Einfluss von genetischen Faktoren auf die Hitzeresistenz von Deinococcus radiodurans

Kurzfassung

To study the role of different DNA repair mechanisms and the impact of pigmentation in the resistance of Deinococcus radiodurans to wet and dry heat, wild-type cells and cells from knockout strains, deficient in RecA, PprA IrrE, and CrtB, were exposed as cells in phosphate buffered saline and air-dried cells to different temperatures (40, 60, 80 and 100 °C) and survival kinetics were determined. It can be shown that all strains were more sensitive to wet than to dry heat. Especially, the strain deficient in recA was highly sensitive to dry and wet heat compared to the wild-type, while pprA and irrE exhibit same response as the wild–type strain. Interestingly, D. radiodurans cells with a deficiency in the carotinoid synthesis of deinoxyanthin (crtB–knockout) were significantly more sensitive to dry heat then recA-deficient strain, indicating the important role of pigmentation in the dry heat resistance of D. radiodurans. These results suggest that the repair of DNA lesions e.g. double strand breaks is essential after exposure to heat stress. The protection from free radical species stress by carotinoids may also play an important role in the cell survival after wet and dry heat exposure. Additionally, to the survival analyses by colony formation, control and heat-treated cells were investigated by fluorescence microscopy to visualize changes in the cell wall, membrane and DNA. To monitor damage in the membrane potential cells were stained with DiBac4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol). Other staining approaches were also conducted with commercial Live/Dead staining Kits, including propidium iodide, and syto9 to give further insights of heat exposure to D. radiodurans cells. In direct comparison, it can be clearly shown that both survival data and information gathered by fluorescence microscopy are in good agreement. One further aim of this thesis was the study of the role of rifampicin-resistance of D. radiodurans cells to heat stress. Ten spontaneous rifampicin-resistant D. radiodurans cells were characterized on their response to 80 °C dry and wet heat stress.