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).