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

Abstract

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.

FIG. 1.

Chip assay signals detected from spots corresponding to 16S rRNA and mcyE probe pairs when DNA or cDNA from Microcystis aeruginosa PCC 7806, Anabaena sp. 90, or a mix of the two strains was used as a target. Each bar represents the average signal intensity of the four replicate spots as a percentage of the average signal intensity of the hybridization control spots (100%).

FIG. 2.

Chip assay signals detected from spots corresponding to 16S rRNA (A) or mcyE (B) probe pairs when DNA and cDNA isolated from Lake Tuusulanjärvi water samples were used as targets. The bars represent the average signal intensity of each spot (four replicate spots) as a percentage of the average signal intensity of the hybridization control spots (100%).

FIG. 3.

Microcystin (MC) variants detected in Lake Tuusulanjärvi samples in summer 2006 by LC/MS. The quantities of total microcystins and different variants are given on top and on the right side of the bars, respectively. For the first two samples, the quantity of only demethylated (dm) MC-RR is given. In addition, MC-LR (1 ng liter⁻¹), dmMC-LR (1 ng liter⁻¹), and MC-WR (0.1 ng liter⁻¹) were detected in the June sample, and MC-LR (1 ng liter⁻¹) and MC-RR (2 ng liter⁻¹) were detected in the 10 July sample.

FIG. 4.

Anabaena sp. and Microcystis sp. mcyE gene (DNA) and transcript (cDNA) copy numbers and total microcystin (MC) concentrations in Lake Tuusulanjärvi water samples from June to September 2006, determined by quantitative real-time PCR and LC/MS. The error bars represent standard deviations for three replicate samples.