Analysis of a Cyanobacterial UV-Sensitive Sensor Kinase Expressed in Escherichia coli

Exposure to ultraviolet radiation (UVR) has been shown to cause cellular damage in cyanobacteria. In response to UVR exposure, some cyanobacteria produce scytonemin, an indole-alkaloid sunscreen capable of absorbing long-wavelength UVA radiation. Previous genomic and transcriptomic analyses have determined that the production of scytonemin is controlled by a two-component regulatory system (TCRS), encoded by Npun_F1277 and Npun_F1278 in the cyanobacterium Nostoc punctiforme ATCC 29133. This TCRS is thought to not only regulate scytonemin biosynthesis, but also other responses to light and UVR stimuli. To better understand the functionality of the sensor kinase (SK) Npun_F1277 and to determine if it could activate alternative UVR protection pathways, the SK was expressed in Escherichia coli. The first objective of this study was to observe and quantify the level of fitness conferred to E. coli expressing Npun_F1277 from N. punctiforme (strain SKE) when exposed to white light, UVA, and UVB stress. Results from these experiments do not indicate that expression of the N. punctiforme SK conferred an advantage to E. coli under white light, UVA, or UVB stress based on growth alone. Therefore, the second objective was to study the expression of regulatory genes, such as response regulators, in E. coli that are homologs to those associated with the SK Npun_F1277 in N. punctiforme using quantitative-PCR. Expression of the selected genes was measured following exposure to white light and UVA after 30 and 60 minutes as well as UVB after 15 and 30 minutes. Comparison of SKE to empty-vector (EV) control cells exposed to the same stress showed that there were significant changes in the expression of important regulatory genes (e.g. recA, spoT, relA) in the SKE strain. Moreover, when comparing SKE cells exposed to the same conditions above to unstressed SKE cells, a similar result was seen for SKE cells exposed to UVA and UVB as was found in the studies comparing SKE to EV cells. These results suggest that the SK Npun_F1277 may play a role in multiple defense mechanisms of N. punctiforme in addition to initiation of the scytonemin biosynthesis pathway.