Cry1 is important for ROS resistance in V. cholerae.
To investigate additional physiological functions of Cry1 beyond its known role as a photolyase, we compared resistance to reactive nitrogen species (RNS) and reactive oxygen species (ROS) between wildtype and Δcry1 . When we exposed V. cholerae to high concentrations of DEA NONOate (with a 2-minute half-life) and diethylenetriamine (DETA) NONOate (with a 20-hour half-life), we observed minimal growth differences between the wildtype and Δcry1 mutants (Fig. 4A).
Next, we employed a disc diffusion assay to assess the susceptibility ofΔcry1 mutants to hydrogen peroxide (H2O2). Notably, the diameter of the inhibition zone for Δcry1 mutants was significantly larger compared to that of the wildtype (Fig. 4B). The introduction of wildtypecry1 in Δcry1 mutants restored hydrogen peroxide susceptibility to levels comparable to the wildtype (Fig. 4B). In the liquid culture experiment, V. cholerae was cultivated to the mid-log growth phase and exposed to 1 mM of H2O2. We report an approximate 99% reduction in the survival rate of Δcry1 mutants, while the impact on the survival of the wildtype and complemented strains was relatively minor (Fig. 4C). Moreover, in response to H2O2 exposure, Δcry1 mutants exhibited elevated expression of recA , whereas no such increase was observed in the wildtype strain (Fig. 4D). These data collectively suggest that Cry1 plays a crucial role in conferring resistance to oxidative stress in V. cholerae .
While V. cholerae Cry1 primarily serves as ssDNA photolyase, Cry1 also harbors a FAD-binding domain in the fully reduced two-electron FADH2 form (Worthington et al. , 2003) (Fig. 4E). It has been shown that the FAD-binding domain has important regulatory roles for cryptochromes and photolyases and has the inherent capability to function as catalysts themselves (Massey, 1994). Reduced flavoproteins can react with various forms of molecular oxygen and may produce the observed ROS-protective effect. Cahoon, et al. reported that the mutation in a photolyase orthologue PhrB in Neisseria gonorrhoeae exhibits increased sensitivity to oxidative killing (Cahoonet al. , 2011). To explore whether other Cry1 homologs contribute to ROS resistance, we assessed ROS resistance in a phrB mutant ofE. coli . Notably, E. coli PhrB is involved in blue-light-dependent repair of cyclobutane pyrimidine dimers (Sancar & Sancar, 1988), thus we compared H2O2resistance between E. coli wildtype and phrB mutants. Strikingly, similar to V. cholerae Δcry1 mutants, E. coli ΔphrB mutants exhibited increased susceptibility to H2O2 (Fig. 4F). These findings imply that Cry1-like photolyase proteins in diverse bacteria may play important roles in conferring resistance against ROS.
ChrR-RpoE mediated regulation of cry1.
Next, we investigated the mechanism underlying the induction ofcry1 expression by NO. Previous studies have elucidated the key role of transcriptional regulator NorR, which senses NO and activates genes responsible for detoxifying of NO (Stern et al. , 2012, Stern et al. , 2013). To test whether NorR is also essential forcry1 transcriptional activation, we evaluated cry1-lacZexpression in ΔnorR mutants in the presence of NO. We observed NO-induced cry1 expression in ΔnorR mutants (Fig. 5A, left panel), mirroring the pattern observed in the wildtype strain (Fig. 1C). These results suggest that the activation of cry1 by NO is not dependent on NorR.
RNA sequencing revealed sigma factor E (RpoE) and anti-sigma factor (ChrR) play a pivotal role in orchestrating transcriptional induction for over 150 genes in response to blue light, including cry1 inV. cholerae (Tardu et al. , 2017). To investigate the potential involvement of RpoE and ChrR in NO induced cry1expression, we measured cry1-lacZ in ΔrpoE andΔchrR mutants in the presence of NO. Strikingly, we observed a complete loss of induction of cry1 in the ΔrpoE mutant (Fig. 5A, mid panel), and the constitutive expression in theΔchrR mutant (Fig. 5A, right panel). These data suggest that the ChrR-RpoE regulatory pathway governs the NO-triggered induction ofcry1 .
V. cholerae ChrR is an ortholog to ChrR from the photosynthetic bacterium Rhodobacter sphaeroides . In R. sphaeroides , ChrR functions as a sensor for singlet oxygen stress arising from photosynthesis, leading to the release of its associated sigma factor, RpoE. This cascade results in the activation of RpoE regulon (Anthonyet al. , 2005, Ziegelhoffer & Donohue, 2009). Both V. cholerae ChrR and R. sphaeroides ChrR belong to the family of zinc-binding anti-sigma factor (ZAS) proteins, featuring a conserved HisXXXCysXXCys sequence motif (Kanget al. , 1999). In R. sphaeroides , these two cysteine residues play a critical role in zinc binding, singlet oxygen sensing, and anti-sigma E activity (Newman et al. , 2001). To investigate the significance of the cysteine residues in this motif of V. cholerae ChrR, we introduced site-directed mutations to replace the coding sequences of ChrR C33 and C36 with serine residues. These mutated constructs were then cloned into a Ptac -controlled plasmid and introducedΔchrR mutants to assess their functionality by measuringcry1-lacZ expression. We found that complementation with wildtype ChrR (ChrRWT) led to a reduction in the basal level expression of cry1-lacZ , with subsequent induction by NO (Fig. 5B). In the strain expressing ChrRC33S, cry1expression was elevated regardless of NO exposure, while in the ChrRC36S-expressing strain, the NO-induced cry1pattern resembled that of the wildtype (Fig. 5B). Correspondingly, upon exposure to blue light, cry1 induction occurred in thechrR WT andchrR C36S-expressed strains, whereas constitutive expression was observed in the vector control andchrR C33S strains (Fig. 5C). These findings collectively suggest that in V. cholerae , the C33 residue plays a pivotal role in mediating ChrR’s anti-Sigma E activity, as well as sensing reactive nitrogen species (RNS) and blue light-induced reactive oxygen species (ROS) signals.