Figure 2.

Toxicity of RecF over-expression from the chromosomal locus. Wild type RecF over-expression was realized from a chromosomal construct in which the wild type promoter at the normal recF locus was substituted by the araBAD promoter. (A) Schematic description of the chromosomal over-expression strains. The regulation of recF (EAW1130) or recF_K36R (EAW1148) genes is placed under the control of the araBAD promoter as a termination sequence was added after dnaN, the 3′end of recF encoding gyrB promoter has been duplicated downstream of the Kan cassette. The parental strain carrying the native promoter was used as control. (B) Expression of RecF and GyrB following 6h incubation with increased concentration of arabinose was determined by Western-Blot anti-RecF and anti-GyrB. Similar samples were loaded but the immunoblots are from different membranes. Representative membranes of biological triplicate were used. (C) Determination of the arabinose concentration required to detect RecF over-expression toxicity. Cells were serially diluted and spotted on LB plates after culturing cells for 16 h in presence of the indicated concentration of arabinose. The addition of 10% arabinose caused a dramatic toxicity for those strains over-expressing RecF, whereas no toxicity was observed for the parental or the inducible RecF_K36R. (D) SOS response was monitored over time after RecF over-expression using a PrecN-sfgfp reporter. The fluorescence signal was plotted relative to absorbance at 600 nm under over-expression. Signal started to be recorded 2 h after the inoculation at the 1:1000 ratio in presence of absence of arabinose. RecF over-expression led to a significant increase in the SOS response. Values represented are the mean ± SD of biological triplicates.