Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Harmful algal blooms in Cayuga lake, NY: From microbiome analysis to eDNA monitoring.

The global increase in harmful algal blooms (HABs) has become a growing concern over the years, and New York State (NYS) is no exception. The Finger Lakes region in NYS has been identified as a hotspot for HABs, with Cayuga Lake having the highest number of blooms reported. The Cayuga Lake HABs Monitoring Program has been tracking cHABs (dominant bloom taxa, chlorophyll A, and microcystin levels) since 2018. However, limited research has been conducted on the microbiome of HABs in this region. In this study, the microbiome of HABs in the Cayuga Lake was surveyed and compared with non-HAB baseline samples. Using 16S rDNA community analysis, common bloom-forming cyanobacteria, were identified, with Microcystis being the dominant taxa in high toxin blooms. Further, this study evaluated the ability of Microcystis mcyA qPCR to detect elevated levels of potential toxigenic Microcystis in water samples using both benchtop and handheld qPCR devices. The results showed good performance of the qPCR assay as a screening for high toxin versus low/no toxin blooms. Additionally, the handheld qPCR device holds potential for in-field rapid (<1 h) screenings for high toxin blooms. This study provides insights into the microbiome of HABs in Cayuga Lake and offers a potential tool for rapid screening of high toxin blooms.

High Structural Diversity of Aeruginosins in Bloom-Forming Cyanobacteria of the Genus Planktothrix as a Consequence of Multiple Recombination Events.

Many compounds produced by cyanobacteria act as serine protease inhibitors, such as the tetrapeptides aeruginosins (Aer), which are found widely distributed. The structural diversity of Aer is intriguingly high. However, the genetic basis of this remains elusive. In this study, we explored the genetic basis of Aer synthesis among the filamentous cyanobacteria Planktothrix spp. In total, 124 strains, isolated from diverse freshwater waterbodies, have been compared regarding variability within Aer biosynthesis genes and the consequences for structural diversity. The high structural variability could be explained by various recombination processes affecting Aer synthesis, above all, the acquisition of accessory enzymes involved in post synthesis modification of the Aer peptide (e.g., halogenases, glycosyltransferases, sulfotransferases) as well as a large-range recombination of Aer biosynthesis genes, probably transferred from the bloom-forming cyanobacterium Microcystis. The Aer structural composition differed between evolutionary Planktothrix lineages, adapted to either shallow or deep waterbodies of the temperate climatic zone. Thus, for the first time among bloom-forming cyanobacteria, chemical diversification of a peptide family related to eco-evolutionary diversification has been described. It is concluded that various Aer peptides resulting from the recombination event act in chemical defense, possibly as a replacement for microcystins.

From in-silico screening to in-vitro evaluation: Enhancing the detection of Microcystins with engineered PP1 mutant variants.

Cyanotoxins produced during harmful algal blooms (CyanoHABs) have become a worldwide issue of concern. Microcystins (MC) are the most ubiquitous group of cyanotoxins and have known carcinogenic and hepatotoxic effects. The protein phosphatase inhibition assays (PPIAs), based on the inhibition of Protein Phosphatase 1/2A (PP1/PP2A) by MC, are one of the most cost-effective options for detecting MC. In this work, we aimed to design in-silico and evaluate in-vitro mutant variants of the PP1 protein, in order to enhance their capabilities as a MC biosensor. To this end, we performed an in-silico active site-saturated mutagenesis screening, followed by stability and docking affinity calculation with the MCLR cyanotoxin. Candidates with improved both affinity and stability were further tested in a fully flexible active-site docking. The best-scored mutations (19) were individually analysed regarding their locations and interactions. Four of them (p.D197F; p.Q249Y; p.S129W; p.D220Q) were selected for in-vitro expression and evaluation. Mutant p.D197F, exhibited a significant increment in inhibition by MCLR with respect to the WT, while showing a non-significant difference in stability nor activity. This successful PP1 inhibition enhancement suggests the potential of the p.D197F variant for practical MC detection applications.

Cyanotoxins, biosynthetic gene clusters, and factors modulating cyanotoxin biosynthesis.

Cyanobacterial harmful algal blooms (CHABs) are a global environmental concern that encompasses public health issues, water availability, and water quality owing to the production of various secondary metabolites (SMs), including cyanotoxins in freshwater, brackish water, and marine ecosystems. The frequency, extent, magnitude, and duration of CHABs are increasing globally. Cyanobacterial species traits and changing environmental conditions, including anthropogenic pressure, eutrophication, and global climate change, together allow cyanobacteria to thrive. The cyanotoxins include a diverse range of low molecular weight compounds with varying biochemical properties and modes of action. With the application of modern molecular biology techniques, many important aspects of cyanobacteria are being elucidated, including aspects of their diversity, gene-environment interactions, and genes that express cyanotoxins. The toxicological, environmental, and economic impacts of CHABs strongly advocate the need for continuing, extensive efforts to monitor cyanobacterial growth and to understand the mechanisms regulating species composition and cyanotoxin biosynthesis. In this review, we critically examined the genomic organization of some cyanobacterial species that lead to the production of cyanotoxins and their characteristic properties discovered to date.

JNK regulates the Nrf2/NQO1-ARE pathway against Microcystins-Induced oxidative stress in freshwater mussel Cristaria plicata.

In response to stress, cells can utilize several processes, such as the activation of the Nrf2/Keap1 pathway as a critical regulator of oxidative stress to protect against oxidative damage. C-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is involved in regulating the NF-E2-related nuclear factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. NAD(P)H quinone redox enzyme-1 (NQO1), a downstream target gene of the Nrf2 pathway, plays a vital role in removing peroxide and providing resistance to oxidative injury. We found that microcystins (MCs) stimulated CpNrf2 to express and increase anti-oxidative enzyme activities in a previous experiment. In our current study, the full-length cDNAs of JNK and NQO1 from Cristaria plicata (designated CpJNK and CpNQO1) were cloned. The relative levels of CpJNK and CpNQO1 were high in hepatopancreas. Upon MCs induction, the relative level of CpNQO1 was increased, whereas that of CpJNK was decreased significantly. In contrast, CpNrf2 knockdown upregulated the expression of CpJNK mRNA and phosphorylation of CpJNK protein (Cpp-JNK), but inhibited CpNQO1 expression. Additionally, we found that JNK inhibitor SP600125 stimulated expression of CpNQO1 and CpNrf2 upon exposure to MCs, and we further confirmed that CpNrf2 protein combined with the ARE element in CpNQO1 gene promoter in vitro, and increased CpNQO1-ARE-luciferase activity in a CpNrf2-dependent manner. These findings indicated C. plicata effectively alleviated MC-induced oxidative injury through JNK participated in regulating the Nrf2/NQO1-ARE pathway.

The thioredoxin expression of Cristaria plicata is regulated by Nrf2/ARE pathway under microcystin stimulation.

Thioredoxin plays an important role in inhibiting apoptosis and protecting cells from oxidative stress. This study was aimed to clarify how the expression of Trx from Cristaria plicata is regulated by Nrf2/ARE pathway. The expression of CpTrx mRNA was significantly up-regulated in gill and kidney tissues under microcystin stress. The Nrf2 gene of Cristaria plicata was identified to possess an auto active domain bit. While CpNrf2 was knocked down by specific small RNA, CpTrx mRNA expression was significantly down-regulated. The promoter of CpTrx gene had high transcriptional activity, and this basic transcriptional activity persisted after ARE element mutation. The region of promoter -206 to +217 bp was a core promoter region and had forward regulatory elements. Gel shift Assay exhibited that the CpTrx promoter could bind to the purified proteins CpNrf2 and CpMafK in vitro. The binding phenomenon disappeared after the ARE element mutation in promoter region. Subcellular localization experiments displayed that fluorescence overlap between CpNrf2 and Trx promoter increased under microcystin toxin stress. These results suggested that Trx expression was regulated by Nrf2/ARE pathway under oxidative stress.

Metagenomic mapping of cyanobacteria and potential cyanotoxin producing taxa in large rivers of the United States.

Cyanobacteria and cyanotoxin producing cyanobacterial blooms are a trending focus of current research. Many studies focus on bloom events in lentic environments such as lakes or ponds. Comparatively few studies have explored lotic environments and fewer still have examined the cyanobacterial communities and potential cyanotoxin producers during ambient, non-bloom conditions. Here we used a metagenomics-based approach to profile non-bloom microbial communities and cyanobacteria in 12 major U.S. rivers at multiple time points during the summer months of 2019. Our data show that U.S. rivers possess microbial communities that are taxonomically rich, yet largely consistent across geographic location and time. Within these communities, cyanobacteria often comprise significant portions and frequently include multiple species with known cyanotoxin producing strains. We further characterized these potential cyanotoxin producing taxa by deep sequencing amplicons of the microcystin E (mcyE) gene. We found that rivers containing the highest levels of potential cyanotoxin producing cyanobacteria consistently possess taxa with the genetic potential for cyanotoxin production and that, among these taxa, the predominant genus of origin for the mcyE gene is Microcystis. Combined, these data provide a unique perspective on cyanobacteria and potential cyanotoxin producing taxa that exist in large rivers across the U.S. and can be used to better understand the ambient conditions that may precede bloom events in lotic freshwater ecosystems.

Reconstitution and expression of mcy gene cluster in the model cyanobacterium Synechococcus 7942 reveals a role of MC-LR in cell division.

Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. Microcystin-LR (MC-LR) produced by Microcystis aeruginosa is the most common cyanotoxins. Due to the limitation of isolation, purification, and genetic manipulation techniques, it is difficult to study and verify in situ the biosynthetic pathways and molecular mechanisms of MC-LR. We reassembled the biosynthetic gene cluster (mcy cluster) of MC-LR in vitro by synthetic biology, designed and constructed the strong bidirectional promoter biPpsbA2 , transformed it into Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 d-1 . We found the expression of MC-LR led to abnormal cell division and cellular filamentation, further using various methods proved that by irreversibly competing its GTP-binding site, MC-LR inhibits assembly of the cell division protein FtsZ. The study represents the first reconstitution and expression of the mcy cluster and the autotrophic production of MC-LR in model cyanobacterium, which lays the foundation for resolving the microcystins biosynthesis pathway. The discovered role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their nonblooming counterparts.

Identification of Cyanobacteria and Its Potential Toxins in the Joanes I Reservoir, Bahia, Brazil.

The Joanes I Reservoir is responsible for 40% of the drinking water supply of the Metropolitan Region of Salvador, Bahia, Brazil. For water sources such as this, there is concern regarding the proliferation of potentially toxin-producing cyanobacteria, which can cause environmental and public health impacts. To evaluate the presence of cyanobacteria and their cyanotoxins in the water of this reservoir, the cyanobacteria were identified by microscopy; the presence of the genes of the cyanotoxin-producing cyanobacteria was detected by molecular methods (polymerase chain reaction (PCR)/sequencing); and the presence of toxins was determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The water samples were collected at four sampling points in the Joanes I Reservoir in a monitoring campaign conducted during the occurrence of phytoplankton blooms, and the water quality parameters were also analysed. Ten cyanobacteria species/genera were identified at the monitoring sites, including five potentially cyanotoxin-producing species, such as Cylindrospermopsis raciborskii, Cylindrospermopsis cf. acuminato-crispa, Aphanocapsa sp., Phormidium sp., and Pseudanabaena sp. A positive result for the presence of the cylindrospermopsin toxin was confirmed at two sampling points by LC-MS/MS, which indicated that the populations are actively producing toxins. The analysis of the PCR products using the HEPF/HEPR primer pair for the detection of the microcystin biosynthesis gene mcyE was positive for the analysed samples. The results of this study point to the worrisome condition of this reservoir, from which water is collected for public supply, and indicate the importance of the joint use of different methods for the analysis of cyanobacteria and their toxins in reservoir monitoring.

A Novel Microcystin-Producing Cyanobacterial Species from the Genus Desmonostoc, Desmonostoc alborizicum sp. nov., Isolated from a Water Supply System of Iran.

A qanat or kariz is a slightly sloping underground aqueduct used to transport water from wells or aquifers to the surface for irrigation and drinking supply. A cyanobacterial strain was isolated from a cyanobacterial mat colonizing the wall of a qanat in Golestan province, Gorgan City, Iran. Fragments of 16S rRNA, mcyG, and mcyD genes were amplified and sequenced, as well as the 16S-23S internal transcribed spacer (ITS). After microscopic examination, the isolate was related to a morphotype of Nostoc sensu lato group, with similar characteristics to Desmonostoc. The 16S rRNA phylogenetic analysis placed the isolate into the typical cluster of the recently proposed genus Desmonostoc. Morphological analysis revealed distinctive characteristic and secondary 16S-23S rRNA structures derived from comparative analysis, which did not match known species of Desmonostoc. These results lead us to propose a novel Desmonostoc species, Desmonostoc alborizicum, which was described and compared with similar taxa. Furthermore, for the first time a potentially toxic species of Desmonostoc was isolated from a water supply, since the mcyD and mcyG genes of the microcystin synthetase (mcy) cluster were successfully sequenced. Using mass spectrometry, detectable amounts of the hepatotoxin microcystin-LR and -RR, along with demethylated variants, were present in cell extracts of the Desmonostoc strain. Our findings contribute to a deeper understanding of the diversity, systematics, and occurrence of the genus Desmonostoc.

Shinella lacus sp. nov., a novel microcystin-degrading alphaproteobacterium containing the bla carbapenemase gene.

A Gram-stain-negative, rod-shaped, microcystin-degrading bacterium, designated as CPCC 100929T, was isolated from a fresh water reservoir in Sichuan Province, PR China. This isolate grew well at 4-37 °C and pH 6.0-8.0, with optimal growth at 28-32 °C and pH 7.0, respectively. The major cellular fatty acids were C18:1 ω7c/C18:1 ω6c, C16:0, C18:1 ω7c 11-methyl and C19:0 cyclo ω8c. The predominant respiratory quinone was Q-10. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylcholine were detected in the polar lipids extraction. The 16S rRNA gene sequence of strain CPCC 100929T was closely related to those of members of the genus Shinella, with the highest similarity of 98.6 % to Shinella zoogloeoides DSM 287T and 97.4-98.4 % with other identified Shinella members. In the phylogenetic trees based on 16S rRNA gene sequences and the core-genes analysis, strain CPCC 100929T was included within the clade of the genus Shinella. The values of average nucleotide identity (81.4-86.7 %) and digital DNA-DNA hybridization (25.4-44.6 %) between strain CPCC 100929T and other Shinella species were all below the thresholds for bacterial species delineation, respectively. The genomic DNA G+C content of strain CPCC 100929T was 63.6 %. The genomic sequence analysis indicated that this species contained genes encoding peroxidase, bla carbapenemase and the key enzyme for microcystin bio degradation, as well as rich carbohydrate-active enzyme coding genes, which might endow the micro-organism with properties to adapt to diverse environments. Based on its phenotypic and genetic properties, we propose that strain CPCC 100929T (=T1A350T=KCTC 72957T) is the type strain of a novel species with the name Shinella lacus sp. nov.

New Multidrug Efflux Systems in a Microcystin-Degrading Bacterium Blastomonas fulva and Its Genomic Feature.

A microcystin-degrading bacterial strain, Blastomonas fulva T2, was isolated from the culture of a microalgae Microcystis. The strain B. fulva T2 is Gram-stain-negative, non-motile, aerobic, non-spore-forming and phototrophic. The cells of B. fulva T2 are able to grow in ranges of temperature from 15 to 37 °C, with a pH of 6 to 8 and a salinity of 0 to 1% NaCl. Here, we sequenced the complete genome of B. fulva T2, aiming to better understand the evolutionary biology and the function of the genus Blastomonas at the molecular level. The complete genome of B. fulva T2 contained a circular chromosome (3,977,381 bp) with 64.3% GC content and a sizable plasmid (145.829 bp) with 60.7% GC content which comprises about 3.5% of the total genetic content. A total of 3842 coding genes, including 46 tRNAs and 6 rRNAs, were predicted in the genome. The genome contains genes for glycolysis, citric acid cycle, Entner-Doudoroff pathways, photoreaction center and bacteriochlorophylla synthesis. A 7.9 K gene cluster containing mlrA, mlrB, mlrC and mlrD1,2,3,4 of microcystin-degrading enzymes was identified. Notably, eight different efflux pumps categorized into RND, ABC and MFS types have been identified in the genome of strain T2. Our findings should provide new insights of the alternative reaction pathway as well as the enzymes which mediated the degradation of microcystin by bacteria, as well as the evolution, architectures, chemical mechanisms and physiological roles of the new bacterial multidrug efflux system.

Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embayment.

Planktothrix agardhii is a filamentous cyanobacterial species that dominates harmful algal blooms in Sandusky Bay, Lake Erie and other freshwater basins across the world. P. agardhii isolates were obtained from early (June) blooms via single filament isolation; eight have been characterized from 2016, and 12 additional isolates have been characterized from 2018 for a total of 20 new cultures. These novel isolates were processed for genomic sequencing, where reads were used to generate scaffolds and contigs which were annotated with DIAMOND BLAST hit, Pfam, and GO. Analyses include whole genome alignment to generate phylogenetic trees and comparison of genetic rearrangements between isolates. Nitrogen acquisition and metabolism was compared across isolates. Secondary metabolite production was genetically explored including microcystins, two types of aeruginosin clusters, anabaenopeptins, cyanopeptolins, microviridins, and prenylagaramides. Two common and 4 unique CRISPR-cas islands were analyzed for similar sequences across all isolates and against the known Planktothrix-specific cyanophage, PaV-LD. Overall, the uniqueness of each genome from Planktothrix blooms sampled from the same site and at similar times belies the unexplored diversity of this genus.

A new strain of Neowestiellopsis (Hapalosiphonaceae): first observation of toxic soil cyanobacteria from agricultural fields in Iran.

BACKGROUND: In the present research, challenges arose when many reports have been published on the poisoning of humans due to the ingestion of crops of Crataegus plants contaminated with cyanobacterial toxins. The discovery of several poisonings around agricultural zones prompted us to study the toxic compounds in a strain of Neowestiellopsis which is the most abundant in the agricultural zones of Kermanshah province of Iran, using a polyphasic approach. Molecular procedure was followed to study these strains deeply. MATERIAL AND METHODS: To elucidate their systematic position, besides the 16S rRNA gene, the analyses of molecular toxicity markers, namely nos, mcy G, mcy D and internal transcribed spacer (ITS), were also used. RESULTS: Based on the results, for the first time, we record the presence of a gene cluster coding for the biosynthesis of a bioactive compound (Nostopeptolides) that is very rare in this family and the presence of toxic compounds (microcystin), which might account for the poisoning of humans. CONCLUSIONS: This case is the first observation of a toxic soil strain from the genus Neowestiellopsis from agricultural fields in Iran.

Metagenomic and Metatranscriptomic Insights into Population Diversity of Microcystis Blooms: Spatial and Temporal Dynamics of mcy Genotypes, Including a Partial Operon That Can Be Abundant and Expressed.

Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [mcyA-J]), partial (truncated mcyA, complete mcyBC, and missing mcyD-J), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ, suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.

Cyanotoxin-encoding genes as powerful predictors of cyanotoxin production during harmful cyanobacterial blooms in an inland freshwater lake: Evaluating a novel early-warning system.

Freshwater harmful cyanobacterial blooms (HCBs) potentially produce excessive cyanotoxins, mainly microcystins (MCs), significantly threatening aquatic ecosystems and public health. Accurately predicting HCBs is thus essential to developing effective HCB mitigation and prevention strategies. We previously developed a novel early-warning system that uses cyanotoxin-encoding genes to predict cyanotoxin production in Harsha Lake, Ohio, USA, in 2015. In this study, we evaluated the efficacy of the early-warning system in forecasting the 2016 HCB in the same lake. We also examined potential HCB drivers and cyanobacterial community composition. Our results revealed that the cyanobacterial community was stable at the phylum level but changed dynamically at the genus level over time. Microcystis and Planktothrix were the major MC-producing genera that thrived in June and July and produced high concentrations of MCs (peak level 10.22 µg·L-1). The abundances of the MC-encoding gene cluster mcy and its transcript levels significantly correlated with total MC concentrations (before the MC concentrations peaked) and accurately predicted MC production as revealed by logistic equations. When the Microcystis-specific gene mcyG reached approximately 1.5 × 103 copies·mL-1 or when its transcript level reached approximately 2.4 copies·mL-1, total MC level exceeded 0.3 µg L-1 (a health advisory limit) approximately one week later (weekly sampling scheme). This study suggested that cyanotoxin-encoding genes are promising predictors of MC production in inland freshwater lakes, such as Harsha Lake. The evaluated early-warning system can be a useful tool to assist lake managers in predicting, mitigating, and/or preventing HCBs.

Effective Early Treatment of Microcystis Exponential Growth and Microcystin Production with Hydrogen Peroxide and Hydroxyapatite.

Mitigating cyanotoxin production is essential to protecting aquatic ecosystems and public health. However, current harmful cyanobacterial bloom (HCB) control strategies have significant shortcomings. Because predicting HCBs is difficult, current HCB control strategies are employed when heavy HCBs have already occurred. Our pilot study developed an effective HCB prediction approach that is employed before exponential cyanobacterial growth and massive cyanotoxin production can occur. We used a quantitative polymerase chain reaction (qPCR) assay targeting the toxin-encoding gene mcyA to signal the timing of treatment. When control measures were applied at an early growth stage or one week before the exponential growth of Microcystis aeruginosa (predicted by qPCR signals), both hydrogen peroxide (H2O2) and the adsorbent hydroxyapatite (HAP) effectively stopped M. aeruginosa growth and microcystin (MC) production. Treatment with either H2O2 (10 mg·L-1) or HAP (40 µm particles at 2.5 g·L-1) significantly reduced both mcyA gene copies and MC levels compared with the control in a dose-dependent manner. While both treatments reduced MC levels similarly, HAP showed a greater ability to reduce mcyA gene abundance. Under laboratory culture conditions, H2O2 and HAP also prevented MC production when applied at the early stages of the bloom when mcyA gene abundance was below 105 copies·mL-1.

Quorum-Sensing Signals from Epibiont Mediate the Induction of Novel Microviridins in the Mat-Forming Cyanobacterial Genus Nostoc.

The regulation of the production of oligopeptides is essential in understanding their ecological role in complex microbial communities, including harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the microbial community harbored as epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. Here, we present insight into the role of a bacterial epibiont in regulating the production of novel microviridins isolated from Nostoc, an ecologically important cyanobacterial genus. Microviridins are well-known elastase inhibitors with presumed antigrazing effects. Heterologous expression and identification of specific signal molecules from the epibiont suggest the role of a quorum-sensing-based interaction. Furthermore, physiological experiments show an increase in microviridin production without affecting cyanobacterial growth and photosynthetic activity. Simultaneously, oligopeptides presenting a selective inhibition pattern provide support for their specific function in response to the presence of cohabitant epibionts. Thus, the chemical interaction revealed in our study provides an example of an interspecies signaling pathway monitoring the bacterial flora around the cyanobacterial filaments and the induction of intrinsic species-specific metabolic responses. IMPORTANCE The regulation of the production of cyanopeptides beyond microcystin is essential to understand their ecological role in complex microbial communities, e.g., harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. The frequency of cyanopeptide occurrence also demonstrates the need to understand the contribution of cyanobacterial peptides to the overall biological impact of cyanopeptides on aquatic organisms and vertebrates, including humans. Our results shed light on the epibiont control of microviridin production via quorum-sensing mechanisms, and we posit that such mechanisms may be widespread in natural cyanobacterial bloom community regulation.

Variability in microcystin quotas during a Microcystis bloom in a eutrophic lake.

Microcystis is a bloom-forming genus of cyanobacteria with some genotypes that produce highly toxic microcystin hepatotoxins. In waterbodies where biological and physical factors are relatively homogenous, toxin quotas (the average amount of toxin per cell), at a single point in time, are expected to be relatively constant. In this study we challenged this assumption by investigating the spatial distribution of microcystin quotas at a single point in time on two separate occasions in a lake with a major Microcystis bloom. Microcystis cell concentrations varied widely across the lake on both sampling occasions (730- and 137-fold) together with microcystin quotas (148- and 362-fold). Cell concentrations and microcystin quotas were strongly positively correlated (R2 = 0.89, P < 0.001, n = 28; R2 = 0.67, P < 0.001, n = 25). Analysis of Microcystis strains using high-throughput sequencing of the 16S-23S rRNA intergenic spacer region showed no relationship between microcystin quota and the relative abundance of specific sequences. Collectively, the results of this study indicate an association between microcystin production and cell density that magnifies the potential for bloom toxicity at elevated cell concentrations.