Responses of the hyperthermophilic archaeon Sulfolobus solfataricus to UV-light vom Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Doctor rerum naturalium genehmigte DISSERTATION Vorgelegt von Dipl. Biol. Sabrina Fröls aus Frankfurt am Main Darmstadt 2008 D17 Tag der Einreichung: 09. April 2008 Tag der mündlichen Prüfung: 17. Juni 2008 1. Berichterstatterin extern : Prof. Dr. Christa Schleper 2. Berichterstatterin: Prof. Dr. Felicitas Pfeifer 3. Berichterstatter: Dr. habil. Arnulf Kletzin Cellular aggregation of S. solfataricus after UV-irradiation. (DAPI stained, fluorescence micrograph) “Nothing in an organism makes sense except in the light of the functional context.” (Jan-Hendrik S. Hofmeyr) Index I Index Chapter 1 Summary 1 Chapter 2 General Introduction 4 2.1 Archaea - the third domain of life 4 2.2 Archaea as models for the central information processing in Eukarya 6 2.3 Sulfolobus solfataricus and its virus SSV1 9 2.4 Sulfolobus solfataricus - an archaeal model system 11 2.4.1 Cell cycle properties 12 2.4.2 UV-light induced DNA damage and repair 13 2.5 Aims of this study 14 Chapter 3 Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA-microarrays 3.1 Abstract 16 3.2 Introduction 17 3.3 Results 19 3.3.1 UV-effects on growth of host and virus 19 3.3.2 Analysis of the transcription cycle 21 3.3.3 Transcriptional activity in the non-induced state and effects on the host 25 3.3.4 Differences in the transcriptional host reaction after UV-treatment 26 3.4 Discussion 29 3.4.1 SSV1 exhibits a chronological transcription cycle 29 3.4.2 Regulation of SSV1 30 3.4.3 Reaction of the host to virus induction 31 3.5 Materials and Methods 33 3.6 Supplementary data 38 3.7 References 40 Chapter 4 Protein-protein interactions of the Sulfolobus shibatae virus 1 (SSV1) 4.1 Abstract 44 4.2 Introduction 45 4.3 Results and Discussion 46 4.3.1 Interactions involving proteins with known or putative function 46 4.3.2 Interactions involving proteins with unknown function 48 4.4 Materials and Methods 49 4.5 Supplementary data 51 4.6 References 52 Index II Chapter 5 Response of the hyperthermophilic archaeon Sulfolobus solfataricus to UV-damage 5.1 Abstract 54 5.2 Introduction 55 5.3 Results 57 5.3.1 Survival and growth of S. solfataricus cells after exposure to UV-light 57 5.3.2 UV-exposure induces the formation of cell aggregates 58 5.3.3 Analysis of cellular DNA content by flow cytometry 60 5.3.4 Formation of double-strand breaks 61 5.3.5 General transcriptional response 62 5.3.6 Differential reaction of the three cdc6 genes in Sulfolobus 65 5.3.7 UV-induced transcriptional response in S. solfataricus is limited to 55 genes 65 5.3.8 Proteins potentially involved in the repair of DNA-damage 67 5.4 Discussion 69 5.4.1 Growth inhibition, cell death and cell cycle perturbation 70 5.4.2 Formation of double-strand breaks and induction of the recombinational repair system 71 5.4.3 Formation of cell-to-cell contacts – an indication for conjugation ? 72 5.4.4 Conclusion 72 5.5 Materials and Methods 73 5.6 Supplementary data 77 5.7 References 86 Chapter 6 UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation 6.1 Abstract 90 6.2 Introduction 91 6.3 Results 94 6.3.1 UV-inducible Induction of operon SSO0120 94 6.3.2 Maturation of prepilins 96 6.3.3 UV-induced pili formation 97 6.3.4 Cellular aggregation after UV-treatment 98 6.3.5 The gene products of the UV-inducible pili operon are responsible for pili formation and mediate cellular aggregation 100 6.3.6 Cellular aggregation is not inducible by other environmental stressors or in late growth phases 104 6.3.7 Cellular aggregation is induced through treatment with DSB-inducing argents 104 6.3.8 UV-light induced conjugation in S. solfataricus 106 6.4 Discussion 108 6.4.1 UV-inducible pili mediate cellular aggregation 108 6.4.2 UV-inducible cellular aggregation is highly dynamic 109 6.4.3 UV-light is the only identified stressor to induce auto-aggregation 110 6.4.4 Cellular aggregation is induced by double-strand breaks and might mediate a recombinational repair system via conjugation 111 6.5 Materials and Methods 113 6.6 Supplementary data 119 6.7 References 123 Index III Chapter 7 General Discussion 128 7.1 The UV-induced proliferation of the Sulfolobus shibatae virus 1 129 7.1.1 The UV-induced proliferation of SSV1 is correlated with the host response 129 7.1.2 T-ind may be involved in the UV-induced transcription and replication 130 7.1.3 SSV1 probably encodes its own transcriptional repressor 130 7.1.4 First insights into the virus-host interaction after UV-induction 131 7.1.5 Conclusions and outlook 131 7.2 The transcriptional and cellular reactions of S. solfataricus to UV-light 132 7.2.1 Genes involved in replication and transcription 132 7.2.2 Transcriptional reactions of the predicted UV-damage repair genes in S. solfataricus 133 7.2.3 Comparison of DNA-microarray analyses in S. solfataricus 135 7.2.4 UV-irradiation causes double-strand breaks in DNA 138 7.2.5 How is UV-damage sensed in Sulfolobus ? 139 7.2.6 The UV-induced cellular aggregation and conjugation of S. solfataricus 140 7.2.7 Conclusions 141 7.2.8 Outlook 142 Chapter 8 Literature 144 Chapter 9 Abbreviations 151 Chapter 10 Eidesstattliche Erklärung 152 Chapter 11 Curriculum Vitae 153 Chapter 12 Acknowledgments 155 Chapter 1 – Summary 1. Summary 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 UV- treatment. 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 DNA- microarray 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 1 Chapter 1 – Summary 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 UV- irradiation. 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.