Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells.

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN's lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

A study of polymethoxy-1-alkenes in Raphidiopsis (Cylindrospermopsis) raciborskii and Aphanizomenon gracile isolated in Poland.

Previous studies indicated that teratogenic polymethoxy-1-alkenes (PMAs) are produced by phylogenetically diverse cyanobacteria taxa, however corresponding studies on the occurrence of PMAs in European cyanobacteria are lacking. Herein, the presence of PMAs in strains of Raphidiopsis raciborskii and Aphanizomenon gracile isolated from surface waters in Poland was studied using nuclear magnetic resonance and mass spectrometry. No PMAs were detected in any of the strains investigated, indicating that production of these compounds may be geographically diversified. Further studies are necessary to elucidate mechanisms of cyanobacterial PMAs synthesis.

Is the presence of Central European strains of Raphidiopsis (Cylindrospermopsis) raciborskii a threat to a freshwater fish? An in vitro toxicological study in common carp cells.

As yet European strains of Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) have not been found to produce known cyanotoxins although their extracts have caused adverse effects in mammals, as shown using in vitro and in vivo experimental models. The present study investigated whether R. raciborskii isolated from Western Poland and Ukraine can affect fish cells using in vitro exposures of hepatocytes and red blood cells (RBC), and brain homogenates obtained from common carp (Cyprinus carpio) to 1.0% and 0.1% extracts of 7 strains. The studied extracts evoked different responses of catalase activity in hepatocytes with both increase and decrease observed under low and high concentrations. The cellular thiol pool was also altered with most extracts inducing a decrease in the activity of glutathione-S-transferase, and Ukrainian strains leading to an increase in glutathione level and a decrease in metallothionein content. All the studied extracts induced comparable reactive oxygen species formation, lipid peroxidation, protein carbonylation and DNA fragmentation in hepatocytes, and all but one increased the activity of caspase-3. Only one extract caused lysosomal membrane destabilization as measured by neutral red retention in RBC. In contrast to extracts of Ukrainian isolates, exposure of brain homogenates to extracts of Polish strains induced an increase in acetylcholinesterase activity suggesting the neurotoxic action of their exudates. The results indicate that both Polish and Ukrainian strains of R. raciborskii may pose a toxicological risk to freshwater fish, and further, that Polish strains may produce compound(s) evoking neurotoxic effects.

Behavior of Cylindrospermopsis raciborskii during coagulation and sludge storage - higher potential risk of toxin release than Microcystis aeruginosa?

Owing to the global warming and its strong adaptability, Cylindrospermopsis raciborskii has spread world-wide. However, as one of toxic cyanobacteria in many drinking water sources, it has not been drawn proper consideration in drinking water treatment plants so far. The investigation aimed at unveiling the fate of C. raciborskii during polyaluminum ferric chloride (PAFC) coagulation and sludge storage, revealing its differences from Microcystis aeruginosa. Results showed that C. raciborskii cells were effectively removed intactly under optimum coagulation conditions, but PAFC at higher dosages (>10 mg/L) triggered additional cylindrospermopsins release. In sludge storage, coagulated C. raciborskii cells suffered severe oxidative damage, leading to significant cylindrospermopsins release after day 6. C. raciborskii manifested different behaviors from M. aeruginosa which cells didn't release much microcystins during coagulation and sludge storage. This was mostly due to their differences in physiology and morphology. In flocs, M. aeruginosa could be enveloped by coagulant which can protect cells against the nasty attack from outside, whereas C. raciborskii with long filaments was hard to be wrapped and prone to suffering oxidative damage. These results confirmed C. raciborskii had a higher risk of toxin release in water production process than M. aeruginosa, which should deserve more attention.

First report of neurotoxic effect of the cyanobacterium Cylindrospermopsis raciborskii on the motility of trematode metacercariae.

Cylindrospermopsis raciborskii (Woloszynska) is a photosynthetic cyanobacterium that can produce cytotoxic (cylindrospermopsin) and neurotoxic cyanotoxins (saxitoxins). In Brazil the strains of C. raciborskii are reported to produce only saxitoxins (STX) and their effect on fish parasites has not been tested to date. The fish Poecilia vivipara Bloch and Schneider is a common host for the trematode Pygidiopsis macrostomum Travassos off the coast of Rio de Janeiro, and this fish-parasite interaction is a model for behavioural and ecotoxicological studies. The aim of this work was to evaluate the motility of metacercariae of P. macrostomum from P. vivipara exposed to 40 mg l-1 and 400 mg l-1 of crude lyophilized extract of the cyanobacterium C. raciborskii (CYRF-01) for 48 h. The fish were separated into groups of ten individuals and, after exposure, five fish from each group were dissected for counting and checking the motility of metacercariae. The other five fish were dissected after 48 h in clean water. The detection and quantification of STX in the solutions of cyanobacteria, and the gills and guts of fish, were performed by an enzyme-linked immunosorbent assay. The crude extract of C. raciborskii caused temporary paralysis in metacercariae of P. macrostomum after exposure of fish to both concentrations, and the motility recovered after the fish were kept for 48 h in clean water. STX was detected in the guts and gills of all fish analysed, suggesting that this toxin is involved in the paralysis of metacercariae. This is the first report on the action of neurotoxins in metacercariae of fish.

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.

Effects of crude extracts of a saxitoxin-producer strain of the cyanobacterium Cylindrospermopsis raciborskii on the swimming behavior of wild and laboratory reared guppy Poecilia vivipara.

The cyanobacterium Cylindrospermopsis raciborskii is an invasive species in water supply reservoirs worldwide, which can produces cylindrospermopsins and saxitoxins. In the wild, guppy (Poecilia vivipara) can be exposed to cyanotoxins, but those born and reared in laboratory are free of this contact. The aim of this paper was to comparatively measure the locomotor activity of 'wild' and 'lab' P. vivipara before and after exposure to crude extracts of two different cultures of C. raciborskii (CYRF-01), a saxitoxin-procucer strain. The movement of each fish was recorded using an image monitoring system (Videomex V®) before and after 48 h exposure to cyanobacterial extracts. Each experiment was performed during 4 h, with 1 h acclimation and 3 h recording period of the parameters Distance performed (DP), Swimming time (SwT), Stereotypic time (StT), Resting time (RT) and Average speed (AS). The quantification of saxitoxin in the solutions was performed by the enzyme-linked immunosorbent assay (ELISA). The weight or the total length did not influence the locomotor activity of fish in any of the experiments. The saxitoxin value was similar for both cultures (Culture 1: 7.3 µg L-1 and Culture 2: 8.6 µg L-1). However, in experiments with Culture 1 an increased activity in most parameters was observed, while in Culture 2, a decreased activity was observed only in 'lab' fish. Wild fish was less affected, showing higher resistance to both cyanobacterial crude extracts. This study showed that different cultures of the same strain of C. raciborskii and with similar contents of saxitoxin are able to change the locomotor activity of P. vivipara, contributing to the validation of the use of behavioral parameters to the evaluation of sublethal effects of toxic cyanobacteria on fish.

Toxicity Overrides Morphology on Cylindrospermopsis raciborskii Grazing Resistance to the Calanoid Copepod Eudiaptomus gracilis.

Toxicity and morphology may function as defense mechanisms of bloom-forming cyanobacteria against zooplankton grazing. Yet, the relative importance of each of these factors and their plasticity remains poorly known. We tested the effects of chemical and morphological traits of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii on the feeding response of the selective feeder Eudiaptomus gracilis (Calanoida, Copepoda), using a saxitoxin-producing strain (STX+) and a non-saxitoxin (STX-)-producing strain as food. From these two chemotypes, we established cultures of three different morphotypes that differed in filament length (short, medium, and long) by incubating the strains at 17, 25, and 32 °C. We hypothesized that the inhibitory effects of saxitoxins determine the avoidance of C. raciborskii, and that morphology would only become relevant in the absence of saxitoxins. Temperature affected two traits: higher temperature resulted in significantly shorter filaments in both strains and led to much higher toxin contents in the STX+ strain (1.7 µg eq STX L(-1) at 17 °C, 7.9 µg eq STX L(-1) at 25 °C, and 25.1 µg eq STX L(-1) at 32 °C). Copepods strongly reduced the ingestion of the STX+ strain in comparison with STX- cultures, regardless of filament length. Conversely, consumption of shorter filaments was significantly higher in the STX- strain. The great plasticity of morphological and chemical traits of C. raciborskii and their resultant contrasting effects on the feeding behavior of zooplankton might explain the success of this cyanobacterium in a variety of aquatic environments.

Simulation of saxitoxins adsorption in full-scale GAC filter using HSDM.

Many different species of cyanobacteria capable of producing saxitoxins have been identified as a threat to the safety of drinking water supplies worldwide. Removal of these contaminants can be accomplished by adsorption on granular activated carbon (GAC) but little is yet known about the kinetics of this process. This research investigated adsorption kinetics and diffusion behaviour of decarbomoyl saxitoxin (dc-STX) and carbamate saxitoxin (STX) on a GAC sample and simulated a full-scale GAC filter using batch experimental data and the homogeneous surface diffusion model (HSDM). HSDM was able to successfully describe batch adsorption of STX and dc-STX onto GAC sample and the surface diffusion coefficient was identified as the main adjustment parameter for this model. Different scenarios of STX and dc-STX removal in a GAC filter were simulated, offering engineers and scientists an option for the design of GAC full-scale filters, bench or pilot-scale experiments.

Combining immunolabeling and catalyzed reporter deposition to detect intracellular saxitoxin in a cyanobacterium.

We combined the use of polyclonal antibodies against saxitoxin with catalyzed reporter deposition to detect production of saxitoxin by the cyanobacterium Cylindrospermopsis raciborskii. The procedure is simple, allows detection of intracellular saxitoxin in cyanobacteria filaments by confocal laser microscopy and is a promising tool to study toxin production and metabolism.

Study of carotenoids in cyanobacteria by Raman spectroscopy.

Cyanobacteria have established dominant aquatic populations around the world, generally in aggressive environments and under severe stress conditions, e.g., intense solar radiation. Several marine strains make use of compounds such as the polyenic molecules for their damage protection justifying the range of colours observed for these species. The peridinin/chlorophyll-a/protein complex is an excellent example of essential structures used for self-prevention; their systems allow to them surviving under aggressive environments. In our simulations, few protective dyes are required to the initial specimen defense; this is an important data concern the synthetic priority in order to supply adequate damage protection. Raman measurements obtained with 1064 and 514.5 nm excitations for Cylindrospermopsis raciborskii and Microcystis aeruginosa strains shows bands assignable to the carotenoid peridinin. It was characterized by bands at 1940, 1650, 1515, 1449, 1185, 1155 and 1000 cm(-1) assigned to ν(C=C=C) (allenic vibration), ν(C=C/CO), ν(C=C), δ(C-H, C-18/19), δ(C-H), ν(C-C), and ρ(C-CH3), respectively. Recognition by Raman spectroscopy proved to be an important tool for preliminaries detections and characterization of polyene molecules in several algae, besides initiate an interesting discussion about their synthetic priority.

Constitutive cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2 partial pressure conditions.

Cylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwater Cylindrospermopsis raciborskii. C. raciborskii is an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO2 affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO2 affect CYN and dCYN pool size as well as the expression of the key genes, cyrA and cyrK, involved in CYN biosynthesis in a toxic C. raciborskii strain. For cells growing at different light intensities (10 and 100 µmol photons m(-2) s(-1)), we observed that the rate of CYN pool size production (µCYN) was coupled to the cell division rate (µc) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (QCYN) and dCYN (QdCYN) are fixed. Moreover, a lack of correlation between expression of cyrA and total CYN cell quotas (QCYNs) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO2 (1,300 ppm), we observed minor effects on QCYN and no effects on expression of cyrA and cyrK. We conclude that the CYN pool size is constitutive and not affected by light and CO2 conditions. Thus, C. raciborskii bloom toxicity is determined by the absolute abundance of C. raciborskii cells within the water column and the relative abundance of toxic and nontoxic strains.

Toxic potencies of metabolite(s) of non-cylindrospermopsin producing Cylindrospermopsis raciborskii isolated from temperate zone in human white cells.

Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) has worldwide distribution and is well known for producing the toxic alkaloid, cylindrospermopsin (CYN). Strains unable to synthesize this compound but potentially toxic were recently identified in Europe. Here, for the first time the effect of cell-free extracts of a non-CYN-producing strain of C. raciborskii was studied in human cells (neutrophils and lymphocytes) isolated from healthy donors. The observed effects were compared to those induced by CYN (1.0-0.01 µg mL(-1)). Short-term (1h) extract treatments resulted in altered viability of cells demonstrated by increased necrosis and apoptosis in neutrophils and elevated apoptosis in lymphocytes. CYN did not induce similar effects, regardless of the toxin concentration. Exposure of T-lymphocytes to 100% C. raciborskii extract in isolated and whole-blood 72 h cultures resulted in decrease of proliferation by 20.6% and 32.5%, respectively. In comparison, exposure to 1.0 µg mL(-1) of CYN caused lymphocytes proliferation to be inhibited by 91.0% in isolated cultures and 56.5% in whole-blood assay. Significant antiproliferative properties were also found for 0.1 µg mL(-1) of CYN in whole-blood culture. From the results we conclude that strains occurring in temperate zones may pose a threat to human health through the production of hitherto unknown metabolites that reveal a toxic pattern different to that of CYN. At the same time our study demonstrates that CYN is a powerful but slowly-acting toxin in human immune cells.

In vivo genotoxicity of treated water containing the cylindrospermopsin-producer Cylindrospermopsis raciborskii.

Cylindrospermopsin (CYN) is an alkaloid commonly produced by some cyanobacteria that has been implicated in outbreaks of human illness. The aim of this study was to investigate the genotoxicity of Cylindrospermopsis raciborskii cellular content (including CYN) and its byproducts resulting from chlorination during water treatment. DNA damage in blood and liver cells was analysed by the comet assay and micronucleus test (MN). Mice were injected intraperitoneally with the following treatments: (a) physiological saline, (b) treated water, (c) treated water plus C. raciborskii extract (CYN producer strain, CYPO-011 K), (d) C. raciborskii extract (CYN producer strain, CYPO-011 K), (e) C. raciborskii extract (CYN non producer strain), and (f) treated water plus C. raciborskii extract (CYN non producer strain) extract. After 48 h, samples were taken to perform tests (blood and liver cells to the comet assay and bone marrow to MN test). The CYPO-011 K had a genotoxic and mutagenic effects on liver and bone marrow cells. The group that received chlorine-treated water plus CYPO-011 K also exhibited genotoxic effects in the liver, as well as in the blood, and a mutagenic effect in blood marrow cells. The results emphasise the need of improving CYN monitoring in waters bodies in order to reduce the risk of human exposure.

Effects of Cylindrospermopsis raciborskii strains (Woloszynska, 1912) Senayya & Subba Raju on the mobility of Daphnia laevis (Cladocera, Daphniidae).

Cylindrospermopsis raciborskii is a cyanobacterium distributed worldwide that is known to produce cyanotoxins. Some of the Brazilian strains can produce saxitoxins (STXs), which are classified as neurotoxins and can paralyze cladocerans .Daphnia laevis is a cladoceran with a wide distribution in the Americas and has been studied as a possible test-organism in toxicity bioassays. The present work tested the acute effect on D laevis mobility when fed a saxitoxin-producing (STX and neoSTX) C. raciborskii strain, CYRF-01, and compared the results with the effects of a non-toxic strain (NPCS-1). Neonates (6-24 hours after birth) were exposed to concentrations of C. raciborskii varying from 102 to 106 cells·mL-1 of each strain for up to three hours. The cladocerans were then transferred to a medium without toxic filaments for 24 hours. Only the organisms exposed to the STX-producing strain showed signs of the immobilization of swimming movements, confirming the effects of the toxins. There was a linear correlation between the time required to induce stopping the swimming movement, with a shorter time to needed to induce immobilization at a higher the concentration; this correlation was inverse to the time required to recover the swimming movements (longer at higher concentrations, p < 0.1). D. laevis is a tropical and subtropical species with great potential for use in toxicity tests for the detection of STXs, despite being native to and found in a great array of freshwater bodies. This is the first assay testing STX-producing and non-producing C. raciborskii strains on D. laevis, species that are both found in Brazilian ecosystems.

Effects of Cylindrospermopsis raciborskii strains (Woloszynska, 1912) Senayya & Subba Raju on the mobility of Daphnia laevis (Cladocera, Daphniidae) / Efeitos de cepas de Cylindrospermopsis raciborskii (Woloszynska, 1912), Senayya & Subba Raju na mobilidade de Daphnia laevis (Cladocera, Daphniidae)

Cylindrospermopsis raciborskii is a cyanobacterium distributed worldwide that is known to produce cyanotoxins. Some of the Brazilian strains can produce saxitoxins (STXs), which are classified as neurotoxins and can paralyze cladocerans .Daphnia laevis is a cladoceran with a wide distribution in the Americas and has been studied as a possible test-organism in toxicity bioassays. The present work tested the acute effect on D laevis mobility when fed a saxitoxin-producing (STX and neoSTX) C. raciborskii strain, CYRF-01, and compared the results with the effects of a non-toxic strain (NPCS-1). Neonates (6-24 hours after birth) were exposed to concentrations of C. raciborskii varying from 102 to 106 cells·mL–1 of each strain for up to three hours. The cladocerans were then transferred to a medium without toxic filaments for 24 hours. Only the organisms exposed to the STX-producing strain showed signs of the immobilization of swimming movements, confirming the effects of the toxins. There was a linear correlation between the time required to induce stopping the swimming movement, with a shorter time to needed to induce immobilization at a higher the concentration; this correlation was inverse to the time required to recover the swimming movements (longer at higher concentrations, p < 0.1). D. laevis is a tropical and subtropical species with great potential for use in toxicity tests for the detection of STXs, despite being native to and found in a great array of freshwater bodies. This is the first assay testing STX-producing and non-producing C. raciborskii strains on D. laevis, species that are both found in Brazilian ecosystems.

Cylindrospermopsis raciborskii é uma espécie de cianobactéria, difundida mundialmente, conhecida como produtora de cianotoxinas. Algumas linhagens brasileiras são conhecidas como produtoras de saxitoxinas (STXs), as quais são classificadas como neurotoxinas e podem induzir a paralização dos movimentos natatórios em cladóceros. Daphnia laevis é um cladócero de ampla distribuição nas Américas e tem sido estudado como um organismo teste para uso em ensaios de toxicidade. Neste sentido, o presente trabalho avaliou os efeitos agudos de C. raciborskii, de uma cepa produtora de STXs (STX e neoSTX) - CYRF-01 e outra não produtora de cianotoxinas (NPCS-1) sobre a mobilidade de D. laevis. Para tanto, neonatas entre 6-24 horas de idade foram submetidas a concentrações de C. raciborskii variando de 102 a 106 céls.mL–1 de ambas as cepas, por um período de 3 horas, e transferidos posteriormente para um meio isento de filamentos tóxicos por 24horas. Apenas os organismos expostos à cepa produtora de STXs apresentaram paralisia dos movimentos natatórios, confirmando o efeito da cianotoxina. Houve uma correlação linear entre o tempo de paralização e as concentrações, isto é, nas concentrações mais altas, os organismos paralisaram num curto período de tempo, e conseqüentemente, levaram mais tempo para se recuperar (p < 0,1). D.laevis é uma espécie de região tropical e subtropical com potencial uso em bioensaios para detecção de STXs. Trata-se dos primeiros relatos acerca da resposta de D.laevis quando submetida às cepas de C. raciborskii produtora e não produtora de STXs, ambas isoladas de ecossistemas brasileiros.

Comparative genomics of Cylindrospermopsis raciborskii strains with differential toxicities.

BACKGROUND: Cylindrospermopsis raciborskii is an invasive filamentous freshwater cyanobacterium, some strains of which produce toxins. Sporadic toxicity may be the result of gene deletion events, the horizontal transfer of toxin biosynthesis gene clusters, or other genomic variables, yet the evolutionary drivers for cyanotoxin production remain a mystery. Through examining the genomes of toxic and non-toxic strains of C. raciborskii, we hoped to gain a better understanding of the degree of similarity between these strains of common geographical origin, and what the primary differences between these strains might be. Additionally, we hoped to ascertain why some cyanobacteria possess the cylindrospermopsin biosynthesis (cyr) gene cluster and produce toxin, while others do not. It has been hypothesised that toxicity or lack thereof might confer a selective advantage to cyanobacteria under certain environmental conditions. RESULTS: In order to examine the fundamental differences between toxic and non-toxic C. raciborskii strains, we sequenced the genomes of two closely related isolates, CS-506 (CYN+) and CS-509 (CYN-) sourced from different lakes in tropical Queensland, Australia. These genomes were then compared to a third (reference) genome from C. raciborskii CS-505 (CYN+). Genome sizes were similar across all three strains and their G + C contents were almost identical. At least 2,767 genes were shared among all three strains, including the taxonomically important rpoc1, ssuRNA, lsuRNA, cpcA, cpcB, nifB and nifH, which exhibited 99.8-100% nucleotide identity. Strains CS-506 and CS-509 contained at least 176 and 101 strain-specific (or non-homologous) genes, respectively, most of which were associated with DNA repair and modification, nutrient uptake and transport, or adaptive measures such as osmoregulation. However, the only significant genetic difference observed between the two strains was the presence or absence of the cylindrospermopsin biosynthesis gene cluster. Interestingly, we also identified a cryptic secondary metabolite gene cluster in strain CS-509 (CYN-) and a second cryptic cluster common to CS-509 and the reference strain, CS-505 (CYN+). CONCLUSIONS: Our results confirm that the most important factor contributing to toxicity in C. raciborskii is the presence or absence of the cyr gene cluster. We did not identify any other distally encoded genes or gene clusters that correlate with CYN production. The fact that the additional genomic differences between toxic and non-toxic strains were primarily associated with stress and adaptation genes suggests that CYN production may be linked to these physiological processes.

Cylindrospermopsis raciborskii (Cyanobacteria) exudates: chemical characterization and complexation capacity for Cu, Zn, Cd and Pb.

Cylindrospermopsis raciborskii is a cosmopolitan and potentially toxic planktonic Cyanobacteria that produces and exudes copious amounts of dissolved organic materials. This organism dominates the eutrophic reservoir Barra Bonita (Brazil), where it normally blooms throughout the year. This investigation focused on the characterization of such exudates analyzing their capacity to complex copper, zinc, lead and cadmium through the determination of ligand concentration (CL) and conditional stability constant (logK'ML), as well as elemental composition (C, H, N and S), the content of carbohydrates, proteins, lipids and dissolved organic carbon (DOC). The dissolved organic material was fractionated into 3 molecular weights (>30 kDa; 30-10 kDa; 10-3 kDa) and each fraction was analyzed. The results showed that in the >30 kDa and 30-10 kDa fractions carbohydrates dominate over proteins and lipids. Different CL and logK'ML were obtained for the different molecular weight fractions of the excreted organic materials, suggesting high diversity of ligands. In the >30 kDa, there were more complexing sites (CL) for Cu, but higher affinity (K') for Zn. In the 30-10 kDa fraction, the higher CL was for Cd, but the greatest affinities were for Cu and Zn. In the 10-3 kDa fraction, higher CL was obtained for Cd and Zn, while Cu and Cd had the highest strengths of association. In the environment, such diversity of ligands and strengths of association can result in a displacement of metals weakly bound to the EOM, and increase metal buffering capacity of the environment, supporting higher metal inputs before toxic effects are detected in the biota.

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening.

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

In silico analysis of putative paralytic shellfish poisoning toxins export proteins in cyanobacteria.

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na(+) channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H(+)) or sodium (Na(+)) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na(+) and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na(+) recognition domain was conserved in both SxtF/M, indicating that Na(+) can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na(+) incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na(+) in toxin export, as well as the motifs L(398)XGLQD(403) (SxtM) and L(390)VGLRD(395) (SxtF) in toxin recognition.