Author: Dr. Tanya Soule Assistant Professor, Department of Biology Indiana University - Purdue University Fort Wayne
Some cyanobacteria can synthesize and accumulate sunscreen pigments. One such sunscreen, the indole-alkaloid scytonemin, is found exclusively among the cyanobacteria as an eco-friendly, photo-stable sunscreen that is produced in response to long-wavelength UVA irradiation (320 to 400 nm wavelength). Furthermore, it has also been documented that scytonemin has anti-cancer and anti-inflammatory properties. Previous studies have focused on the biosynthesis and physical properties of scytonemin, while research on the regulation of scytonemin biosynthesis has been lacking. In our work we evaluated scytonemin regulation at the molecular level, focusing on two genes which encode proteins which collectively function as a two-component regulatory system (TCRS). In general, TCRSs are composed of a sensor/histidine kinase (SK/HK) and a response regulator (RR) protein. When an environmental stimulus, such as UVA radiation, is received, the TCRS responds by autophosphorylation of the HK followed by a phospho-transfer to the RR. The phosphorylated form of the RR usually acts as a transcriptional regulator and activates or represses transcription of a gene or set of genes by binding near the start site of transcription.
Figure 1. Scytonemin biosynthetic gene cluster and associated two-component regulatory system in N. punctiforme. The break represents genes Npun_R1270 through Npun_R1260 and arrows show the direction of transcription. Image is not drawn to scale.
Upstream and adjacent to the genes which encode for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme is a putative TCRS which we hypothesized was involved in regulating scytonemin biosynthesis (Figure 1). Since scytonemin is produced in response to UVA radiation, we wanted to see how this TCRS responded to not only UVA radiation, but also UVB (280 to 320 nm), high light, and oxidative stress (a by-product of UVA radiation). We used quantitative-PCR (qPCR) to measure the expression response of both TCRS genes in N. punctiforme following exposure to UVA, UVB, high light, and oxidative stress for 20, 40, and 60 minutes (Janssen and Soule, 2016). We found that both genes responded similarly to all four conditions and that their response was to generally increase in expression when exposed to UVA, UVB, or high light. Under oxidative stress, however, the genes responded with a slight decrease in expression when compared to the control cultures that were not stressed. This study provided evidence that this TCRS not only responded to UVA stress, which is the main environmental stimulus for scytonemin biosynthesis, but also to similar light-related conditions such as UVB radiation and high light (Figure 2).
Figure 2. Temporal gene expression fold change of Npun_F1277 (diamonds) and Npun_F1278 (squares) from cells exposed to (a) UVA, (b) UVB, (c) high light, and (d) oxidative stress. Error bars represent the standard error of triplicate samples with letters denoting values that are statistically similar (p ≤ 0.05; determined by post-hoc analysis) for each treatment. Note that there were no significant differences between time points for cells under oxidative stress.
The next study was designed to provide direct evidence that the TCRS regulated scytonemin biosynthesis. To do this we focused on deleting the RR gene from the Nostoc punctiforme genome (Naurin et al, 2016). As hypothesized, the mutant strain with the deleted RR gene was unable to produce scytonemin following exposure to UVA radiation. Using qPCR, it was also confirmed that the scytonemin biosynthetic genes were not expressed in the mutant strain under UVA radiation. To further evaluate the phenotype of the mutant strain, we measured the growth rate and pigment profiles of the mutant and compared them to the wild type strain. It was found that the deletion of the RR not only affected scytonemin biosynthesis, but the mutant also produced more of the light-harvesting pigment phycocyanin than the wild type. This suggests that the RR may regulate more than just scytonemin biosynthesis and future studies will focus on characterizing the range of conditions and physiological processes affected by this RR.
Overall we were able to not only confirm the role of the RR in scytonemin biosynthesis directly through the generation of a mutant strain, but we also found that the TCRS involved in scytonemin biosynthesis is also responsive to a variety of light-associated conditions. By further defining the transcriptional regulation of scytonemin biosynthesis, we can progress towards constitutive or controlled expression of this system and enhance efforts towards mass production of scytonemin for biomedical or industrial applications.
Janssen, J. and T. Soule (2016) Gene expression of a two-component regulatory system associated with sunscreen biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133. FEMS Microbiology Letters 363: fnv235
Naurin, S., J. Bennett, P. Videau, B. Philmus, and T. Soule (2016) The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133. Journal of Phycology doi:10.1111/jpy.12414