Spermine nonoate

For this study, overnight cultures of S. Tm containing their respective pZS-Ptet-mCherry-TGA-YFP-X-ECFP were 1:100 diluted in either LB (pH 7.0), acidic LB (pH adjusted to 4.5 by HCl) or LB supplemented with H₂O₂ at 500 µM (ThermoFisher Scientific), Spermine-NONOate at 0.5 mM (ThermoFisher Scientific) and grown for 24 h at 37°C. Cultures were harvested by centrifugation and resuspended in 20 μl LB. A volume of 1 μl of the resulting cultures were placed on a 1.5% agarose LB pad on a 12-well slide. The bacteria were then imaged by wide-field fluorescence microscopy (see procedure below).

Two 36- to 48-h 5% sheep blood agar (BBL) plates of H. pylori cells grown at 37°C, 5% CO₂ were harvested into 3 mL of SFBB/chol/pH 7 broth. Initial broth cultures in SFBB/chol/pH 7 were inoculated to an OD₆₀₀ of 0.2 and grown overnight at 37°C, CO₂ to reach an OD₆₀₀ of 1.5 to 2.5.
Each culture was then subcultured and allowed to grow until mid-log phase of growth (OD₆₀₀ of 0.8 to 1.2). The cultures were subjected to a 10 μM NO (spermine NONOate; ThermoFisher) challenge or vehicle control (PBS) treatment for 4 h at 37°C, 5% CO₂, 150 rpm. Cells were harvested by collecting 1 OD₆₀₀ unit (~10⁹ cells) and centrifugation, and the cells were resuspended in 1 mL of RNAzol RT (Molecular Research Center Inc). Once resuspended, each sample was frozen at −80°C for subsequent RNA extraction and cDNA synthesis.