A possible functional link between RNA degradation and transcription in Bacillus subtilis Dissertation for the award of the degree “Doctor of Philosophy” Division of Mathematics and Natural Sciences of the Georg-August-University Göttingen within the program “Microbiology and Biochemistry” of the Georg-August University School of Science (GAUSS) submitted by Martin Benda from Prague (Czech Republic) Göttingen 2020 Thesis Committee Prof. Dr. Jörg Stülke (Supervisor and 1st Reviewer) Institute for Microbiology and Genetics, Department of General Microbiology, Georg-August-University Göttingen Prof. Dr. Rolf Daniel (2nd Reviewer) Institute for Microbiology and Genetics, Department of Genomic and Applied Microbiology, Georg-August- University Göttingen Prof. Dr. Fabian M. Commichau Institute for Biotechnology, Department of Synthetic Microbiology, Brandenburg University of Technology Cottbus-Senftenberg Additional members of the Examination Board Prof. Dr. Markus Bohnsack Department of Molecular Biology, University Medical Center Göttingen Prof. Dr. Patrick Cramer Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Stefanie Pöggeler Institute for Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg- August-University Göttingen Date of oral examination: September 17th, 2020 I hereby declare that this Ph.D. thesis entitled “A possible functional link between RNA degradation and transcription in Bacillus subtilis” has been written independently and with no other sources and aids than quoted. Martin Benda Acknowledgements First of all, I would like to express my deep thanks to my supervisor Prof. Dr. Jörg Stülke for the great guidance during this work. He put his full trust in me and gave me the opportunity to work on this intricate, but engaging topic of RNA degradation. I could always turn to him with questions, I have been encouraged in moments of despair and supported in developing my own scientific ideas. My immense thanks go to Prof. Dr. Fabian Commichau and Prof. Dr. Rolf Daniel for being members of my Thesis Advisory Committee and for broadening our view of the topic with meaningful comments and ideas. I would also like to thank Dr. Katrin Gunka who introduced me both to the project and to the lab and made the transition to a new working environment easier for me. I thank G2L Göttingen and especially Dr. Anja Poehlein for all the sequencing performed and discussion of the results. Many thanks go to Julia Busse and Gabriele Beyer for their help with the experimental work. Importantly, huge thanks go to Silvia Carillo-Castellón, all the work on this thesis would have been much more difficult without a constant supply of sterile tips, tubes or freshly prepared media. I would also like to thank my students Simon Wölfel, Jonas Jennrich, Leon Daniau, Melin Güzel, Fabian Fiedler and Maxim Wintergoller, not only for their contribution to this thesis, but mainly for the great time I had working with all of them. I learned many new skills during that time and realized, quite unexpectedly, how much I like teaching. My huge thanks go to all former and current members of the Stülke, Commichau and Rismondo labs I have met here. There were many of you and yet everyone kind, helpful in the laboratory and creating warm and friendly atmosphere not only in the laboratory but also outside. I would also like to express my gratitude to Dr. Libor Krásný and all members of his lab. I learned the essential basics of laboratory and scientific work during my undergraduate time there and when this doctoral thesis turned its way towards his expertise, I received warm and great support again. Finally and above all, I would like to thank my wife Klára for the daily unconditional support in all aspects of life. She always stands by my side and supports me, even if it meant moving abroad to a completely new environment and brought inconvenience to her own career. Without such a great support, I would have never been able to complete this thesis. Big thanks go also to my parents and my whole family, they have always been there for me since my first steps and only thanks to that I got up all the way to this thesis. Table of contents Table of contents ......................................................................................................................................... I List of abbreviations .................................................................................................................................. III Summary .................................................................................................................................................... V 1 Introduction ....................................................................................................................................... 1 1.1 mRNA degradation and RNA degradosomes in bacteria ............................................................... 2 1.2 mRNA degradation and degradosome-like network of B. subtilis ................................................. 6 1.2.1 RNase Y .............................................................................................................................. 9 1.2.2 RNases J1 and J2............................................................................................................... 11 1.2.3 Polynucleotide phosphorylase (PNPase) ........................................................................... 13 1.2.4 CshA, a DEAD-box RNA helicase ........................................................................................ 14 1.2.5 Enolase and phosphofructokinase .................................................................................... 15 1.3 Essentiality and RNase Y ........................................................................................................... 16 1.4 Natural competence in B. subtilis .............................................................................................. 17 1.5 Aims of this thesis ..................................................................................................................... 19 2 Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis .............................................................................................................................................. 20 3 The YtrBCDEF ABC transporter is involved in the control of social activities in Bacillus subtilis ........ 46 4 Discussion......................................................................................................................................... 63 4.1 Suppressor mutant screen revealed initiation of bulk mRNA degradation as the pivotal function of RNase Y ............................................................................................................................................. 63 4.2 Analysis of the rny suppressor mutants brings new insights into the regulation of the RNA polymerase ........................................................................................................................................... 70 4.3 Loss of RNase Y leads to phenotypic effects independent of the total mRNA accumulation........ 73 5 References........................................................................................................................................ 78 6 Appendix ........................................................................................................................................ 101 6.1 Supplementary material.......................................................................................................... 101 6.2 Bacterial strains, plasmids and oligonucleotides ...................................................................... 110 7 Curriculum Vitae............................................................................................................................. 123 I II List of abbreviations General A adenosine NTD N-terminal domain ABC ATP-binding cassette OD optical density Aco aconitase Orn oligoribonuclease AMP adenosine monophosphate P phosphate aphA3 kanamycin resistance gene P promoter Asn asparagine PNPase polynucleotide phosphorylase ATP adenosine triphosphate PCR polymerase chain reaction B. Bacillus PFK phosphofructokinase C cytosine pH power of hydrogen C. Caulobacter RBS ribosomal binding site cat chloramphenicol resistance gene rev reverse CTD C-terminal domain RNA ribonucleic acid DLN degradosome-like network RNase ribonuclease DNA deoxyribonucleic acid rRNA ribosomal RNA E. Escherichia S. Staphylococcus e. g. exempli gratia (for example) S. Streptococcus Eno enolase spc spectinomycin resistance gene ermC erythromycin resistance gene sRNA small RNA et al. et alia (and others) SRP signal recognition particle FDR false discovery rate T thymine Fig. figure Term. transcriptional terminator fwd forward tRNA transfer RNA G guanosine Trp tryptophan Glu glutamic acid Tyr tyrosine H. Helicobacter vs. versus i. e. id est (that is) Δ deletion IPTG isopropyl β-D-1-thiogalactopyranoside kan kanamycin Prefixes LB lysogeny broth Leu leucine k kilo LFH long flanking homology m milli mRNA messenger RNA µ micro nt nucleotide n nano III Units °C degree Celsius bp base pair g gram g standard gravity h hour l liter min minute mol mol M molar s second IV Summary Cellular levels of RNA depend on the rate of its synthesis and degradation. While synthesis is performed by RNA polymerase conserved