Assessing the role of a putative response regulator in sunscreen biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Under exposure to UV radiation, some cyanobacteria can produce scytonemin, which is a yellow to brown, lipid-soluble, non-fluorescent, very stable sunscreen compound. A genomic region associated with scytonemin biosynthesis has been identified in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 (PCC 73102) 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 and are expected to be involved directly in scytonemin biosynthesis. Further genomic characterization of this gene cluster has revealed a putative two-component regulatory system (NpF1277 and NpF1278) upstream from the cluster that is conserved and is likely involved in scytonemin regulation. In this study, we will inactivate the putative response regulator NpF1278 to determine its relationship with the scytonemin biosynthetic gene region. To accomplish this, we prepared a plasmid construct by fusion PCR which contains an in-frame knock out of NpF1278. The sequence of the knock out on the plasmid has been confirmed and we are currently inserting the plasmid into N. punctiforme through bi-parental conjugation. Once the mutant strain is complete we will observe the effect of knocking out this gene on sunscreen production. Furthermore, to observe the interaction of NpF1278 with the putative promoter region of the scytonemin gene cluster, we have expressed the protein with IPTG and are now in the process of purification followed by an electrophoretic mobility shift assay (EMSA). If the protein binds to the promoter region then it is likely that NpF1278 initiates synthesis of scytonemin.http://opus.ipfw.edu/stu_symp2014/1052/thumbnail.jp