Abstract (eng)
Most living entities are not able to survive under vacuum (10-4 Pa), extensive UV and ionizing radiation, combined with temperature variations between -100 °C and +100 °C. These conditions are present at the low Earth orbit (LEO), the location of the International Space Station (ISS). However, some microorganisms manage to withstand such harsh environmental conditions for years. One example is the polyextremophile bacterium Deinococcus radiodurans, which was sent to the ISS for exposure between one and three years on the Japanese Exposure Module Kibō. Molecular analysis of the cells after exposure may help understanding how organisms can survive long term exposure to space conditions. Analysing tools include survival assays, cellular characterization by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and interpretation of molecular adaptions via transcriptomic, proteomic and metabolomic approaches. Results of the conducted experiments showed that D. radiodurans indeed can survive long-term exposure outside the ISS and after a short lag phase of few hours in complex medium, it proliferates again. This indicates that an interplanetary travel of certain organisms might be possible. Results presented in this dissertation can be interesting for future space exploration and planetary protection, however, as it similarly important for areas involving stress response from extreme environmental conditions, such as vacuum, radiation and temperature cycles.
The present work comprises different stress response experiments on D. radiodurans. Apart from LEO exposure, several other experiments were conducted with simulated LEO conditions. D. radiodurans was exposed solely to vacuum, to vacuum and UVC radiation, and a combination of vacuum, UVC radiation and temperature cycles, mirroring conditions present outside the ISS. In addition, the response of D. radiodurans to growth under simulated microgravity conditions was studied.
An integrative extraction protocol for limited amounts of D. radiodurans was established to extract mRNA, extra- and intracellular proteins and polar metabolites simultaneously from each replicate. Transcripts were measured on an Illumina HiSeq, proteins in a shotgun proteomics approach on a LC-orbitrap and polar metabolites on a GC-TOF. All applied methods were used for quantitative approaches.
Neither simulated nor real space exposure caused visible damage to the cell wall in D. radiodurans. However, exposure experiments reduced survivability compared to control cells and induced molecular alterations. All applied factors, except microgravity, induced the expression of genes related to DNA repair, especially the UvrABC excision repair mechanism appears as a major component of the repairment of nucleic acid damage after exposure to LEO conditions. Furthermore, proteins to alleviate oxidative stress, for instance catalases, peroxidases or related proteins, were higher abundant after exposure. Generally, the overall level of amino acids and organic acids was lower in D. radiodurans cells, which were exposed to harsh environmental conditions, as they might be used as intermediates in repair processes or as carbon energy source. Moreover, induced expression levels of different regulators were observed, for instance histidine kinases, which may be involved in activation of stress responses. Ultimately, statistical analysis showed that S-layer proteins of D. radiodurans are likely involved in stress response after LEO exposure.
In conclusion, multiple alterations on various molecular levels were observed after LEO or simulated LEO exposure. The combination of several experimental, methodological and bioinformatical strategies presented in this work provide new insights into complex stress response mechanisms, which are triggered through exposure to outer space conditions.