Regulatory Mechanisms Of Protein Phosphorylation And Toxin-antitoxin System In Response To Stress In Deinococcus Radiodurans

Deinococcus radiodurans is an extreme microorganism with strong resistance to ionizing radiation,ultraviolet,drying,high temperature and a variety of chemical mutagens.As one of the model organisms to study the survival mechanisms and DNA damage repair ability under extreme environment,the in-depth study on the stress response mechanism of D.radiodurans will be of great interests to explore the unique life processes of organisms under extreme environment.In this study,two important post transcriptional regulatory systems,namely phosphorylation system and toxinantitoxin system,were explored through proteomics,transcriptomes,and molecular biological methods.Proteomics and bioinformatics methods were used to investigate the overall phosphorylation proteomes of D.radiodurans.A total of 265 phosphorylation sites from 135 phosphorylated proteins were identified and annotated in D.radiodurans.Through GO enrichment analysis,KEGG pathway analysis and subcellular localization analysis,we found that phosphorylation modification was involved in a variety of biological processes in D.radiodurans,especially metabolism-related pathways including glycolysis and TCA cycle.By mapping the interaction network of these proteins,they were highly linked with DNA/RNA related protein classification.The overall phosphorylation level and S/T/Y site distribution of D.radiodurans were similar to those of other bacteria.By comparing the phosphorylation profiles of the two group of cells under normal condition and after irradiation,18 proteins with significant differences stand out with their phosphorylation levels.The results combining EMSA,D-loop binding and ATPase experiments and structural analysis showed that the phosphorylation of a newly identified phosphorylation site S174 of Rec A protein directly altered its binding ability to single stranded DNA and double stranded DNA as well as D-loop activity,while had no effect on its ATPase activity.These results suggest that the protein phosphorylation plays an important role in DNA damage response in D.radiodurans.Structural biology,transcriptome analysis and biochemical experiments were used to explore the possible mechanism of maz EF-dr system responding to DNA damage in D.radiodurans.The Maz F-dr protein of D.radiodurans was expressed and purified in E.coli.And the Maz F-dr crystals were obtained and the structure was solved.Maz F-dr protein has a typical Maz F/Ccd B fold of Maz F family,which is very similar to the homologous protein structure including a conserved catalytic site.However,the ?1-?2loop located at the interface of dimer interaction is different from that in E.coli Maz F.By comparing the transcriptome data,650 genes were screened out as possible substrates of Maz F in response to stress.Bioinformatics enrichment results showed that many genes were enriched in metal ion transportation,osmotic stress and cell division.These genes may be linked to the maz EF-dr system,which acts as a global regulator for DNA damage response of D.radiodurans.Biochemical results showed that in the presence of DNA damaging agent,not only the content of iron in cells significantly increased,but also elevated levels of ROS accumulation and protein carbonylation.However,such phenomenon was not found with Maz EF knockout strain.These results suggest that Maz EF-dr system may increase cellular ROS level by accumulating intracellular iron concentration,which ultimately lead to cell death after stress in D.radiodurans.