Saxitoxin Production Research Articles

Growth and saxitoxin production responses to copper (CuCl2) exposure by the cyanobacterium Raphidiopsis raciborskii

Copper (Cu2+) is an essential micronutrient for cyanobacteria, but it has a toxic effect above a certain threshold. The presence of Cu2+ in water is usually related to human activity due to it being used in pesticides, fertilizer, and algaecides. Previous studies observed that high Cu2+ concentrations stimulated toxin synthesis in cyanobacteria and microalgae. Furthermore, saxitoxins (STXs) can bind to Cu2+ transporters in microorganisms, decreasing the Cu2+ uptake and consequently reducing Cu toxicity. Therefore, considering the invasive capacity of the cyanobacterium Raphidiopsis raciborskii and its potential for STXs production, this study aimed to evaluate the effects of different Cu2+ concentrations in the production of STXs and growth of R. raciborskii. In acclimatized growth conditions, cultures of R. raciborskii strain (ITUC01) were exposed to four different copper concentrations for 20 days (0.8, 8, 80, and 800 × 10−3 μmol L−1 of CuCl2). Raphidiopsis raciborskii growth and physiological responses were evaluated measuring the cell concentration, cell volume, biovolume, chlorophyll a levels, and STXs concentration. Comparing the lowest and highest Cu2+ concentration (0.8 and 800 × 10−3 μmol L−1), it was observed that the increment of Cu2+ in the medium led to a reduced maximum growth rate (μmax), cell concentration, biovolume, and chlorophyll a levels, while the cell volume increased. Despite the low cell concentration and biovolume in the highest Cu2+ condition, it was observed that the STXs volumetric concentration was significantly high on day 5, which is indicative of the fact that increased Cu2+ concentration might induce STXs production in early growth. In addition, our results revealed that STXs production was uncoupled with growth and a reduction of R. raciborskii toxicity from day 5 to 20 was observed. Therefore, the present study identified some of the survival responses of R. raciborskii in Cu-stressed condition and suggested that Cu2+ might be one of the factors that can affect R. raciborskii bloom toxicity.

Monitoring of saxitoxin production in lakes in Denmark by molecular, chromatographic and microscopic approaches

Diversity of phytoplankton in three eutrophic and two mesotrophic lakes in Denmark was determined by microscopic and HPLC pigment analyses to identify and quantify potential saxitoxin (STX) producing cyanobacteria. Potential dominant STX-producers were identified to the filamentous genera Dolichospermum, Cuspidothrix, Phormidium and Planktolyngbya. Presence of STX production was documented by extraction of five intracellular STXs that included (in declining concentration in the cyanobacteria) dc-neo-STX, neo-STX, dc-STX, STX and GTX. Total concentrations of the five STXs varied from 9 to 142 fg per potential STX producer, corresponding to 87 to 985 ng L−1 in the lakes. For molecular detection of the STX-producers, a quantitative PCR method was developed by design of a new robust primer set with broad coverage to target the sxtA gene that is common to all STX-producing cyanobacteria. After validation, copy numbers of the sxtA gene were determined to vary from about 104 (mesotrophic lakes) to 108 per mL (the most eutrophic lake). A moderate but significant correlation was observed between abundance of the sxtA copies and concentrations of the five intracellular STXs. The qPCR assay was found to be a rapid and robust procedure for quantification of STX producers. Saxitoxin and its analogs appeared not to cause health concerns in the lakes, but commercial fishing for pike perch in the most eutrophic lake should be monitored to test for food web accumulation of STXs.

Strong constitutive expression divergence among strains but no evidence of differential expression associated with sexual reproduction in Alexandrium minutum

Sexual reproduction remains poorly characterized in dinoflagellates. This is especially the case at the molecular level. Here crossing experiments were performed among strains of the toxic dinoflagellate Alexandrium minutum belonging to two genetically divergent groups. Gene expression was compared between sexually compatible and incompatible crosses at the time of gamete fusion and resting cyst (~zygote) formation. Not a single transcript was identified as differentially expressed between compatible and incompatible crosses at these two crucial time points of the dinoflagellate life cycle. However, several thousands of transcripts displayed constitutive expression differences between strains. This was especially the case between the strains belonging to the genetically divergent groups. A few hundreds of transcripts were also identified as differentially expressed between strains belonging to opposite mating types. Some of these transcripts displayed homology with the SxtA protein, known to be involved in saxitoxin production in cyanobacteria, as well as with proteins potentially involved in mating in fungi.

Changes in pH and dissolved inorganic carbon in water affect the growth, saxitoxins production and toxicity of the cyanobacterium Raphidiopsis raciborskii ITEP-A1

Raphidiopsis raciborskii is a widely distributed, potentially toxic cyanobacterium described as a tropical-subtropical species. However, its occurrence in temperate regions has been expanding. Understanding the environmental factors underlying the expansion and colonization success of Raphidiopsis has been the object of numerous studies. However, less is known regarding its responses to pH and inorganic carbon in water. Thus, the aim of the present study was to investigate the effects of changes in pH and dissolved inorganic carbon on growth and saxitoxins production in the strain R. raciborskii ITEP-A1. We incubated batch cultures with different unbuffered and buffered pH (neutral-acid and alkaline) and inorganic carbon availability (CO2-rich air bubbling and the addition of NaHCO3) to assess the effect of these factors on the growth, toxin production as well as saxitoxins composition of the cyanobacterium. The carbon concentrating mechanism (CCM) system of ITEP-A1 was also characterized by an in silico analysis of its previously sequenced genome. The growth and saxitoxins production of R. raciborskii were affected. The addition of sodium bicarbonate and air bubbling enhanced the growth of the cyanobacterium in alkaline pH. In contrast, saxitoxins production and relative toxicity were decreased. Moreover, significant changes in the cellular composition of saxitoxins were strongly related to pH changes. ITEP-A1 potentially expresses the low-flux bicarbonate transporter BicA, an efficient CCM which uptakes most of its carbon from HCO3−. Hence, increasing the diffusion of CO2 in alkaline eutrophic lakes is likely to increase R. raciborskii dominance, but produce less toxic blooms.

Physiological responses of Raphidiopsis raciborskii (Cyanobacteria) strains to water conductivity: effect of sodium and magnesium ions

Cyanobacterial blooms dominated by Raphidiopsis raciborskii, a potential producer of saxitoxin (STX), are commonly reported in continental water bodies of semiarid regions with high conductivity (> 1000 µS cm−1). STX production is proposed as a cellular protection mechanism against salt stress. Here, we test the hypothesis that high conductivity favors R. raciborskii growth and that STX is advantageous under this condition. We included four tropical strains (two STX+ and two STX−) cultured for 12 days in control (450 µS cm−1) or in media with NaCl (10 mM) or MgCl2 (5 mM) (1500 µS cm−1). High conductivity (either NaCl or MgCl2) had neutral or positive effects on growth for all strains. Total STX content did not differ in response to salt concentrations. All strains could tolerate higher conductivity without signals of stress, as noted for stable photosynthetic parameters and protein expression profiles. For the strain with higher growth in salt, accumulation of intracellular carbohydrates occurred in early times (3 days). In conclusion, high conductivity is a favorable condition for R. raciborskii, with no clear relation with the ability to produce STX. Pronounced intraspecific variability was observed, a fact that must be considered for a better understanding of this species.

Transcriptome survey and toxin measurements reveal evolutionary modification and loss of saxitoxin biosynthesis genes in the dinoflagellates Amphidinium carterae and Prorocentrum micans.

In the present study, we characterized the potential toxin genes for polyketide synthase (PKS) and saxitoxin (STX) biosynthesis using the transcriptomes of two non-STX producing dinoflagellates Amphidinium carterae and Prorocentrum micans. RNA sequencing revealed 94 and 166 PKS contigs in A. carterae and P. micans, respectively. We first detected type III PKS, which was closely related to bacteria. In addition, dozens of homologs of 20 STX biosynthesis genes were identified. Interestingly, the core STX-synthesizing genes sxtA and sxtB were only found in P. micans, whereas sxtD was detected in A. carterae alone. Bioinformatic analysis showed that the first two core genes (sxtA and sxtG) had a low sequence similarity (37.0-67.6%) and different domain organization compared to those of other toxigenic dinoflagellates, such as Alexandrium pacificum. These might result in the breakdown of the initial reactions in STX production and ultimately the loss of the ability to synthesize the toxins in both dinoflagellates. Our findings suggest that toxin-related PKS and sxt genes are commonly found in non-STX producing dinoflagellates. In addition to their involvement in the synthesis of toxins, our result indicates that genes may also have other molecular metabolic functions.

Comparative transcriptomics of toxin synthesis genes between the non-toxin producing dinoflagellate Cochlodinium polykrikoides and toxigenic Alexandrium pacificum

In the present study, we extensively characterized potential toxin-related genes, including polyketide synthase (PKS), saxitoxin (STX) and fatty acid synthase (FAS) from the non-toxin producing marine dinoflagellate Cochlodinium polykrikoides, comparing to those of a toxigenic dinoflagellate Alexandrium pacificum. RNA sequencing revealed 50 and 271 PKS contigs from C. polykrikoides and A. pacificum, respectively. According to domain constitute and amino acid alteration, we further classified the dinoflagellate type I PKS genes into 4 sub-groups. Type III PKS was first identified in C. polykrikoides. Interestingly, we detected a large number (242 and 288) of homologs of 18 sxt genes from two studied dinoflagellates. Most of the eight key genes (sxtA, sxtB, sxtD, sxtG, sxtH/T, sxtI, sxtS and sxtU) for STX synthesis were detected in both dinoflatellates, whereas a core STX biosynthesis gene sxtG was not detected in C. polykrikoides. This may partially explain the absence of saxitoxin production in C. polykrikoides. In addition, we identified several type I and type II FAS genes, including FabD, FabF, FabG, FabH, FabI, and FabZ, whereas FabB was not found in C. polykrikoides. Overall, the numbers of the toxin-related genes in C. polykrikoides were less than that of A. pacificum. Phylogenetic analyses showed that type I PKS/FASs of dinoflagellates had close relationships with apicomplexans and bacteria. These suggest that the toxin-related PKS and sxt genes are commonly present in toxigenic and non-toxin producing dinoflagellates, and may be involved not only in the toxin synthesis, but also in other related molecular metabolic functions.

Effect of nutrient concentration on growth and saxitoxin production of Raphidiopsis raciborskii (Cyanophyta) interacting with Monoraphidium contortum (Chlorophyceae)

Raphidiopsis raciborskii is the dominant species in several subtropical aquatic ecosystems. This study addressed the influence of nutrient concentration on cyanobacteria growth and saxitoxin synthesis. Therefore, we performed bioassays of interaction between the Chlorophyceae Monoraphidium contortum and R. raciborskii simulating oligotrophic and supereutrophic environments. Experiments were carried out in a climatized room for 15 days in pure cultures of each species (control) and mixed (interaction). The biomass growth (biovolume) and specific growth rates were measured. Saxitoxin was analyzed using the ELISA biochemical method. In the oligotrophic environment, the cell volume of R. raciborskii decreased. This species also showed senescence in interaction with M. contortum. However, there were no statistical differences in the saxitoxin synthesis in both conditions. In the supereutrophic environment, the growth of R. raciborskii and saxitoxin production was similar in both the control and interaction conditions. However, M. contortum growth decreased in interaction with the cyanobacteria. The increase in trophic status from oligo- to supereutrophic contributes to the growth of M. contortum in subtropical aquatic ecosystems, but the decrease in the area/volume ratio of cyanobacteria and the saxitoxin synthesis is an evident survival strategy in oligotrophic environments.

Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12β-deoxygonyautoxin 3 (12β-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC–MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

Transcriptional and physiological responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for nutrient uptakes and assimilation

The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3− or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.

Combined Effect of Light and Temperature on the Production of Saxitoxins in Cylindrospermopsis raciborskii Strains.

Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium that can tolerate a wide range of light and temperature. Due to climatic changes, the interaction between light and temperature is studied in aquatic systems, but no study has addressed the effect of both variables on the saxitoxins production. This study evaluated the combined effect of light and temperature on saxitoxins production and cellular quota in C. raciborskii. Experiments were performed with three C. raciborskii strains in batch cultures under six light intensities (10, 40, 60, 100, 150, and 500 μmol of photons m−2 s−1) and four temperatures (15, 20, 25, and 30 °C). The growth of C. raciborskii strains was limited at lower temperatures and the maximum growth rates were obtained under higher light combined with temperatures equal or above 20 °C, depending on the strain. In general, growth was highest at 30 °C at the lower light intensities and equally high at 25 °C and 30 °C under higher light. Highest saxitoxins concentration and cell-quota occurred at 25 °C under high light intensities, but were much lower at 30 °C. Hence, increased temperatures combined with sufficient light will lead to higher C. raciborskii biomass, but blooms could become less toxic in tropical regions.

Growth of Raphidiopsis raciborskii and Monoraphidium contortum from a subtropical Brazilian Reservoir: interaction, saxitoxin production and response to the trophic state

In order to investigate the influence of trophic state from a subtropical reservoir in the state of São Paulo, Brazil, in the competition between the species dominant, Raphidiopsis raciborskii and Monoraphidium contortum, an assay was carried out of interaction between these microorganisms simulating the reservoir in its current conditions, mesotrophic, and another in a supereutrophic condition, as well as detect the production of saxitoxin by cyanobacteria. The experiments were conducted in a climate-controlled room for 15days in triplicate for pure and mixed cultures of each species. Growth curves were developed according to the biovolume-to-biomass and were calculated the growth rates. The saxitoxin synthesis was determined by ELISA. When simulating the mesotrophic and supereutrophic environments, R. raciborskii did not show any differences in its growth and in the saxitoxin production in the interaction with M. contortum, when compared to its control. Concerning M. contortum, your growth was affected in the interactions with cyanobacteria in both trophic state, although the chlorophyceae presented an increasing specific growth rate. The reduction of the growth of M. contortum in interaction with R. raciborskii shows the increase in trophic state can increase the competitiveness of R. raciborskii in the reservoir and to favor your dominance.

Biotechnological potential of Neolyngbya (Cyanobacteria), a new marine benthic filamentous genus from Brazil

Marine filamentous cyanobacteria are sources of biologically active secondary metabolites with potential biotechnological application. Despite the extension and environmental variety of the Brazilian coast, floristic surveys of filamentous cyanobacteria are scarce. Studies on their biotechnological potential are even more limited. In this work, we sampled Neolyngbya (Oscillatoriaceae) populations along the Brazilian coast and evaluated the antimicrobial, antioxidant, and cytotoxic activities of their organic extracts. We also assessed the strains' genetic potential for production of cyanotoxins. The strains were cultivated in SWBG-11 medium. Extraction of biomass was carried out with methanol, ethanol, and acetic acid. Antimicrobial (against seven bacterial strains and the yeast Candida albicans), antioxidant (DPPH and ABTS radicals), and cytotoxic (brine shrimp) potential were evaluated. PCR reactions were performed to analyze the genetic potential for the production of microcystin, saxitoxin, and cylindrospermopsin. Brazilian marine filamentous cyanobacteria presented relevant antimicrobial capacity. Seven extracts from four Neolyngbya strains inhibited growth of almost all bacteria, while nine extracts from four strains inhibited C. albicans, with a minimum inhibitory concentration (MIC) range from 2.5 to 10.0 mg·mL−1. Methanolic extracts presented the greatest antioxidant potential for both DPPH and ABTS radicals, ranging from 50.0 to 98.3% at a concentration of 10 mg·mL−1. Cytotoxicity assays showed a LC50 ranging from 0.8 to 6.6 mg·mL−1. We detected toxic genotypes in eight of the analyzed strains, in which the most frequent gene clusters were mcyG (microcystin) and cyrJ (cylindrospermopsin). All Neolyngbya strains screened in this study have potential for biotechnological application, which highlights the economical and pharmacological importance of studying this underestimated biodiversity in tropical areas.

Application of metagenome analysis to characterize the molecular diversity and saxitoxin-producing potentials of a cyanobacterial community: a case study in the North Han River, Korea

A wide variety of cyanobacterial species that inhabit freshwater systems are known to produce diverse toxins and off-flavor compounds during the development of environmentally harmful blooms. However, cyanobacterial community development and toxin production potential have not been well studied. In this study, we examined the taxonomic diversity and saxitoxin production potential of cyanobacteria in the water and sediments of a large river, the North Han River in South Korea, by metagenome analysis using next-generation sequencing (NGS) and molecular biological approaches, respectively. NGS revealed that the entire cyanobacterial community in the study area consisted of 39 genera and 47 species. The most abundant genera were Microcystis, Anabaena, Cyanobium, and Synechococcus, which accounted for more than 90% of the entire community. The saxitoxin production potential of the cyanobacterial community was assessed by detecting the sxtA and sxtG genes related to saxitoxin production. Eleven sxtA and 24 sxtG genes were identified through molecular cloning and sequencing. Phylogenic analysis revealed that three sxtA genes that grouped in one phylogenic branch with Scytonema sp. were distinctly separated from the sxtA genes of Anabaena, Aphanizomenon, Lyngbya, and Cylindrospermopsis. Sixteen of the detected sxtG genes were phylogenically similar to those of Anabaena circinalis (Dolichospermum circinale), Aphanizomenon gracile, and Aphanizomenon flos-aquae. Our study demonstrates the utility of the metagenomics approach for characterizing the natural community structure of cyanobacteria containing diverse and even rare species, and the evaluation of saxitoxin-producing potential in the cyanobacterial community.

Temperature Influences the Production and Transport of Saxitoxin and the Expression of sxt Genes in the Cyanobacterium Aphanizomenon gracile.

The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM, sxtPer) were assessed in Aphanizomenon gracile UAM529 cultures under temperatures covering its annual cycle (12 °C, 23 °C, and 30 °C). Temperature influenced saxitoxin production being maximum at high temperatures (30 °C) above the growth optimum (23 °C), concurring with a 4.3-fold increased sxtA expression at 30 °C. Extracellular saxitoxin transport was temperature-dependent, with maxima at extremes of temperature (12 °C with 16.9% extracellular saxitoxin; and especially 30 °C with 53.8%) outside the growth optimum (23 °C), coinciding with a clear upregulation of sxtM at both 12 °C and 30 °C (3.8–4.1 fold respectively), and yet with just a slight upregulation of sxtPer at 30 °C (2.1-fold). Nitrate depletion also induced a high extracellular saxitoxin release (51.2%), although without variations of sxtM and sxtPer transcription, and showing evidence of membrane damage. This is the first study analysing the transcriptional response of sxtPer under environmental gradients, as well as the effect of temperature on putative saxitoxin transporters (sxtM and sxtPer) in cyanobacteria in general.

Influence of environmental variables on saxitoxin yields by Cylindrospermopsis raciborskii in a mesotrophic subtropical reservoir.

Saxitoxins are a class of toxins produced by at least two groups of evolutionarily distant organisms (cyanobacteria and dinoflagellates). While the toxicity of these toxins is relatively well characterized, to date little is known about their drivers and ecological functions, especially in lower latitude tropical and subtropical freshwater ecosystems. In the present study, we aimed to obtain a better understanding of the main drivers of saxitoxin concentrations in aquatic environments. We investigated the relationships among saxitoxin concentrations in a mesotrophic subtropical reservoir dominated by the cyanobacteria Cylindrospermopsis raciborskii with physical, chemical and biological water variables. The highest saxitoxin concentrations were 0.20 μg·L-1, which occurred in the samples with the highest densities of C. raciborskii (maximum of 4.3 × 104 org·mL-1) and the highest concentration of dissolved nutrients (nitrate from 0.2 to 0.8 μg·L-1, ortophosphate from 0.3 to 8.5 μg·L-1). These correlations were confirmed by statistical analyses. However, the highest saxitoxin relative concentrations (per trichome) were associated with lower C. raciborskii densities, suggesting that saxitoxin production or the selection of saxitoxin-producing strains was associated with the adaptation of this species to conditions of stress. Our results indicate that C. raciborskii toxin yields vary depending on the enrichment conditions having potential implications for reservoir management.

Evolutionary processes and cellular functions underlying divergence in Alexandrium minutum.

Understanding divergence in the highly dispersive and seemingly homogeneous pelagic environment for organisms living as free drifters in the water column remains a challenge. Here, we analysed the transcriptome-wide mRNA sequences, as well as the morphology of 18 strains of Alexandrium minutum, a dinoflagellate responsible for harmful algal blooms worldwide, to investigate the functional bases of a divergence event. Analysis of the joint site frequency spectrum (JSFS) pointed towards an ancestral divergence in complete isolations followed by a secondary contact resulting in gene flow between the two diverging groups, but heterogeneous across sites. The sites displaying fixed SNPs were associated with a highly restricted gene flow and a strong overrepresentation of nonsynonymous polymorphism, suggesting the importance of selective pressures as drivers of the divergence. The most divergent transcripts were homologs to genes involved in calcium/potassium fluxes across the membrane, calcium transduction signal and saxitoxin production. The implication of these results in terms of ecological divergence and build-up of reproductive isolation is discussed. Dinoflagellates are especially difficult to study in the field at the ecological level due to their small size and the dynamic nature of their natural environment, but also at the genomic level due to their huge and complex genome and the absence of closely related model organism. This study illustrates the possibility to identify the traits of primary importance in ecology and evolution starting from high-throughput sequencing data, even for such organisms.

Influence of nitrogen availability on the expression of genes involved in the biosynthesis of saxitoxin and analogs in Cylindrospermopsis raciborskii

The development of cyanobacterial blooms in inland aquatic ecosystems is greatly promoted by nutrient availability, especially nitrogen and phosphorous. When blooms are dominated by toxigenic species the harmful effects of nutrient loading becomes particularly relevant. Among toxic species, Cylindrospermopsis raciborskii found in South American ecosystems is characterized by the production of saxitoxin and analogs (Paralytic Shellfish Poisoning, PSP), for which the factors that trigger their production have not been elucidated. In this study, the effect of nitrate availability on the relative transcript abundance of two genes (sxtU and sxtI), both involved in different steps of PSP biosynthetic pathway, was addressed in C. raciborskii MVCC19 by qPCR. The relative transcript abundance of both genes significantly increased from the beginning to the end of growth, independent of nitrate availability in the culture medium. Differences between the genes in terms of the levels of relative expression were also found, implying that during growth in nitrate-rich or nitrate-deprived conditions C. raciborskii MVCC19 has the ability to produce different kind of PSP molecules. The presence of nifH transcripts in the nitrogen-depleted treatment confirmed that in the absence of nitrate C. raciborskii fixed atmospheric N2. Moreover, after transferring filaments to nitrate-rich conditions the synthesis of nifH mRNA continued for few hours, suggesting that cell adjustments enabling the utilization of soluble nitrogen sources are not immediate. Our results show that biosynthesis of saxitoxin and analogs in C. raciborskii is not related to nitrate availability, but rather is linked to cyanobacteria growth rate.

Abiotic variables affect STX concentration in a meso-oligotrophic subtropical coastal lake dominated by Cylindrospermopsis raciborskii (Cyanophyceae)

The cyanobacterium Cylindrospermopsis raciborskii is capable of producing toxins including saxitoxin (STX). Few studies have verified the influence of environmental variables on the production of STX and most have only been studied in the laboratory. The goal of this work was to identify the abiotic variables related to STX concentration in situ. The relationship among STX concentration and the physical variables, nutrients and chlorophyll-a (chl-a) concentration was examined in a meso-oligotrophic subtropical coastal lake dominated by C. raciborskii. A generalized linear model was developed, incorporating all variables measured monthly over a 45-month monitoring period. Conductivity and dissolved inorganic nitrogen (DIN) concentration provided the greatest explanatory power for STX concentration in situ. Previous studies suggested that C. raciborskii cells exposed to stress associated with higher ionic concentrations appear to activate the biosynthesis of STX suggesting that STX can elicit changes cell permeability and may contribute to the homeostasis of this organism. An increase of DIN concentration results in a higher concentration of STX which may be related to a reduced metabolic demand, since the uptake of inorganic nitrogen requires less energy than N2-fixation. Thus, increased DIN can favor the growth of C. raciborskii population or improve cellular homeostasis, both potentially increasing STX concentration in the aquatic system, which was observed through a delayed response pattern. The developed model, while providing only a moderate predictive power, can assist in the understanding of the environmental variables associated with increases in STX concentration, and in monitoring and minimizing the risks of toxic blooms.

Proteogenomics of a saxitoxin-producing and non-toxic strain of Anabaena circinalis (cyanobacteria) in response to extracellular NaCl and phosphate depletion.

In Australia, saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model saxitoxin-producing A. circinalis AWQC131C and non-toxic A. circinalis AWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria.