Figure 4

Expression of PvuII methyltransferase causes chromosome methylation and mcrBC-dependent chromosome breakage and cell death. (a) Confirmation of chromosome methylation. BIK18260 (mcrB1) cells carrying pEF24 (pvuIIM under the pBAD promoter; see Table 1), were grown in LB broth under antibiotic selection to the mid-exponential phase, diluted to OD600 = 0.1, and further grown in the presence of 0.002% or 0.002% arabinose (ara) to induce expression of M.PvuII. At the indicated time intervals (in minutes), chromosomal DNA was prepared, digested with PvuII endonuclease (TaKaRa Bio), and subjected to pulsed-field agarose gel electrophoresis. M, λ DNA ladder. (b) Chromosome DNA in BIK18261 (mcrBC⁺) carrying pEF24 after induction of PvuII methyltransferase. (c) Ethidium-bromide fluorescence in the well was measured for the experiments in (b). (d) Loss of cell viability. The number of viable cells was monitored in duplicate in two independent experiments. Each value was divided by the value at time zero. (e) Cell shape. The cells were recovered 60 minutes after addition of a higher (0.002%) concentration of arabinose. They were stained with DAPI to visualize nucleoids and were observed by phase-contrast (left) and fluorescence (right) microscopy. The scale bar indicates 10 μm.