Figure 2.

The MTase pre-expression is vital only at the stage of the Csp231I R-M system transfer into a new E. coli host, and C regulator does not play a role in this context. A two-plasmid system was generated. The first one was from series of plasmids harboring Csp231I R-M system variants: p18 (WT C+R+M+), p30 (ΔCR+M+) p18DA (C+R-M+), or pBRtet as a no R-M system control. The second plasmid (pHGMCsp) carried an additional, separate MTase gene on the thermo-sensitive pSC101 replicon. Cell survival after loss of the thermosensitive MTase plasmid was measured using a spotting assay and calculating CFUs. Dilutions of the cultures were spotted onto an agar plates for incubation at a permissive temperature for replication of pHGMCsp (30°C, grey dots) or at a non-permissive temperature, where MTase production is lost (43°C, black dots). To prove the MTase carrying plasmid is lost, the cell death due to lack of bla gene expression at 43°C on ampicillin supplemented plates (white dots) is shown. The average from four replicates is indicated by black bar.