Intracellular DNA Cleavage Assay in Bacteria

ICL activity was assessed as described previously [15 (link)]. Briefly, 3×10⁶E. coli cells or 6×10⁶Erwinia oleae cells pre-grown for 3.5 h in Dulbecco's modified Eagle medium (DMEM) with 25 mM HEPES (Invitrogen) were mixed with EDTA (1 mM) and 400 ng of linearized plasmid pUC19 DNA and the mixtures were incubated for 40 min at 37 °C. After pelleting the bacteria, the DNA was purified from the supernatant and analysed by electrophoresis on denaturing (40 mM NaOH – 1 mM EDTA) 1 % agarose gels. ICL activity of Erwinia oleae was also tested in the presence of 400 nM 6-histidine-ClbS, which was purified with HisPur nickel-nitrilotriacetic acid (Ni-NTA) agarose (Thermo Scientific) from a culture of BL21(DE3) strain hosting the plasmid pET28a-ClbS-His, as described previously [15 (link)].