Interactions between intracellular Na levels and saxitoxin ... - CiteSeerX

Microbiology (2004), 150, 455–461

DOI 10.1099/mic.0.26350-0

Interactions between intracellular Na+ levels and saxitoxin production in Cylindrospermopsis raciborskii T3 Francesco Pomati,1 Carlo Rossetti,2 Gianluca Manarolla,2 Brendan P. Burns1 and Brett A. Neilan1 1

Cyanobacteria and Astrobiology Research Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, NSW, Australia

Correspondence Brett A. Neilan

2

[email protected]

DBSF, University of Insubria, via J. H. Dunant 3, 21100 Varese, Italy

Received 14 March 2003 Revised 20 October 2003 Accepted 18 November 2003

Saxitoxin (STX) is the most potent representative among the paralytic shellfish poisoning (PSP) toxins, which are highly selective Na+ channel-blocking alkaloids. This study investigated, in cultures of the cyanobacterium Cylindrospermopsis raciborskii T3, the effects of pH, salt, amiloride and lidocaine hydrochloride on total cellular levels of Na+ and K+ ions and STX accumulation. Both Na+ levels and intracellular STX concentrations increased exponentially in response to rising alkalinity. NaCl inhibited cyanobacterial growth at a concentration of 10 mM. In comparison with osmotically stressed controls, however, NaCl promoted STX accumulation in a dose-dependent manner. A correlation was seen in the time-course of both total cellular Na+ levels and intracellular STX for NaCl, amiloride and lidocaine exposure. The increase in cellular Na+ induced by NaCl at 10 mM was coupled with a proportional accumulation of STX. The two Na+ channel-blocking agents amiloride and lidocaine had opposing effects on both cellular Na+ levels and STX accumulation. Amiloride at 1 mM reduced ion and toxin concentrations, while lidocaine at 1 mM increased the total cellular Na+ and STX levels. The effects of the channel-blockers were antagonistic and dependent on an alkaline pH. The results presented suggest that, in C. raciborskii T3, STX is responsive to cellular Na+ levels. This may indicate that either STX metabolism or the toxin itself could be linked to the maintenance of cyanobacterial homeostasis. The results also enhance the understanding of STX production and the ecology of PSP toxin-producing cyanobacteria.

INTRODUCTION The paralytic shellfish poisoning (PSP) toxins are wellknown natural bioactive compounds due to their accidental consumption in contaminated seafood (Zingone & Enevoldsen, 2000). These molecules, of which the most potent representative is saxitoxin (STX), are a class of neurotoxic alkaloids comprising different isoforms and with varied toxicities (Strichartz, 1981). PSP toxins selectively block voltage-gated Na+ channels in excitable cells (Catterall, 1980), thereby affecting the impulse generation in animals which can lead, in extreme cases, to death (Zingone & Enevoldsen, 2000). STX binds to nerve membranes with high affinity (dissociation constant Kd=2 nM), with a single toxin molecule interacting with a single Na+ channel (Catteral et al., 1979). PSP toxins block resting, active or inactive Na+ channels equally well, through a receptor site named as site 1, occupied by the so-called ‘pore-blocking’ toxins (Ceste`le & Catterall, 2000). These toxins act from the Abbreviations: PSP, paralytic shellfish poisoning; STX, saxitoxin.

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extracellular side of the plasma membrane by occluding the entry of the Na+ channel pore. The positively charged guanidinium group of PSP toxins interacts with negatively charged carboxyl groups at the mouth of the channel (Ceste`le & Catterall, 2000). 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 STXproducing 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 455

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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 toxinproducing 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 channelblockers (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.

METHODS Growth conditions and cyanobacterial cultures. Cylindrospermopsis raciborskii strain T3 was obtained from Sandra Azevedo (Federal University of Rio de Janeiro, Brazil). The cyanobacterium was maintained in ASM-1 medium (Gorham et al., 1964) adjusted to pH 9?5. Cultures were grown in glass 250 ml flasks in a cabinet at a constant temperature of 26 uC and under continuous irradiance of cool white light at an intensity of 15 mmol photons m22 s21. Cultures were monitored spectrophotometrically by recording the OD750 with a Lambda 10 UV/VS spectrometer (Perkin Elmer), and microscopically by viewing viable cells under an Olympus EHA phase-contrast microscope. For the growth experiments, a culture in late-exponential growth phase was used as inocula for new batch cultures exposed to 0 (control), 1 and 10 mM NaCl. The initial cell density was adjusted to approximately 0?1 OD750 and then measured once every 24 h for 7 days. Short-term experiments were carried out for 2 h or 4 h with cultures in standard growth conditions at pH 9?5 or at pH 7?5, when needed. For all experiments, cultures in the late-exponential growth phase were exposed to NaCl (1, 5, 10 mM), lidocaine at 1 mM and amiloride at 1 mM. These concentrations of lidocaine and amiloride were chosen based on previous studies on cyanobacteria (Pomati et al., 2003a) and general physiological investigations on animal sodium homeostasis (Kim & Smith, 1986). To evaluate the effect of pH on total Na+ and K+ cellular levels and STX accumulation, aliquots of the same culture were adjusted to different pH and analysed after 2 h. All experiments were performed in triplicate or quadruplicate. 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 (25 min) and immediately after the addition of the agents (0 min). All sampled pellets were 456

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 mm 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 220 uC 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 (15064?6 mm, i.d. 5 mm) (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 min21. 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 SigmaAldrich. Lidocaine hydrochloride and amiloride solutions (100 mM

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 ana-

lyses 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).

RESULTS Effects of pH on Na+ and K+ levels and STX content Total cellular Na+ and K+ levels were monitored in C. raciborskii T3 by flame photometry; the results are summarized in Fig. 1(a). At low pH (3?1), total cellular contents of both Na+ and K+ were at their lowest. In response to rising alkalinity, K+ levels reached their maximum at optimal growth pH (7–10?5), but decreased for pH values higher than 11. In contrast, cellular Na+ content increased Microbiology 150

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Fig. 1. (a) Total cellular Na+ (&) and K+ (#) concentrations measured by flame photometry (means±SE in the final cell suspension) in C. raciborskii T3 cell suspensions adjusted to different pH and analysed after 2 h. (b) Intracellular STX concentrations in cultures adjusted to different pH and analysed by HPLC after 2 h. Data were normalized by the OD750 of the corresponding culture sample. Solid line, Gauss fit; dashed lines, exponential fit.

exponentially with the rising alkalinity of the medium, achieving the highest concentration at pH 12. Intracellular STX concentrations, as measured by HPLC, also increased exponentially in response to the rising pH of the culture medium (Fig. 1b), with the highest rate for pH >9. The maximum cyanobacterial STX content was observed at the highest pH tested (10?0), a value 69-fold greater than the amount recorded at pH 7?5. 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. http://mic.sgmjournals.org

Fig. 2. (a) Effect of NaCl at 1 mM (%) and 10 mM (#) on the photoautotrophic growth of C. raciborskii T3. Data points represent the OD750 ratio of the test sample over the control (0 mM NaCl), expressed as mean±SE. (b) Comparison of the intracellular STX concentrations of cultures exposed to 0 (control), 1, 5, 10 and mM NaCl, 20 mM sorbitol, and 5 mM MgCl2 for 2 h at pH 9?5. All data were normalized by the OD750 of the corresponding culture sample.

To investigate the ability of Na+ ions to promote intracellular STX accumulation in C. raciborskii T3, cyanobacterial cultures grown at pH 9?5 were exposed for 2 h to NaCl at 1, 5 and 10 mM, sorbitol at 20 mM (osmotic stress control), or MgCl2 at 5 mM (ionic stress control) (Fig. 2b). Treatment of cultures with increasing concentrations of NaCl stimulated STX accumulation in a dose-dependent manner compared both to the unexposed and the osmotic/ ionic stress controls. Cultures exposed to MgCl2 at 5 mM and sorbitol at 20 mM showed no statistically significant difference in STX accumulation compared with untreated controls. By ANOVA analysis and post-hoc comparison using the LSD test, the mean STX concentration corresponding to cultures dosed with NaCl at 10 mM was demonstrated to be significantly distinct from mean control levels. STX values recorded in the 5 and 10 mM NaCl experiments were instead statistically different from the mean concentration recorded after exposing cells to MgCl2 at 5 mM (P¡0?05). Intracellular STX levels, normalized against the hyperosmotic effect induced by sorbitol on cyanobacterial cells, increased by 24?4±5?6 % for 5 mM and 29±6?6 % after the addition of 10 mM Na+. The above results suggested that STX accumulation in the cyanobacterial cells was promoted by Na+ ion stress. The 457

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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 225±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

Fig. 3. (a) Time-course of total cellular Na+ and K+ levels in C. raciborskii T3 cultures exposed to 10 mM NaCl for 2 h (&, Na+; $, K+), in comparison with untreated samples (%, Na+; #, K+). (b) Time-course of the intracellular (&; left axis) and extracellular (n; right axis) STX concentrations in cultures exposed to 10 mM NaCl for 4 h. Intracellular values were normalized by the OD750 of the corresponding culture sample; extracellular concentrations were normalized by the total protein content (mg ml”1) of the corresponding filter-sterilized culture medium. All values are expressed as mean percentile variation over the sample at 0 min±SE. 458

lysis. The extracellular concentration of STX represented 25–30 % of the total STX content of the cultures (which ranged from 179 to 247 mg l21). Extracellular levels, after normalizing for the total protein concentration in the cellfree 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. Effects of channel-blockers on Na+ and K+ levels and STX accumulation To confirm the observed correlation between changes in cellular Na+ levels and STX concentrations, we utilized amiloride and lidocaine to alter Na+ and K+ fluxes in C. raciborskii T3 over 2 h of exposure. The two Na+ channelblockers elicited a rapid response by ion cycles, as seen by comparison of Na+ and K+ values at 25 min and 0 min (i.e. immediately after addition of the blockers). Amiloride at 1 mM induced a decrease in total cellular Na+ and K+ levels (Fig. 4a). However, Na+ values diminished by only 21±3?1 % after 2 h, while K+ content decreased by 76± 1 % compared with samples at 25 min. Lidocaine hydrochloride at 1 mM, in contrast, promoted an increase in the levels of both Na+ and K+ within 30 min (Fig. 4b). The K+ content eventually decreased to control levels after 60 min. The highest and lowest values reached for Na+ and K+, compared with samples at 25 min, were 42±11 % and 215±1?6 % at 60 and 120 min, respectively.

Fig. 4. Effects of (a) amiloride at 1 mM and (b) lidocaine hydrochloride at 1 mM on the total cellular Na+ (&) and K+ (#) levels in C. raciborskii T3 cultures at pH 9?5. Values are expressed as mean percentile variation over the sample at ”5 min±SE. Microbiology 150

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As regards the intracellular STX concentration, at pH 7?5, amiloride only slightly inhibited the accumulation of STX by C. raciborskii T3 after 4 h exposure (Fig. 5a). The effect detected at pH 9?5, however, was markedly unambiguous. At alkaline pH, amiloride significantly reduced STX intracellular levels (233?5±4?3 %) after 2 h exposure and the STX content remained at this lower level during the next 2 h (234?1±4?3 %). Lidocaine at 1 mM and pH 9?5 promoted intracellular STX accumulation by C. raciborskii T3 over the 4 h time-course. STX concentrations (Fig. 5b) increased linearly with time, reaching levels 43±14 % greater than the sample at 0 min. Lidocaine-induced STX accumulation was, however, suppressed by the addition of 1 mM amiloride (Fig. 5b), and displayed the same temporal pattern as seen in Fig. 5(a) for pH 9?5. The positive effect of lidocaine on STX production prevailed during the first hour (18±1?5 % and 14?5±0?7 %, after 30 and 60 min, respectively), while at 2 and 4 h STX levels dropped compared to control values (221±1?5 %), a change similar to the trend observed in Fig. 5(a).

DISCUSSION In a previous study (Pomati et al., 2003a), we investigated the effect of lidocaine hydrochloride, a synthetic Na+

Fig. 5. (a) Comparison of the effect of amiloride 1 mM at pH 7?5 (#) and 9?5 (&) on the time-course of intracellular STX accumulation in C. raciborskii T3. (b) Time-course of the intracellular STX concentration in C. raciborskii T3 cultures exposed at pH 9?5 to 1 mM lidocaine hydrochloride (n) and 1 mM lidocaine+1 mM amiloride (.). All values were normalized by the OD750 of the corresponding culture sample and expressed as the percentile mean increase over the sample at 0 min±SE. http://mic.sgmjournals.org

channel-blocker (Suzuki et al., 2000), on growth and STX intracellular accumulation in the freshwater cyanobacterium C. raciborskii T3. This strain was isolated in Brazil from a neurotoxic bloom and found to produce the PSP toxins STX and C-toxins (C1/C2) (Lagos et al., 1999). Lidocaine is a local anaesthetic used in medical and veterinary practice and its physiological effect is related to the binding of Na+ channels, although it has been shown to interfere with the activity of Na+/H+ antiporters in several organisms (Shibamoto et al., 1990; Nosaka et al., 1992; Bidani & Heming, 1997). Focusing on the metabolism of STX, we previously found that lidocaine stimulated intracellular STX accumulation, and that the increase in STX content induced by this agent was dependent both on the concentration of Na+ ions in the culture medium and on alkaline pH (Pomati et al., 2003a). In the present study, the alkaline pH reliance of lidocaine effects was accompanied by a similar dependence of the inhibition of STX accumulation by amiloride (Fig. 5a). Amiloride is a Na+ channel-blocker that has been associated with the blockage of Na+ channels utilized in the maintenance of pH and Na+ homeostasis in eukaryotes (Schaefer et al., 2001) and prokaryotes (Rowbury et al., 1994), including the cyanobacterium Synechocystis PCC 7120 (Maestri & Joset, 2000). Furthermore, here we documented that the accumulation of STX itself is dependent upon alkaline pH (Fig. 1b). 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 459

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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 timecourse 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 460

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 STXproducing 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.

ACKNOWLEDGEMENTS The authors are grateful to A. M. Martinengo for the helpful experimental assistance and to I. Couperwhite for support and encouragement. Research scholarships to F. P. from the University of New South Wales and the School of Biotechnology and Biomolecular Sciences are also acknowledged.

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