Fig 4. Expression of the N-terminal DNA binding domain of Ref induces the SOS response.
A. Induction of the SOS response in P1 lysogens by 8 ng/mL ciprofloxacin. E. coli strains EAR15, EAR16, and EAR17 (non-lysogen, P1ref+lysogen, and P1Δref lysogen, respectively) were grown at 30°C. Each strain contained a plasmid (pEAW903) expressing SuperGlo GFP from the recN promoter, allowing the SOS response to be reported as fluorescence. After reaching log phase, ciprofloxacin (8 ng/mL) or water was added (time = 0) and growth continued under the same conditions. Δfluorescence (as calculated in Methods) average and standard deviation of three biological and four technical replicates is reported. B. SOS induction by strains expressing Ref in the absence of phage P1. E. coli strains EAR86 (EV), EAR87 (pRef) EAR88 (pRefΔC110), EAR 120 (pRefΔN76), EAR121 (pRefnuc-) and EAR123 (pRbsR) harboring protein expression plasmids and an SOS reporter plasmid were grown to log-phase at 30°C. Cultures were treated with 1% arabinose to induce protein expression (time = 0). Fluorescence and optical density were measured as in panel A. Normalized fluorescence average and standard deviation of at least two biological and two technical replicates is reported for each condition. C. Microscopy of E. coli cells expressing Ref variants. Log phase cultures of EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), EAR98 (pRefΔN76), and EAR105 (pRefnuc-) at 1x108 CFU/mL were treated with 1% arabinose. Cells were outgrown for 4 hours and incubated with DAPI before imaging at 600x magnification using brightfield and fluorescence channels. Scale bar = 10 μm, representative images shown. D. Quantification of cell length data from (C) was obtained using the MicrobeTracker plugin for MatLab [66]. Each counted cell is represented by a single data point with the average and standard deviation for the data shown. At least 100 cells were measured for each condition, except WT Ref in which only 39 cells could be found. **** = p-value <0.0001.