FIGURE LEGENDS
Fig. 1. Nitric oxide induces VC1812-VC1815 cluster, includingcry1 (VC1814). A. Genetic arrangement of VC1812-VC1814.B. RNA sequence read analysis of VC1812-VC1814. V.
cholerae C6706 was grown in modified M9 medium at 37ºC stationarily
until mid-log phase. 50 µM NO donor DEA NONOate was then added and
incubated for an additional 30’. Total RNA was extracted for RNA
sequencing and analysis. The data represent means ± SD from three
independent RNA sequence readings. *, P<0.05; ****,
P<0.0001 (determined by two-way ANOVA). C. The effect
of NO on the expression of two other photolyase genes. The ratio was
calculated by dividing +NO reads over -NO reads. D.cry1-lacZ . Overnight
cultures of wildtype containing a Pcry1-lacZ transcriptional
reporter plasmid were inoculated into modified M9 medium, without or
with 30 µM NO donors DEA NONOate and DETA NONOate. These cultures were
then grown at 37°C stationarily for 6 hrs.
Miller units were used to
determine promoter activity. The
data shown are means ± SD from six independent experiments. **** ,P < 0.0001 (determined by Unpaired t -test).
Fig. 2. Blue light induction of cry1 and its effect onV. cholerae responses. A. Activation of cry1 by blue
light. Overnight cultures of wildtype containing a Pcry1-lacZplasmid were inoculated into modified M9 medium. The cultures were
either shielded (-BL control) or exposed to blue light (+BL). Cells were
collected at the indicated time points for β-galactosidase activity
assays. The data shown are means ± SD from four independent experiments.
***, P < 0.0005; ****, P <
0.0001(determined by Unpaired t -test). B. Blue
light-induced cellular DNA damage.
Overnight cultures of wildtype orΔcry1 mutants containing chromosomal
Ptet -mCherry and
PrecA -gfp reporter plasmids were diluted
into artificial seawater. The cultures were either shielded or exposed
to blue light for 16 hrs. Individual V. cholerae cell intensity
of green fluorescent protein (GFP) and mCherry was quantified using a
Nikon NiU fluorescence microscope. Around 50 cells were analyzed for
each condition, and the red lines represent the mean GFP intensity
normalized to mCherry intensity. ns, non-significance; **,
P<0.005; ***, P<0.0005, ****; P<0.0001
(determined by one-way ANOVA). C. Intracellular ROS
accumulation. Mid-log phase cultures of wildtype or Δcry1 mutants
containing chromosomal Ptet -mCherryreporters were resuspended in PBS buffer containing 10 µM of DCFDA dye.
The mix was either covered (-BL) or exposed to blue light (+BL) for 2
hrs. Fluorescent signals and mCherry intensity of each V.
cholerae cell were measured using a Nikon NiU fluorescence microscope.
Approximately 60 cells were analyzed for each condition, and the red
lines are the mean of fluorescent intensity normalized against mCherry
intensity. ***, P<0.0005, ****; P<0.0001 (determined
by one-way ANOVA). D. Cry1 impact on V. cholerae blue
light survival. Wildtype(vector), Δcry1 (vector), and ∆cry1(Ptac -∆cry1 ) were grown in LB containing
appropriate antibiotics and 1 mM IPTG at 37°C stationarily for 16 hrs.
The cultures were then diluted into artificial seawater and exposed to
blue light. Viable cells were determined at the time points indicated by
serial dilutions and plating. The data shown represent means ± SD from
four repeats. **, P < 0.005; ****, P <
0.0001 (determined by t test and compared to wild type at each time
point).
Fig. 3. Influence of Cry1 pre-induction on V. choleraesurvival under blue light exposure. A. In vitro . WT (vector),∆cry1 (vector), and ∆cry1(Ptac -∆cry1 ) were incubated in LB media
supplemented with 1 mM IPTG, with or without 100 µM of DEA NONOate and
DETA NONOate. After stationary incubation at 37°C for 16 hrs, cultures
were diluted 1000-fold into artificial seawater, shielded or exposed to
blue light for 8 hrs. Percentage survival was determined by normalizing
the exposed cells to non-exposed cells. The data shown represent means ±
SD from three repeats. ns, non-significant; **, P <
0.005; ***, P < 0.0005; ****, P <
0.0001 (determined by two-way ANOVA). B. Schematic diagram of
the mouse experiments. ABX water includes 1 mg/ml streptomycin and 0.5%
aspartame. AG: aminoguanidine, administered at 1 mg/ml in water with
daily 1 mg/mouse intragastrical inoculation. C. Competitive
index. Fecal pellets were collected and purified two days after V.
cholerae mouse inoculation. Approximately 106 CFU/mlV. cholerae cells were then dilutedinto artificial seawater. The
cultures were either covered or exposed to blue light for 6 hrs, and
viable cells were quantified.
Competitive index was calculated
as ∆cry1 /WT. ns, non-significant **, P < 0.005
(determined by t -test).
Fig. 4. Impact of Cry1 on ROS resistance. A. NO
resistance. Wildtype and Δcry1 mutants were grown in modified M9
medium at 37°C stationarily, either without or with 750 µM DEA NONOate
and DETA NONOate. OD600 was recorded. The data shown
represent means ± SD from four repeats. B. Disc diffusion
assays. Approximately 108 overnight cultures of WT
(vector), ∆cry1 (vector), and ∆cry1(Ptac -∆cry1 ) were added to 0.4% soft agar
supplemented with 500 µM IPTG. 4 µl of 9.8 M
H2O2 was placed on a disk at the center
of each plate. After incubation 37°C for 16 hrs, the inhibition zone was
measured. Photographed plates were pre-incubated at 37°C for 1.5 hr
before photographed and clear zone measured. Representative images are
displayed. The data shown represent means ± SD from four repeats. ns,
non-significant; ****, P < 0.0001. C. Liquid
assays. Mid-log phase cultures of WT (vector), ∆cry1 (vector),
and ∆cry1 (Ptac -∆cry1 ) were treated
with 1 mM of H2O2 for 1 hr. Viable cells
were enumerated and percentage survival was determined by normalizing
against non-treated cultures. The data shown represent means ± SD from 7
repeats. ns, non-significance; ****, P < 0.0001
(determined by one-way ANOVA), D. ROS-induced cellular DNA
damage. Overnight cultures of wildtype or Δcry1 mutants
containing chromosomal Ptet -mCherry and
PrecA -gfp reporter plasmids were
inoculated into modified M9 medium and grown until mid-log phase. 1 mM
H2O2 was then added to half of the
cultures and continued to incubate at 37ºC for 1 hr. Individual V.
cholerae cell intensity of green fluorescent protein (GFP) and mCherry
was quantified using a Nikon NiU fluorescence microscope. Around 120
cells were analyzed for each condition, and the red lines represent the
mean GFP intensity normalized to mCherry intensity. ns,
non-significance; ****, P<0.0001 (determined by one-way
ANOVA). E. Cry1 Domain structure. F. ROS sensitivity
of E. coli Δphr B mutants. Mid-log phase cultures of E.
coli WT (MG1655) and ΔphrB were treated with 15 mM of
H2O2 for 1 hr. Viable cells were
enumerated and percentage survival was determined by normalizing against
non-treated cultures. The data shown represent means ± SD from 9
repeats. *, P < 0.05 (determined by t -test).
Fig. 5. Regulations of NO-mediated cry1 induction. A.Involvement of NorR, RpoE, and ChrR. Overnight cultures of ΔnorR ,ΔrpoE , and ΔchrR containing cry1-lacZ reporter
plasmids were inoculated into modified M9 with or without 30 µM DEA
NONOate and DETA NONOate, incubated stationarily at 37°C for 6 hr.β- galactosidase assay was then performed. The data shown
represent means ± SD from three repeats. ns, non-significant; *,P < 0.05 (determined by Unpaired t -test).B & C. Role of cysteine residues in ChrR functions. Overnight
cultures of ΔchrR (vector), ΔchrR
(Ptac-chrR WT and cysteine→serine derivatives)
harboring a cry1-lacZ reporter plasmid were inoculated in
modified M9 medium supplemented with 5 mM IPTG. The cultures were
incubated with or without 30 µM DEA NONOate and DETA NONOate
(B ) or exposed to blue light (C ) for 6 hrs. The data
shown represent means ± SD from four repeats. **, P <
0.005; ***, P<0.0005; ****, P<0.0001 (determined by
Unpaired t -test).
Fig. 6. Enhanced environmental survival of V.
cholerae through in vivo-induced Cry1: a working model. DuringV. cholerae infection, the activation of cry1 by
host-derived RNS occurs via the ChrR-RpoE pathway. This activation
primes V. cholerae to confront aquatic environmental challenges,
including blue light exposure, by engaging Cry1-mediated DNA repair
mechanisms and bolstering resistance against (ROS).