Fig. 3

5-azaC reduces cytosine DNA methylation of the osmE promoter in Escherichia coli. E. coli wild-type strain BW25113 was grown in LB at 37 °C in the absence of 5-azaC and presence of 5 μg/mL 5-azaC. DNA was isolated after two hours (logarithmic phase) and eight hours (early stationary phase). DNA samples were treated with sodium bisulfite. The osmE locus was amplified by PCR using sodium bisulfite-treated DNA as a template. PCR products were inserted into the plasmid pGEM-T Easy, and analyzed by Sanger sequencing. a) logarithmic phase DNA in untreated cells, b) logarithmic phase DNA in 5-azaC treated cells, c) early stationary phase DNA in untreated cells, and d) early stationary phase DNA in 5-azaC treated cells. The numbers represent the position of the cytosines with respect to the transcription start site (+1). The cytosines in the 5′CCWGG3′ site are at positions −13 and −12. Open boxes represent Cs in the osmE sequence (Ts after bisulfite sequencing), and closed boxes represent 5-MeCs in the osmE sequence (Cs after bisulfite sequencing). Rows represent different clones. Xs represent ambiguous sequence information