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.