METHODS AND MATERIALS
Strains and Culture Conditions. V. cholerae El Tor C6706
(Zhu et al. , 2002) was used as the wildtype strain throughout
this study. Both V. cholerae and E. coli were propagated
in LB (Luria-Bertani) Miller medium with appropriate antibiotics at 37°C
respectively, unless otherwise noted. The minimal medium used in this
study was a modified M9 medium. It contained the standard M9 medium
components supplemented with 0.2% glucose, 10 mM HEPES (pH 7.4), and
0.1% casamino acids. Artificial seawater used in this study were
described previously . For supplying NO donors in cultures, either
diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA
NONOate)(half-life of 2 minutes at 37°C) or
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate
(DETA NONOate)(half-life of 20 hours at 37°C) (Cayman Chemical) was
used.
In-frame deletions of cry1 (VC1814), chrR (VC2301), andrpoE (VC2302) were generated using the multiplex genome editing
by natural transformation (MuGENT) method (Dalia et al. , 2014).
Complementation of cry1 was constructed by PCR amplification ofcry1 coding sequences, followed by cloning it into the pSRKTc
vector, which harbors a lacIQ and a
Ptac promoter (Khan et al. , 2008).
Similarly, Ptac -controled chrR wildtype
and its cysteine-to-serine derivatives were constructed by cloning the
respective sequences into pSRKTc. The cry1-lacZ transcriptional
reporter was constructed by cloning the cry1 promoter region into
pAH6 (Zhou et al. , 2021). V. cholerae containing a
constitutive Ptet-mCherry were constructed via
homologous recombination of PtetmCherry into the
intergenic region between VCA0104-VCA0105 (Chen et al. , 2022).
The PrecA -gfp plasmid reporter wasgenerated by cloning the promoter region of recA into pUA66
(Zaslaver et al. , 2006). The norR deletion construct was
described in (Stern et al. , 2012). The deletion of E. coliphrB (EG10736) was sourced from the Keio collection and
introduced via transduction into MG1655 (Baba et al. , 2006).
Comprehensive protocols for generating these strain constructs and
primer sequences employed are available upon request.
Transcriptomic analysis of NO-regulated genes in V.
cholerae. Wildtype V. cholerae were inoculated into modified M9
medium and incubated without shaking for 4 hrs at 37ºC. One set of
cultures were then exposed to 50 µM DEA NONOate for 30 mins. RNA was
then purified using TRIzol1(ThermoFisher Sci) and RNeasy purification
kits (Qiagen). RNA sequencing was performed by PrimBio Research
Institute LLC (Exton, PA, USA). The detailed procedures and analyses
will be described in another study.
Blue light radiation of bacterial cultures. The blue light
setup was created using 11 strips cut from a 5 m single-color COB blue
LED strip, each measuring 12 cm in length. These strips were linked
together to ensure even light distribution and attached to a glass sheet
with adhesive backing. The apparatus covered an irradiance area of 12 cm
x 18.5 cm, emitting around 200 mW/cm² of 465 nm blue light. Positioned
12 cm above white 96-well plates, it regulated irradiance and minimized
heat-related interference. Continuous operation yielded a temperature of
approximately 30°C. For non-blue light control cultures, 3 layers of
tape were applied to shield the wells. All materials for constructing
the blue light apparatus were sourced from Super Bright LEDs.
V. cholerae mouse colonization. All animal experiments
were performed in strict accordance with the animal protocols that were
approved by the IACUC of the University of Pennsylvania.
The streptomycin-treated adult mouse model (Chen, 2022) was used to
examine the effect of cry1 preinduction in vivo . Briefly,
six-week-old CD-1 mice were used. 0.5 % (wt/vol) streptomycin and 0.5
% aspartame were added to the drinking water throughout the experiment.
After 3 days of streptomycin treatment, 1 mg/ml aminoguanidine (AG,
Acros Organics) was added to the drinking water of +AG group of mice
throughout the remainder of the experiment. Additionally, the equivalent
of 1 mg of AG was orally gavaged into each mouse in the +AG group daily,
throughout the remainder of the experiment. One day after starting the
AG treatment, approximately 108 CFU of each of the two
differentially labeled strains (wildtype was kanamycin-resistant andΔcry1 was spectinomycin-resistant) were mixed at a 1:1 ratio and
intragastrically administered to each mouse. Fecal pellets were
collected from each mouse at the indicated time points, resuspended in
PBS buffer; fecal debris was removed, and bacterial cells were
inoculated into artificial seawater for blue light exposure experiments.
Simultaneously, the samples were serially diluted, and then plated on
plates containing appropriate antibiotics.
DNA damage-induced SOS responses. V. cholerae harboring
chromosomal Ptet-mCherry and
PrecA -gfp plasmids were exposed to blue
light for 16 hrs or treated with 1 mM
H2O2 for 1 hr. Cells were washed with
PBS and affixed to 1% agarose pads on microscope slides. Fluorescence
was measured through a Nikon Eclipse Ni-U microscope using a DS-Qi2
camera. Multi-filter composite images composed of phase contrast, GFP,
and mCherry filter cubes were taken through the Plan Apo 60x oil
objective and Ph3 condenser settings, and analysis was conducted on a
per-cell basis using the Nikon NIS Elements imaging software. Single
cell GFP fluorescence was normalized to the mCherry background of
individual cells.
Intracellular ROS accumulation.Intracellular ROS levels ofV. cholerae were measured by staining bacteria with the
redox-sensitive, cell-permeable dye 2’,7’-dichlorodihydrofluorescein
diacetate (DCFDA). Mid-log phase cultures of wildtype and Δcry1containing a chromosomal Ptet -mCherry were
resuspended in PBS buffer with 10 µM DCFDA (Abcam). Cells were then
covered or exposed to blue light for 2 hrs. Fluorescent signals and
mCherry intensity of each V. cholerae cell were measured using a
Nikon NiU fluorescence microscope.
H2O2 disc diffusion assays.Overnight cultures of V. cholerae wildtype, Δcry 1, andΔcry 1 (Ptac -cry1 ) were added to
individual tubes of 0.4% soft agar containing 500 µM IPTG. Discs were
then placed onto the soft agar and 4 µl of 9.8 M of
H2O2 was added. Plates were incubated
overnight at 30°C. Photographs were taken and zone of inhibition was
measured.