Document Type

Master's Research

Degree Name

Master of Science

Department

Biology

Advisor(s)

Tanya T. Soule

Date of Award

5-2015

Abstract

Under exposure to long-wavelength ultraviolet radiation (UVA), some cyanobacteria can produce scytonemin, a yellow to brown, lipid-soluble, non-fluorescent, stable sunscreen compound. A genomic region associated with scytonemin biosynthesis has been identified in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 that contains 18 adjacent genes transcribed in a single direction. Most of the genes in the upstream region of the cluster code for unique proteins involved directly in scytonemin biosynthesis. Further genomic characterization of this gene cluster in N. punctiforme has revealed a conserved putative two-component regulatory system (TCRS; NpF1277 and NpF1278) upstream and adjacent to the biosynthetic cluster that is likely involved in scytonemin regulation. In this study, it is hypothesized that if NpF1278, the putative response regulator, regulates scytonemin biosynthesis in N. punctiforme, there will be a change in the production of scytonemin following UVA radiation when the NpF1278 gene is deleted. To knock out NpF1278 from N. punctiforme, an in-frame marker-less deletion fragment complementary to the 5’ and 3’ ends of NpF1278 which lacks the internal sequence of the gene was constructed and inserted into N. punctiforme through conjugation. The mutant strain was found incapable of producing scytonemin following UVA radiation as assessed using spectroscopy. To further study this system, the interaction of the NpF1278 protein with the promoter region of the scytonemin biosynthetic genes was assessed using electrophoretic mobility shift assays (EMSA). If the NpF1278 protein is associated with the production of scytonemin, it is expected to bind to the promoter region of the gene cluster. Thus far, binding has not been observed. However, failure to bind to the promoter region can occur due to various factors. Preliminary results of this study suggest that the putative response regulator NpF1278 regulates scytonemin biosynthesis in Nostoc punctiforme ATCC 29133. Understanding these regulatory elements may enable future genetic manipulation to artificially control the amount of sunscreen produced for biomedical and industrial purposes.