Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp

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.

FIG. 1.

(A) Standard curves (black symbols) for both the PC operon (circles) and mcyB (squares) based on predetermined cell concentrations of Microcystis HUB524 by relating the known DNA concentrations (in cell equivalents) to the C_t of the diluted samples. In addition, both TNAs were tested in the presence of a 1:100 dilution (6.6 × 10⁻⁴ mm³, white symbols) and a 1:1,000 dilution (6.6 × 10⁻⁵ mm³, dotted symbols) of a natural background (containing other cyanobacteria but no Microcystis) from Lake Wannsee. All data are means of three parallels ± 1 standard error (error bars that are not visible are hidden behind the symbols; however, error bars have been omitted for outliers shown at 10⁵ cells). (B) Comparison between cell numbers estimated for both genes by TNA from the standard curve in the absence (white columns) or in the presence of 6.6 × 10⁻⁴ mm³ and 6.6 × 10⁻⁵ mm³ of background. (C) Number of cells estimated in the presence of a natural background divided by the cell number estimated in the absence of a natural background for both the PC operon (circles) and mcyB (squares) and both background dilutions (6.6 × 10⁻⁴ mm³ [black symbols] and 6.6 × 10⁻⁵ mm³ [white symbols]).

FIG. 2.

(A) Cell numbers of Microcystis HUB524 grown under high-light conditions in batch culture and quantified via electronic particle counting and TNA over 4 months (mean ± 1 standard error). Data are from two independent batch culture experiments. For reasons of clarity, only the cell numbers quantified with the PC gene are shown; for details on statistical correlation between cell numbers and both the PC gene and mcyB, see the text. The region within the dotted lines is considered the transition phase from logarithmic growth to the stationary phase. (B) Ratio of cell numbers determined by TNA to cell numbers determined by the particle counter during the same batch culture experiment (mean ± 1 standard error). The transition from the exponential growth phase to the stationary phase differed significantly (P < 0.001, Mann-Whitney rank sum test) from the earlier and later growth phases. (C) Regression of cell numbers (mean ± 1 standard error) determined by the TNA (PC operon) versus cell numbers determined by the electronic particle counter during the same batch culture experiment. Error bars that are not visible are hidden behind the symbols.

FIG. 3.

(A) Cell numbers of 10 Microcystis strains grown under high-light conditions in batch culture and quantified by the electronic particle counter and by the TNA (mean ± 1 standard error). Data are from two independent parallels. (B) Ratios of cell numbers determined by TNA to cell numbers determined by the particle counter observed among strains and during the stationary phase of strain HUB524. The boxes show the median (line) and the 25th and the 75th percentiles, and the whiskers indicate the 5th and the 95th percentiles.

FIG. 4.

(A) Numbers of Microcystis cells in Lake Wannsee from June 1999 to October 2000, determined by counting under an inverted microscope or by TNA with the PC gene (mean ± 1 standard error). (B) Proportion of mcyB genotypes during the same period (one outlier on 8 June 1999, 114%, was omitted). The solid line indicates the mean water temperature integrated every meter over the total water column. (C) Dependence of cell numbers determined by TNA of the mcyB gene on cell numbers determined by TNA for PC. Error bars show the mean ± 1 standard error. (D) Comparison between cell number determined in the microscope and determined via TNA for PC (black) and mcyB (white) during the same study period. For details on statistical regression parameters, see the text.