Figure 2.

Deletion of rnl sensitizes D. radiodurans to gamma irradiation. (A) Diagram of the rnl–DR_B0095 locus in wild-type D. radiodurans (WT) and after homologous recombination with the aadA streptomycin resistance deletion cassette (Δrnl). The co-oriented ORFs encoding DraRnl (in yellow fill) and DR_B0095 (in gray fill) are depicted as horizontal arrows. The aadA cassette (white fill) disrupts the rnl locus; the Δrnl strain retains 96 bp at the 3΄ end of the rnl ORF. Positions of the diagnostic PCR primers used for genotyping are shown. (B) Left panel. PCR confirmation of the Δrnl mutant using the diagnostic primers. The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The positions and sizes (kbp) of linear DNA size markers are indicated on the left. Right panel. Western blot of whole cell extracts of equivalent aliquots of exponentially growing WT and Δrnl cells using an anti-DraRnl antibody. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (C) WT and Δrnl cells were irradiated on ice in 500 μl volumes using a ¹³⁷Cs gamma ray source. Aliquots were removed at the indicated doses, diluted, and plated on TGY agar. Survivors were scored by counting colonies after 3–4 days growth at 30°C. Survival (normalized to mock-treated controls for each strain, defined as 100%) is plotted as a function of IR dose. Each datum is the average survival of three separate irradiated cell aliquots ± SEM.