GBF Gesellschaft für Biotechnologische Forschung mbH, Division of Molecular Biotechnology, Mascheroder Weg 1, D-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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In a previous paper, we showed that P. aeruginosa PAO1 can strongly reduce the rate of O2 transfer from the gas phase into the culture, causing oxygen limitation and simply blocking the supply of oxygen for the formation of reactive oxygen intermediates (Sabra et al., 2002). Under these oxygen-limited or microaerobic conditions, P. aeruginosa PAO1 itself grew effectively and appeared to be more pathogenic (Sabra et al., 2002
). This possibly represents a new and more efficient defence strategy of P. aeruginosa PAO1 against oxidants. However, little is known about the factors causing the blockage of oxygen transfer in the P. aeruginosa culture.
It is known that iron and iron-containing proteins play important roles in the growth and pathogenesis of P. aeruginosa, especially in its defence against oxidative stress (Vasil & Ochsner, 1999). Many proteins involved in respiration (e.g. ferredoxins and other ironsulphur proteins) and degradation of H2O2 and
(e.g. haem catalase, iron superoxide dismutase and peroxidase) require iron for functionality. However, iron in an aerobic environment exists mainly in the form of Fe3+ which is extremely insoluble at neutral pH. Thus, increased oxygen tension in the culture can reduce the availability of iron. This was considered as the main reason why aerobic bioprocesses normally require a much higher concentration of usable iron compared to microaerophilic or anaerobic processes (Andrews, 1998
; Vasil & Ochsner, 1999
). In this connection it is worth mentioning that in the lung, which normally has a highly oxygenated environment, the iron concentration is very low (Griffiths et al., 1988
; Stintzi et al., 1998
). Low-iron solubility, together with the process of withholding iron from infecting bacteria by the host through iron complexing with proteins such as transferrin and lactoferrin, is an important strategy in host defence (Griffiths et al., 1988
; Stintzi et al., 1998
; Ratledge & Dover, 2000
). However, the interplay between high oxygenation and low-iron concentration, and its implications for pathogenhost interactions, have not been studied to our knowledge.
The current study was undertaken to examine whether iron concentration in the growth medium influences the transfer rate of oxygen in P. aeruginosa cultures and how the physiology of P. aeruginosa is affected under these conditions.
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METHODS |
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Electron microscopy.
Transmission electron microscopy (TEM) was used to detect the possible presence of polysaccharide capsule on the surface of cells grown in iron-rich and iron-limited media. Cells were taken directly from bioreactor cultures controlled at a pO2 of 10 % air saturation. Embedding and ultrathin sectioning of P. aeruginosa were as described previously for Azotobacter vinelandii (Sabra et al., 2000).
Biochemical analysis.
The total extracellular protein in cell-free supernatants was determined by the method of Lowry. Elastase activity was determined in a spectrophotometric assay using Elastin-Congo red (Sigma) as substrate as described by Kessler et al. (1993). Siderophores (pyoverdine and pyochelin) were measured with a microtitre plate fluorometer (MFX Microtiter Plate Fluorometer; Dynex Technologies). Fluorescence was determined by exciting the culture supernatant 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
).
Determination of iron concentration.
The concentration of iron in culture supernatants was determined as Fe2+ or Fe3+ by spectrophotometric assay using iron test kits (Merck). Briefly, Fe2+ in the sample was reacted with 1,10-phenanthroline to form a red complex that was determined photometrically. Fe3+ was first reduced to Fe2+ by ascorbic acid and the total amount of Fe2+ was measured as above. The detection limit of iron concentration was given as 0·01 mg l-1 for the test kits.
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RESULTS |
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Both P. aeruginosa PAO1 NCCB 2452 and ATCC 15692 grew somewhat faster in the iron-rich medium than in the low-iron medium. Growth in the low-iron medium showed a relatively long lag phase (35 h), whereas almost no lag phase was observed in the iron-rich medium. However, the biomass concentrations reached in both media are comparable, indicating an effective utilization of iron in the cultures grown in the low-iron medium. Calculation of the specific growth rates (µ) during the different phases of the low-iron culture revealed a higher growth rate during the period of iron deficiency than in the iron non-deprived period, indicating an effective adaptation of the organism to the low-iron or iron-deficient conditions. For example, the growth rate of strain ATCC 15692 increased from 0·026 h-1 in the first 5 h of cultivation, to 0·07 h-1 in the 711 h period, to 0·16 h-1 during the period of iron deficiency (about 1316 h, Fig. 2b). The µmax reached in the low-iron culture was the same as the µmax (0·16 h-1) of this strain reached in the iron-rich medium, although the former had a very slow growth rate in the initial period of cultivation compared to the iron-rich culture. A similar effect on the growth rate was observed with strain NCCB 2452, where the µmax reached in the low-iron medium was 0·30 h-1, somewhat lower than the µmax reached in the iron-rich medium (0·40 h-1).
The oxygen limitation observed in Figs 1(b) and 2(b) was not caused by a high oxygen consumption rate of the cells (Geckil et al., 2001
; Sabra et al., 2002
). In fact, the biomass concentrations in these cultures (<1·6 g l-1; see Fig. 1b
for NCCB 2452) were not high. For strain ATCC 15692, the biomass concentration during the oxygen limitation period in the low-iron culture (Fig. 2b
) was even lower than that in the late exponential phase of the iron-rich culture (Fig. 2a
). The measurement of kLa in cell-free supernatants from NCCB 2452 cultures under different iron concentrations revealed the reason for oxygen limitation in the low-iron culture. As depicted in Fig. 3
, both cultures had a relatively low kLa value after inoculation with the iron-exhausted seed culture which had poor oxygen transfer properties. The kLa value increased during the first few hours of cultivation in both iron-rich and low-iron cultures. Whereas the increase of kLa continued in the culture with sufficient iron (Fig. 3a
), the kLa value of the low-iron culture levelled off and significantly decreased after about 9 h of cultivation (Fig. 3b
), namely at about the same time as the onset of iron depletion (Fig. 1b
). The reduction of the kLa value was much more drastic in our previous study with strain NCCB 2452 grown under different preset pO2 values (Sabra et al., 2002
). This decrease in oxygen transfer efficiency from the gas to the liquid phase could explain why the pO2 value reached zero even though the O2 content in the inlet gas was increased to 100 % oxygen under iron-deficient conditions (Figs 1b and 2b
). This study clearly shows that the reduction of the oxygen transfer rate is mediated by conditions of iron deficiency.
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DISCUSSION |
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In this work we showed using two P. aeruginosa PAO1 isolates that this pathogenic organism can apply a hitherto unreported strategy to deal with iron limitation, namely a significant reduction of the oxygen transfer rate from the gas phase into the liquid phase as reflected by the kLa value in Fig. 3, thereby causing oxygen-limited or microaerobic conditions in the culture (Figs 1 and 2
). These results explain well the phenomenon of oxygen limitation reported in our previous study (Sabra et al., 2002
) in which we used the same low-iron medium for cultivating P. aeruginosa PAO1 NCCB 2452 under different preset values of pO2. In all the previous cultivations with this strain at different preset pO2 values, a strong oxygen limitation occurred 68 h after inoculation despite vigorous aeration of the bioreactor with pure oxygen. The exact mechanism used by P. aeruginosa to reduce the oxygen transfer rate is not known. In our previous study we showed that the production of biosurfactants such as rhamnolipid may contribute to this phenomenon (Sabra et al., 2002
). Whatever the mechanism is, the physiological consequences of this reduced oxygen transfer rate are obvious. First, the oxygen-limited or microaerobic conditions can greatly increase the usability of the remaining iron through the transformation of Fe3+ to the more soluble Fe2+. Second, the reduced oxygen transfer rate and thus pO2 can better protect the cells from the formation of oxidative radicals, especially under low-iron conditions. It is known that the synthesis and functionality of many enzymes involved in defence against oxidative radicals such as catalase and dismutase require iron (Frederick et al., 2001
). Furthermore, under oxygen limitation, the respiration rate is reduced. This in turn can result in a reduction of endogenous generation of oxidative radicals such as H2O2 and
. These favourable conditions may be the reason why P. aeruginosa PAO1 grew even faster under apparently iron-deficient conditions (e.g. cultivation time after 12 h in Fig. 2b
, µ=0·16 h-1) than before iron limitation (e.g. cultivation time between 6 and 11 h in Fig. 2b
, µ=0·07 h-1). This indicates that P. aeruginosa PAO1 can effectively adapt to environments of iron deficiency. However, it is not known if the improved availability of iron or the reduced formation of oxidative radicals is the main contributor to the improved growth of P. aeruginosa PAO1 under microaerobic conditions. Microaerobic conditions are known to dominate in biofilms, the preferred mode of growth of P. aeruginosa in the lung of CF patients, and in biofouling of different systems (Costerton et al., 1999
; 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 real reason for the improved growth is not clear and deserves more detailed study. This may give useful hints to better combat the infection and contamination by P. aeruginosa.
Oxygen limitation triggered by iron deficiency resulted in a dual limitation in the culture of P. aeruginosa PAO1 in the later phase of cultivation (Figs 1 and 2). Under these conditions P. aeruginosa PAO1 showed a drastic increase in secretion of proteins (Fig. 5
). One of these proteins is elastase (Fig. 6
). An enhanced activity of elastase at decreased iron concentration has been reported (Bjorn et al., 1979
; Brumlik & Storey, 1992
; Storey et al., 1992
). In host cells, elastase is able to specifically cleave transferrin, an animal iron carrier. Following cleavage of the iron carrier, the iron can be used by the bacterial cells. The increased elastase synthesis has been considered to be one of the strategies of iron acquisition by P. aeruginosa under iron-limited conditions (Wolz et al., 1994
). The formation of the two siderophores, e.g. pyoverdine (Fig. 7
) and pyochelin which can chelate iron, was also found to increase significantly under iron-limited conditions. This may contribute to the complete consumption of Fe3+ in the late phase of cultivation (Fig. 2
). Both elastase and siderophores can help P. aeruginosa to remove iron from host sources and enhance growth (Cox, 1982
). Under the bioreactor cultivation conditions applied in this study, no iron resource other than FeSO4 was present. It would be of interest to know if the oxygen limitation caused by iron deficiency, which is reported for the first time in this study and suggested as a new strategy for P. aeruginosa to combat iron limitation and oxidative stress, also takes place when iron-containing proteins exist as in the lung of CF patients.
In fact, the environment of the lung of many CF patients is quite similar to the culture conditions applied in this study in at least two important aspects, i.e. low iron concentration and high oxygenation. In this connection there is another important observation in this study (Fig. 4), namely the formation of alginate and the occurrence of a polysaccharide capsule on the cell surface is mainly related to iron limitation rather than to oxidative stress. The latter was previously considered as the main reason for an enhanced formation of an alginate capsule on the surface of PAO1 cells (Sabra et al., 2002
). The enhanced formation of alginate may be explained by the iron regulation circuit that is known to be activated under oxygen stress and iron deprivation (Vasil & Ochsner, 1999
). In view of the importance of alginate in the pathogenicity (e.g. through the formation of biofilm and avoidance of encounters with phagocyte-derived reactive oxygen intermediates) of P. aeruginosa (Govan & Deretic, 1996
; Miller & Britigan, 1997
), the finding presented in Fig. 4
represents an important extension of our previous knowledge. The high mortality of CF patients infected by P. aeruginosa is often due to biofilm formation and respiratory failure. Oxygen limitation and the formation of an alginate capsule on the cell surface due to iron deficiency as shown in this work may therefore play an important role and deserve more detailed study.
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ACKNOWLEDGEMENTS |
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Received 31 January 2003;
revised 19 May 2003;
accepted 12 June 2003.