UC San Diego

Depletion of the stratospheric ozone layer is increasing the level of harmful ultraviolet (UV) radiation reaching the earth's surface. The environmental impacts of this radiation are largely unknown; however UV-induced DNA damage may lead to effects on primary productivity in plants and the prevalence of skin cancer. Cyanobacteria are photosynthetic prokaryotes with an evolutionary history that precedes the development of atmospheric ozone protection. These factors make cyanobacteria a valuable resource for the study of adaptations to UV radiation, including the production of UV absorbing secondary metabolites. One of these metabolites is scytonemin, a yellow-brown pigment found in the sheaths of many cyanobacteria. Its unique dimeric indolic-phenolic structure, powerful UV-A absorbing properties, and biological activity in biomedical assays prompted our study of its biosynthesis. This thesis involved a multidisciplinary investigation of the biosynthesis of scytonemin. Preliminary evidence for genes involved in scytonemin biosynthesis were identified through bioinformatic analyses and semi-quantitative reverse transcriptase PCR using the N. punctiforme ATCC 29133 genome. These genes are shown to be a part of a transcriptional gene cluster that is upregulated after exposure to UV radiation. Examination of this gene cluster across cyanobacterial lineages reveals unique genetic characteristics in individual clusters, and further suggests ancient evolutionary history for its biosynthesis. Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF) mass spectrometry technique was utilized in a novel method to understand the biosynthetic precursors used for scytonemin biosynthesis through stable isotope incubation studies. The MALDI-TOF technique also allowed a unique glimpse into the near-real-time induction and rate of scytonemin biosynthesis. The final study reported in this thesis reveals the function of a gene uniquely present in the scytonemin gene cluster of N. punctiforme ATCC 29133. This gene is shown to encode a protein with in vitro oxygenase activity, similar to the well characterized mushroom tyrosinase