Analysis Of Deinococcus Radiodurans Regulator Related With Antioxidation And Transcriptional Response To UV Radiation

Deinococcus radiodurans is characterized by its extraordinary resistance to all kinds of DNA damaging agents, such as ionizing radiation, hydrogen peroxide, UV radiation, desiccation and other physical and chemical DNA damaging agents However, to date the molecular mechanism responsible for the astonishing resistance is not well understand. It has been demonstrated that damaging effects of ionizing radiation are due to the indirect oxidative stress to DNA. This indicates that the antioxidation ability is importantly contributed to the resistance to ionizing radiation. OxyR which belongs to the LysR family of transcriptional regulator widely exit in bacteria. E. coli OxyR regulates expression of the majority of genes response to hydrogen peroxide. In this study, the function of D. radiodurans OxyR2 (DRA0336) were investigated. In addition, the genome transcriptional profiling of D. radiodurans following 800 J/m2 UV radiation was studied. The results as below:1. Analysis of the D. radiodurans R1 genome using the BLAST program with the E. coli OxyR amino acid sequence as a query sequence revealed the presence of another putative homolog DRA0336 (OxyR2). Multiple amino acid sequence alignment indicated amino acid residues of OxyR2 involved in DNA binding domain, protein tetramerization, possible activating region and key cysteine residue C208 are conserved. It is first found that there exit two homologues in one bacterium. To investigate the function, we constructed the deletion mutant of oxyR2 and the double mutant of oxyR oxyR2/oxyR2. MOxyR2-C strains and MOxyR2-CΔN strains, complemented by full OxyR2 or OxyR2 with N terminal absence of 16 amino acids respectively, were also constructed. PCR site-direct mutagenesis was performed to construct MOxyR2-MC228, MOxyR2-MC272和MOxyR2-MC290 strains.2. All mutant strains and complementary strains were characterized. Survival rate assay with H2O2 treatment indicated that MOxyR2 was significantly more sensitive to H2O2 compared to the wild type. The complementation experiment showed that tans-expression of the OxyR2 or OxyR2 with N terminal absence of 16 amino acids in MOxyR2 could fully restore the H2O2 resistance. C228 is a crucial site for survival in H2O2 stress. The double mutant DM was significantly more sensitive to H2O2 than any of the single mutants. MOxyR2 accumulated much more ROS than the wild type R1 and DM accumulated the highest level of ROS. Deletion of oxyR2 attenuates the enzymatic activity of catalase and DM had almost no increase after the treatment with H2O2. These results demonstrate that the oxyR2 gene is responsible for the sensitivity to hydrogen peroxide.3. To test the role of OxyR2 in D. radiodurans under H2O2 treatment, the whole genome expression profile of MOxyR2 was performed in comparison with R1 after treatment with 20 mM H2O2 during the exponential growth phase. The result showed that total 103 genes changed the expression pattern and exhibited response to ROS emergency. One obvious character was that many genes related with production of ROS, antioxidation and iron metabolism significantly down-regulated.4. To better understand the response mechanism how D. radiodurans coped with the UV irradiation, the investigation of the global gene expression profile in response to UV irradiation was performed. Overall,167 of the total predicted D. radiodurans genes represented on the chip were pronouncedly differentially expressed following UV irradiation of 800 J/m2 after 30 min of post-irradiation incubation. DNA repair gene including uvsE, uvrA2, uvrB, uvrC, uvrD, pprA and recA were induced. The microarray results indicate that there may be no SOS response after UV radiation in D. radiodurans. DNA replication initiation factor dnaA and minE related with mitosis were obviously down-regulated. These indicated that there may exit a period of stasis characterized by repair phase.