Identification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339.

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

Transcriptome analysis of changes in M. aeruginosa growth and microcystin production under low concentrations of ethinyl estradiol.

Ethinyl estradiol (EE2) is a synthetic environmental estrogen with considerable estrogenic activity. It has been found to consequently pose a significant threat to the aquatic ecosystem. Harmful algal blooms are a major aquatic ecological issue. However, the relationship between EE2 and cyanobacterial bloom is mainly unknown. In this study, the physiological and molecular responses of Microcystis aeruginosa to EE2 exposure were investigated. A low level of EE2 (0.02 µg/L) significantly enhanced the growth of algal cells (P < 0.05), whereas higher concentrations of EE2 (0.2-200 µg/L) inhibited it. EE2 at doses ranging from 0.02 to 200 µg/L promoted the production of microcystins (MCs), with genes mcyABD playing a key role in the regulation of MC synthesis. The alterations of chlorophyll-a, carotenoid, and phycocyanin contents caused by EE2 showed the same trend as cell growth. At the molecular level, 200 µg/L EE2 significantly down-regulated genes in photosynthetic pigment synthesis, light harvesting, electron transfer, NADPH, and ATP generation. High concentrations of EE2 caused oxidative damage to algal cells on the 4th d. After 12d exposure, although there was no significant change in superoxide dismutase (SOD) content and no damage observed in membrane lipids, genes related to SOD and glutathione were changed. In addition, due to the down-regulation of pckA, PK, gltA, nrtA, pstS, etc., carbon fixation, glycolysis, TCA cycle, nitrogen and phosphorus metabolism were hindered by EE2 (200 µg/L). Gene fabG in fatty acid biosynthesis was significantly up-regulated, promoting energy storage in cells. These findings provide important clues to elucidate the effects and mechanisms of cyanobacterial blooms triggered by EE2 and help to effectively prevent and control cyanobacterial blooms.

Application of a Mechanistic Model for the Prediction of Microcystin Production by Microcystis in Lab Cultures and Tropical Lake.

Microcystin is an algal toxin that is commonly found in eutrophic freshwaters throughout the world. Many studies have been conducted to elucidate the factors affecting its production, but few studies have attempted mechanistic models of its production to aid water managers in predicting its occurrence. Here, a mechanistic model was developed based on microcystin production by Microcystis spp. under laboratory culture and ambient field conditions. The model was built on STELLA, a dynamic modelling software, and is based on constitutive cell quota that varies with nitrogen, phosphorus, and temperature. In addition to these factors, varying the decay rate of microcystin according to its proportion in the intracellular and extracellular phase was important for the model's performance. With all these effects, the model predicted most of the observations with a model efficiency that was >0.72 and >0.45 for the lab and field conditions respectively. However, some large discrepancies were observed. These may have arisen from the non-constitutive microcystin production that appear to have a precondition of nitrogen abundance. Another reason for the large root mean square error is that cell quota is affected by factors differently between strains.

Microcystis Sp. Co-Producing Microcystin and Saxitoxin from Songkhla Lake Basin, Thailand.

The Songkhla Lake Basin (SLB) located in Southern Thailand, has been increasingly polluted by urban and industrial wastewater, while the lake water has been intensively used. Here, we aimed to investigate cyanobacteria and cyanotoxins in the SLB. Ten cyanobacteria isolates were identified as Microcystis genus based on16S rDNA analysis. All isolates harbored microcystin genes, while five of them carried saxitoxin genes. On day 15 of culturing, the specific growth rate and Chl-a content were 0.2-0.3 per day and 4 µg/mL. The total extracellular polymeric substances (EPS) content was 0.37-0.49 µg/mL. The concentration of soluble EPS (sEPS) was 2 times higher than that of bound EPS (bEPS). The protein proportion in both sEPS and bEPS was higher than the carbohydrate proportion. The average of intracellular microcystins (IMCs) was 0.47 pg/cell on day 15 of culturing, while extracellular microcystins (EMCs) were undetectable. The IMCs were dramatically produced at the exponential phase, followed by EMCs release at the late exponential phase. On day 30, the total microcystins (MCs) production reached 2.67 pg/cell. Based on liquid chromatograph-quadrupole time-of-flight mass spectrometry, three new MCs variants were proposed. This study is the first report of both decarbamoylsaxitoxin (dcSTX) and new MCs congeners synthesized by Microcystis.

Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC-MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake.

Cell density-dependent regulation of microcystin synthetase genes (mcy) expression and microcystin-LR production in Microcystis aeruginosa that mimics quorum sensing.

As the secondary metabolites of cyanobacterial harmful algal blooms (Cyano-HABs), microcystins (MCs) were generated under various environmental and cellular conditions. The understanding of the causes of MCs generation is of great interest in the field of water treatment and environmental science. In this work, we studied how Microcystis aeruginosa (FACHB-905) cell densities affect the MCs synthetase genes (mcy) expression, microcystin-LR (MC-LR) and quorum sensing molecules (Acyl-homoserine lactones (AHLs)) production. An electrochemical sensor was developed here for sensitive and quantitative detection of MC-LR that cultured at different cell densities. The results showed that mcy expression and MC-LR concentration started to increase when the cell density reached ca. 22 × 106 cells/mL, and was significantly increased with increasing cell densities. Moreover, the up-regulation of AHLs with increasing cell densities revealed that MC-LR is quorum sensing-mediated. Our results undoubtedly confirmed that MC-LR was produced in a cell density-dependent way that mimics quorum sensing, and the minimum cell density (ca. 22 × 106 cells/mL) that was required to produce MC-LR was provided and offered a reference standard for the prevention and control of MCs pollution in the actual water environment.

Mechanisms for the stimulatory effects of a five-component mixture of antibiotics in Microcystis aeruginosa at transcriptomic and proteomic levels.

Antibiotic contaminants could promote the formation of harmful cyanobacterial blooms through hormetic stimulation, but the mechanisms underlying these stimulatory effects remain unclear. This study investigated the biochemical, transcriptomic, and proteomic responses of a dominant bloom-forming cyanobacterium, Microcystis aeruginosa, to a five-component mixture of frequently detected antibiotics at current contamination levels. The growth rate of M. aeruginosa presented a U-shaped dose-response to 50-500 ng L-1 of mixed antibiotics. Alterations in the transcriptome of M. aeruginosa suggested the excitation of both photosynthesis and carbon metabolism, increasing energy generation in response to oxidative stress induced by low-dose antibiotics, and thus contributing to the significant (p < 0.05) increase in growth rate, Fv/Fm, and cell density. Comparison between transcriptomic and proteomic responses further confirmed the action mode of the mixed antibiotics. Proteins and their corresponding genes related to ROS scavenging, photosynthesis, carbon fixation, electron transport, oxidative phosphorylation, and biosynthesis, showed consistent expression tendencies in response to 200 ng L-1 of mixed antibiotics, which were credible action targets of mixed antibiotics in M. aeruginosa. Mixed antibiotics stimulated microcystin synthesis by upregulating a microcystin synthetase and its encoding gene (mcyC), which could increase the hazard of M. aeruginosa in aquatic environments.

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.

Growth, physiological responses and microcystin-production/-release dynamics of Microcystis aeruginosa exposed to various luteolin doses.

By testing time-dependent IC50 of luteolin against Microcystis growth, this study revealed 6.5 mg/L as nearly IC50 value during prolonged stress until day 14, and explored chlorophyll-a (CLA) and phycobiliproteins (PBPs) contents, antioxidant responses and microcystin (MC)-production/-release dynamics at rising luteolin doses (0.5~2-fold IC50). Growth inhibition ratio (GIR) generally rose at rising luteolin dose, while at each dose GIR firstly increased and then leveled off or dropped. In early stage, CLA, allophycocyanin (APC), phycoerythrin (PE) and glutathione (GSH) contents, and superoxide dismutase (SOD) and catalase (CAT) activities, were increasingly stimulated at rising luteolin dose to enhance energy yield and antioxidant defense, but Microcystis was damaged more severely at rising dose, due to stress-repair imbalance. Such more severe damage in early stage, coupled with stronger PBPs-inhibition in mid-late stage, at rising dose could jointly account for rising GIR at rising dose. The CAT/GSH-stimulation persisting until late stage could alleviate cell damage in late stage, which explained for why GIR no longer increased in late stage at each luteolin dose. Besides, more MCs were produced and retained in cell to exert protective roles against luteolin-stress in early stage, but intracellular MCs decreased following inhibited MC-production by prolonged stress to decrease cell protectant. Extracellular MCs detection showed that less MCs amount existed in water phase than control along luteolin-stress, implying luteolin as eco-friendly algaecide with promising potential to remove MPM blooms and MC-risks. This is the first study to reveal the effect of various luteolin doses on MC-production/release and PBP-synthesis dynamics of Microcystis during prolonged stress. The findings shed novel views in anti-algal mechanisms of luteolin, and provided direct evidence for luteolin applied as safe agent to remediate Microcystis-dominant blooms.

Effects of CeO2 Nanoparticles on Microcystis aeruginosa Growth and Microcystin Production.

The interaction between metal oxide nanoparticles and toxin-producing cyanobacteria is relatively unknown. The present work exposed Microcystis sp.7806 to different concentrations of cerium oxide nanoparticles (CeO2 NPs) (1 mg/L, 10 mg/L and 50 mg/L), and evaluated the growth, photosynthetic activity, reactive oxygen species level, and the extra-(intra-) cellular microcystin-LR (MC-LR) contents. The particle size, zeta potential and cerium ions released into the medium were analyzed. Results showed 10 mg/L NP treatment promoted algae growth but slightly inhibited the photosynthetic yield of algae, and the 50 mg/L treatment reduced algae biomass. The algal cells remarkably responded to oxidative stress at higher concentrations (10 mg/L and 50 mg/L). CeO2 NPs largely increased the intracellular MC-LR content at 50 mg/L, and significantly reduced the extracellular MC-LR content at any concentration. This demonstrates CeO2 NPs may pose an ecological risk potential during harmful algal blooms by stimulating toxin production.

Antibiotics induced alterations in cell density, photosynthesis, microcystin synthesis and proteomic expression of Microcystis aeruginosa during CuSO4 treatment.

Antibiotic contaminants have the potential to interfere with the control of cyanobacterial bloom through generating hormesis in cyanobacteria at current contamination level of ng L-1. This study investigated the influence of a mixture of four frequently detected antibiotics, amoxicillin, ciprofloxacin, sulfamethoxazole and tetracycline, during the treatment of Microcystis aeruginosa by copper sulfate (CuSO4) algaecide. CuSO4 significantly (p <  0.05) inhibited cell density, growth rate, Fv/Fm value, chlorophyll a content and microcystin production ability of M. aeruginosa in a dose-dependent manner at application doses of 0.01-0.05 mg L-1. Besides, CuSO4 inhibited oxidation-reduction process, photosynthesis and biosynthesis in M. aeruginosa at the proteomic level. Preventative application of CuSO4 to a low density (4 × 105 cells mL-1) of M. aeruginosa effectively prevented the formation of bloom at low CuSO4 doses, which is a possible route for eliminating the negative effects of CuSO4 algaecide in aquatic environments. The presence of mixed antibiotics alleviated the toxicity of CuSO4 in M. aeruginosa, through the downregulation of cation transport proteins and the upregulation of proteins related with chlorophyll a synthesis, photosynthesis, gene expression and oxidation-reduction. Mixed antibiotics also promoted microcystin synthesis in CuSO4 treated cells through the upregulation of microcystin synthetases. Mixed antibiotics significantly (p <  0.05) increased cell density, growth rate, Fv/Fm value, chlorophyll a content and microcystin production ability in CuSO4 treated cells at test concentrations of 80 and 200 ng L-1. A no-impact threshold of 20 ng L-1 for mixed antibiotics (5 ng L-1 for each antibiotic) was suggested for eliminating the interference of antibiotic contaminants on cyanobacterial bloom control.

Suppression of water-bloom cyanobacterium Microcystis aeruginosa by algaecide hydrogen peroxide maximized through programmed cell death.

The global expansion and intensification of toxic cyanobacterial blooms require effective algaecides. Algaecides should be selective, effective, fast-acting, and ideally suppress cyanotoxin production. In this study, whether both maximum growth suppression and minimal toxin production can be simultaneously achieved was tested with a selective algaecide H2O2, through its ability to induce apoptosis-like programmed cell death (AL PCD) in a common bloom species Microcystis aeruginosa. Under doses of 1-15 mg L-1, non-monotonic dose-response suppression of H2O2 on M. aeruginosa were observed, where maximal cell death and minimal microcystin production both occurred at a moderate dose of 10 mg L-1 H2O2. Maximal cell death was indeed achieved through AL PCD, as revealed by integrated biochemical, structural, physiological and transcriptional evidence; transcriptional profile suggested AL PCD was mediated by mazEF and lexA systems. Higher H2O2 doses directly led to necrosis in M. aeruginosa, while lower doses only caused recoverable stress. The integrated data showed the choice between the two modes of cell death is determined by the intracellular energy state under stress. A model was proposed for suppressing M. aeruginosa with AL PCD or necrosis. H2O2 was demonstrated to simultaneously maximize the suppression of both growth and microcystin production through triggering AL PCD.

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.

Structural Diversity, Characterization and Toxicology of Microcystins.

Hepatotoxic microcystins (MCs) are the most widespread class of cyanotoxins and the one that has most often been implicated in cyanobacterial toxicosis. One of the main challenges in studying and monitoring MCs is the great structural diversity within the class. The full chemical structure of the first MC was elucidated in the early 1980s and since then, the number of reported structural analogues has grown steadily and continues to do so, thanks largely to advances in analytical methodology. The structures of some of these analogues have been definitively elucidated after chemical isolation using a combination of techniques including nuclear magnetic resonance, amino acid analysis, and tandem mass spectrometry (MS/MS). Others have only been tentatively identified using liquid chromatography-MS/MS without chemical isolation. An understanding of the structural diversity of MCs, the genetic and environmental controls for this diversity and the impact of structure on toxicity are all essential to the ongoing study of MCs across several scientific disciplines. However, because of the diversity of MCs and the range of approaches that have been taken for characterizing them, comprehensive information on the state of knowledge in each of these areas can be challenging to gather. We have conducted an in-depth review of the literature surrounding the identification and toxicity of known MCs and present here a concise review of these topics. At present, at least 279 MCs have been reported and are tabulated here. Among these, about 20% (55 of 279) appear to be the result of chemical or biochemical transformations of MCs that can occur in the environment or during sample handling and extraction of cyanobacteria, including oxidation products, methyl esters, or post-biosynthetic metabolites. The toxicity of many MCs has also been studied using a range of different approaches and a great deal of variability can be observed between reported toxicities, even for the same congener. This review will help clarify the current state of knowledge on the structural diversity of MCs as a class and the impacts of structure on toxicity, as well as to identify gaps in knowledge that should be addressed in future research.

Algicidal characterization and mechanism of Bacillus licheniformis Sp34 against Microcystis aeruginosa in Dianchi Lake.

Microcystis aeruginosa blooms are a worldwide serious environmental problem and bloom control with bacteria is promising. In this study, a Bacillus licheniformis strain Sp34 with potent algicidal and inhibitory effects on the microcystins synthesis against fast-growing M. aeruginosa was isolated from Dianchi Lake. Sp34 killed the bloom-causing algal strain M. aeruginosa DCM4 of Dianchi Lake with an initial Chlorophyll-a concentration of 2.0 mg/L at a cell density of no less than 1.35 × 105 CFU/ml. It can also efficiently kill some other harmful algal species, such as M. wesenbergii and Phormidium sp. The algicidal activity of Sp34 relied on the release of algicidal substances, which had good heat (-20°C to 121°C) and acid-base (pH 3-11) resistance. In addition, the high algicidal activity depended on the good growth of algae indicated by the significantly positive correlations between algal growth and algicidal ratio (p < .001). The algicidal effect of Sp34 involved causing oxidative stress, lipid peroxidation, and morphological injury of algal cells, along with DNA damage and dysfunction of DNA-repair function, weakening the photosynthesis system, and inhibiting microcystin synthesis. In general, Sp34 can kill fast-growing M. aeruginosa and inhibit algal microcystin synthesis efficiently, so, it is a promising biocontrol agent to mitigate cyanobacterial blooms.

Insights into the Diversity of Secondary Metabolites of Planktothrix Using a Biphasic Approach Combining Global Genomics and Metabolomics.

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with diverse chemical structures and potent biological activities and toxicities. The chemical identification of these compounds remains a major bottleneck. Strategies that can prioritize the most prolific strains and novel compounds are of great interest. Here, we combine chemical analysis and genomics to investigate the chemodiversity of secondary metabolites based on their pattern of distribution within some cyanobacteria. Planktothrix being a cyanobacterial genus known to form blooms worldwide and to produce a broad spectrum of toxins and other bioactive compounds, we applied this combined approach on four closely related strains of Planktothrix. The chemical diversity of the metabolites produced by the four strains was evaluated using an untargeted metabolomics strategy with high-resolution LC-MS. Metabolite profiles were correlated with the potential of metabolite production identified by genomics for the different strains. Although, the Planktothrix strains present a global similarity in terms of a biosynthetic cluster gene for microcystin, aeruginosin, and prenylagaramide for example, we found remarkable strain-specific chemodiversity. Only few of the chemical features were common to the four studied strains. Additionally, the MS/MS data were analyzed using Global Natural Products Social Molecular Networking (GNPS) to identify molecular families of the same biosynthetic origin. In conclusion, we depict an efficient, integrative strategy for elucidating the chemical diversity of a given genus and link the data obtained from analytical chemistry to biosynthetic genes of cyanobacteria.

Long-term exposure to antibiotic mixtures favors microcystin synthesis and release in Microcystis aeruginosa with different morphologies.

The ecological risks of antibiotics in aquatic environments have raised great concerns worldwide, but the chronic effect of antibiotic contaminants on cyanotoxin production and release remains unclear. This study investigated the long-term combined effects of spiramycin (SP) and ampicillin (AMP) on microcystin (MC) production and release in both unicellular and colonial Microcystis aeruginosa (MA) through semi-continuous exposure test. At exposure concentration of 300 ng L-1, MA growth rates were stimulated till the end of exponential phase accompanied with the up-regulation of photosynthesis-related gene. The exponential growth phases of unicellular and colonial MA were prolonged for 2 and 4 days, respectively. The stimulation rate of growth rate and MC content in unicellular MA were significantly higher than that in colonial MA. The highest concentrations of intracellular MC (IMC) and extracellular MC (EMC) were observed in the binary mixture at equivalent SP/AMP ratio (1:1). The promotion of IMC concentration was in consistent with the stimulated expression of MC-synthesis-related gene and nitrogen-transport-related gene. The malondialdehyde content and activities of superoxide dismutase and catalase in unicellular MA were significantly higher than those in colonial MA. The EMC concentration and the antioxidant responses of both unicellular and colonial MA significantly increased with exposure time. Long-term exposure to mixture of SA and AMP at environmentally relevant concentrations would aggravate the disturbance to aquatic ecosystem balance through the stimulation of MA proliferation as well as the promotion of MC production and release.

The Biosynthesis of Rare Homo-Amino Acid Containing Variants of Microcystin by a Benthic Cyanobacterium.

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic strain Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.

Effects of titanium dioxide nanoparticles on Microcystis aeruginosa and microcystins production and release.

Due to growing production and use, release of nanoparticles (NPs) into the aquatic environment may pose a hazard to ecosystem. In this study, Microcystis aeruginosa was exposed to different concentrations (0.1, 1, 10, 50, 100 mg/L) of titanium dioxide (TiO2) NPs to assess their impact on algae. Meanwhile, the production and release of microcystins (MCs) was determined. Results showed that TiO2 NPs significantly decreased the maximal photochemical efficiency of photosystem II, and thus inhibited the photosynthetic activity of M. aeruginosa. They also increased the content of reactive oxygen species (ROS) and malondialdehyde (MDA), indicating their oxidative damage on algae. Besides, TiO2 NPs at high concentrations (50 and 100 mg/L) aggregated on the algal surface and block the light, herein inhibited algae growth (16.03%±2.50% and 54.13%±0.93%) but induced the production (25.02%±1.23% and 114.43%±2.96%) and release (20.96%±13.30% and 12.10%±8.80%) of MCs. These results indicated that high concentrations of TiO2 NPs increased MCs concentration in water system, which may be harmful to aquatic ecosystem.

Study on the cyanobacterial toxin metabolism of Microcystis aeruginosa in nitrogen-starved conditions by a stable isotope labelling method.

In this study, the biosynthesis of microcystins (MCs) was investigated after long-term nitrogen-starved conditions in cyanobacterium Microcystis aeruginosa. The results demonstrated that the algal cells were able to survive in a non-growing state with nitrogen starvation for more than one month. The physiological properties of the algal cells were studied to elucidate the mechanisms of viability under nitrogen-deprivation conditions. After the state of nitrogen chlorosis, new toxins could be resynthesized and tracked using 15N-stable isotope-labelled nitrogen. Nitrogen starvation of nutritionally replete cells resulted in a significant increase of microcystin-LY (MC-LY), thereby suggesting that MC-LY may undergo catabolism to provide nitrogen or that MC-LY may be produced to play an important role in the cell in response to nitrogen deprivation. The rank order of different types of nitrogen in algal cells assimilation was N-ammonium > N-urea > N-nitrate > N-alanine. The relationship between the production of toxin variants and various environmental conditions is an interesting issue for future research and may help improve the understanding of the ecological role of cyanobacterial toxins.