Blue light-induced Cry1 contributes to blue light protection.
As Cry1 repairs cellular DNA damage resulting from exposure to UV/ blue light, we then selected blue light to study how it induces cry1promoter. We constructed an apparatus delivering 465 nm of uniform blue light with an irradiance of approximately 200 mW/cm2. We inoculated overnight cultures of wildtype V. choleraeharboring a Pcry1 -lacZ transcriptional fusion reporter plasmid into M9 minimal media and subsequently exposed them to blue light. In the absence of blue light, cry1 expression remained at a low level (Fig. 2A, black line), however; upon exposure to blue light, the induction of cry1 was observed (Fig. 2A, blue line).
Next, we examined the effect of Cry1 on blue light-induced DNA damage repair in V. cholerae . We constructed an in-frame deletion ofcry1 . We then introduced a recA-gfp transcriptional fusion plasmid in the wildtype and Δcry1 backgrounds to monitor DNA damage (Chen et al. , 2022). In the absence of blue light,recA expression in Δcry1 was higher than that in wildtype (Fig. 2B). Blue light exposure further induced recA expression inΔcry1 but not in wildtype (Fig. 2B). These results suggest that blue light may cause elevated DNA damage in Δcry1 cells. We also measured intracellular reactive oxygen species (ROS) accumulation using the redox-sensitive, cell-permeable dye 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA). Compared to wildtype, more ROS was accumulated inΔcry1 cells regardless of blue light exposure (Fig. 2C). Similar to the recA induction, blue light exposure further increased ROS accumulation in Δcry1 cells (Fig. 2C).
Next, we examined whether Cry1 is important for V. choleraesurvival under blue light exposure. We diluted V. cholerae into artificial seawater (Joelsson et al. , 2007) and subjected the bacteria to varying exposure times of blue light. We found that compared to that of wildtype, viable Δcry1 cells significantly decreased in survivability as blue light exposure time increased (Fig. 2D). Whencry1 was introduced on a plasmid in the Δcry1 mutant, the complemented strain exhibited comparable survivability to the wildtype when exposed to blue light (Fig. 2D). These data suggest that under this experimental condition, Cry1 is important for protection from blue light.