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
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ABSTRACT |
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INTRODUCTION |
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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 hostpathogen 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.
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METHODS |
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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 l1 was used for low-iron medium and 7 mg FeSO4.7H2O l1 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 l1) and low (0·6 mg l1) 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 h1 as described by Sabra et al. (2003)
. The agitation speed was constant at 300 r.p.m. pO2 was controlled at various ranges of 0220 % 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 l1) 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 min1) 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
).
-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 0·20·8. Chloroform and 0·1 % SDS were added to this suspension and mixed vigorously. Preheated (28 °C) o-nitrophenyl -D-galactoside (0·2 ml, 4 mg ml1) 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.
-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 310. 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 310NL (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.
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RESULTS |
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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.
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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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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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DISCUSSION |
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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.
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Received 18 August 2004;
revised 20 December 2004;
accepted 21 December 2004.
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