsR, vqsR gene mutant P. aeruginosa strain.
Spot Annotation
no.
Protein or homologue
Proteome
(this study:
iron-limited
culture)
pI
Mol.
mass
(kDa)
Score
PMF Sequence
match/ coverage
(%)
search
(1)
Microbiology 151
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
PA1249
PA1249
PA2939
PA1092
PA1092
PA1092
PA1092
PA0852
PA0852
PA0852
PA0852
PA0852
PA0852
PA0852
PA0852
PA0852
PA0852
PA3724
PA3724
PA3724
PA3724
PA4175
PA4175
AprA: alkaline metalloproteinase precursor
Probable aminopeptidase
FliC: B-type flagellin
CpbD: chitin-binding protein CbpD precursor
LasB: elastase (neutral metalloproteinase)
PrpL: Pvds-regulated endoprotease, lysyl class
+
+
+
2
2
2
2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
4?28 50?402
5?02 57?818
5?40 49?213
6?38 42?347
6?28 53?882
6?45 48?582
95
184
106
99
202
209
247
58
100
46
59
70
95
61
96
79
65
155
73
179
83
146
161
8/16
11/22
10/33
9/19
13/21
14/26
15/22
5/12
8/17
4/10
3/6
7/25
8/17
5/11
7/11
7/17
6/16
14/28
7/12
19/41
9/20
10/17
11/25
24
33
29
23
31
32
33
16
33
12
8
25
22
17
14
14
12
28
17
35
19
27
33
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 21:30:48
Transcriptome (literature)
Proteome
(literature)
QS regulated
+
+
+
2
2
2
2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(2)
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Iron
limited
(3)
QS regulated
DvqsR
(4)
(5)
(6)
(7)
(8)
+
+
+
ND
+
+
+
+
ND
ND
+
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
+
ND
2
E.-J. Kim and others
1134
Table 1. Identification of secreted proteins in P. aeruginosa cultures by PMF from MALDI-TOF MS analysis (P<0?05)
http://mic.sgmjournals.org
Table 1. cont.
Spot Annotation
no.
Protein or homologue
Proteome
(this study:
iron-limited
culture)
pI
Mol.
mass
(kDa)
Score
PMF Sequence
match/ coverage
(%)
search
(1)
PA4175
PA3190
26
27
28
29
30
31
PA3190
PA0958
PA0958
PA0026
PA3923
PA3909
32
33
PA4370
PA1094
34
35
36
37
38
39
40
41
42
43
44
45
46
47
PA1493
PA0283
PA1725
PA0973
PA4922
PA0225
PA0423
PA3230
PA0572
PA5310
PA0888
PA3433
PA4016
PA0456
+
+
Probable binding protein component of ABC
ugar transporter
OprD: porin D precursor
Hypothetical protein
Hypothetical protein
Hypothetical protein
Extracellular nuclease (by similarity)
Outer-membrane metalloproteinase precursor
FliD: B-type flagellar hook-associated protein 2
(flagellar cap rotein)
CysP: sulfate-binding protein of ABC transporter
Sbp: sulfate-binding protein precursor
PscL: type III export protein PscL
OprL: outer-membrane protein OprL precursor
Azu: azurin precursor
Probable transcriptional regulator
Hypothetical protein
Hypothetical protein
Hypothetical protein
Conserved hypothetical protein
AotJ: arginine/ornithine binding protein
Probable transcriptional regulator
Hypothetical protein
Probable cold-shock protein
+
+
+
+
+
2
+
+
+
+
NC
+
2
2
+
+
+
+
2
ND
ND
ND
+
37
18
28
18
19
24
ND
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
ND
ND
+
+
+
ND
2
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
8/18
5/10
19
17
ND
ND
+
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
7/12
11/18
6/47
3/4
4/18
4/17
5/11
6/24
10/17
4/4
6/14
6/42
7/28
5/21
26
34
28
13
36
39
28
14
15
5
28
25
14
40
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
101
155
54
56
54
52
76
55
96
60
73
44
50
57
4?68
6?52
(8)
ND
7?77 36?495
8?45 36?411
4?95 24?000
5?95 17?971
6?39 16?169
6?39 19?981
6?09 20?764
5?83 42?585
6?13 100?607
9?4 59?220
6?43 28?106
5?73 33?012
5?66 62?793
8?09
7?702
+
+
(7)
+
47?430
49?420
36?809
69?775
83?143
(6)
ND
10/28
6/10
9/24
6/19
9/16
17/21
5?30
4?94
5?03
(5)
+
93
78
81
66
96
219
709
79
62
48?331
(4)
ND
19
19
4?96
+
+
+
+
+
+
DvqsR
+
7/14
6/14
45?331
QS regulated
ND
88
70
5?68
(2)
Iron
limited
(3)
1135
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On: Fri, 16 Jun 2017 21:30:48
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
ND
+
+
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Effects of iron and oxygen on lasR expression
24
25
Transcriptome (literature)
Proteome
(literature)
QS regulated
E.-J. Kim and others
Wagner et al., 2003) (Table 1). Several of the up-regulated
extracellular proteins (e.g. AprA, LasB, PrpL) under iron
limitation are in fact QS-regulated proteins as shown in
Table 1. Overall, the proteomic analysis also strongly
supports the notion that iron availability can affect the
regulation of QS-related genes.
DISCUSSION
The results of this study demonstrate that the expression
of the QS regulator LasR is affected by the availability of
iron and oxygen in cultures of P. aeruginosa irrespective of
the cell density. The expression of lasR as measured by a
reporter enzyme (b-galactosidase) and the formation of
elastase, which is considered to be controlled by QS, do
not always closely correlate with the cell density. This was
first shown with batch cultures (Fig. 2). The results are
consistent with the data of Bollinger et al. (2001) regarding
the effects of iron limitation on the expression of lasI. In
our study, oxygen limitation seems also to enhance the
expression of lasR. However, the inherent effects of iron
and O2 cannot be separately assessed in batch cultures
because of the concurrent changes of culture conditions
during the cultivation in general, and in particular due
to the accompanying oxygen limitation caused by iron
limitation in P. aeruginosa cultures (Fig. 1b). Therefore, we
used continuous culture (chemostat) conditions to study
the effects of each parameter separately. In addition to the
possibility to control the individual culture parameter
exactly, the chemostat culture has another important
advantage, namely a relatively constant cell density at
different steady states (Fig. 4). This makes it particularly
suited for studying cellular responses that might be affected
by the cell density such as the expression of lasR, which
encodes the QS regulator. The results in Fig. 4 clearly show
that the expression of lasR is significantly enhanced by
iron limitation independent of the cell density. The effect
of O2 concentration on the expression of lasR is more
complex (Fig. 5). In general, the expression of lasR is
slightly decreased by decreasing O2 concentration with the
exception that a dual limitation by both iron and O2 can
reverse the declining trend (Fig. 5a). The steady state with
dual iron and O2 limitation mimics the situation of the
late phase of the batch culture with low iron concentration
(Fig. 1b) in which the lasR expression was higher.
Several large-scale transcriptomic and proteomic studies
of QS and iron limitation in P. aeruginosa have been
published recently (Ochsner et al., 2002; Arevalo-Ferro et al.,
2003; Nouwens et al., 2002, 2003; Palma et al., 2003; Wagner
et al., 2003; Schuster et al., 2003, 2004; Juhas et al., 2004;
Wolfgang et al., 2004; Cornelis & Aendekerk, 2004). We
compared the regulation of secreted proteins detected in
our work by proteomic analysis with those from the transcriptomic and proteomic studies reported in the literature (Table 1). Except for the protein FliC, there is a
good correlation among the expression patterns of ironregulated and QS-regulated genes. These results strongly
1136
support the notion of Whiteley et al. (1999) and Cornelis
& Aendekerk (2004) that there are links between the QS
and iron-regulatory systems.
In summary, this study clearly demonstrated the inherent
effects of iron and O2 on the expression of lasR, which
encodes the key regulatory protein of QS in P. aeruginosa.
In particular, the dual limitations by iron and O2 have
the strongest effect. This was not obvious by solely
examining shake-flask or even controlled batch cultures,
but was revealed by careful studies with chemostat cultures.
In this respect, it is worth mentioning that apparent celldensity-dependent phenomena sometimes reported in the
literature can in fact be due to the different availability of
nutrients or changes of other environmental conditions,
as demonstrated in this work and previously quantitatively
shown for the so-called cell-density effect on the metabolism of animal cells by mathematical modelling (Zeng,
1996). This possibility has so far received little attention
in the study of QS but is highly relevant. In general, little
is known about the mechanisms of regulation of LasR by
different environmental factors and how the effects are
transmitted to other parts of the cellular machinery
through LasR, which is embedded in a large and complex
regulatory network. On the other hand, many genes are
involved in iron and O2 regulation (Vasil & Ochsner, 1999;
Hassett et al., 2002). The interplays of LasR, QS and the
regulatory components related to iron and oxygen deserve
further study. To this end, a quantitative and systematic
approach is important as shown in this work for the effects
of iron and O2 on the expression of lasR.
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