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Ambros et al. Microbiome Res Rep 2023;2:34 https://dx.doi.org/10.20517/mrr.2023.18 Page 3 of 19

METHODS
Bacterial cultivation
[26]
L. curvatus bacterial glycerol stocks (1:1,000 final dilution) were inoculated in fresh MRS broth , in which
ammonium citrate (mMRS) was substituted for di-ammonium hydrogen citrate (Carl Roth). The overnight
cultures were incubated static at 30 °C with a closed lid until the next day.

Prophage induction
For induction with UV light, overnight cultures were first diluted 1:50 in 50 mL fresh, preheated (30 °C)
mMRS medium and incubated at 30 °C, static, with a closed lid, until an optical density (OD ) of 0.3-0.4
600
was reached. 10 mL of bacterial suspension was transferred into sterile 100 mL Erlenmeyer flasks and
irradiated for 4 min with a UVT-28 M (Herolab) transilluminator, featuring eight 8 W UV-B light tubes
(Herolab; Cat. No. 2984400; spectrum optimum at 312 nm wavelength). Homogeneous UV light exposure
was ensured by shaking the cultures periodically during radiation treatment.

For mitomycin C induction, 15 mL preheated mMRS medium was set to an OD = 0.05 with overnight
600
cultures. When an OD = 0.1 was reached, mitomycin C from Streptomyces (Sigma-Aldrich) was added in
600
different concentrations (20, 10, 5, 0.5, 0.2, and 0 µg/mL).

Growth after induction was monitored at 30 °C in a FLUOstar Omega reader (BMG LABTECH GmbH) for
12 h. Measurements were taken every 5 min in a 48-well microplate format with 1 mL sample volume per
well and double orbital shaking at 200 rpm prior to each measurement.

Phage purification
Virions containing bacterial cell lysates were harvested 24 h after induction by sterile filtration using
Filtropur S 0.45 µm filters (Sarstedt). Virions were precipitated by adding 4% (w/v) PEG 8000 (Sigma-
Aldrich) and 0.5 M NaCl (Carl Roth) (both final concentrations) and incubating for 1 h on ice. After
centrifugation (1.5 mL lysate volume: 4 °C; 13,000 × g; 10 min), the supernatant was discarded and phage
pellets were solubilised in 1/10th of the former sample volume with SM buffer with gelatine . For 1 L SM
[27]
buffer with gelatine, 5.8 g NaCl (Carl Roth), and 2.0 g MgSO ·7 H O (Sigma-Aldrich) were dissolved in
4
2
800 mL demineralised water and 50 mL 1 M Tris-Cl and 5 mL gelatine solution [aqueous solution with 2%
w/v gelatine (Carl Roth)] were added before adjusting the volume to 1 L. For 1 L Tris-Cl, 121.1 g/L Tris base
(MP Biomedicals) was dissolved in 800 mL water, and after adjusting the pH to 7.5 with concentrated HCl
(Carl Roth), the volume was adjusted to 1 L with demineralised water. Sterilisation by autoclaving was
performed at 121 °C for 15 min. Enriched phage samples were stored at 4 °C.

Electron microscopy
Negative staining transmission electron microscopy (TEM) of enriched phage solutions was performed as
[28]
described previously . For this, 5 µL of the sample was applied to glow-discharged and carbon-coated
copper grids. After blotting on filter paper, samples were washed twice with double-distilled water. After
negative staining with 2% uranyl acetate for 20 s, samples were blotted again and air-dried. Electron
micrographs were generated using a Zeiss EM912 (operated at 80 kV in the zero-loss mode) with an
integrated Omega filter (Zeiss), utilising a 2 k × 2 k CCD camera (TRS).

Cell disruption and DNA extraction
Bacterial genomic DNA was extracted from 0.8 to 1.5 mL of an overnight culture using the “E.Z.N.A. ®
Bacterial DNA Kit” (Omega Bio-Tek) according to the manufacturer’s information. For cell lysis, 220 µL
lysozyme solution [10 mg/mL lysozyme (Omega Bio-Tek)] solubilised in TE-buffer [1 mM EDTA-
dihydrate (VWR™), 10 mM Tris (Gerbu), pH 8.0] was added to each centrifuged cell pellet, followed by