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.

Non-autonomous transposable elements associated with inactivation of microcystin gene clusters in strains of the genus Anabaena isolated from the Baltic Sea.

Microcystins are potent peptide toxins produced by a range of distantly related cyanobacteria. They are assembled on a large enzyme complex encoded by the 55 kb microcystin synthetase (mcy) gene cluster. Here we report two strains of the genus Anabaena isolated from the Baltic Sea, which contain the entire mcy gene cluster but do not produce microcystins. Transcription analysis demonstrated that mcy genes were not expressed in these strains. We identified short insertion elements interrupting the mcyA, mcyD and mcyE genes in the two strains isolated in different years from different parts of the Baltic Sea. The 126-207 bp insertion elements encoded both terminal inverted repeats and direct repeats but lacked transposases. However, we found evidence for transposition of these elements despite the absence of genes encoding transposases, suggesting that they are non-autonomous mobile miniature inverted-repeat transposable elements (MITEs) recently described from cyanobacteria. The MITE insertion elements were present in mcyD genes amplified directly from the cyanobacterial community present in the Baltic Sea blooms from 2005. The results demonstrate that mcy gene cluster mutants can make up a stable proportion of the mcy gene pool in the Baltic Sea population.

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.