Genotype determined toxin content in cyanobacteria

The toxic heptapeptide microcystin (MC) has gained much attention as bloom forming cyanobacteria are known to contain high amounts of microcystin posing a considerable health risk to humans worldwide. While the influence of environmental factors on the net production of MC has been studied during the last years, the genotype determined content of MC is not understood. For example, under identical environmental conditions the MC content among strains can differ more than tenfold and this difference has been reproducible shown over years. In this project it is proposed to use the filamentous microcystin producing strain Planktothrix agardhii CYA126/8 as a model system to investigate factors regulating the genotype determined MC content in cyanobacteria. For this purpose we sequenced the genome of this strain as it is amenable to genetic manipulation. Analysis of the genome data revealed three additional gene clusters that are responsible for the biosynthesis of peptides related to MC (anabaenopeptin, cyanopeptolin and aeruginoside). It is aimed to elucidate (1) functional consequences of mutations on MC content that have been described to occur in the MC biosynthesis genes of different strains, (2) the influence of additional peptide synthetase gene clusters on MC biosynthesis, (3) the influence of availability of building block precursors for the biosynthesis of MC. As mutations have been observed within the gene cluster encoding MC biosynthesis of different strains we will experimentally analyse their consequences. Single enzymes with high mutational rates will be heterologously expressed and biochemically characterized. The influence of genetically inactivated peptide synthetases related to MC synthesis will be analyzed in growth experiments under limiting and nonlimiting environmental conditions. Feeding precursors to the axenic model strain e.g. amino acids which are known to be incorporated into MC will elucidate whether precursors potentially limit the MC content. The results are of crucial relevance to understand genotype dependent determination of MC production in water.

Harmful algal blooms are increasing worldwide in freshwater and marine ecosystems due to eutrophication of water bodies and changes in water temperature. Typically those bloom-forming organisms consist of a diversity of genotypes which cause the enormous variety of toxins and related bioactive peptides. For example, within the bloom-forming cyanobacterium Planktothrix (Oscillatoria) individual strains producing the hepatotoxin microcystin (MC) differ in their MC content by an order of magnitude. The aim of this project was to identify factors leading to the observed differences in MC content within forty MC-producing strains of Planktothrix isolated from European lakes. For the first time it could be shown that the regulation of particular genes involved in toxin synthesis not only differs between strains but depends on particular mutations in the intergenic spacer regions of the corresponding operon. The differences in transcript amounts between strains can explain a significant part of the variation in MC content among strains. For example, strains carrying a 128-bp deletion in one of the intergenic spacer regions differed in transcript amount from other low MC-producing strains and showed highest MC contents. Further it could be shown that specific enzymatic domains of one toxin synthesis pathway reveal an unexpected promiscuity that can explain the high diversity of structural variants of certain toxin (peptide) families such as the MCs occurring in waterbodies. This finding is new, as analogous enzymatic domains from heterotrophic bacteria or fungi did not show this promiscuity but rather predictable substrate specificity. From an evolutionary point of view, these promiscuous enzyme domains might represent a transitional state in the process of reshaping certain peptide groups towards a modified/novel function as enzyme inhibitors. For the first time the genome of a Planktothrix model strain that is amenable to genetic manipulation has been assembled to one contiguous molecule and five plasmids. Two of the plasmids have been transformed to biparental plasmids replicating both in Escherichia coli and Planktothrix. The resulting plasmids were successfully shuttled between Escherichia coli and Planktothrix. The availability of shuttle vectors is considered of relevance to study the consequences of introducing foreign genes or investigate the function of an unknown gene by overexpression or complementation. In this project the specificity of an associated tailoring enzyme (a type II thioesterase) on the MC synthesis rate could be unequivocally demonstrated. Unexpectedly the experimental generation of mutants that are unable to produce a specific peptide did not show significant alteration in MC transcript amount or MC content or in the content of related peptides either under light-reduced or under nutrient-reduced (nitrate, phosphate) conditions. These results imply that the individual peptide synthesis pathways do not depend on common limiting resources such as precursors but rather are regulated independently on the transcriptional level. Consequently the presence/absence of one specific peptide synthesis gene cluster cannot be expected to have a measurable effect on the synthesis rate of other peptides. This would allow for a high flexibility in the exchange or combination of different peptide synthesis pathways by means of horizontal gene transfer.