Subcellular Localization of Toxin production

Content: Harmful algal blooms formed by cyanobacteria (cyanoHABs) not only deteriorate ecosystem services, but cause significant economic losses because of managing and treating drinking water and food supplies. Among toxins produced by cyanobacteria, most prominent are the microcystins (MCs) and related peptides, the anabaenopeptins (APs) inhibiting eukaryotic protein phosphatases of higher organisms. Intracellular toxic peptides can be released into the surrounding environment either through cell lysis or through active transport out of the cell. In general ecophysiological variation in cyanotoxin peptide production has been studied in the field and in the laboratory by calculating average contents from a large number of cells. This approach ignores the variability on the individual cell level which is induced, for example, by transposable elements. One possibility to investigate cyanotoxins on the individual cell level is the so-called bioorthogonal labeling. Labeling of cyanotoxins/peptides in real time is based on the discovery of unspecific key enzymes involved in the synthesis pathway of those compounds which also can use non-natural functional groups as precursors. The resulting modified molecule is subsequently labeled by a fluorophore through a so-called click chemistry reaction. Hypotheses: In this project we aim to localize toxic peptides in the cells to investigate the underlying peptide storage and transport mechanism: (1) localize peptides intracellularly and identify the responsible cellular compartments, (2) describe the intercellular variability in peptide synthesis as a result of spontaneous mutations (e.g. transposases), (3) find out whether genotype-dependent release of peptides can explain the large variability in extracellular toxin contents, and (4) analyze the influence of physiological stress conditions on peptide storage and release. Methods: We will perform real time cyanotoxin/peptide labeling and high resolution imaging to localize, quantify and reveal inter/intracellular peptide storage and release using various isolates varying substantially in intra- and extracellular toxic peptide content (0-60% of the total content). Labeled peptides will be quantified by advanced imaging as well as flow cytometry taking into account genetic factors causing intercellular variation as well as physiological factors causing intra- and extracellular variation. Pulsed feeding experiments will be used to understand the fate of labeled peptides over time under both maximum growth rate and physiological stress conditions which will lead to an increased understanding on MC and AP peptide storage in the cells. Innovation: For the first time we will localize cyanotoxins/peptides using the advanced techniques of molecule imaging in real time at the single cell level. This exciting approach has the potential to change our view on driving forces for toxic peptides release into the environment and to offer a new perspective on future water management for toxic cyanobacterial blooming.