GENOME AND TRANSCRIPTOME ARCHITECTURE IN PYROCOCCUS FURIOSUS DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Felix Grünberger aus Hutthurm Im Jahr 2020 Das Promotionsgesuch wurde eingereicht am: 18.09.2020 Die Arbeit wurde angeleitet von: PD Dr. Winfried Hausner Unterschrift: …….………………………………….. Felix Grünberger References of Manuscripts This thesis is composed of the following manuscripts: 1. Grünberger, F., Reichelt, R., Bunk, B., Spröer, C., Overmann, J., Rachel, R., et al. (2019). Next Generation DNA-Seq and Differential RNA-Seq Allow Re-annotation of the Pyrococcus furiosus DSM 3638 Genome and Provide Insights Into Archaeal Antisense Transcription. Front. Microbiol. 10. doi:10.3389/fmicb.2019.01603 2. Grünberger, F., Knüppel, R., Jüttner, M., Fenk, M., Borst, A., Reichelt, R., et al. (2020a). Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing. bioRxiv, 2019.12.18.880849. doi:10.1101/2019.12.18.880849. 3. Grünberger, F., Reichelt, R., Waege, I., Ned, V., Bronner, K., Kaljanac, M., et al. (2020b). CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus. bioRxiv, 2020.08.14.251413. doi:10.1101/2020.08.14.251413. Personal Contributions The personal contribution from Felix Grünberger (FG) in the three manuscripts has been adapted from the author contributions section in the manuscripts: 1. RoR prepared the RNA from Pyrococcus. BB, CS, and JO performed the PacBio and Illumina sequencing and FG the nanopore sequencing. The bioinformatical analysis was carried out by FG and RoR. FG, RoR, DG, and WH wrote the manuscript. WH, RR, and DG coordinated and supervised the work. All authors approved the final version of the manuscript (Grünberger et al., 2019). 2. FG established the nanopore workflow and performed all the bioinformatic analysis. FG, R.K., M.J., R.R. and A.B. performed RNA extractions. M.F. helped to optimize the RNA treatment protocol. FG carried out library preparations and performed sequencing. FG, R.K., M.J. carried out H. volcanii wildtype/ΔksgA library preparations and sequencing. M.F. and R.R. performed transcription assays. R.K. and S.F.-C. generated the KsgA deletion strain. R.K. performed primer extension analysis. FG, S.F.-C. and D.G. designed the study, analysed and interpreted the data, and wrote the manuscript with the input of all authors. J.S., W.H., S.F.-C. and D.G. supervised the experiments. S.F.-C. and D.G. initiated and supervised the project (Grünberger et al., 2020a). 3. FG did the DGE and the complete bioinformatic analysis, RR constructed the Pyrococcus deletion strain, IW did the in vitro transcription and the footprinting experiments. VN and LK performed the gel shift assays, KB the ChIP-seq experiments and the qPCR assays. MK, NW, ZE, GM and CZ did the negative-stain TEM imaging. FG, DG and WH wrote the manuscript and DG and WH coordinated and supervised the work. All authors agreed to the final version of the manuscript (Grünberger et al., 2020b). Die vorliegende Arbeit wurde im Zeitraum von Februar 2016 bis September 2020 am Lehrstuhl für Mikrobiologie des Institutes für Biochemie, Genetik und Mikrobiologie der Fakultät für Biologie und Vorklinische Medizin der Universität Regensburg unter Anleitung von PD Dr. Winfried Hausner angefertigt. Table of Contents i Table of Contents List of Main Figures .......................................................................................................... iii List of Supplementary Figures .......................................................................................... iv List of Tables ...................................................................................................................... v List of Supplementary Tables ............................................................................................. v Abstract ............................................................................................................................ vii CHAPTER I General Introduction ...................................................................................... 1 1. Three generations of sequencing technologies .......................................................... 3 2. Archaea: Evolution, model organisms & genomes ................................................. 13 3. The mosaic nature of archaeal transcription ......................................................... 20 4. Scope of this thesis ................................................................................................ 31 CHAPTER II Publications ................................................................................................. 33 Next Generation DNA-Seq and Differential RNA-Seq Allow Re-annotation of the Pyrococcus furiosus DSM 3638 Genome and Provide Insights Into Archaeal Antisense Transcription .................................................................................................... 35 1. Abstract ................................................................................................................. 36 2. Introduction ........................................................................................................... 37 3. Material and Methods ........................................................................................... 40 4. Results and Discussion .......................................................................................... 46 5. Conclusion ............................................................................................................. 60 Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing .......................................... 63 1. Abstract ................................................................................................................. 64 2. Introduction ........................................................................................................... 65 3. Material and Methods ........................................................................................... 67 4. Results ................................................................................................................... 76 5. Discussion .............................................................................................................. 96 CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus ........................................................................................................................... 105 1. Abstract ............................................................................................................... 106 2. Introduction ......................................................................................................... 107 ii Table of Contents 3. Material and Methods .......................................................................................... 109 4. Results ................................................................................................................. 119 5. Discussion ............................................................................................................ 127 CHAPTER III General Discussion .................................................................................... 135 1. Comprehensive summary ..................................................................................... 135 2. Dissecting archaeal transcription ......................................................................... 139 3. Regulation beyond basal transcription ................................................................. 153 4. Conclusion: Perspectives ...................................................................................... 162 Zusammenfassung ........................................................................................................... 163 Bibliography .................................................................................................................... 167 Appendix ......................................................................................................................... 191 Supplementary Figures ............................................................................................... 192 Supplementary Tables: ................................................................................................ 210 Acknowledgements .......................................................................................................... 211 List of Figures iii List of Main Figures FIGURE 1 | GRAPHICAL ABSTRACT. ....................................................................................................................... IX FIGURE 2 | 1ST GENERATION SHOTGUN SANGER SEQUENCING. ................................................................................... 5 FIGURE 3 | 2ND GENERATION MASSIVELY PARALLEL SEQUENCING USING ILLUMINA TECHNOLOGY. ...................................... 7 FIGURE 4 | 3RD GENERATION LONG-READ SEQUENCING TECHNOLOGIES. ................................................................... 10 FIGURE 5 | HISTORY OF ARCHAEA IN A GENOMICS CONTEXT. .................................................................................. 14 FIGURE 6 | GENOMIC FEATURES AND OPERON ORGANISATION. ..............................................................................