EVIDENCE THAT MICROCYSTIN IS A THIO-TEMPLATE PRODUCT

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectiv ely termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resemble small, cyclic pe ptide secondary metabolites from fungi and eubacteria. Many of these m etabolites are manufactured via a nonribosomal thio-template mechanism . We submit evidence that microcystin is synthesized by a similar mech anism. The organism used in this study was Microcystis aeruginosa PCC7 820. Using the traditional ATP-(PPi)-P-32 exchange assay for thio-temp late activity, we found activity in the presence of the substrate D-am ino acids occurring in microcystin. Thio-template mechanism are known to be unaffected by protein synthesis inhibitors such as chloramphenic ol. We subjected cultures in exponential and stationary growth to chlo ramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remainin g cells increased. We developed an in vitro assay to measure microcyst in synthesis in cell lysates in the presence of chloramphenicol. By su pplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.