DFG Priority Program 1115: Mars and the terrestrial planets: Adaptive strategies of microorganisms to extreme environmental conditions: Investigation of the molecular mechanisms of desiccation and UV radiation resistance in the extremophile bacterium Deinococcus radiodurans

Kurzfassung

Deinococcus radiodurans belongs to one of the most ionizing radiation resistant families of bacteria discovered so far and can be regarded as a prime model organism for the investigation of putative life on Mars. It seems to employ only typical prokaryotic repair genes to encounter ionizing radiation induced DNA damage. The molecular basis for its high resistance is still unknown. In this project we could show that D. radiodurans can survive every applied space parameter alone or in combination with the exception of solar ultraviolet (UV) radiation. UV radiation is the biocidal parameter that is present on every solar body. During biological evolution strategies have been developed by all terrestrial organisms to combat this stressor. We could demonstrate that D. radiodurans exhibits a high resistance to monochromatic (λ = 254 nm) and polychromatic UV radiation (λ = 200 – 400 nm). It was hypothesized that the known UV-damage repair pathways like nucleotide excision (ER) and homologous recombination (HR) repair, amongst others, may be highly effective due to D. radiodurans’ particular success to recruit the basic involved components in time at the appropriate spot. First proprietary studies on the distribution pattern of UV-induced bipyrimidine dimers of DNA repair-defective D. radiodurans mutants in comparison to the wild-type strain R1, already succeeded in hypothesizing that D. radiodurans encodes an as yet undefined repair pathway. It is apparent only when the DNA repair pathways ER and HR are inactivated. For the comparison of the post-irradiation DNA repair kinetics of the three selected D. radiodurans strains the UVC-induced photoproducts were isolated after different post-irradiation incubation times and measured using HPLC (high performance liquid chromatography) coupled with tandem mass spectrometry. Astonishingly, a recovery time of 0.5 h was sufficient to give first clues to the UV-repair process flow: The ER efficiently removes both cyclobutane pyrimidine dimers (CPD) and 6-4 bipyrimidine adducts. HR assists removal of CPD from UV-irradiated DNA, but plays a minor role in removal of 6-4 adducts. Though the ER is not existent in the ER-deficient mutant, up to 10 % of both bipyrimidine photoproduct types were equally repaired. Hence, an additional pathway must be available to contribute to the removal of 6-4 adducts. Preliminary DNA microarray data analysis identified 41 genes that are either upor down-regulated in both repair-deficient mutant strains after UVC exposure. Of these, 18 were identified to belong to the top 100 UV-induced and repressed genes in either strain. Due to the genes opposed induction behaviour in the UV-sensitive strains, we come to the following conclusions: a. Six of the 18 genes are part of a HR bypass pathway because they are upregulated in the ER-deficient but down-regulated in the recA-deficient strain. b. Five are part of a regulatory or signalling reaction system only apparent, if both ER and HR are intact, because they are down-regulated in both mutant strains. c. Six up regulated genes function as players of the yet undefined but ER- and HRindependent pathway, which we postulated when establishing the UV-damage distribution patterns of D. radiodurans. The individual role of each of these genes has to be investigated in detail in future experiments.