GBF Gesellschaft für Biotechnologische Forschung mbH, Microbial Systems, Mascheroder Weg 1, 38124 Braunschweig, Germany1
Author for correspondence: An-Ping Zeng. Tel: +49 531 6181 188. Fax: +49 531 6181 751. e-mail: AZE{at}GBF.de
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ABSTRACT |
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Keywords: oxygen transfer, pathophysiology, polysaccharide capsule, microaerophilic growth, virulence factor
Abbreviations: CF, cystic fibrosis; kLa, oxygen transfer coefficient; pO2, dissolved oxygen tension
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INTRODUCTION |
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Numerous cell-associated factors, including alginate, pili and lipopolysaccharide, are important in P. aeruginosa virulence. Furthermore, many P. aeruginosa virulence factors, including toxins (exotoxin A and exoenzyme S), proteases (elastase, LasA protease and alkaline protease) and haemolysins (phospholipase and rhamnolipid), are released from cells of the organism (Kamath et al., 1998 ; Rumbaugh et al., 1999
). These factors contribute to the virulence of P. aeruginosa in animal models and have been the focus of in vitro studies with clinical isolates (Pearson et al., 1997
).
The pathogenic roles of alginate, in particular in relation to the formation of the mucoid phenotype in P. aeruginosa strains, have received much attention (see review by Govan & Deretic, 1996 ). It is now recognized that oxygen plays an important role in the formation of alginate and the appearance of the mucoid phenotype in P. aeruginosa (Krieg et al., 1986
; Leitäo & Sä-Correia, 1997
; Sato et al., 1984
; Xu et al., 1998
). Oxygen-dependent upregulation of transcription of alginate genes was normally found in highly mucoid strains of P. aeruginosa (Leitäo & Sä-Correia, 1997
). In an experiment with a mixture of mucoid and non-mucoid P. aeruginosa strains (1:1), Krieg et al. (1986)
found that aeration of cultures favourably selected for the growth of mucoid strains. Non-mucoid strains of P. aeruginosa were reported to be sensitive to oxygen. In previous studies with another alginate-producing bacterium (Azotobacter vinelandii) grown under controlled oxygen tension in a bioreactor system, we have shown that the formation of alginate exopolysaccharide on the cell surface of this organism is dependent on the oxygen tension (Sabra et al., 2001
, 1999
). Hence, alginate formation represents an important mechanism for protecting bacterial cells from oxidative stress, especially under conditions of diazotrophical growth.
In this work, we have quantitatively studied the physiological responses of P. aeruginosa PAO1 to oxidative stress in computer-controlled microaerobic and aerobic cultures. In addition to cell growth, attention was paid to polysaccharide formation and the release of proteins (virulence factors) by the bacterium under these conditions. For the first time, we report that cultures of P. aeruginosa PAO1 exhibit the unusual property of dramatically reducing the efficiency of oxygen transfer and thus the dissolved oxygen concentration in the culture broth. The reasons and consequences for these unusual physiological responses to oxidative stress are discussed.
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METHODS |
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A shake-flask-culture study was done in 100 ml Erlenmeyer flasks containing 40 ml medium A (Mian et al., 1978 ). In experiments for evoking mucoidy in PAO1 by oxidative stress, the cells were exposed to 30 mM H2O2 in the early-exponential phase of growth and shaken vigorously at 37 °C.
Bioreactor and control.
Cultivation of PAO1 was carried out in a 2·5 l stirred tank bioreactor (Applikon) with a working volume of 1·5 l. The agitation speed was kept constant at 500 r.p.m. by a proportional-integral-differential (PID) controller installed in the control unit of the bioreactor. Temperature was kept at 37 °C by a single-loop PID controller. An autoclavable pH electrode (Mettler-Toledo) was used to measure the pH, which was kept at 7·0±0·2 by the automatic addition of HCl (2 M) or NaOH (2 M) to the bioreactor. The pO2 was measured by using an autoclavable pO2 electrode (Mettler-Toledo).
The bioreactor was aerated with a fine-porous gas distributor. Thermal mass flow meters/controllers were used for the supply of gases. The total aeration rate was kept at a constant value (1·5 l min-1) by the real-time operation computer control system UBICON (Universal Bioprocess Control System, ESD, Hanover, Germany). pO2 was controlled over the range 150±1·5% of air saturation by mixing nitrogen and pure oxygen in the inlet gas using a PID controller defined through the UBICON facilities.
Analytical methods.
Biomass dry weight of PAO1 was determined gravimetrically as described previously (Sabra et al., 2000 ). The alginate concentration in the culture supernatant was determined by precipitation of the compound with 3 vols of ice cold 2-propanal. The alginate pellet was suspended in water, reprecipitated with 2-propanal and resuspended in 2·5 ml water. The amount of alginate recovered from the culture supernatant was determined using a borate/carbazole method (Knutson & Jeanes, 1968
), with algal alginate used for calibration of the standard curve.
The total extracellular protein content of the cell-free supernatant was determined by using the Lowry method. Elastase activity present in the cell-free supernatant was determined in a spectrophotometric assay using Elastin/Congo red (Sigma) as a substrate, as described by Kessler et al. (1993) . The amount of rhamnolipid present in the cell-free supernatant was determined by a duplicate orcinol assay compared to a rhamnose standard (Kock et al., 1991
). Rhamnolipid was determined by using the relationship 0·1 mg of rhamnose corresponds to 2·5 mg of rhamnolipid.
Electron microscopy.
Transmission electron microscopy was used to detect the alginate capsule on the cell surface of P. aeruginosa cells that had been taken directly from the controlled bioreactor or uncontrolled flask cultures. Embedding and ultra-thin sectioning of P. aeruginosa were done as described previously for A. vinelandii (Sabra et al., 2000 ).
Oxygen transfer coefficient (kLa) determination.
For determination of the volumetric kLa, the static method of gassing-out was used (Stanbury & Whitaker, 1987 ). The equation applied for the determination of the kLa is as follows:
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where C* is the oxygen saturation concentration of the liquid with the aeration gas, CL is the concentration of oxygen in the liquid and t is the time in seconds.
Cell-free culture supernatants contained within 50 ml flasks were first sparged with nitrogen to get a pO2 value of zero; air was then supplied to the supernatants at a constant aeration rate and a constant agitation. An oxygen tension electrode connected to the UBICON system monitored the change in the dissolved oxygen concentration in the liquid. The increase in the dissolved oxygen concentration can be described by the above equation related with kLa. Temperature as well as agitation speed was fixed for all of the samples. The mean results of at least two measurements are presented.
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RESULTS |
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The reason for the strong reduction in the oxygen transfer rate in cultures of P. aeruginosa PAO1 is not clear. It is possible that this phenomenon could be due to the production of biosurfactants by P. aeruginosa. P. aeruginosa is known to produce several kinds of amphiphilic compounds, such as rhamnolipid, under oxidative stress. The production of these compounds can result in heavy foam formation and in significant alterations in the surface and interfacial tensions of water (Chayabutra et al., 2001 ; Guerra-Santos et al., 1986
; Haba et al., 2000
; Kock et al., 1991
; Maier & Soberon-Chavez, 2000
; Rocha et al., 1992
). The hydrophobic part of these amphiphilic compounds can bind onto the gas bubble, thereby forming an extra boundary layer and reducing the availability of oxygen for transfer to the bacterial cells. To assess this issue, we measured the level of rhamnolipid production in three P. aeruginosa cultures (Fig. 4
). The amounts of rhamnolipid produced by the cultures increased successively in all three cultures and reached more than 1 g rhamnolipid l-1. The specific formation rate of rhamnolipid was greater for cells grown at higher pO2 values. However, the absolute amount of rhamnolipid produced by the cultures was not significantly affected by the concentration of dissolved oxygen (Fig. 4
). This is also in agreement with the results of kLa measurements taken for supernatants from all of the cultures tested (Fig. 3
). Whatever the mechanism involved in the reduction of oxygen transfer, it can be stated that this reduction of transfer in the culture broth is a general property of P. aeruginosa PAO1 cultures when grown under the experimental conditions applied in this work.
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Occurrence of polysaccharide capsules on PAO1 cells as a response to oxidative stress
P. aeruginosa PAO1 has been reported to be a non-mucoid clinical strain (Mathee et al., 1999 ). We investigated the possible appearance of a polysaccharide layer on the surface of PAO1 cells grown under different pO2 levels, by examining ultra-thin sections of negatively-stained PAO1 cells using transmission electron microscopy. No obvious mucoid polysaccharide layer was observed on cells taken from a shake-flask culture (after 20 h incubation) (Fig. 5a
). It is worth mentioning that an on-line measurement of pO2 values during shake-flask cultivation of P. aeruginosa PAO1 revealed oxygen-limited growth (i.e. pO2=0) of the organism 34 h after inoculation of the medium (data not shown). Since almost all of the work reported in the literature for P. aeruginosa PAO1 has been done using shake-flask cultures, the observed non-mucoidy of this strain may reflect a phenotype of this organism only seen when it is grown under oxygen-limited conditions. In fact, cells of PAO1 grown under controlled oxygen tension displayed a markedly different morphology to those grown under no oxygen control. The cells grown under controlled oxygen tension had a clear thick polysaccharide layer on their surfaces (Fig. 5b
). A low amount of alginate (0·10·3 g alginate l-1) was also detected in the culture supernatant of strain PAO1 when it was grown under these conditions. As inferred from other mucoid strains of P. aeruginosa, the mucoid polysaccharide layer present on the surface of P. aeruginosa PAO1 is very likely to consist primarily of alginate (Mathee et al., 1999
). The occurrence of this layer in controlled microaerobic and aerobic cultures of P. aeruginosa PAO1 seems to represent a physiological response of this strain to oxidative stress.
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DISCUSSION |
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Microaerobic conditions are known to dominate in biofilms, the preferred mode of growth for P. aeruginosa in the lungs of CF patients (Costerton et al., 1999 ; de Beer et al., 1994
; Xu et al., 1998
). Microaerobic conditions have also been reported to be optimal for the growth of P. aeruginosa on hydrocarbons (Chayabutra & Ju, 2000
). The preference of P. aeruginosa for microaerophilic growth is in accordance with recent findings from genomic analysis of this species (Croft et al., 2000
). Analysis of the genomic DNA sequences of P. aeruginosa PAO1 has shown that this strain possesses a 15 kb cluster of genes that encodes a micro-oxic respiration system, similar to that used by nitrogen-fixing bacteria, and which includes genes that encode homologues of nitrogen fixation/micro-oxic regulatory cascade proteins (fixL, fixJ and fixK/anr). These genes enable nitrogen-fixing bacteria such as A. vinelandii to grow microaerobically and they may help P. aeruginosa to survive the unusual conditions in biofilms.
A second remarkable physiological and morphological change in P. aeruginosa PAO1 under oxidative stress is the occurrence of a polysaccharide on the cell surface (Fig. 3). This polysaccharide may be primarily composed of alginate, as found for several other strains of P. aeruginosa (Krieg et al., 1986
; Mathee et al., 1999
). In fact, alginate was also released into the culture broth by PAO1. Earlier studies have shown that P. aeruginosa PAO1 converts to the mucoid phenotype when cultures are treated with levels of H2O2 similar to those that can be normally released by polymorphonuclear leukocytes in the pulmonary tract of CF patients (Mathee et al., 1999
). The strong oxygen limitation observed in liquid cultures of PAO1 may explain the non-mucoid appearance of this strain in cells from shake-flask cultures (Fig. 5a
).
The known pathogenic roles of alginate in P. aeruginosa, in the form of a mucoid bacterial coating or as a free substance, have been reviewed by several authors (e.g. Govan & Deretic, 1996 ; Miller & Britigan, 1997
). Besides its possible roles in adhesion and colonization (biofilm formation), this polysaccharide layer may also function as a physical barrier against the diffusion of oxygen into the cells, as has been suggested for the alginate-forming bacterium A. vinelandii (Sabra et al., 2000
). A possible taxonomic relationship between the nitrogen-fixing bacterium A. vinelandii and the opportunistic pathogen P. aeruginosa has been discussed previously (Fyfe & Govan, 1983
). A common feature of A. vinelandii and P. aeruginosa is the formation of the microbial polysaccharide alginate by these organisms under certain conditions (Sabra et al., 2001
, 1999
).
Another important role of the exopolysaccharide of P. aeruginosa is the avoidance of encounters with phagocyte-derived reactive oxygen intermediates (Miller & Britigan, 1997 ). The formation of reactive oxygen and reactive nitrogen intermediates is an important innate immune response of host cells upon their infection by microbial pathogens (Bogdan et al., 2000
; Nathan & Shiloh, 2000
). Pathogenic bacteria have developed several mechanisms of defence against these toxic reactive intermediates. In addition to the formation of a polysaccharide coat, other non-enzymic and enzymic means of removing these intermediates from the surroundings have been discussed for different pathogens (Miller & Britigan, 1997
). For example, P. aeruginosa has been reported to have highly specific enzymic pathways for oxidant inactivation, including those catalysed by superoxide dismutase, catalase and peroxidase. In this work, we postulate a possible new and more efficient defence strategy of pathogen cells against oxidants and related nitric oxide derivatives. For the first time, we have shown that P. aeruginosa PAO1 can strongly reduce oxygen transfer to the culture, causing oxygen limitation and thus simply blocking the sources for the formation of reactive oxygen intermediates. It would be interesting to investigate whether P. aeruginosa PAO1 could also cause oxygen limitation in its surrounding environment in the lungs of CF patients by such a mechanism, thus avoiding the toxicity of reactive oxygen species. In fact, P. aeruginosa PAO1 itself grows best under these oxygen-limited (microaerobic) conditions and seems to be more toxic (see below).
A third remarkable physiological response of P. aeruginosa PAO1 to oxidative stress is its enhanced release of proteins (Fig. 6b). The mechanisms and intrinsic physiological consequences of these enhanced secretions are basically unknown. So far, little work has been done on this enhanced protein secretion, particularly with respect to the release of different virulence factors as a response to oxidative stress. Indeed, the identification of these excreted proteins will be interesting, since the proteins excreted under different pO2 levels are highly heterogeneous. For example, elastase activity was almost only recorded at the lowest pO2 value tested (Fig. 7
). Moreover, the formation of pyocyanine by PAO1 was also enhanced by the microaerophilic conditions. This pigment can inhibit the capacity of macrophages to produce reactive nitrogen intermediates without altering cell viability (Shellito et al., 1992
). Culture supernatants are currently being examined for their toxicity towards mammalian cells and analysed by two-dimensional electrophoresis for identification of the proteins secreted at different dissolved oxygen concentrations. Preliminary studies with supernatants taken from two different cultures (pO2=1 and 10%) of P. aeruginosa showed considerable differences in the toxicity of these supernatants towards mammalian cell cultures (Bi et al., 2001
). This variable toxicity may be due to the formation of different proteins by the two cultures, as revealed by two-dimensional gel analysis (unpublished data).
In conclusion, P. aeruginosa PAO1 prefers microaerobic conditions for growth and can create these environmental conditions itself through at least two mechanisms: (i) blockage of oxygen transfer and (ii) formation of a polysaccharide layer. In particular, the blockage of oxygen transfer by this species may represent a new and efficient mechanism for the defence of the organism against reactive oxygen and nitrogen intermediates or it may be relevant to the hostpathogen relationship. More research is needed to investigate the mechanism(s) of blockage of oxygen transfer and the pathogenicity of P. aeruginosa under microaerobic conditions.
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ACKNOWLEDGEMENTS |
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Received 11 January 2002;
revised 26 April 2002;
accepted 11 June 2002.