title: Pyrazine_437Treatment
design: A pyr K. oxytoca glycerol stock was streaked onto an LB-Agar plate supplemented with spectinomycin (50 g/mL) and incubated aerobically at 30C for 24 hrs in a stationary incubator. Single colonies were grown for 24 hrs aerobically in 5 mL of M9 minimal medium supplemented with 5 g/L casamino acids and 0.4% D-galactose at 30 C and 250 rpm. Each pyr K. oxytoca overnight cultures was diluted 1:1000 into fresh medium (5 mL) and incubated aerobically at 30 C and 250 rpm to mid-exponential growth (OD600 = 0.8). These conditions abrogated both detectable pyrazine and pyrazinone production. At this point, 10 M of 3 or 7, in DMSO, were added to the cultures; an equal volume of DMSO was added to cultures without 3 or 7 as a solvent vehicle control. The cultures were incubated under aerobic conditions with the compounds for an hour at 30 C and at 250 rpm. After incubation, the cultures were centrifuged, the supernatants were decanted, and the cell pellets were quickly resuspended in 5 mL of Bacterial RNAProtect (Qiagen). The samples were centrifuged again, and the pellets were frozen on dry ice and stored at -80 C until RNA extraction.