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 Kim1, Wei Wang2, Wolf-Dieter Deckwer2 and An-Ping Zeng1

1 Division of Molecular Biotechnology, GBF – German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany
2 Group of TU-BCE, GBF – German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany

Correspondence
An-Ping Zeng
AZE{at}GBF.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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. {beta}-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{approx}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{approx}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.


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


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.

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 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 Arevalo-Ferro 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 iron-regulated were found to be repressed in a vqsR mutant, among which some were also regulated by QS.

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).

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 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 –324 to –4 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 µF; pulse controller parallel resister, 200 {Omega}. After electroporation, transformed cells were selected on LB agar plates containing 300 µg carbenicillin ml–1. Cells were cultivated in a modified glucose minimal medium described previously (Sabra et al., 2002). The medium contained 300 µg carbenicillin ml–1 and varied concentration of FeSO4.7H2O. Seed culture was prepared in medium A without iron (Mian et al., 1978) and shaken vigorously at 37 °C.

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 l–1 was used for low-iron medium and 7 mg FeSO4.7H2O l–1 for iron-rich medium for growth cultivation. For batch cultures, conditions were as described for the recombinant strain batch cultivation. Late-exponential-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 l–1) and low (0·6 mg l–1) 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 stirred-tank bioreactor with a working volume of 1·0 l at a constant dilution rate of 0·2 h–1 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 l–1) in the feed medium were used for the continuous culture under microaerobic conditions (pO2 air saturation{approx}0 %). The total aeration rate was kept at a constant value (1 litre min–1) 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 elastin-Congo 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).

{beta}-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 2-mercaptoethanol) 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 °C) o-nitrophenyl {beta}-D-galactoside (0·2 ml, 4 mg ml–1) was added to the above mixture and incubated for 30 min at 37 °C. The reaction was then stopped by adding 1 M Na2CO3. The mixture was centrifuged to remove cell debris, and the A420 measured. {beta}-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 °C and collected by centrifugation (18 000 r.p.m. for 30 min at 4 °C) (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 –80 °C until analysed.

For 2-DE analysis protein pellets were solubilized in a 2-DE-compatible 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 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 °C 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-of-flight 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 computer-control 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 of {beta}-galactosidase, followed similar trends as the cell density in the two cultures.



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Fig. 1. Control of pO2, growth parameters and LasR ({beta}-galactosidase) expression in batch cultures of recombinant P. aeruginosa PAO1 (plasR–lacZ) grown in (a) iron-rich medium and (b) low-iron medium. *, {beta}-Galactosidase expression level; {bullet}, biomass; –, pO2; {blacksquare}, inlet flow rate of O2; {square}, Fe2+ concentration; {blacktriangleup}, Fe3+ concentration. Each point represents the mean±SD of three separate measurements.

 
The above results seem to fit well with the generally accepted conjecture that the expression of the QS transcriptional-regulatory 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 {beta}-galactosidase activity appeared to be higher under iron-limiting conditions (i.e. for cell densities>0·30 g l–1). 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.



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Fig. 2. {beta}-Galactosidase expression level as a function of biomass for batch cultivation of recombinant P. aeruginosa PAO1 (plasR–lacZ). {triangledown}, Iron-rich medium; {bullet}, low-iron medium. Each point represents the mean±SD of three separate measurements.

 
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 low-iron culture.



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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 ({square}) and low-iron medium ({blacksquare}). Each point represents the mean±SD of three separate measurements.

 
Effect of iron concentration on lasR expression in continuous cultures excluding influences of cell density and oxygen concentration
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 continuous culture had a constant dilution rate of 0·2 h–1 (i.e. a constant growth rate) and a negligible percentage of air in the inlet flow gas, resulting in pO2{approx}0 %. Fig. 4 shows the activity of {beta}-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 l–1), which is significantly lower than that (0·33–0·44 g l–1) reached in the batch cultures (Figs 1 and 2). The activity of {beta}-galactosidase significantly decreased with increasing iron concentration in the cultures. The {beta}-galactosidase activity was maximal under iron-deficient conditions. Secretion of proteins showed similar trends to lasR expression (data not shown).



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Fig. 4. {beta}-Galactosidase activity ({blacksquare}), iron concentration ({blacktriangleup}) and biomass ({circ}) in glucose- and oxygen-limited chemostat culture with different iron inlet concentrations. {blacktriangledown}, outlet iron concentration; {blacksquare}, {beta}-galactosidase expression level; {circ}, biomass. Each point represents the mean±SD of three separate measurements.

 
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 h–1 but varied pO2 in the range of nearly zero to as high as 200 % air saturation (sparging with pure O2).

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{approx}0 %, where the cell density was at a minimum.



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Fig. 5. {beta}-Galactosidase activity (bars) and biomass ({blacksquare}) in (a) low-iron and glucose-limited, and (b) iron-rich and glucose-limited chemostat cultures at steady states under different oxygen stresses. Each point represents the mean±SD of three separate measurements.

 
Such an increase of lasR expression at pO2{approx}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 and the highest biomass concentration was observed under the high oxygen stress (pO2{approx}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 low-iron 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 iron-rich 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), sulfate-binding protein (Sbp), sulfate-binding protein of ABC transporter (CysP), flagellin typeB (FliC), flagellar cap protein (FliD), porin protein (OprD), outer-membrane 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.



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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.

 
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 iron-limited 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).

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 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 shake-flask 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; Arevalo-Ferro 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.



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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.

 
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; 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.


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Table 1. Identification of secreted proteins in P. aeruginosa cultures by PMF from MALDI-TOF MS analysis (P<0·05)

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; –, down-regulation; ND, not determined; NC, not changed; {Delta}vqsR, vqsR gene mutant P. aeruginosa strain.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 ({beta}-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 iron-regulated and QS-regulated genes. These results strongly 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 cell-density-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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 18 August 2004; revised 20 December 2004; accepted 21 December 2004.



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