Staphylococcus aureus after mutagenesis
We used a Newman strain with a chromosomal integration of a transcriptional fusion that reports for ComK activity (amyE::PcomG-yfp). This strain was grown in 10 ml of TSB medium at 37C with 200 rpm agitation until the culture reach stationary phase. Cells were collected by centrifugation and resuspended in 5 ml solution of 0,85% trisodium citrate pH = 5 and 50 g/ml of N-methyl N-nitro N-nitrosoguanidine (NG). Cells were incubated for 1h at 37C, washed in PBS buffer and resuspended in 10 ml of TSB medium. Cultures were incubated at 37C with 200 rpm agitation until they reached 2x of the initial OD600. Serial dilutions of the cultures were performed before plating in TSB agar. Colonies that grew on the agar where monitored for fluorescence using a Bio-rad chemi doc system equipped with fluorescence detector.