1 Cyanobacteria and Astrobiology Research Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, NSW, Australia
2 DBSF, University of Insubria, via J. H. Dunant 3, 21100 Varese, Italy
Correspondence
Brett A. Neilan
b.neilan{at}unsw.edu.au
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ABSTRACT |
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INTRODUCTION |
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STX and its analogue compounds have been reported to occur naturally in marine dinoflagellates (Shimizu, 1977; Catterall, 1980
; Harada et al., 1982
), filamentous cyanobacteria (Alam et al., 1973
; Humpage et al., 1994
; Carmichael et al., 1997
; Lagos et al., 1999
; Pomati et al., 2000
), and certain heterotrophic bacteria (Gallacher & Smith, 1999
). In freshwater environments, PSP toxins are almost exclusively associated with cyanobacteria, and the occurrence of STX-producing neurotoxic cyanobacterial blooms has been increasingly reported (Kaas & Henriksen, 2000
; Pereira et al., 2000
; Pomati et al., 2000
).
Although much is known about their pharmacology and their chemistry, PSP toxins have rarely been studied as regards their metabolism, including the physiology of STX-producing cyanobacteria. The stimuli inducing or repressing STX production in cyanobacteria are currently unknown, as is as the metabolic role of PSP toxins within the producing micro-organisms. Laboratory studies documented that cyanobacterial isolates preferentially produce PSP toxins under conditions which are most favourable for their growth (Sivonen & Jones, 1999). In PSP toxin-producing dinoflagellates, however, high salinity has been found to increase cell toxicity (Ogata et al., 1987
; Hwang & Lu, 2000
). The effects of salt stress and pH variations on cyanobacterial PSP toxins production have yet to be investigated, and alkalinity of waters is one of the main features characterizing toxic cyanobacterial blooms. This parameter, combined with high salinity, has been reported in correlation with blooms of PSP toxin-producing species such as Anabaena circinalis in Australia (Bowling & Baker, 1996
).
Here we describe the effects of pH, salt and two channel-blockers (amiloride and lidocaine) on the total cellular content of Na+ and K+, and STX accumulation, in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3. The results presented suggest that in C. raciborskii T3, STX production is responsive to changes in intracellular sodium levels. The present study also indicates a possible correlation between STX metabolism and cyanobacterial homeostasis.
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METHODS |
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Flame photometry analysis.
Total cellular Na+ and K+ levels in cyanobacteria were assayed by flame photometry. Two-millilitre aliquots of C. raciborskii T3 cultures were collected by centrifugation in 2 ml plastic tubes at 11 000 g for 15 min. Samples were harvested immediately after exposure (0 min) and at 30, 60 and 120 min. The control culture (unexposed) was monitored for an additional sample at 90 min. In the lidocaine and amiloride experiments, culture aliquots were withdrawn prior to exposure (-5 min) and immediately after the addition of the agents (0 min). All sampled pellets were resuspended in 0·5 ml diluent flame solution (3 mM Li in Milli-Q water) and analysed for the total cellular content of Na+ and K+ using an FLM3 flame photometer (Radiometer).
HPLC analysis.
STX concentrations were measured using HPLC. Cyanobacteria (100 ml culture) were harvested after 0, 30, 60, 120 or 240 min by centrifugation (15 min, 4000 g), cells resuspended for extraction in 3 ml Milli-Q water and lysed by sonication (3 min, 100 W). The supernatant (growth medium) was either discarded or filter-sterilized (0·2 µm membrane), freeze-dried and analysed after resuspension in 2 ml Milli-Q water. Aqueous cellular extracts were prepared by centrifugation (10 min, 13 000 r.p.m.) to remove cell debris and stored frozen at -20 °C until HPLC analysis. Screening for PSP toxins was performed by prechromatographic oxidation with H2O2 followed by HPLC separation. Chromatography was carried out according to the method of Lawrence et al. (1996), using a Waters 600 HPLC apparatus coupled with a Waters 470 fluorescence detector (Millipore). The column used was a Supelcosil LC-18 (150x4·6 mm, i.d. 5 µm) (Supelco). The samples and standard mixture were oxidized as previously described (Lawrence et al., 1996
; Pomati et al., 2003a
) using H2O2 prior to injection. Oxidation products were eluted under isocratic conditions with 1 % acetonitrile (v/v) in 0·1 M ammonium formate, pH 6·0, at a flow rate of 1·0 ml min-1.
Concentrations of STX in the culture samples were calculated by comparing the peak area corresponding to STX in the cyanobacterial extracts to that of the STX standard solution. When necessary, H2O2 was removed from the oxidant solution to verify the oxidation dependence of HPLC peaks. In this study, no auto-fluorescent peaks with the same retention time as STX standard solution were detected. STX data were normalized by the OD750 of the culture sample to account for differences in cell numbers of the experimental replicates.
Total protein content.
Protein concentration in the C. raciborskii T3 culture medium was determined by means of the method of Bradford (1976), using bovine serum albumin as a standard.
Reagents and standards.
All reagents were obtained from Sigma-Aldrich. Lidocaine hydrochloride and amiloride solutions (100 µM and 100 mM, respectively) were prepared freshly in Milli-Q water prior to each experiment and diluted in culture medium to obtain the final concentrations required. Certified standard calibration solutions for analysis of PSP toxins (PSP-1C) were obtained from the Institute of Marine Bioscience (IMB), National Research Council of Canada, Halifax, NS, Canada.
Statistical analyses.
All graphical and descriptive statistical analyses were performed using the software for PC Origin 5.0 (Microcal Software). ANOVA and post-hoc analysis of means using the least significant difference (LSD) test were carried out with Statistica software for Windows, release 4.3 (Osiris Technology Systems).
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RESULTS |
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Effect of NaCl on growth, Na+ and K+ levels and STX accumulation
The effect of NaCl at 1 and 10 mM on the growth of C. raciborskii T3 was investigated for 7 days (Fig. 2a). A decreased growth rate was evident at day 6 for NaCl at 10 mM based on the cell density ratio of the experimental cultures compared to the controls. During the course of the following experiments, cultures of C. raciborskii T3 exposed to the different agents and conditions were also monitored spectrophotometrically and microscopically for short-term variations in the density and morphology of the cyanobacterial cells. In this study, no significant changes in OD750, mean cell size or trichome structure were evident after either 2 h or 4 h of treatment.
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The above results suggested that STX accumulation in the cyanobacterial cells was promoted by Na+ ion stress. The effects of 10 mM NaCl on total cellular Na+ and K+ levels and STX accumulation were therefore investigated (Fig. 3). In comparison with untreated cyanobacteria, exposure to NaCl at 10 mM resulted in an increase of total Na+ levels coupled with a corresponding decrease in cellular K+ (Fig. 3a
). The highest and lowest percentage changes for the two ions over the samples at 0 min were 36±10·6 % and -25±4·6 % for Na+ and K+, respectively. In C. raciborskii T3, intracellular STX accumulation also increased as a consequence of Na+ stress, with the greatest increment of change during the first 2 h after initial exposure (45·8±4·9 % versus time 0) (Fig. 3b
). These levels decreased slightly between 2 and 4 h (30·1±1·9 %). In order to evaluate the possible involvement of extracellular transport in the observed trend of intracellular STX accumulation under Na+ stress, the time-course of extracellular STX was also studied. Total protein concentrations were quantified in the filter-sterilized culture medium, as an indication of cell lysis. The extracellular concentration of STX represented 2530 % of the total STX content of the cultures (which ranged from 179 to 247 µg l-1). Extracellular levels, after normalizing for the total protein concentration in the cell-free culture medium, were found to remain constant over time (Fig. 3b
). These results suggested that no active extracellular transport of STX was involved in the effect seen for NaCl in C. raciborskii T3.
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DISCUSSION |
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The majority of freshwater cyanobacteria, including C. raciborskii T3, are alkaliphilic, growing naturally and preferentially at pH >8. In alkaliphilic bacteria, the principal active process employed for the maintenance of cytoplasmic pH neutrality involves the cycling of ions (mainly Na+ and K+) across cell membranes (for reviews, see Horikoshi 1991; Krulwich et al., 2001
). In this study, the predicted imbalance of total cellular Na+ and K+ induced by applied pH and Na+ stresses was verified (Figs 1
3). In cyanobacteria, however, K+ is thought to play a minor role and intracellular pH neutrality is achieved by net H+ accumulation coupled to Na+ efflux as mediated by the Na+/H+ antiporter (Lengeler et al., 1999
; Maestri & Joset, 2000
; Waditee et al., 2001
). This process is energized by an imposed proton-motive force (Apte & Thomas, 1986
; Sonoda et al., 1998
), with uptake of Na+ required in alkaline conditions. Na+ uptake can be achieved by general Na+/solute symporters, cation channels (Miller et al., 1984
; Krulwich et al., 2001
) or pH-gated Na+ channels (Lengeler et al., 1999
). Therefore, to study in detail the interaction between cellular Na+ levels and STX accumulation, for subsequent experiments we chose the strain's optimal conditions for growth and STX production, corresponding to pH 9·5, conditions which are also associated with active Na+ homeostasis.
The possibility of an extracellular transport of STX cannot be refuted or supported by the data presented in this study (Fig. 3b). However, based on the time-course of extracellular concentration of the toxin under salt stress, no evidence was found to indicate that this process was involved in the changes of STX content shown by C. raciborskii T3. This consideration is consistent with previously published data indicating no export of PSP toxins by the cyanobacterium Anabaena circinalis (Negri et al., 1997
). Given the promoting effect of NaCl on intracellular STX accumulation (Fig. 2
) and the strong correlation seen between total cellular Na+ and intracellular STX over time (Fig. 3
), our results suggest that STX metabolism may somehow be regulated by Na+ levels within the cyanobacterial cell.
To verify this hypothesis, amiloride and lidocaine hydrochloride were employed to modulate cellular Na+ and K+ levels. According to the model of alkaline pH homeostasis previously discussed, the inhibition of Na+/H+ antiporters would result in a net Na+ intracellular accumulation, while the blockage of Na+ uptake would lead to a concomitant decrease in the intracellular level of this ion. Here we confirmed that amiloride and lidocaine can interfere with cyanobacterial Na+ uptake and Na+ export mechanisms, respectively, affecting the total cellular Na+ concentration in opposite ways (Fig. 4). In addition, such variations in total cellular Na+ levels were observed to be coupled with corresponding changes in intracellular STX, as verified by simultaneous exposure of cyanobacterial cells to both the compounds (Fig. 5
).
Curiously, Na+ stress, lidocaine and amiloride affected STX accumulation to similar extents (a variation of between 30 and 40 % compared to the controls). This indicated that, at a given pH, threshold levels of intracellular STX may exist. Exposure to salts and channel-blocking agents did not affect the HPLC profile of toxins produced by C. raciborskii T3, compared to otherwise stimulated or control cultures. This also indicated that no detectable toxin transformations were involved in the observed variations in STX levels. The time-course of STX production, under the different conditions studied, however, resembled regulation of a metabolic pathway. Strong changes in ion fluxes, as shown here, are known to cause up- or down-regulation of genes involved in the maintenance of cell homeostasis (Maestri & Joset, 2000).
The possibility of a regulation of STX metabolism in C. raciborskii T3 in response to cellular Na+ levels may suggest either that STX biosynthesis is influenced by certain processes involved in cyanobacterial homeostasis, or that the toxin itself could play a role in the maintenance of cell functions. In consequence of Na+ stress, cyanobacteria are known to activate the production of amine osmolites, such as proline, that are synthesized from arginine via the urea cycle (Quintero et al., 2000). Arginine is also the principal biosynthetic precursor of the STX perhydropurine skeleton (Shimizu, 1996
). Enhanced production of osmolytes could, through increased availability of arginine, also favour STX biosynthesis. This process has already been documented in plants, where amine-derived alkaloids increase under salt stress (Ali, 2000
). Alternatively, STX may interact with membrane ion fluxes in C. raciborskii T3, preventing the deleterious effect of intracellular Na+ increase in this freshwater cyanobacterium. Recently the blockage of Na+ uptake by STX has been demonstrated in strains of C. raciborskii and A. circinalis (Pomati et al., 2003b
). This inhibition of Na+ uptake by STX raises questions regarding the potential advantage that PSP toxin-producing cyanobacteria could have over other non-toxic species under conditions of high pH or salt stress. Similar circumstances may prevail in subtropical and temperate regions during natural cycles of flood and drought periods, or as a consequence of human exploitation. During 1991 high pH and elevated water conductivity were associated with the most extensive STX-producing bloom of A. circinalis in Australia (Bowling & Baker, 1996
). The same circumstances correlated with the dominance of a neurotoxic strain of C. raciborskii in Brazilian freshwaters (S. Azevedo, personal communication).
Conclusions
The present study demonstrates a strong correlation between variations in cellular Na+ levels and STX production in the cyanobacterium C. raciborskii T3. The evidence reported suggests that either STX metabolism or the toxin itself could be linked to the maintenance of cyanobacterial homeostasis under alkaline pH or Na+ stress conditions. The model proposed could also apply to other PSP toxin-producing bacteria, cyanobacteria or dinoflagellates, and may represent an important element in understanding the ecology of PSP toxin-producing micro-organisms. In addition, the results reported here will be important for further physiological, biochemical or gene expression studies of STX and related compounds in cyanobacteria and other micro-organisms.
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ACKNOWLEDGEMENTS |
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Received 14 March 2003;
revised 20 October 2003;
accepted 18 November 2003.
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