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Keywords:

  • Aphanizomenon ovalisporum;
  • cylindrospermopsin;
  • sulfate and phosphate starvation;
  • ATP sulfurylase;
  • alkaline phosphatase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The effect of sulfate and phosphate deprivation on cell growth and cylindrospermopsin level was studied in Aphanizomenon ovalisporum ILC-164. Sulfate starvation induced a characteristic reduction of cylindrospermopsin pool size on the basis of cell number and unit of dry mass of culture. Phosphorous starvation of A. ovalisporum cultures induced a lesser reduction of cylindrospermopsin pool size. This divergence in the pool size of cylindrospermopsin may be the consequence of different growth rate. To show the metabolic changes concomitant with reduction of cylindrospermopsin pool size were obtained by measurement of ATP sulfurylase and alkaline phosphatase activity. The present study is the first concerning the cylindrospermopsin content under sulfate starvation and discusses it in relation to phosphorous starvation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bloom-forming toxic cyanobacteria have caused increasing concern over the last years both in fresh water and in marine environments (Falconer, 1993; Chorus & Bartram, 1999; Dow & Swoboda, 2000). Toxic metabolites of cyanobacterial origin, called cyanotoxins are a diverse group of natural toxins both from the toxicological and the chemical points of view (Carmichael, 1992; Bell & Codd, 1994). Toxic cyanobacteria are known to produce harmful metabolites such as hepatotoxins, neurotoxins, lipopolysaccharides, gastroenteral toxins and a variety of other, as yet unidentified toxic compounds. In more recent years cylindrospermopsin (CYN), a sulfur containing hepatotoxin (tricyclic guanidyl hydroxymethyluracil) known to be produced by Cylindrospermopsis raciborskii, Umezakia natans, Aphanizomenon ovalisporum, A. flos-aquae, Raphidiopsis curvata was concerned in the worst known case of human poisoning in Australia (Banker et al., 1997; Chorus & Bartram, 1999).

The availability of nutrients is recognized as major limitation for proliferation of phytoplankton in water habitats. Nitrogen, phosphorous and sulfur were identified as main limiting nutrients, whose relative contribution varies in different habitats (Smith, 1983). In spite of the importance of mineral availability for proliferation of photosynthetic organisms, including cyanobacterial blooms there is a shortage of studies about the interference of main nutrient limitations with cyanotoxin content. In addition, existing literature provides data that under natural conditions not all cyanobacterial blooms or isolated strains have the same toxicity and that individual blooms or strains can vary in toxicity in time (van der Westhuizen & Eloff, 1983; Codd & Poon, 1988; Saker & Griffiths, 2000). However, how the sulfur starvation interferes with cyanotoxin content no data are available. Therefore, cylindrospermopsin a sulfur-containing cyanotoxin provides a realistic experimental system to analyze and understand the interference of sulfur starvation with cyanotoxin content. As nitrogen fixing cyanobacteria are frequently prominent members of undesirable blooms in natural waters, in which phosphate availability is a common regulating factor it deserves considerable attention to compare the consequences of sulfur and phosphorous starvation on cylindrospermopsin content of A. ovalisporum, a nitrogen-fixing cyanobacterium.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Strain and growth conditions

Cylindrospermopsin producing A. ovalisporum strain ILC-164 from Lake Kinneret, Israel was grown as described (Allen, 1968; Vasas et al., 2002, 2004). The cultures were kept in glass flasks thermostatically maintained at 28°C, illuminated with cool white fluorescent light (80 μmol photons m−2 s−1) and bubbled with sterile air. Growth of the cultures was monitored by measurement of chlorophyll a (Bendall et al., 1988), cell number and dry mass content. Cell number per mL was determined from 5 μL culture samples in triplicate (all cells of filaments were counted microscopically). For dry mass measurements, 3 mL samples were centrifuged and the pellets were lyophilized, weighed and cell mass per mL calculated. All measurements were in triplicates.

Sulfate and phosphate starvation of A. ovalisporum cultures

Exponentially growing A. ovalisporum cultures were centrifuged (6000 g for 10 min, 25°C) and the cell pellet was carefully washed twice with sterile nutrient-free medium as it required. At zero time the washed cells were resuspended at the same densities in sterile prewarmed full medium (controls) or in media lack of sulfur or phosphorous, respectively. The resuspended cultures were further cultivated under otherwise unchanged conditions. In the case of sulfate starvation, the cultures were grown in sulfate-free medium for 48 h and afterward the culture was supplemented with MgSO4 solution. In the case of phosphate starvation, the cultures were grown in Pi-free medium for 168 h and then the culture was supplemented with K2HPO4 solution. As an internal control nutrient starvations were induced at the middle of experiments as well (for sulfate starvation at 48 h, for phosphate deficiency at 168 h, respectively). At predetermined time (each day) samples were collected and used for assays as described above.

Enzyme assays

The ATP-sulphurylase enzyme (ATPS) activity was measured by molybdate-dependent formation of pyrophosphate method of Lappartient & Touraine (1996) with minor modification. Two milliliter samples were centrifuged (6000 g, 3 min) and the pellet was resuspended in 100 μL of 50 mM Tris-HCl buffer (pH 8.0). The cell suspension was frozen and thawed three times to damage cell wall and after centrifugation protein content of the supernatant was used as an enzyme extract for ATP-sulfurylase assay and assayed for protein (Bradford, 1976). Briefly, the 500 μL enzyme reaction mixture contained 80 mM Tris-HCl buffer (pH=8.0), 7 mM MgCl2, 0.2 mM Na2ATP, 1 U inorganic pyrophosphatase (from Escherichia coli, Fluka, Germany) and 12 μL crude extract. The enzyme assay mix was incubated for 1 h at 37°C and the reaction was stopped by addition of 100 μL of 20% SDS. The liberated inorganic phosphate was assayed by a modified version of molibdenate-based method of Fiske and Subbarow as described in Cooper (1977).

The measurement of in situ alkaline phosphatase (AP) activity was based upon the in situ method of Ihlenfeldt & Gibson (1975). One hundred microliters of samples were collected from the cultures and 500 μL of 0.4 M Tris HCl buffer (pH=9.0) and 500 μL 8 mM p-nitrophenyl-phosphate was added and incubated for 10 min at 25°C in darkness. The reaction was stopped by adding 500 μL of 0.2 M Na2HPO4 in 1 M NaOH. The mixtures were centrifuged (1000 g for 1 min) and liberation of p-nitrophenol was measured at 400 nm and μM pNP mg−1 protein h−1 calculated. In enzyme assays (ATPS, AP) the complete reaction system stopped at zero time served as blank.

Quantitation of cylindrospermopsin content of A. ovalisporum cells

Three milliliters of culture samples were centrifuged (6000 g for 3 min) and the pellets and supernatants were lyophilized separately. The cyanotoxin (cylindrospermopsin) content was determined with the help of capillary electrophoresis as described by our laboratory earlier (Vasas et al., 2002, 2004). The obtained data were normalized to the zero time controls which were chosen to 100% on basis of dry mass or cell number.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sulfate and phosphate starvation altered the growth of A. ovalisporum

Growth curves from representative experiments of sulfate- and phosphate-starved A. ovalisporum cultures are shown in Figs 1 and 2. It is obvious, the sulfate-deprived cultures markedly stopped accumulating Chl-a, dry mass and cell number per mL (Figs 1a–c), irrespective of that the starvation started at the beginning of experiment (Fig. 1; squares and circles) or after 48 h (Fig. 1; triangles) of culturing in full medium. However, when sulfate was added to cells starving 48 h for sulfur, rapid cell growth was resumed after a significant lag period of about 48–50 h (Figs 1a–c; open squares). Cells transferred to medium lacking sulfur after 48 h of culturing in full medium stopped their growth (Figs 1a–c, open triangles).

image

Figure 1.  Growth of Aphanizomenon ovalisporum culture on sulfate deprivation. An exponential phase culture grown in Allen medium was centrifuged, gently washed with sulfate-free medium and divided into parts (zero time) and further cultured in full medium (control, –◆–), starved for sulfate throughout the experiment (S−, –○–), sulfate starved for 48 h and afterward supplemented with sulfate (S−S+, –□–), grown in full medium for 48 h and afterward starved for sulfate (S+S−, –▵–). Growth was monitored by measuring chlorophyll a content (μg mL−1, a); dry mass (mg mL−1, b); cell number (cell number mL−1, c). The vertical arrow shows the time when the shift down or shift up was performed.

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image

Figure 2.  Growth of Aphanizomenon ovalisporum culture on phosphate deprivation. An exponential phase culture grown in Allen medium was centrifuged, gently washed with Pi-free medium and divided into parts (zero time) and further cultured in full medium (control, –◆–), starved for phosphate throughout the experiment (P−, –•–), phosphate starved for 168 h and afterward supplemented with phosphate (P−P+, –▪–), grown in full medium for 168 h and afterward starved for phosphate (P+P−, –▴–). Growth was monitored by measuring chlorophyll a content (μg mL−1, a); dry mass (mg mL−1, b); cell number (cell number mL−1, c). The vertical arrow shows the time when the shift down or shift up was performed.

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Phosphate-deprived cultures slowed down the growth and reduced accumulating Chl-a, dry mass and cell number per mL irrespective of that the starvation started at the beginning of experiment (Fig. 2; squares and circles) or after 168 h (Fig. 2, triangles) of culturing in full medium (Figs 2a–c). When Pi was added to cells starving for a period of time (168 h) rapid cell growth was resumed (Fig. 2; squares). Pi-grown cells transferred to medium lacking phosphate (after 168 h) reduced their growth without delay as compared with sister cultures (Fig. 2; triangles).

Alteration of ATP sulfurylase and AP activity of sulfate- and phosphate-starved A. ovalisporum cultures

When A. ovalisporum cells were transferred from sulfate-efficient medium to sulfate deficient one sulfur starvation induced an activity increase of ATPS (Fig. 3). The crude extract of sulfate efficient A. ovalisporum cells had pH optima of ATPS activity at 6.0 and 8.0. However, the enzyme activity at pH 8.0 was significantly higher and sulfate deprivation induced of this activity by several fold, therefore we measured activity changes at this pH (Fig. 3; see inset). In addition, the enzyme activity at pH 6.0 cannot be altered by sulfur starvation (Fig. 3, inset).

image

Figure 3.  Effect of sulfate starvation on ATP sulfurylase activity in Aphanizomenon ovalisporum cultures. An exponential phase culture grown in Allen medium was centrifuged, gently washed with sulfate-free medium and divided into parts (zero time) and further cultured in full medium (control, –♦–), starved for sulfate throughout the experiment (S−, –○–), sulfate starved for 48 h and afterward supplemented with sulfate (S−S+, –□–), grown in full medium for 48 h and afterward starved for sulfate (S+S−, –▵–). ATP sulfurylase activity was measured as described in Materials and methods. The inset shows the pH optima of ATP sulfurylase enzymes extracted from cells grown in full medium (–◆–) and extracted from sulfate-starved cells (–○–). The vertical arrow shows the time when the shift down or shift up was performed.

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From zero time sulfate deprivation induced a significant increase of the ATPS activity with a maximal activity at 56 h of starvation (Fig. 3, open circles). This high activity decreased afterward and showed a more or less stabile but increased level (four- to five-folds) throughout the experiment (Fig. 3, open circles). In sister culture where sulfur starvation started at zero time for 48 h and at this point (after 48 h sulfur starvation) sulfate added, the induction of ATPS activity was comparable to the early period (0–48 h) of sulfate deprivation (Fig. 3, open squares). The maximal activity of ATPS (15- to 20-fold increase) coincided with highest activity of from zero time sulfate-starved culture and after sulfate addition, within 50 h the enzyme activity returned roughly to control level (Fig. 3, open squares). After this period of time the level of ATPS activity was practically the same as it obtained at zero time. In cultures where the sulfur starvation started after 48 h of the beginning of experiment the lack of sulfate induced the ATPS activity (Fig. 3, open triangles). As not only the sulfate but also the effects of phosphate starvation were studied we analyzed the alteration of ATPS activity in Pi-starved cultures. There was no equivalent induction of ATPS activity in Pi-deficient medium (data not shown).

Phosphate starvation induced a significant and continuous 30-fold increase of in situ AP enzyme activity (Fig. 4, closed circles). In normal medium 168 h phosphorous deprivation resulted in an immediate continuous increase of AP activity (almost 15-fold; Fig. 4, closed triangles). When Pi was added to 168 h phosphate-starved cells the level of AP activity decreased (Fig. 4, closed squares). Sulfate starvation did not affect AP activity of A. ovalisporum cultures (data not shown). Chloramphenicol, a specific inhibitor of prokaryotic protein synthesis inhibited the increase of ATPS and AP enzyme activity in nutrient starved cultures (data not shown).

image

Figure 4.  Effect of phosphate starvation on AP activity in Aphanizomenon ovalisporum cultures. An exponential phase culture grown in Allen medium was centrifuged, gently washed with Pi-free medium and divided into parts (zero time) and further cultured in full medium (control, –◆–), starved for phosphate throughout the experiment (P−, –•–), phosphate starved for 168 h and afterward supplemented with phosphate (P−P+, –▪–), grown in full medium for 168 h and afterward starved for phosphate (P+P−, –▴–). AP activity was measured as described in Materials and methods. The vertical arrow shows the time when the shift down or shift up was performed.

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Alteration of cylindrospermopsin content of sulfate- and phosphate-starved A. ovalisporum

As the cylindrospermopsin is sulfur containing cyanotoxin we analyzed alterations in cylindrospermopsin content of sulfur-deprived cultures with the help of capillary electrophoresis (Vasas et al., 2002, 2004; Fig. 5). In control cultures cylindrospermopsin content of cells was stable and the amount of cylindrospermopsin did not change on dry mass or cell number base throughout the experiment in A. ovalisporum cells (Figs 5 and 6, diamonds). However, sulfate or phosphate starvation of cells induced a decrease in cylindrospermopsin content but not necessarily at the same rate (Figs 5 and 6, open/sulfate/and filled/phosphate/circles, squares and triangles). As evident from Figs 5a and b, the cylindrospermopsin content of sulfur-deficient cultures decreased dramatically independent of starvation started at zero time (Figs 5a and b, open circles) or after 48 h of culturing (Figs 5a and b, triangles). The decrease rate of cylindrospermopsin content was almost identical in these cultures (Figs 5a and b, open circles and triangles). When sulfate was added to sulfur-starved cultures (after 48 h) the cylindrospermopsin content of cultures started to increase with a significant lag period of about 35–40 h (Figs 5a and b, open squares).

image

Figure 5.  Changes in cylindrospermopsin level on dry mass and cell number base in sulfate-deprived Aphanizomenon ovalisporum cultures. The shift downs and ups were as described in Figs 1 and 2. In the time 0 sample, the basal level of cylindrospermopsin was 5.21 μg mg−1 dry mass (a) and 2.17 ng per cell (b). These values were defined as 100% to calculate the relative concentration of cylindrospermopsin. The vertical arrow shows the time when the shift down or shift up was performed.

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image

Figure 6.  Changes in cylindrospermopsin level on dry mass and cell number base in phosphate-deprived Aphanizomenon ovalisporum cultures. The shift downs and ups were as described in Figs 1 and 2. In the time 0 sample, the basal level of cylindrospermopsin was 5.12 μg mg−1 dry mass (a) and 2.12 ng per cell. These values were defined as 100% to calculate the relative concentration of cylindrospermopsin. The vertical arrow shows the time when the shift down or shift up was performed.

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Phosphorous starvation induced a moderate decrease in cylindrospermopsin content of cells independent of starvation started at zero time (Figs 6a and b, filled circles and squares) or after 168 h (Figs 6a and b, triangles). The decrease of cylindrospermopsin content was substantially lower on dry mass base in phosphate-deprived cultures (47–48% of zero time level in 2 days) than that noted in sulfate-starved conditions (64–65% of zero time level in 2 days) at otherwise identical conditions and no lag was obtained in the increase of cylindrospermopsin content if phosphate added back after 168 h of starvation (Figs 6a and b, squares). The decline of cyanotoxin content per cell in sulfate-starved cultures was 56% of zero time level within 48 h and in lack of phosphate it reached 58% of zero time level under otherwise unchanged condition.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The goal of this work was to show the possible effects of sulfate and phosphate starvation on cylindrospermopsin content of A. ovalisporum cultures. Cylindrospermopsin is an unusual water soluble compound as it contains a guanidino and a sulfate group and the presence of a uracil side chain adds to the peculiarity of this molecule (Ohtani et al., 1992). As the sulfate limitation stress and its regulation in cyanobacteria may have much common with heterotrophic bacterial sulfur restriction (Kredich, 1996), one would anticipate an influence in sulfate starvation induced perturbation of cyanotoxin metabolism. The sulfate starvation results in an immediate stop of growth, while the phosphate starvation reduces it significantly although that was not completely abolished (Figs 1 and 2). This phenomenon is well-known feature of cyanobacteria, and is in accord with previous findings for these organisms and discussed in the literature (Healey, 1982; Tandeau de Marsac & Houmard, 1993; Grossman et al., 1994). Interestingly, the capillary electrophoretic analysis of crude extracts of sulfate- and phosphate-starved A. ovalisporum cells demonstrates for the first time a characteristic reduction of the pool size of cylindrospermopsin under the changes of environmental conditions (Figs 5 and 6). However, in phosphate-deprived cultures a significant, but smaller decrease of pool size of cylindrospermopsin is observed than that is obtained for sulfate-limited cultures. In addition, the difference observed between the sulfate- and phosphate-limited culture on the basis of dry mass cannot be obtained if changes of cyanotoxin content were counted per cell. The explanation of this phenomenon is not known, but it should be stressed that the growth of phosphate-limited cultures are not completely abolished (Fig. 2). Nonetheless, we are left with the necessity of explaining how the sulfur and phosphate shift downs may regulate and/or interact in cyanotoxin synthesis. It should be noted that the difference in rate of cylindrospermopsin pool size reduction on dry mass base and on cell number may reflect, in total or in part, the metabolic changes induced by sulfate starvation stress. On the basis of physiological studies of cyanobacteria it has been suggested that any shortage of carbon, nitrogen, sulfur, light etc. will transiently affect the amount of phycobiliproteins and may produce carbohydrate storage materials, polyphosphate bodies and a unique nitrogenous compound known as cyanophycin (Stevens & Paone, 1981; Allen, 1984; Allen et al., 1984; Page-Sharp et al., 1988; Shively et al., 1988; Mackerras et al., 1990a, b). Thus the unbalanced growth of starved cells results in a significant increase of dry mass without altering considerably the cell number. Therefore the present finding, the difference in reduction rate of cylindrospermopsin based on dry mass and cell number upon nutrient deprivation (sulfate and phosphate, respectively) may be the consequence of the rearrangement of starved cell metabolism.

It is pertinent to emphasize, the alteration of physiological parameters like ATPS and AP enzyme activities in nutrient-limited cells may support this idea. Our results clearly showed that lack of sulfate induces a significant increase of ATPS activity, one of the key enzyme of sulfate metabolism in filamentous A. ovalisporum cultures with a pH optimum of 8.0 (Fig. 4, inset). In filamentous cyanobacteria almost no data available on sulfur deficiency beside the phycobiliprotein degradation under these conditions (Mishra & Schmidt, 1992; Grossman et al., 1994; Bhaya et al., 2000). The biochemical nature of ATPS enzyme of A. ovalisporum was in accord with previous findings published for Spirulina platensis (Menon & Varma, 1978) and Anabaena cylindrica (Sawhney & Nicholas, 1977). Whereas detailed analyses are available concerning the sulfur, nitrogen and phosphorous metabolism of unicellular cyanobacteria (Sawhney & Nicholas, 1977; Grossman et al., 1994; Sperling et al., 1998; Richaud et al., 2001), beside the early data of Schmidt laboratory (Schmidt & Christen, 1978; Schmidt, 1988; Mishra & Schmidt, 1992) no study was published on ATPS alteration in cyanobacteria. In cyanobacteria AP enzyme system is inducible by phosphate deficiency (Healey, 1982; Tandeau de Marsac & Houmard, 1993; Grossman et al., 1994). The only aim of measuring AP activity is to have an inducible system to which the alteration of cyanotoxin production can be compared in nutrient deprived A. ovalisporum cells. Consequently, data reported here would be of interest not only from the viewpoint of cyanotoxicology but it may provide a ‘physiological’ tool to study the biochemistry of stress responses of toxic cyanobacteria.

These data, taken together with earlier conclusions drown from other studies by Orr & Jones (1998), lend to further support to the conclusion that ‘there is a direct linear correlation between the cell division and toxin production rates regardless of environmental factors that limiting the cell division’. Concerning the cyanotoxin production in nutrient-limited cultures the findings of this study are in accordance with various interpretations of Orr & Jones (1998); Sivonen (1990) and Rapala et al. (1997) as their data were correlated with changes of growth, dry mass and cell number, respectively. Therefore the alteration of cylindrospermopsin in A. ovalisporum is the first clear example of the probable involvement of sulfate starvation in the determination of cyanotoxin pool size, in a filamentous cyanobacterium, and would be a system for detailed study of sulfur limitation of this kind of organisms. Our consideration of the results reported here leads us to the expectation that the sulfate and phosphate limitation in A. ovalisporum cells may have pleiotropic effects on cyanobacterial toxin metabolism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work has been supported by Hungarian National Research Foundation Grants OTKA T22988, T5235, F046493 and GVOP-3.2.1.-2004-04-0110/3.0 which is greatly acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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