Response of deinococcus radiodurans to directly damaging genotoxic stressors is regulated through RDR regulon

The Deinococcus radiodurans bacterium is highly resistant to very high doses of ionizing radiation, UV and many genotoxic chemical reagents. The organism responds to the radiation stress through the coordinated expression of genes belonging to Radiation and Desiccation Response (RDR) regulon operated through cis-acting sequence RDRM (Radiation Desiccation Response Motif), trans-acting repressor DdrO and protease PprI (also called IrrE). The role of the RDR regulon in other DNA damaging stresses such as UV rays, chemical mutagens, DNA intercalating agents, oxidative stress, metal stress, etc. is explored by monitoring activation of key RDRM-promoters tagged to GFP reporter. The bacterial strains used were E. coli (DH5α) and D. radioduransR1. The cloning and over expression were done using standard molecular biology techniques. The D. radiodurans clones carrying pKG with different deinococcal RDRM-promoters (P_gyrB, P_ddrB and P₁₁₄₃) were subjected to various genotoxic stresses and the promoter activity was monitored through Gfp fluorescence for 6h during post stress. DdrO abundance in different stresses was detected by immunoblotting using anti-DdrO antibodies. The present study evaluated whether RDR regulon controls the response of D. radiodurans to various DNA damaging stressors, which it is resistant to, such as UV rays, mitomycin C (MMC), methyl methanesulfonate (MMS), ethidiumbromide (EtBr), ROS generating stresses (H₂O₂, MV and metal ions). Activation of 3 RDR regulon genes (ddrB, gyrB and DR1143) was studied by tagging their promoter sequences with a highly sensitive GFP reporter. Here we demonstrated that all the DNA damaging stressors elicited activation of RDR regulon of D. radiodurans in a dose-dependent and RDRM-/IrrE-dependent manner. However, ROS-mediated indirect effects [induced by hydrogen peroxide (H₂O₂),methyl viologen (MV), heavy metal/ metalloid (zinc or tellurite), etc.] did not activate RDR regulon. The level of RDRM-promoter activation was inversely proportional to the cellular abundance of repressor DdrO. The stresses that caused direct DNA damage or distortion in DNA structure were found to be regulated through RDR regulon. The ROS generating stresses that cause indirect damage to DNA did not elicit RDR regulon genes in D. radiodurans. (author)