Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

Proteomic mechanisms for the stimulatory effects of antibiotics on Microcystis aeruginosa during hydrogen peroxide treatment.

Application of low dosage of H2O2 at early stage of cyanobacterial life cycle is a promising route for cyanobacterial bloom mitigation, which could minimize adverse effects on non-target organisms. Besides, influence of co-existing contaminants on cyanobacterial bloom mitigation under combined pollution conditions remains unclear. This study assessed the influence of a mixture of four frequently detected antibiotics (tetracycline, sulfamethoxazole, ciprofloxacin and amoxicillin) during H2O2 treatment of Microcystis aeruginosa at early growth stage. H2O2 significantly (p < 0.05) inhibited growth rate, chlorophyll a content, Fv/Fm and rETRmax in a dose-dependent manner at low doses of 0.25-1 mg L-1, through downregulating proteins involved in cell division, cellular component organization, gene expression and photosynthesis. Although H2O2 increased microcystin content in each cyanobacterial cell through the upregulation of microcystin synthetases (mcyC and mcyF), total microcystin concentration in H2O2 treated groups was significantly (p < 0.05) reduced due to the decrease of cell density. Existence of 80 and 200 ng L-1 mixed antibiotics during H2O2 treatment facilitated the scavenging of ROS by antioxidant enzymes and significantly (p < 0.05) stimulated growth, photosynthesis, microcystin synthesis and microcystin release in H2O2 treated cells, through the upregulation of proteins involved in photosynthesis, oxidation-reduction process, biosynthesis, gene expression and transport. Mixed antibiotics increased the hazard of M. aeruginosa during H2O2 treatment, through the stimulation of microcystin synthesis and release at the proteomic level. Each target antibiotic should be controlled below 5 ng L-1 before the application of H2O2 for eliminating the interference of antibiotics on cyanobacterial bloom mitigation.

Role of illumination intensity in microcystin development using Microcystis aeruginosa as the model algae.

Microcystis aeruginosa (M. aeruginosa) is one of the most common genera of cyanobacteria in algal blooms. In the present work, the impact of the illumination intensity on the growth of M. aeruginosa has been studied and a grinding method for the extraction of intracellular microcystins (MCs) was developed. The variations of algal density, pH, total phosphorus (TP), and total nitrogen (TN) have been investigated during MCs' culturing period. Results showed that the extraction efficiency of MC-YR by the grinding method was 275% higher than the sonication method, and the extraction efficiencies of MC-RR and MC-LR by the grinding method were similar to the sonication method. The optimal illumination intensity for M. aeruginosa was found to be 19-38 µmol m-2 s-1 with suitable pH range of 7.5-10.5. Active release of extracellular MCs was not significantly observed when illumination intensities were ≤ 38 µmol m-2 s-1. Furthermore, the intracellular MC yields under different illumination intensities were found to be a relatively stable level. However, excess illumination intensity (≥ 47 µmol m-2 s-1) led to the lysis of algal cell and increased the concentrations of extracellular MCs, with MC-RR as the dominant compound. The calculated intracellular/extracellular MCs ratios for MC-RR, MC-LR, and MC-YR were 2.38 (N = 100, SD = 2.44), 2.68 (N = 64, SD = 3.48), and 1.25 (N = 30, SD = 1.64), respectively. Strong illumination intensity and cell lysis were found to be the two major factors influencing the release of extracellular MCs.

Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes.

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics.

Could the presence of larger fractions of non-cyanobacterial species be used as a predictor of microcystin production under variable nutrient regimes?

The occurrence of cyanobacteria and microcystin is highly dynamic in natural environments and poses one of the biggest challenges to water resource management. While a number of drivers are known to be responsible for the occurrence of cyanobacterial blooms, the drivers of microcystin production are not adequately known. This study aims to quantify the effects of the changes in the structures of phytoplankton and cyanobacterial communities on the dynamics of microcystin production under highly variable nutrient concentration. In our study, nutrient variability could explain 64% of the variability in microcystin production. When changes in the fractions of non-cyanobacteria versus cyanobacteria genera were additionally included, 80% of the variability in microcystin production could be explained; under high nutrient concentrations, non-cyanobacterial phytoplankton groups were dominant over cyanobacteria and cyanobacteria produced more toxins. In contrast, changes in the cyanobacterial community structures could only explain a further 4% of the dynamics of microcystin production. As such, the dominance of non-cyanobacterial groups appears to be a useful factor to explain microcystin occurrence in addition to traditionally used factors such as absolute cyanobacterial cell numbers, especially when the nutrient regime is taken into account. This information could help to further refine the risk assessment frameworks which are currently used to manage the risk posed by cyanobacterial blooms.

Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Controlling toxic cyanobacteria: effects of dredging and phosphorus-binding clay on cyanobacteria and microcystins.

Sediment dredging and Phoslock(®) addition were applied individually and in combination in an enclosure experiment in a Dutch hypertrophic urban pond. These measures were applied to control eutrophication and reduce the risk of exposure to cyanobacterial toxins. Over the 58 days course of the experiment, cyanobacteria (predominantly Microcystis aeruginosa) gradually decreased until they dropped below the level of detection in the combined treated enclosures, they were reduced in dredged enclosures, but remained flourishing in controls and Phoslock(®) treated enclosures. Cyanobacteria were, however, less abundant in the enclosures (medians chlorophyll-a 30-87 µg l(-1)) than in the pond (median chlorophyll-a 162 µg l(-1)), where also a thick surface scum covered one-third of the pond for many weeks. Soluble reactive phosphorus (SRP), total phosphorus and total nitrogen concentrations were significantly lower in the combined dredged and Phoslock(®) treated enclosures than in controls. Median SRP concentrations were 24 µg P l(-1) in the combined treatment, 58 µg P l(-1) in dredged enclosures, and 90 µg P l(-1) in controls and 95 µg P l(-1) in Phoslock(®) treated enclosures. Hence, the combined treatment was most effective in decreasing SRP and TP, and in lowering cyanobacterial biomass. Microcystin (MC) concentrations were analyzed by LC-MS/MS. MC concentrations and cyanobacterial biomass were positively correlated in all treatments. Mean MC concentrations in controls (71 µg l(-1)), Phoslock(®) treated enclosures (37 µg l(-1)) and dredged enclosures (25 µg l(-1)) exceeded the provisional guideline of 20 µg l(-1), whereas mean MC concentrations were 13 µg l(-1) in the combined treated enclosures. All samples contained the MC variants dmMC-RR, MC-RR, MC-YR, dmMC-LR and MC-LR; traces of MC-LY and nodularin were detected in few samples. The different treatments did not change the relative contribution of the variants to the MC pool; MC profiles in all treatments and the pond showed dominance of MC-RR followed by MC-LR. In the surface scum of the pond, total MC concentration was extremely high (64000 µg l(-1) or 1300 µg g(-1) DW), which poses a serious health hazard to children playing on the banks of the pond. Based on our results and pond characteristics, we propose combined sediment dredging and Phoslock(®) addition, fish removal and strong reduction of duck feeding by the neighborhood as most promising measures controlling cyanobacterial hazards in this pond.

Dynamics of microcystin production and quantification of potentially toxigenic Microcystis sp. using real-time PCR.

Cyanobacterial blooms in eutrophied water body are generally composed of various genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is a need for quantification of potent toxin producing strains. The present study aimed at identifying microcystin variants and its producer strains in Durgakund pond, Varanasi, India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using real-time PCR and LC-MS. Increase in microcystin concentrations was correlated with increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and carotenoids). Also, selected environmental factors (water temperature, light irradiance, rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR) were also assessed in samples during May 2010 to April 2011 to establish the possible correlation among these parameters. Nutrients favored cyanobacterial bloom but it could not be correlated with the levels of microcystin variants and seemed to be geographically specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to 14%. Such studies paved the way to identify and quantify the most potent microcystin producer in the tropical aquatic body.

[Effect of phosphorus on the production of microcystin].

Effect of phosphorus on the production of microcystin was researched. The effects of soluble reactive phosphorus (SRP) on the growth of cells and on the production of Microcystin were studied. In addition, the efficiency of four different phosphorus compounds was researched. The results showed that microcystin increased with the increase of SRP, and c(TP) = 0.55 mg x L(-1) was the best growth concentration. When c(TP) < or = 0.55 mg x L(-1), the microcystin production increased with the increase of phosphorus concentration and was the lowest without phosphorus. Moreover, when c(TP) > 0.55 mg x L(-1), the microcystin was restrained by the content of phosphorus. At the same time, the effects of three inorganic substance of different phosphorus forms (K3PO4, K2HPO4, and KH2PO4) were no significant difference, but their effects on the production of microcystis were larger than organic phosphorus of sodium beta-glycerophosphate (GP).

[Effect of nitrogen and phosphorous on the production of microcystin under laboratory conditions].

OBJECTIVE: To explore the effect of phosphorus, nitrogen on the production of microcystin under specific laboratory condition. METHODS: The microcystis were ampliatively cultured for three times in N and P free culture. Then the microcysitis were inoculated in the culture at the concentrations of 0, 0.5, 1.0, 5.0 and 10.0 mg/L P for 20 days. The microcystis were inoculated in the BG-11 cultures at the concentrations of 0.05 mg/L and 5.0 mg/L P, NaNO3 were added in the culture according to the mol ratios of N/P were 5:1, 10:1, 20:1, 50:1, 100:1 and were cultured for 20 days. The changes of the count of the microcystis were observed. The microcystis cell were breaked at the 8th, 12th, 16th and 20th days after the beginning of culturing, the the microcystin were extracted and detected by HPLC. RESULTS: When the concentrations of phosphorus were lower than 5.0 mg/L, The productions of microcystin increased with the phosphorus concentrations. But when the concentrations of phosphorus were 10.0 mg/L, the productions of microcystin significantly decreased. In the culture at the concentrations of 0.05 mg/L P, the microcystin concentration per cell (MCYST fg/cell) and the microcystin concentrations per milliliter (MCYST microg/ml culture) presented the greatest value when the N/P ratio was 50:1. But, In the culture at the concentrations of 5.0 mg/L P, the microcystin concentrations per cell (MCYST fg/cell) and the microcystin concentrations per milliliter (MCYST microg/ml culture) presented the greatest value when the N/P ratio was 20:1. CONCLUSION: P concentrations significantly incluence the production of microcystin. The P concentrations in water should be controlled through different way to control the production of microcystins.

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta).

Environmental factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA(3) on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA(3) treatment. GA(3) increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA(3) was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-linked immunosorbent assay (ELISA) increased in GA(3) treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA(3) increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin.