Whole-Genome Sequence of Toxic Freshwater Cyanobacterium Chrysosporum ovalisporum Strain UAM-MAO.

Here, we report the complete nucleotide sequence of Chrysosporum ovalisporum UAM-MAO, a filamentous, cylindrospermopsin-producing cyanobacterium involved in bloom forming in freshwater systems worldwide. It was isolated from an artificial pond in Madrid, Spain. The genome sequence contains 336 contigs, consisting of 7,478,035 bp and 2,851 putative protein-coding genes.

The first detection of potentially toxic Microcystis strains in two Middle Atlas Mountains natural lakes (Morocco).

Aguelmam Azizgza (LAZ) and Dayet Afourgah (DAF) are two Moroccan natural lakes located in a humid hydrographic basin of the Middle Atlas Mountains. Both are considered important reservoirs of plant and animal biodiversity. In addition, they are extensively used for recreational and fishing activities and as a water source for irrigation of agricultural crops. Recurrent cyanobacteria scum episodes in the two water bodies have been reported, Microcystis being the main genus in the scums. Here, we report on the toxic potential of three Microcystis aeruginosa strains isolated from those lakes: Mic LAZ and Mic B7 from LAZ and Mic DAF isolated from DAF. The toxic potential was checked by their microcystin (MC) content and the presence of mcy genes involved in MC synthesis. The identification and quantification of MC variants were performed by high-performance liquid chromatography-photo-diode array. The detection of mcy genes was achieved by whole-cell multiplex PCR that allowed the simultaneous amplification of DNA sequences corresponding to specific mcy regions. MC content of cultured cells, as MC-LR equivalents per gram cell biomass, was slightly higher in Mic LAZ (ca. 860) than in Mic B7 (ca. 700) and Mic DAF (ca. 690). Four MC variants were identified in the three isolates: MC-WR, MC-RR, MC-DM-WR, and MC-YR. The presence of toxic Microcystis strains in the two studied lakes may be regarded as an environmental and health hazard, especially during periods of bloom proliferation. It would be recommended the use of two complementary techniques, as those utilized herein (HPLC and mcy detection) to alert on highly probable toxicity of such lakes.

First evidence of accumulation in cyanobacteria of guanidinoacetate, a precursor of the toxin cylindrospermopsin.

Guanidinoacetate (GAA) is one of the most extensively studied toxic guanidine compounds. Changes in GAA can affect the nervous system and induce hyperhomocysteinemia, representing a risk factor for cardiovascular diseases. In cyanobacteria, GAA is thought to be an intermediate in the synthesis of the toxin cylindrospermopsin (CYN), one of the most common known cyanotoxins that affects multiple organs and functions in animals and plants. In spite of the evidence supporting GAA toxicity and its role in CYN synthesis, no data have been reported on the accumulation of GAA in any cyanobacterium. We have analyzed and compared the content of GAA in cultures of diverse cyanobacteria types, both cylindrospermopsin producing (CYN(+)) and not producing (CYN(-)). The results obtained show that GAA accumulates in the majority of the strains tested, although the highest content was found in one of the CYN(+) strain, Aphanizomenon ovalisporum UAM-MAO. In this strain, both GAA and CYN can be located within and out the cells. In conclusion, GAA appears to be a general cyanobacterial metabolite that due to its proven toxic should be considered when studying and managing cyanobacteria toxicity.

Characterization of Aphanizomenon ovalisporum amidinotransferase involved in cylindrospermopsin synthesis.

An increasing abundance of Aphanizomenon ovalisporum in water bodies from diverse world regions has been reported in the last few years, with the majority of the isolated strains producing the toxin cylindrospermopsin (CYN), leading to a rise in ecological and health risks. The understanding of CYN synthesis is crucial in the control of CYN production. An amidinotransferase (AMDT) seems to be the first enzyme involved in the synthesis of CYN. In this study, we have cloned and overexpressed the aoaA gene from the constitutive CYN producer A. ovalisporum UAM-MAO. The recombinant purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT. It shows an optimal activity between 32 and 37°C, at pH from 8 to 9. The activity exhibits a mixed (ping-pong/sequential) kinetic mechanism, and is inhibited by the reaction product guanidine acetate (GAA) in a noncompetitive manner. Mg(2+) stimulates AoaA activity while Co(2+) and Mn(2+) inhibit it. AoaA conserves the critical residues of the catalytic site and substrate specificity of AMDTs, as the previously reported AMDT from Cylindrospermopsis raciborskii Cyr. Both proteins can be included in a new group of prokaryotic AMDTs involved in CYN production.

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions.

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.

Global warming and hepatotoxin production by cyanobacteria: what can we learn from experiments?

Global temperature is expected to rise throughout this century, and blooms of cyanobacteria in lakes and estuaries are predicted to increase with the current level of global warming. The potential environmental, economic and sanitation repercussions of these blooms have attracted considerable attention among the world's scientific communities, water management agencies and general public. Of particular concern is the worldwide occurrence of hepatotoxic cyanobacteria posing a serious threat to global public health. Here, we highlight plausible effects of global warming on physiological and molecular changes in these cyanobacteria and resulting effects on hepatotoxin production. We also emphasize the importance of understanding the natural biological function(s) of hepatotoxins, various mechanisms governing their synthesis, and climate-driven changes in food-web interactions, if we are to predict consequences of the current and projected levels of global warming for production and accumulation of hepatotoxins in aquatic ecosystems.

Identification of microcystins from three collection strains of Microcystis aeruginosa.

Microcystins (MCs) are toxic cyclic heptapeptides produced by various cyanobacteria genera, especially Microcystis. We identified 10 out of 12 MCs produced by three Microcystis aeruginosa strains from cyanobacteria collections, UTEX 2666, UTEX 2670 and UAM 1303, by using two analytical methods: Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) and HPLC Photodiode Array Detector coupled to a hybrid Quadrupole Time of Flight Mass Spectrometry (HPLC-PDA-QTOF/MS). MALDI-TOF/MS failed to detect non-polar MCs, such as MC-LY and MC-LW. HPLC-QTOF/MS permitted the accurate identification of most MCs present in methanolic extracts. Besides, three new MCs, namely: [D-Glu(OCH3)6, D-Asp3] MC-LAba, MC-YL and MC-YM were detected by HPLC-QTOF/MS.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii.

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa.

Several physiological parameters related to oxidative stress, which is a characteristic of plants exposed to toxic metals, were studied in 3-week-old alfalfa plants treated with cadmium (Cd) or mercury (Hg) at doses of 0, 3, 10 and 30 microM for 7d. The concentrations of biothiols, glutathione (GSH), homoglutathione (hGSH) and phytochelatins (PCs) increased dramatically in metals-treated plants, in particular in the presence of Cd. This was accompanied by a remarkable up-regulation of gamma-glutamyl cysteine synthetase gene, probably in response to the higher demand for GSH|hGSH needed for PC synthesis. The presence of metals enhanced lipid peroxidation in shoots, while chlorophyll content declined in a concentration dependent manner. Ascorbate peroxidase (APX) activity increased moderately in roots of Cd-exposed plants, and a new basic root peroxidase isoform was found in both Cd- and Hg-treated plants. Glutathione reductase (GR) activity was enhanced in shoots of plants exposed to Cd and Hg. However, this enzymatic activity showed a metal dependent response in roots, and was enhanced in Cd-treated plants but was severely inhibited in roots of plants treated with Hg. Inhibition of GR by Hg was confirmed in vitro by incubating a commercially available GR and control shoot extracts with several doses of Hg and Cd. Ascorbate concentrations were elevated with treatments of 3 microM Hg, 10 microM Cd and 30 microM Cd, indicating that this compound is necessary for redox cellular homeostasis. The different responses observed with Cd and Hg treatments might be the basis for specific stress bioindicators.

Physiological changes in Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars, caused by irrigation with water contaminated with microcystins: a laboratory experimental approach.

The aim of the present study was to investigate the effect of exposure to a microcystin (MC)-containing extract from a cyanobacteria bloom on growth, development, mineral nutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisum sativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/or mass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth and development was tested along 30 exposure days. After this period, MC-extract caused a clear reduction in plant growth and productivity, as well as deleterious effects on development and Photosystem II activity, measured by Fv/Fm fluorescence. However, the chlorophyll (a + b) content hardly varied, and the accumulation of Na+, K+, Ca2+, P and N was enhanced. All the effects observed were plant species, MC concentration, and exposure-time dependent. Relative accumulation of each MC variant greatly varied among plant species and plant organ. The data obtained supports the idea that the use of surface water containing MCs for crop irrigation can affect both plant yield and quality, and secondly, that MC accumulation in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings.

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.

Cellular damage induced by cadmium and mercury in Medicago sativa.

Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the kinetics of diverse stress indexes. In the so-called beaker-size hydroponic system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress took place and increased over time, a linear response being observed with Cd but not with Hg. To improve the sensitivity of the stress assays used, a micro-assay system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of metals, quantified as growth inhibition, was observed well before there was any change in the non-protein thiol tissue concentration. When measured with conventional techniques, oxidative stress indexes did not show significant variation. To trace early and small plant responses to Cd and Hg, a microscopic analysis with novel fluorescent dyes, which had not yet been exploited to any significant extent for use in plants, was conducted. These fluorescent probes, which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals to be visualized in the roots of the alfalfa seedlings, were: (i) 2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium iodide that marks nuclei of dead cells. Oxidative stress and cell death increased after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased acutely. This diminution might be the result of direct interaction of GSH/hGSH with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore, both Cd and Hg not only compromised severely the cellular redox homeostasis, but also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine, a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient to promote cell death, and that other phytotoxic mechanisms might be involved.

Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions.

Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing.