Table 2 E. coli and Salmonella mutant strains Salmonella enterica Serovar Enteritidis Mutant Characteristics
Source or reference ΔcyoA SE2472 ΔcyoA::kan This study selleck screening library ΔcyoB SE2472 ΔcyoB::kan This study ΔcyoCD SE2472 ΔcyoCD::kan This study E. coli (from Coli Genetic Stock center) Strain/mutant Strain number Source or reference BW25113 (wild type) CGSC#: 7636  ∆appC JW0960-1  ∆cydB JW0723-2  ∆cyoA JW0422-1  ∆cyoC JW0420-1  ∆cyoD JW0419-1  Culture media Luria Bertani (LB) broth and M9 minimal medium were from BD Diagnostics (Sparks, MD). All bacteria were cultured in LB
broth at 37°C with shaking at 225 rpm or as indicated. Bacterial culture density was measured by OD600nm or by plating serially diluted cultures on LB agar plates and counting colonies after overnight incubation. All chemical reagents were from Sigma Aldrich BX-795 supplier unless otherwise specified. BacTiter-Glo™ Microbial Cell Viability Assay Reagent was from Promega (Madison, WI). Determination of ATP level in bacterial culture Bacteria were cultured in LB broth at 37°C overnight with shaking at 225 rpm. Overnight cultures were diluted 1:100 in fresh LB broth and cultured at 37°C with shaking. Aliquots of cultures were taken after 3, 6, 9, and 24 hours of incubation, and OD600 nm was measured at Gemcitabine each time point. Bacterial cultures were then centrifuged at 16,100 × g for 5 min. Culture supernatant was transferred to a fresh tube and stored at −80°C until assayed. ATP level in bacterial supernatant was determined using BacTiter-Glo™ Microbial Cell Viability Assay
Reagent (Promega, Madison, WI). It is a luciferase – based assay and the ATP level is determined by measuring luminescence levels and comparing to an ATP standard curve. One hundred selleck chemicals microliters of culture supernatant were mixed with an equal volume of BacTiter-Glo™ Microbial Cell Viability Assay Reagent in a 96-well opaque plate and incubated at room temperature for 5 min. After incubation, luminescence was read in a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). ATP standard solutions were prepared using adenosine 5-triphosphate disodium salt hydrate (A2383, Sigma Aldrich, St. Louis, MO) and a standard curve using 10-fold dilutions of ATP standard solutions prepared in H2O was included in each experiment.