Evolution and detection of cyanobacterial hepatotoxin synthetase genes

<p>Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are <em>Microcystis</em>, <em>Anabaena</em>, and <em>Planktothrix</em>. Nodularins are produced by strains of <em>Nodularia</em> <em>spumigena</em> in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. <br />
In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (<em>mcyE</em>) and the orthologous nodularin synthetase gene F (<em>ndaF</em>) sequences. General primers were designed to amplify the <em>mcyE</em>/<em>ndaF</em> gene region from microcystin-producing <em>Anabaena</em>, <em>Microcystis</em>, <em>Planktothrix</em>, and <em>Nostoc</em>, and nodularin-producing <em>Nodularia</em> strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that <em>mcy</em> genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that <em>nda</em> genes evolved from <em>mcy</em> genes. <br />
The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general <em>mcyE</em> primers, and in 91% of the lakes with the three genus-specific <em>mcyE</em> primers. Potential microcystin-producing <em>Microcystis</em> were detected in 70%, <em>Planktothrix</em> in 63%, and <em>Anabaena</em> in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of <em>mcyE</em> genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. <br />
Genus-specific probe pairs for microcystin-producing <em>Anabaena</em>, <em>Microcystis</em>, <em>Planktothrix</em>, and <em>Nostoc</em>, and nodularin-producing <em>Nodularia</em> were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the <em>mcyE</em>/<em>ndaF</em> genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. <br />
Analysis of the <em>mcy</em> and <em>nda</em> gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of <em>Microcystis</em>, <em>Planktothrix</em>, and <em>Anabaena</em>, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.</p>