Black polystyrene plates

Effects of selected nitric oxide donors on light production by a constitutively luminescent Salmonella strain were characterized as indirect assessments of toxicity of the compounds. 1 mL of S. typhimurium 14028 pTIM2442 (harboring the luxCDABE driven by a strong constitutive phage, Alagely et al. 2011 (link)) were washed with BPS and mixed with opportune reagents (BPS, molsidomine, CNC) and grown in black polystyrene plates (Corning, New York, USA). Molsidomine was diluted in PBS (9.89 g L⁻¹) or CNC (Fisher Scientific, Waltham, MA, USA) to final concentrations of 10 µmol L⁻¹. PBS + CNC and PBS alone were used as a control. Luminescence of S. typhimurium 14028 pTIM2442 was measured over time using Victor-2 multimode plate reader (Perkin Elmer, Waltham, MA, USA). Each experiment included 12 replicas.

The tetracycline uptake assay was performed according to the previously described method^{8 (link)}. The tetracycline uptake assay was monitored by fluorescence enhancement of tetracycline after cellular uptake. Cultures of A. baumannii AYE, A. baumannii SDF, E. coli MG1655, and E. coli RPTU54 were grown to OD_600nm ~ 0.6. Cells were pelleted down at 1200 × g for 10 min and resuspended in 10 mM HEPES buffer (pH 7.2) to a final OD_600nm ~ 0.5. Tetracycline was added to the cells at a final concentration of 128 mg/L at this stage. Then, nalidixic acid was added at different concentrations (for A. baumannii AYE 1×, 2×, 4×, and 8× MIC represents 512, 1024, 2048, and 4096 mg/L, respectively; concentrations of nalidixic acid used for A. baumannii SDF, E. coli MG1655, and E. coli RPTU54 is described in Supplementary Fig. 3) in cell suspension and finally pipetted into the black polystyrene plates (Corning®, USA) at 100 µl/well. The fluorescence reading was monitored (at room temperature) using a Synergy^TM H1 Hybrid Multi-Mode fluorescence spectrophotometer (BioTek, USA) at an excitation wavelength of 405 nm and an emission wavelength of 535 nm for 60 min at every 5 min. The fluorescence readings were normalized with respect to tetracycline fluorescence at time zero and plotted against time.

Effects of selected NO donors on light production by a constitutively luminescent Salmonella strain were characterized as indirect assessments of toxicity of the compounds. Two hundred microliters of Luria Bertani broth inoculated with the overnight, 1:50 diluted culture of S. Typhimurium 14028 pTIM2442 (harboring the luxCDABE driven by a strong constitutive phage λ promoter, Alagely et al., [2011 (link)]) were grown in black polystyrene plates (Corning, New York, USA) in presence of serial dilutions of Molsidomine. Molsidomine was diluted in PBS (9.89gL⁻¹) (Fisher Scientific, Waltham, MA, USA) to final concentrations of 10μmolL⁻¹, 10nmolL⁻¹, and 10pmolL⁻¹. PBS was used as a control. Luminescence of S. Typhimurium 14028 pTIM2442 was measured over time using Victor-2 multimode plate reader (Perkin Elmer, Waltham, MA, USA). Each experiment included 12 replicas.