Nitric oxide induces a photolyase gene in V. cholerae.
In order to study how V. cholerae responds to nitrosative stress, we analyzed the transcriptome of V. cholerae grown in the presence or absence of NO using RNA-sequencing. We grew V. cholerae in modified M9 minimal medium until mid-log phase, then exposed cultures to 50 µM diethylamine (DEA) NONOate (an NO donor with a half-life of 2 minutes at 37°C) and further incubated at 37°C for 30 min. Total RNA was then harvested and subjected to subsequent RNA-sequencing. The detailed results will be described in another study. Briefly, approximately 180 genes were differentially regulated by NO by greater than 3-fold. For example, we found that hmpA andnnrS genes were induced 608-fold and 107-fold by NO, respectively, consistent with our previous findings (Stern et al. , 2012, Stern et al. , 2013). Among other genes whose expression was increased in the presence of NO, we focused on VC1814, located in a gene cluster VC1812-VC1815 (Fig. 1A). All these genes were significantly induced by NO (Fig. 1B). Both VC1812 and VC1815 encode putative oxidoreductase and their cellular functions are unknown. VC1814, called Cry1, encodes a single-stranded DNA-specific photolyase that repairs cyclobutane pyrimidine dimers in ssDNA. As V. cholerae resides in aquatic environments and UV/ blue light induces such DNA damage, we focused on characterizing Cry1 in this study. Interestingly, the expression of two other photolyase genes was not affected by NO (Fig. 1C). To confirm the RNA sequencing results, we constructed a cry1-lacZ transcriptional reporter plasmid and introduced it into wildtype V. cholerae . We found thatcry1 was induced after 6 hrs incubation with NO (Fig. 1D). These data suggest that cry1 is induced by NO.