| Deinococcus radiodurans is one of the most radiation-resistant organisms and has been generally considered as an ideal model organism for its extreme resistance to ionizing radiation, ultraviolet (UV) irradiation, desiccation, hydrogen peroxide (H2O2), and other DNA-damaging agents. In the D. radiodurans genome, many open reading frames (ORFs) encode probable transcriptional factors. However, their functions, activities, and binding sites have rarely been elucidated. Cyclic AMP (cAMP) receptor proteins (CRPs) are global transcriptional regulators that widely distributed in bacteria. They play important roles in the regulation of many biological processes. However, there is no report elaborate the direct relation between transcriptional regulator CRP and irradiative resistance.D. radiodurans was predicted to have four CRP family proteins, including DR0997, DR1646, DR2362, and DR0834. Recent research indicated that the transcriptional level of dr0997, also referred to as ddrl (DNA damage response gene I), increased 38-fold after ionizing radiation treatment. We also detected the transcriptional changes of crp genes after hydrogen peroxide treatment. The result shown that the transcript level of dr0997 was increased 4 fold and transcript level of the other two crp genes also increased nearly 2 fold. Those results indicated that CRPs in D. radiodurans were involved in the anti-oxidant process and anti-irradiative process. This thesis focused on the function on irradiative and oxidative resistance. The main results are as follows:1. With application of bio informatics, four homologues were discovered. Using alignment analysis, we found out they have similar cAMP-binding domain and DNA-binding domain with E. coli CRP and contain multiple of conservative amino acid residues involved in signal and DNA binding.2. With gene disruption method, we observed that dr0997 was quite important for cell growth, oxidant resistance and irradiative resistance; dr2362 only contributed to oxidant resistance while dr0834 and dr1646 have no contribution.3. Through quantitate real-time polymerase chain reaction analysis, lots of Catalase and SOD coding genes’ transcriptional levels were obviously attenuated after hydrogen peroxide treatment. And lots of DNA repair relative genes, including recA, recG and priA were all attenuated drastically before and after ionizing radiation, meanwhile other DNA relative genes like ddrO, drRRA, ddrB, ddrC, ddrD, recF, recO were all attenuated in different degrees.4. Taking advantage of the conserved nature of the CRP-binding site and electrophoretic mobility shift assay, we found that the transcription of 18 genes, including those encoding DNA repair protein uvsE (dr1819), pprA (dra0346), and recN (dr1447), antioxidant relative protein nuoA(dr1506) and terB (dr2220), and a series of genes associated with cell physiological activities, like sugar metabolism related genes glpF (dr1929) and glgC(dr1689), Lon protease coding genes dr1974 å'Œ dr0349, cAMP metabolism related genes drA0006 and cpdB (dra0006). Meanwhile, we also found DR0997 have a self-regulative ability.5. Through electrophoretic mobility shift assay and P-galactosidase, we found DR0997 can directly regulated a series of tellurite resistance genes, including terE(dr2217), terB(dr2220), terD(dr2221),terA(dr2223), terZ(dr2224) and terC(dr2226). With gene disruption method, we confirmed TerE, TerB, TerD and TerC have contribution to oxidant resistance.Conclusively, DR0997 is essential for the extreme radio-resistance of D. radiodurans. It is a response regulator with specific DNA-binding activity, and functions mainly through DNA repair genes with direct and indirect regulation. The effect of global regulation is still under investigation. |
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