Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic CyanobacteriumMicrocystissp

Abstract

The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.Water blooms of the cyanobacterium Microcystis sp. have frequently been found to contain the toxic heptapeptide microcystin. In recent decades, environmental problems associated with microcystin in water have been documented (5). It has long been known that microcystin-producing and nonmicrocystin-producing strains can be isolated from one water sample, and the waxing and waning of microcystin-producing versus non-microcystin-producing strains has been suggested as a most important factor regulating net microcystin production in water (26). However, the quantification of microcystinproducers versus non-microcystin-producers was for a long time impeded because of the morphological similarity between strains differing in microcystin production. The genes involved in microcystin synthesis have been identified and sequenced (8,28). On this basis, attempts have been made to study the occurrence of microcystin genotypes directly in the field (14, 15). The first approach consisted of the isolation of individual colonies, subsequent morphological characterization, and PCR analysis to detect the microcystin genes (14). This resulted in significant differences in mcyB distribution between morphologically defined species (as described in reference 11); i.e., 73% of the colonies assigned to Microcystis aeruginosa contained mcyB, while only 16% of the colonies assigned to Microcystis ichthyoblabe and none of the Microcystis wesenbergi...

Variation of Microcystis and microcystins coupling nitrogen and phosphorus nutrients in Lake Erhai, a drinking-water source in Southwest Plateau, China

“…To determine the number of MCM, the mcyB gene was quantitatively amplified using a specific primer set, 16 mcyB-30F (5′-CCTACCGAGCGCTTGGG-3′) and 16 mcyB-108R (5′-GAAAATCCCCTAAAGATTCCTGAGT-3′) (Kurmayer and Kutzenberger 2003;Xu et al 2010). The total cells number of Microcystis was determined using the primer set, 1M-16s209F (5′-ATGTGCCGCGAGGTGAAACCTAAT-3′), and 1M-16s409R (5′-TTACAATCCAAAGACCTTCC TCCC-3′) (Neilan et al 1997) by specially and quantitatively amplifying the 16S rRNA gene of Microcystis.…”

Toxic cyanobacteria in reservoirs in northeastern Brazil: detection using a molecular method

“…A large number of authors have used microcystin synthetase genes (mcy) as molecular markers for microcystinproducing cyanobacteria (Baker et al, 2002;Nonneman and Zimba, 2002;Bittencourt-Oliveira, 2003;Hisbergues et al, 2003), for, in other studies, the presence of microcystin has been correlated, almost without exception, to the presence of mcy genes (Kurmayer and Kutzenberger, 2003;Via-Ordorika et al, 2004;Dittmann and Börner, 2005). There are no morphological differences between toxic and non-toxic cyanobacteria.…”

Towards clarification of the inhibitory mechanism of wheat bran leachate on Microcystis aeruginosa NIES-843 (cyanobacteria): physiological responses

“…Gene expression data from the real-time PCR were evaluated using Ct values. 16S rRNA gene was used as a Urbach et al (1992) b Primer sequence obtained from Nübel et al (1997) c Primers for prx, psbA, recA, grpE and fabZ were obtained from Shao et al (2009) d Primer sequence obtained from Kurmayer and Kutzenberger (2003) housekeeping gene to normalize the expression levels of target genes (Bustin 2000). The induction ratio was calculated using 2 -DDCt where…”