Spontaneous DNA breakage in single living cells of Escherichia coli

Generation of controls with a single DSB in a single chromosome per cell. When shifted to restrictive temperature, cells with a temperature-sensitive replication-initiation defective mutation, such as dnaA(TS), continue to divide until most contain a single chromosome [Supplementary Fig. 1]. We exploited this property to generate cells with a single chromosome so that we could assess the ability of a single I-SceI-generated DSB per cell to induce SOS and be detected in the GFP assay. Earlyto mid-log-phase dnaA46(TS) cells cultured in M9 glucose medium at 30C were shifted to 39C [minimum restrictive temperature for dnaA46(TS)] for 3 hours. At this point, cultures were split and part examined for DNA content while the remainder was induced for ISceI expression, as above. The induced cells were incubated with shaking for 3-4 hours at 39C prior to cytometric analysis. To measure DNA content, cells were fixed in 75% ice-cold ethanol, stained with 2μg/ml of propidium iodide (Sigma), per, and analyzed for red fluorescence with the Epics XL-MCL flow cytometer (Beckman-Coulter, Fullerton, CA). Data showing that most of the cells possess a single chromosome as I-SceI induction began are shown in Supplementary Fig. 1. The “1” value for most cells was verified by counting DAPI-stained nucleoids (bacterial chromosomes) per cell using microscopy.