THE RADIATION TOLERANCE OF IGNICOCCUS SPECIES - THEIR ASTROBIOLOGICAL RELEVANCE AND IMPLICATIONS TO DNA REPAIR PROCESSES

Kurzfassung

The environmental conditions on early Earth were harsh and hostile for life compared to environmental conditions prevailing on present Earth. The atmosphere during the Archaean Age (3.8-2.5 Ga ago) was essentially anoxic and the lack of an UV-absorbing ozone layer enabled the solar ultraviolet radiation spectrum to penetrate Earth´s surface increasing the overall terrestrial UV stress. In addition, elevated radiation levels in terms of ionizing radiation contributed to this rugged terrestrial environment. The Late Heavy Bombardment of the planet took place heaviest until about 3.8 Ga and may have heated up the ocean partially over 100 °C. Nevertheless, life has evolved during the Archaean under these circumstances inhabiting our planet since about 3.8 Ga. The potential setting under which life has evolved fascinates and still encourages humans to think about it. Different ideas, hypothesis and opinions about the Last Universal Common Ancestor (LUCA) and essential abilities needed for the propagation of life are still under debate. The underlying work emphases a hot origin of life and focuses on representatives of the genus Ignicoccus isolated from (deep-sea) hydrothermal vents. All representatives of this genus belong to the crenarchaeal branch and follow a hyperthermophilic, chemolithoautotrophic mode of life, living as obligate anaerobes growing by sulfur reduction. Ignicoccus species are promising candidates for early Earth inhabitants because they combine several abilities which may have been advantageous to withstand early Earth´s harsh environmental conditions including elevated levels of radiation. Results of this work show that Ignicoccus species tend to survive high doses of ionizing radiation. This observation was the starting point to investigate the resistance of all four representatives of this genus with respect to different radiation types, ionizing radiation (Xrays, γ-rays) and non-ionizing radiation (UV-C). All tested species demonstrated similar inactivation tendencies after non-ionizing radiation exposure resulting in a F10-value of ~300 J/m2. Additionally, I. hospitalis and “I. morulus” showed a high tolerance to ionizing radiation exposure with a D10-value of ~5 kGy. Besides this impressive radiation tolerance, it was possible to demonstrate for the first time, that a so called VBNC (viable but nonculturable) state may also exist for Archaea after ionizing radiation exposure. Viable and culturable cells were microscopically observed after exposure to 60Co radiation doses of <19 kGy, passing a transition state, and reaching the VBNC state after doses of >27.2 kGy. This observed VBNC state was ascribed to the ongoing metabolic activity, thus H2S production could be monitored. Additional experiments showed that the ionizing radiation tolerance of I. hospitalis seemed to be unaffected by pre-cultivation temperature and the temperature during radiation exposure. However, the tolerance of I. hospitalis to ionizing radiation accompanied by active repair of radiation induced DNA damages was Abstract XI investigated in more detail. It was shown that the PCR-based randomly amplified polymorphic DNA (RAPD) analysis method was a powerful tool to visualize radiation induced DNA damages thus inferring genomic DNA integrity. This method allowed monitoring the DNA repair over time. It was demonstrated that the overall genome integrity was highly affected by both types of radiation and that RAPD analysis represents an attractive alternative for the commonly used and time consuming pulse-field gel electrophoresis (PFGE). I. hospitalis showed fast DNA repair after ionizing radiation exposure; the repair seemed to be completed within one hour. Due to the fact that I. hospitalis was able to withstand these high radiation doses, it was of great interest to investigate whether classical genes involved in DNA repair (e.g. rad2, rad50, recB and radA) were up- or down-regulated upon irradiation. Gene specific primers were designed for qRT-PCR studies and tested under varying experimental conditions. An upregulation of gene expression was detected for the genes mentioned above after 1500 Gy with I. hospitalis cells, when exposed in their early exponential phase. These promising results gave the first indication in regards to its radiation resistance capabilities and further investigation in terms of transcriptomics is definitely warranted. It is very likely that additional mechanisms may support this unusual high radiotolerance. Post-translational modifications for example would point to a completely new way of thinking in terms of the radiation tolerance of I. hospitalis and would allow the regulation of potentially high levels of repair proteins present due to its hot lifestyle. The surprisingly high radiotolerance may also be supported by a potential polyploidy, an increased genome copy number resulting in an enhanced resistance against DNA-damaging conditions. Nevertheless, I. hospitalis has not yet been observed in terms of post-translational modifications or polyploidy; these are promising experiments for follow-up studies. All underlying results obtained with these studies add new pieces to the puzzle how life on Earth may have evolved and the successful propagation under harsh and life hostile conditions.