Linking CYanoTOcin Production to GENEtic Diversity -CYTOGENE

Cyanobacteria (blue-green algae) frequently form mass developments in surface waters, the majority of which contain toxins - the hepatotoxic microcystins. Due to the wide occurrence of microcystins the WHO has published a threshold of 1 g per liter of microcystin in drinking water and the understanding of microcystin production in water has become an important task of current limnology. While the significance of direct environmental influences on microcystin production (the phenotypic component) has received much attention during the last decades, the impact of the genotypic component on microcystin net production is much less known. Indeed, cyanobacterial populations often not only contain microcystin producing and non-microcystin producing organisms, but even within the group of microcystin producing organisms the quantitative differences in microcystin production may be substantial. So far few studies focused on the genus Planktothrix because these organisms usually do not form dense surface scums. However, Planktothrix occurs in a much broader range of habitats when compared with the scum forming taxa and builds up dense metalimnetic layers also in less eutrophic drinking water reservoirs. This study aims to investigate the genotypic component of microcystin production (independent of the phenotypic influence) in isolates of Planktothrix spp. originating from different lakes in central Europe. The isolates will be phenotypically (morphology, pigments) described and genetically characterised. In particular genetic differences between isolates will be linked to microcystin content quantitatively and qualitatively by considering (1) the influence of physiological differences as shown by maximal specific growth rate; (2) the influence of genetic variability within gene regions encoding the biosynthesis of microcystin; (3) the influence of the production of additional structurally related polypeptides; (4) the influence of ecological factors as indicated by habitat type/life form of the isolates (deep stratified clear lakes vs. shallow turbid more eutrophic systems). The genetic analysis will be done in collaboration with the Institute of Genetics at the Humboldt University in Berlin (Germany). The results of the study will have profound implications for the understanding and for the prediction of microcystin production in nature.

Cyanobacteria (blue green algae) occur frequently in freshwater all over the world and show an impressive diversity in the production of cyclic heptapeptides, the microcystins (MCs) known to be toxic to livestock and man. Typically isolates of the same species differ in the production of MC structural variants or do not produce MC, however, they cannot be discriminated in the microscope. In this project major progress has been made (i) in elucidation of genetic variation, i.e. recombination and mutation of the genes involved in MC synthesis (in total nine genes consisting of 55,000 base pairs) and (ii) in exploring the consequences for MC synthesis. Firstly, recombination processes involving whole enzyme domains probably originating from other peptide synthetases were observed to result in the synthesis of new MC structural variants. Secondly, recombination processes within genes of the MC synthetase also resulted in a significantly altered synthesis of structural variants. Thirdly, within genes involved in MC synthesis large deletions (400-1900 base pairs) or insertions (1400 base pairs) with high similarity to transposases (jumping genes) were detected that both resulted in inactivation of MC synthesis. Progress has further been made with regard to the influence of ecological factors as indicated by the habitat type/life form of cyanobacteria, i.e. the red-pigmented filamentous cyanobacterium Planktothrix rubescens living in deep and physically stratified lakes always contained the genes involved in MC synthesis. In contrast in populations of the green-pigmented filamentous cyanobacterium P. agardhii found in shallow and eutrophic water bodies, MC genotypes co-occurred with genotypes without MC genes. Growing isolates in the laboratory it was found that (i) isolates without MC contained significantly higher amounts of structurally related but less toxic peptides, i.e. the anabaenopeptins and vice versa and (ii) most isolates produced significantly higher amounts of anabaenopeptins when compared with the MCs. The red-pigmented cyanobacterium P. rubescens typically produced significantly higher amounts of anabaenopeptins when compared with MCs while the green-pigmented P. agardhii contained both high anabaenopeptin and high MC producers. The results have important consequences for our ecological understanding of this fascinating variability in synthesis of bioactive secondary metabolites, i.e. in all cyanobacteria MC producers co-occur with non-MC producers due to either the absence or the inactivation of MC genes. The inactivation of MC genes - for example via transposons - might be considered as a first step in evolution of bioactive peptides and our results on genetic variation do provide first insights into the evolutionary origin of structural variability in toxins synthesized by cyanobacteria.