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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.

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0-25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 μg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day−1), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process.

Background While transcriptomics have become a valuable tool for linking physiology and ecology in aquatic microbes, the temporal dynamics of global transcriptomic patterns in Microcystis have rarely been assessed. Furthermore, while many microbial studies have explored expression of nutrient transporter genes, few studies have concurrently measured nutrient assimilation rates. Here, we considered how the global transcriptomic patterns and physiology of the cyanobacterium, Microcystis aeruginosa , changed daily as cells were grown from replete to deficient nitrogen (N) conditions and then back to replete conditions. Results During N deprivation, Microcystis downregulated genes involved in photosynthesis and respiration, carbon acquisition, lipid metabolism, and amino acid biosynthesis while upregulating genes involved in N acquisition and transport. With increasing N stress, both the strength of expression and number of genes being differentially expressed increased, until N was restored at which point these patterns reversed. Uptake of 15 N-labeled nitrate, ammonium and urea reflected differential expression of genes encoding transporters for these nutrients, with Microcystis appearing to preferentially increase transcription of ammonium and urea transporters and uptake of these compounds during N deprivation. Nitrate uptake and nitrate transporter expression were correlated for one set of transporters but not another, indicating these were high and low affinity nitrate transporters, respectively. Concentrations of microcystin per cell decreased during N deprivation and increased upon N restoration. However, the transcript abundance of genes involved in the synthesis of this compound was complex, as microcystin synthetase genes involved in peptide synthesis were downregulated under N deprivation while genes involved in tailoring and transport were upregulated, suggesting modification of the microcystin molecule under N stress as well as potential alternative functions for these genes and/or this toxin. Conclusions Collectively, this study highlights the complex choreography of gene expression, cell physiology, and toxin synthesis that dynamic N levels can elicit in this ecologically important cyanobacterium. Differing expression patterns of genes within the microcystin synthetase operon in response to changing N levels revealed the potential limitations drawing conclusions based on only one gene in this operon.

In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), N-propargyloxy-carbonyl-L-lysine (Prop-Lys), or O-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in Microcystis aeruginosa and AP biosynthesis in Planktothrix agardhii. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging.

The genus Microcystis forms dense cyanobacterial harmful algal blooms (cyanoHABs) and can produce the toxin microcystin, which has been responsible for drinking water crises around the world. While microcystins are of great concern, Microcystis also produces an abundance of other secondary metabolites that may be of interest due to their potential for toxicity, ecological importance, or pharmaceutical applications. In this study, we combine genomic and metabolomic approaches to study the genes responsible for the biosynthesis of secondary metabolites as well as the chemical diversity of produced metabolites in Microcystis strains from the Western Lake Erie Culture Collection. This unique collection comprises Microcystis strains that were directly isolated from western Lake Erie, which experiences substantial cyanoHAB events annually and has had negative impacts on drinking water, tourism, and industry.

The interactive effects of binary antibiotic mixtures of spiramycin (SP) and ampicillin (AMP) on Microcystis aeruginosa (MA) in terms of growth as well as microcystin production and extracellular release were investigated through the response surface methodology (RSM). SP with higher 50 and 5% effective concentrations in MA growth was more toxic to MA than AMP. RSM model for toxic unit approach suggested that the combined toxicity of SP and AMP varied from synergism to antagonism with SP/AMP mixture ratio decreasing from reversed equitoxic ratio (5:1) to equitoxic ratio (1:5). Deviations from the prediction of concentration addition (CA) and independent action (IA) model further indicated that combined toxicity of target antibiotics mixed in equivalent ratio (1:1) varied from synergism to antagonism with increasing total dose of SP and AMP. With the increase of SP/AMP mixture ratio, combined effect of mixed antibiotics on MA growth changed from stimulation to inhibition due to the variation of the combined toxicity and the increasing proportion of higher toxic component (SP) in the mixture. The mixture of target antibiotics at their environmentally relevant concentrations with increased total dose and SP/AMP mixture ratio stimulated intracellular microcystin synthesis and facilitated MA cell lysis, thus leading to the increase of microcystin productivity and extracellular release.

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.

Elevated pCO2 may promote phytoplankton growth, and potentially alleviate carbon limitation during dense blooms. Under nitrogen-limited conditions, elevated pCO2 may furthermore alter the phytoplankton carbon–nitrogen (C:N) balance and thereby the synthesis of secondary metabolites, such as cyanobacterial toxins. A common group of these toxins are the microcystins, with variants that differ not only in C:N stoichiometry, but also in toxicity. Here, we hypothesized that elevated pCO2 will increase the cellular C:N ratios of cyanobacteria, thereby promoting the more toxic microcystin variants with higher C:N ratios. To test this hypothesis, we performed chemostat experiments under nitrogen-limited conditions, exposing three Microcystis aeruginosa strains to two pCO2 treatments: 400 and 1200 μatm. Biomass, cellular C:N ratios and total microcystin contents at steady state remained largely unaltered in all three strains. Across strains and treatments, however, cellular microcystin content decreased with increasing cellular C:N ratios, suggesting a general stoichiometric regulation. Furthermore, as predicted, microcystin variants with higher C:N ratios generally increased with elevated pCO2, while the variant with a low C:N ratio decreased. Thus, elevated pCO2 under nitrogen-limited conditions may shift the cellular microcystin composition towards the more toxic variants. Such CO2-driven changes may have consequences for the toxicity of Microcystis blooms. Elevated pCO2 under nitrogen-limited conditions may shift the cellular microcystin composition towards the more toxic variants. Such CO2-driven changes may have consequences for the toxicity of Microcystis blooms. Graphical Abstract Figure. Elevated pCO2 under nitrogen-limited conditions may shift the cellular microcystin composition towards the more toxic variants. Such CO2-driven changes may have consequences for the toxicity of Microcystis blooms.

The global usage of veterinary antibiotics is significant. Antibiotics can be released into aquatic environments and elicit toxic effects on non-target organisms. In this study, the growth characteristics and toxin release of the cyanobacterium Microcystis aeruginosa (M. aeruginosa) were examined to investigate the physiological effects of tetracycline antibiotics on aquatic life. Results showed that the degree of toxicities of the following target antibiotics was TC (tetracycline hydrochloride) > CTC (chlortetracycline hydrochloride) > OTC (oxytetracycline hydrochloride) in terms of growth parameters, EC10 (0.63, 1.86, and 3.02 mg/L, respectively), and EC20 (1.58, 4.09, and 4.86 mg/L, respectively) values. These antibiotics inhibited the production of microcystin-LR (MC-LR) to varying degrees. CTC interfered M. aeruginosa cells and decreased their ability to release MC-LR, but this antibiotic stimulated the ability of these cells to synthesize MC-LR at 2 and 5 mg/L. OTC elicited a relatively weaker toxicity than CTC did and reduced MC-LR release. TC was the most toxic among the three antibiotics, and this antibiotic simultaneously reduced intracellular and extracellular MC-LR equivalents. Our results helped elucidate the effects of tetracycline antibiotics on M. aeruginosa, which is essential for environmental evaluation and protection. Our results are also helpful for guiding the application of veterinary antibiotics in agricultural settings.