Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystin synthetase (mcy) gene clusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers and TaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The involvement of α-proteobacteria Phenylobacterium in maintaining the dominance of toxic Microcystis blooms in Lake Taihu, China.

Lake Taihu in China has suffered serious harmful cyanobacterial blooms for decades. The algal blooms threaten the ecological sustainability, drinking water safety, and human health. Although the roles of abiotic factors (such as water temperature and nutrient loading) in promoting Microcystis blooms have been well studied, the importance of biotic factors (e.g. bacterial community) in promoting and meditating Microcystis blooms remains unclear. In this study, we investigated the ecological dynamics of bacterial community, the ratio of toxic Microcystis, as well as microcystin in Lake Taihu. High-throughput 16S rRNA sequencing and principal component analysis (PCA) revealed that the bacteria community compositions (BCCs) clustered into three groups, the partitioning of which corresponded to that of groups according to the toxic profiles (the ratio of toxic Microcystis to total Microcystis, and the microcystin concentrations) of the samples. Further Spearman's correlation network showed that the α-proteobacteria Phenylobacterium strongly positively correlated with the toxic profiles. Subsequent laboratory chemostats experiments demonstrated that three Phenylobacterium strains promoted the dominance of the toxic Microcystis aeruginosa PCC7806 when co-culturing with the non-toxic PCC7806 mcyB- mutant. Taken together, our data suggested that the α-proteobacteria Phenylobacterium may play a vital role in the maintenance of toxic Microcystis dominance in Lake Taihu.

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.

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.

Understanding the inhibitory mechanism of antialgal allelochemical flavonoids from genetic variations: Photosynthesis, toxin synthesis and nutrient utility.

Flavonoids are natural polyphenolic compounds from plants. As a new biotechnological algaecide, the molecular mechanism of plant flavonoids on the inhibition of Microcystis aeruginosa is still unknown. Therefore, in this study, we analyzed the variation of expressions of photosynthesis-related genes, microcystin synthesis-related genes and the genes involved in N and P acquisition in M. aeruginosa under the flavonoids stress. The results showed that the expression of psbD1, psaB and rbcL related to photosynthesis were influenced by three flavonoids but with different changing tendencies. The transcription of mcyA, mcyD and mcyH related to microcystin synthesis were decreased after 5-d of exposure, which could block microcystin synthesis. Meanwhile, flavonoids treatments resulted in the inhibition of N and P acquisition related genes transcription to affect the absorption of N and P in algal cells, and further influenced the physiological metabolic process of M. aeruginosa.

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.

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

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

Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix.

BACKGROUND: Bloom-forming cyanobacteria cause toxic algae outbreaks in lakes and reservoirs. We aimed to explore and quantify mutation events occurring within the large mcy gene cluster (55 kbp) encoding microcystin (MC) biosynthesis that inactivate MC net production. For this purpose we developed a workflow to detect mutations in situ occurring anywhere within the large mcy gene cluster as amplified from one single filament of the red-pigmented cyanobacterium Planktothrix rubescens. From five lakes of the Alps eight hundred Planktothrix filaments were isolated and each individual filament was analyzed for mutations affecting the mcy genes. RESULTS: Mutations inactivating MC synthesis were either through an insertion element ISPlr1 or the partial deletion of mcy genes. Neutral mutations not affecting MC biosynthesis occurred within two intergenic spacer regions, either through the insertion of a Holliday-junction resolvase RusA or ISPlr1. Altogether, the insertions affected a few mcy genes only and their location was correlated with regions similar to repetitive extragenic palindromic DNA sequences (REPs). Taking all of the filaments together, the mutations leading to the inactivation of MC synthesis were more rare (0.5-6.9%), when compared with the neutral mutations (7.5-20.6%). On a spatial-temporal scale the ratio of MC synthesis-inactivating vs. neutral mutations was variable, e.g., the filament abundance carrying partial deletion of mcyD (5.2-19.4%) and/or mcyHA (0-7.3%) exceeded the abundance of neutral mutations. CONCLUSIONS: It is concluded that insertion events occurring within the Planktothrix mcy gene cluster are predictable due to their correlation with REPs. The frequency of occurrence of the REPs within the mcy gene cluster of Planktothrix relates to the rather common mutation of mcy genes in Planktothrix. Spatial-temporal variable conditions may favor the emergence of partial mcy deletion mutants in Planktothrix, in particular a higher proportion of genotypes resulting in inactivation of MC synthesis might be caused by increased ISPlr1 activity.

Fluorescence in situ hybridization of Microcystis strains producing microcystin using specific mRNA probes.

Cyanobacteria are ubiquitous micro-organisms that can produce toxic compounds, the cyanotoxins. The monitoring of such producers in the environment is of prime importance for human health. An attractive technology for such monitoring is fluorescence in situ hybridization (FISH), which allows the detection and enumeration of environmental micro-organisms. We present here the application of tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to the detection of microcystin-producing Microcystis strains. We used a 16S rRNA-specific probe, MICR3, to specifically label and observe by epifluorescence microscopy Microcystis aeruginosa strains. Using confocal laser scanning microscopy and a specific probe, MCYA, targeting the mcyA mRNA we have labelled M. aeruginosa PCC 7806, which produces microcystins. Microcystis aeruginosa PCC 7005 which does not produce microcystins is not labelled by this probe. Furthermore, we show here that this specific mRNA labelling in M. aeruginosa PCC 7806 is enhanced in cells illuminated for 1 h just after a dark period of cultivation of 24 h, conditions in which the mcyA gene is up regulated. The data presented here might be applicable to the monitoring of toxic Microcystis strains in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyanobacteria producing toxic compounds (cyanotoxins) are present in the environment and in water bodies. Their presence poses a threat on human and animal health. It is thus important to detect, identify and enumerate these toxic Cyanobacteria. Using tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) and specific probes, with confocal laser scanning microscopy, we have specifically detected Microcystis strains producing microcystin toxins. The data presented here might be applied to the monitoring of water bodies at early stages and all along the formation of Microcystis blooms.

Microcystin-degrading bacteria affect mcyD expression and microcystin synthesis in Microcystis spp.

Cyanobacterial blooms occur increasingly often and cause ecological, economic and human health problems worldwide. Microcystins (MCs) are the dominant toxins produced by cyanobacteria and are implicated in epidemic disease and environmental problems. Extensive research has been reported on the various regulating factors, e.g., light, temperature, nutrients such as nitrogen and phosphorus, pH, iron, xenobiotics, and predators, that influence microcystin (MC) synthesis, but little is known about the effects of cyanobacteria-associated bacteria on MC synthesis. A considerable number of studies have focused on interactions between Microcystis species and their associated bacteria. In this study, we evaluated the effects of MC-degrading bacteria (MCDB) on MC synthesis gene mcyD expression and MC synthesis in axenic strain PCC7806, non-axenic strain FACHB905, and colony strain FACHB1325 of Microcystis by quantitative real-time polymerase chain reaction (RT-PCR) assay and enzyme-linked immunosorbent assay (ELISA). We demonstrate for the first time that MCDB can induce and up-regulate the MC production and transcriptional response of the mcyD gene of toxic Microcystis. On day 4 of the culturing experiment, the intracellular MC concentration and transcriptional response of mcyD of FACHB1325 were up-regulated 1.9 and 5.3-fold over that of the control, and for FACHB905 were up-regulated 1.8 and 4.2-fold over that of the control, respectively. On day 10, the transcriptional response of mcyD was up-regulated 21.3-fold in PCC7806. These results indicate that there are interactions between toxic Microcystis and MCDB, and MCDB may play a role in regulating mcyD expression in toxic Microcystis.