Responses of the hyperthermophilic archaeon Sulfolobus solfataricus to UV-light

In nature UV-light is the most DNA-damaging factor. Photoproducts, that are not removed, result e.g. in the inhibition of replication or can cause lethal mutations. Compared to Eukarya and Bacteria, the DNA-damage response mechanisms in Archaea are not well understood. In particular hyperthermophilic and acidophilic archaea might have to deal with an additional constant challenge to maintain their genomic stability due to their life in harsh environments. This makes them interesting objects to study DNA damage response mechanisms. In this study the transcriptional and cellular reactions of the hyperthermophilic archaeon Sulfolobus solfataricus and its UV-inducible virus (SSV1) to UV-light were investigated by applying a post-genomic approach. SSV1 showed a co-ordinately regulated transcriptional cycle from 0.5 h to 8.5 h after UVtreatment. By using a high-density DNA-microarray approach, the transcripts could be classified into three categories: immediate early (T-ind), early (T5, T6 and T9) and late transcripts (T3, T1/2, T4/7/8 and Tx). The latter were up-regulated upon the onset of viral genome replication. This tightly regulated transcriptional pattern of SSV1 has not been described before for any archaeal virus and is reminiscent of those of many bacteriophages and some eukaryotic viruses. Six host genes were exclusively regulated in an infected strain upon UV-treatment indicating specific virus-host interactions. Among these were genes encoding topoisomerase VI, which probably plays an essential role in the replication of SSV1. All 34 gene products of SSV1 were tested for protein-protein interactions in a yeast two-hybrid approach. Some of the eight observed interactions suggested new putative protein functions involved in the regulation or involved in the particle assembly of SSV1. S. solfataricus exhibited a complex transcriptional and cellular reaction to UV-light. The UV-dependent transcriptional reactions were investigated by a genome-wide DNAmicroarray analysis that extended over 10 time points of two strains. 55 UV-dependently regulated genes that clustered into three major groups were identified. These genes indicated an immediate arrest of replication (cdc6-2, cdc6-1) and a stop in the cell cycle (e.g. soj, ssh7), during the UV-dependent reaction from 1.5 h to 5 h after UV-treatment. In addition potential transcription factors (e.g. tfb-3) were identified, which might be involved in secondary UV-dependent reactions. The induction of an operon involved in homologous recombination (rad50/mre11) indicated the formation of DNA double-strand breaks (DSB). Consistent with this, DNA DSB were observed by pulse-field gel electrophoresis between 2 h and 8 h after UV-treatment, probably as a result of replication stops due to unrepaired photoproducts. Another, rather unexpected finding was the induction of an operon encoding a potential type II/type IV pili biogenesis system (sso0117 through sso0121) for secretion or pili formation. In support of this, a statistical microscopic analysis demonstrated that at least 50-70% of the cells formed aggregates, particularly between 6 h and 8 h after UV-exposure. In addition the study of a deletion mutant verified that the pili are encoded by the potential pili biogenesis operon and that they are essential for mediating the UV-dependent cellular aggregation. Aggregate formation was stimulated by chemically induced DSB in DNA, but not by other environmental stressors, indicating that this reaction is UV-specific. Furthermore, it was shown that UV-light strongly stimulated the conjugation activity of S. solfataricus (with a frequency of up to 10-2), whereas no conjugative activity was observed without UVirradiation. The data of this thesis open new opportunities towards an understanding of the complex mechanisms involved in DNA-repair after UV-damage in Archaea, and provide supporting evidence to a link between recombinational repair via cellular aggregation and subsequent conjugation as major response of S. solfataricus to UV-light damage