Using Microcystin Gene Copies to Determine Potentially-Toxic Blooms, Example from a Shallow Eutrophic Lake Peipsi.

Global warming, paired with eutrophication processes, is shifting phytoplankton communities towards the dominance of bloom-forming and potentially toxic cyanobacteria. The ecosystems of shallow lakes are especially vulnerable to these changes. Traditional monitoring via microscopy is not able to quantify the dynamics of toxin-producing cyanobacteria on a proper spatio-temporal scale. Molecular tools are highly sensitive and can be useful as an early warning tool for lake managers. We quantified the potential microcystin (MC) producers in Lake Peipsi using microscopy and quantitative polymerase chain reaction (qPCR) and analysed the relationship between the abundance of the mcyE genes, MC concentration, MC variants and toxin quota per mcyE gene. We also linked environmental factors to the cyanobacteria community composition. In Lake Peipsi, we found rather moderate MC concentrations, but microcystins and microcystin-producing cyanobacteria were widespread across the lake. Nitrate (NO3-) was a main driver behind the cyanobacterial community at the beginning of the growing season, while in late summer it was primarily associated with the soluble reactive phosphorus (SRP) concentration. A positive relationship was found between the MC quota per mcyE gene and water temperature. The most abundant variant-MC-RR-was associated with MC quota per mcyE gene, while other MC variants did not show any significant impact.

Identification of geosmin and 2-methylisoborneol in cyanobacteria and molecular detection methods for the producers of these compounds.

Geosmin and 2-methylisoborneol (MIB) are muddy/earthy off-flavor metabolites produced by a range of bacteria. Cyanobacteria are the major producers of the volatile metabolites geosmin and MIB which produce taste and odor problems in drinking water and fish worldwide. Here we detected geosmin and MIB by studying 100 cyanobacteria strains using solid phase microextraction gas chromatography mass spectrometry (SPME GC-MS). A total of 21 geosmin producers were identified from six cyanobacteria genera. Two of the geosmin producers also produced MIB. A PCR protocol for the detection of geoA and MIB synthase genes involved in the biosynthesis of geosmin and MIB was developed. The geoA and MIB synthase genes were detected in all strains shown to produce geosmin and MIB, respectively. Cyanobacterial geoA and MIB synthase sequences showed homology to terpene synthases genes of actinobacteria and proteobacteria. Additional off-flavor compounds, nor-carotenoids ß-ionone and ß-cyclocitral, were found from 55 strains among the 100 cyanobacterial strains studied; ß-ionone was present in 45 and ß-cyclocitral in 10 strains. Six of the cyanobacteria which contain off-flavor compounds also produced toxins, anatoxin-a or microcystins. The molecular method developed is a useful tool in monitoring potential cyanobacterial producers of geosmin and MIB.

Genome-derived insights into the biology of the hepatotoxic bloom-forming cyanobacterium Anabaena sp. strain 90.

BACKGROUND: Cyanobacteria can form massive toxic blooms in fresh and brackish bodies of water and are frequently responsible for the poisoning of animals and pose a health risk for humans. Anabaena is a genus of filamentous diazotrophic cyanobacteria commonly implicated as a toxin producer in blooms in aquatic ecosystems throughout the world. The biology of bloom-forming cyanobacteria is poorly understood at the genome level. RESULTS: Here, we report the complete sequence and comprehensive annotation of the bloom-forming Anabaena sp. strain 90 genome. It comprises two circular chromosomes and three plasmids with a total size of 5.3 Mb, encoding a total of 4,738 genes. The genome is replete with mobile genetic elements. Detailed manual annotation demonstrated that almost 5% of the gene repertoire consists of pseudogenes. A further 5% of the genome is dedicated to the synthesis of small peptides that are the products of both ribosomal and nonribosomal biosynthetic pathways. Inactivation of the hassallidin (an antifungal cyclic peptide) biosynthetic gene cluster through a deletion event and a natural mutation of the buoyancy-permitting gvpG gas vesicle gene were documented. The genome contains a large number of genes encoding restriction-modification systems. Two novel excision elements were found in the nifH gene that is required for nitrogen fixation. CONCLUSIONS: Genome analysis demonstrated that this strain invests heavily in the production of bioactive compounds and restriction-modification systems. This well-annotated genome provides a platform for future studies on the ecology and biology of these important bloom-forming cyanobacteria.

Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers.

Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Méjean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.

Molecular methods: chip assay and quantitative real-time PCR: in detecting hepatotoxic cyanobacteria.

Cyanobacterial mass occurrences are widespread and often contain hepatotoxic, i.e. microcystin- and nodularin-producing, species. Nowadays, detection of microcystin (mcy) and nodularin synthetase (nda) genes is widely used for the recognition of toxic cyanobacterial strains in environmental water samples. Chip assay presented here combines ligation detection reaction and hybridization on a universal microarray to detect and identify the mcyE/ndaF genes of five cyanobacterial genera specifically and sensitively. Thus, one chip assay can reveal the co-occurrence of several hepatotoxin producers. The presented quantitative real-time PCR method is used for the detection of either microcystin-producing Anabaena or Microcystis. Determination of the mcyE-gene copy numbers allows the identification of the dominant producer genus in the sample.

Development of a chip assay and quantitative PCR for detecting microcystin synthetase E gene expression.

The chip and quantitative real-time PCR (qPCR) assays were optimized to study the expression of microcystin biosynthesis genes (mcy) with RNA samples extracted from cyanobacterial strains and environmental water samples. Both microcystin-producing Anabaena and Microcystis were identified in Lake Tuusulanjärvi samples. Microcystis transcribed the mcyE genes throughout the summer of 2006, while expression by Anabaena became evident later in August and September. Active mcyE gene expression was also detectable when microcystin concentrations were very low. Detection of Anabaena mcyE transcripts by qPCR, as well as certain cyanobacterial 16S rRNAs with the chip assay, showed slightly reduced sensitivity compared with the DNA analyses. In contrast, even groups undetectable or present in low quantities as determined by microscopy could be identified with the chip assay from DNA samples. The methods introduced add to the previously scarce repertoire of applications for mcy expression profiling in environmental samples and enable in situ studies of regulation of microcystin synthesis in response to environmental factors.