Infinite f200 pro luminometer

The mnimal inhibitory concentrations (MICs) of relacidine A and B were tested with the standard broth dilution method (Wiegand et al., 2008). Polymyxin B was used as a reference. MHB medium was used for all the bacteria tested. Peptides were diluted from 32 μg ml⁻¹ to 0.06 μg ml⁻¹ in a twofold serial dilution and cells were adjusted to a concentration of 5.0 × 10⁵ CFU ml⁻¹. The 96‐well plate was incubated at 28°C for 36 h and the OD₆₀₀ was measured with a Tecan Infinite F200 Pro Luminometer. The lowest concentration that causes invisible growth of bacteria was defined as the MIC values. The experiment was done in quadruplicate for each peptide and each strain.

GLuc bioluminescence was measured 72 h post transfection, employing the Pierce Gaussia Luciferase Glow Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, 20 μL of supernatant from each well was transferred into a flat, white, opaque, 96-well plate (Thermo Scientific, Waltham, MA, USA) and mixed with 50 μL of fresh working solution (i.e., 49.5 μL Gaussia Glow Assay buffer and 0.5 μL 100× Coelenterazine) at room temperature. Bioluminescence (RLU) was measured immediately (integration time 2000 milliseconds) in an Infinite F200 Pro luminometer (Tecan, Männedorf, Switzerland).

The BacTiter‐Glo™ Microbial cell viability assay kit (Promega) was used to determine the intracellular ATP concentration of X. campestris pv. campestris. Cells were grown in LB broth until OD₆₀₀ reached 0.2. Relacidine B was added at a concentration of 0.25 μg ml⁻¹ (1 × MIC) and the DMSO was used as a solvent control. Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP), an uncoupler of oxidative phosphorylation, was used as a positive control (40 μg ml⁻¹). Cells were treated at 28°C for 0, 1, 2, and 3 h. At each time point, 100 μl of cell culture was added to 100 μl BacTiter‐Glo™ reagent and incubated at room temperature for 5 min. Luminescence was with a Tecan Infinite F200 Pro luminometer and the intracellular ATP concentration was calculated with a standard curve made with a commercial ATP solution.

The ATP assay was conducted to measure the ATP level in the cells using the BacTiter‐Glo™ Microbial Cell Viability Assay (Promega). B. subtilis was grown to OD₆₀₀ 0.1 and treated with 1 at 1×MIC, DMSO and CCCP (Carbonyl cyanide m‐chlorophenyl hydrazine, 30 μg/mL). A no‐drug control was also included. Samples were collected after incubation at room temperature for 5, 10, 15 and 30 minutes and snap‐frozen in liquid nitrogen. 100 μL of sample was added to 100 μL of solubilized BacTiter‐Glo Reagent in a 96‐well plate and incubated at room temperature in a shaker for 5 minutes. Luminescence was measured in Tecan Infinite F‐200 Pro luminometer to determine the ATP level. An ATP standard curve in the femtomolar range was also measured with each study. Statistical analysis was done using GraphPad Prism 8. The mean of the amount of ATP for the DMSO (1.25 %) sample for each replicate at each time point was considered as 100 % and the other conditions were expressed as percentages relative to such sample. Ordinary one‐way Anova was performed on the percentage data for all the samples.