Proteomic Studies Of Response Regulator DrRRA And The Exploration Of Targeted Genome Editing System In Deinococcus Radiodurans

Deinococcus radiodurans is one of the most radio-resistant bacteria in the world. It displays remarkable resistance to various ROS-generating agents including ionizing radiation, H2O2 and desiccation. Thus, it became an ideal model organism to investigate the integrity and stability of the genome under stresses. The robust ability of D. radiodurans to survive the extreme stresses was attributed to its distinctive capacity to overcome oxidative damages. On the other hand, a large family of two-component signal (TCS) pathways has been found in D. radiodurans, which play essential role in DNA damage response.Previous studies showed that drRRA, a partner response regulator of TCS, mutation leads to transcriptional down-regulation of numerous genes involved in stress response, DNA replication and repair. The null mutation leads to decreased resistance of D. radiodurans to H2O2 and radiation, suggesting that it is a response regulator with specific DNA-binding activity, and functions mainly through antioxidant pathways. To further clarify the regulatory mechanism of DrRRA, protein expression profiles of the drRRA mutant were analyzed and compared with the wild-type strain under non-stress condition and gamma radiation stress. The main conclusions are:1. A total of 52 proteins displayed significant expression level changes at least 1.5-fold in the mutant relative to wild-type strain, with 31 repressed and 21 induced proteins, indicating that drRRA is involved in cell response of D. radiodurans to gamma radiation at proteomic level.2. The proteins were distributed into functional groups including 7 stress response,16 metabolism regulation, and 20 function unknown proteins.3. Disruptions of several genes encoding the altered proteins including DRA0259 (Catalase E), DR1146 (Conserved hypothetical protein), and DR1538 (Osmotically inducible protein C), reduced the antioxidant activity of D. radiodurans.4. A batch of function unknown proteins were significantly changed in their expressions. To further investigate the function of these genes, we tried to develop a method of the targeted genome editing based on the CRISPR system in D. radiodurans. Preliminary results showed that the Cas9 and the chimeric RNA with strong promoter could be expressed in D. radiodurans. However, the CRISPR system seems not to work at current stage and further optimization are needed.In summary, the different proteomic profile in the drRRA gene mutant compared with wild-type R1 was analyzed. A number of proteins related to cellular resistance to oxidative stress are changed in their translational level in the mutant. Combined with our previous result of transcriptional profile, we further confirmed that inactivation of DrRRA affects the expression of various stress response systems.