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.