Figure 5.

Effect of deleting DR_B0095 and the rnl–DR_B0095 operon. (A) Diagram of the wild-type rnl–DR_B0095 operon and after homologous recombination with a hygromycin-resistance cassette (hyg) that replaces either DR_B0095 (Δ95) or the entire operon (Δrnl Δ95). Positions of the diagnostic PCR primers used for genotyping are shown. (B) PCR confirmation of the Δ95 and Δrnl Δ95 mutants using the diagnostic primer pairs depicted at bottom. The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The positions and sizes (kb) of linear DNA size markers are indicated on the left. (C) Western blot of whole cell extracts of equivalent aliquots of exponentially growing WT, Δ95 and Δrnl Δ95 cells using an anti-DraRnl antibody (top panel). The positions and sizes (kDa) of marker polypeptides are indicated on the left. A western blot of the extracts with anti-GlnA is shown in the bottom panel. (D) WT, Δ95, Δrnl and Δrnl Δ95 cultures were grown as described and then irradiated on ice in 500 μl volumes using a ¹³⁷Cs gamma ray source. 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.