The LiaFSR Three‐Component System of Bacillus subtilis: Mechanism of Stimulus Perception and Signal Transduction Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig‐Maximilians‐Universität München vorgelegt von Karen Schrecke aus Haldensleben München 07. März 2013 Erstgutachter: Prof. Dr. Thorsten Mascher Zweitgutachter: Prof. Dr. Kirsten Jung Tag der mündlichen Prüfung: 23.05.2013 Eidesstattliche Versicherung und Erklärung Hiermit versichere ich an Eides statt, dass die vorliegende Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt wurde. Zudem wurden keine anderen als die angegebenen Quellen verwendet. Außerdem versichere ich, dass die Dissertation keiner anderen Prüfungskommission vorgelegt wurde und ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg unterzogen habe. München, 07.03.2013 __________________________________ Karen Schrecke I Contents Contents Eidesstattliche Versicherung und Erklärung .................................................................................... I Abbreviations ............................................................................................................................................ IV Publications Originating from this Thesis ........................................................................................ V Contributions to Publications .............................................................................................................. VI Summary .................................................................................................................................................... VII Zusammenfassung ................................................................................................................................... IX 1 Introduction ........................................................................................................................................ 1 1.1 Bacterial cell envelope and cell wall biosynthesis – a short overview ............................... 2 1.2 Bacteria and cell envelope stress ....................................................................................................... 3 1.3 LiaFSR‐like systems ................................................................................................................................. 6 1.3.1 The paradigm: LiaFSR of B. subtilis.......................................................................................... 6 1.3.2 Distribution and conservation of LiaFSR‐like regulatory systems ............................ 9 1.3.2.1 LiaFSR‐like systems of group I (liaIH‐liaFSR) in other Firmicutes species…………………………………………………………………………………………9 1.3.2.2 LiaFSR‐like systems of group II (liaFSR) in other Firmicutes species………………………………………………………………………………………10 1.3.3 Stimuli of Lia‐like systems ....................................................................................................... 13 1.4 Physiological role of acetyl phosphate in two‐component signal transduction pathways ................................................................................................................................................... 15 1.4.1 Biosynthesis of acetyl phosphate .......................................................................................... 15 1.4.2 Regulation of the cellular acetyl phosphate pool............................................................ 16 1.4.3 Acetyl phosphate as global signal ......................................................................................... 17 1.4.4 In vivo relevance of acetyl phosphate for the phosphorylation of RRs ................. 17 1.4.5 Acetyl phosphate‐insensitive RRs ......................................................................................... 19 1.5 Aims of this thesis ................................................................................................................................. 20 2 Stoichiometry and perturbation studies of the LiaFSR system of Bacillus subtilis . 21 Schrecke, K., Jordan, S., Mascher, T. (2013). Molecular Microbiology 87(4): 769‐788 II Contents 3 The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter ............................................................................................................................. 42 Toymentseva, A. A., Schrecke, K., Sharipova, M. R., Mascher, T. (2012). Microbial Cell Factories 11: 143 4 Discussion ......................................................................................................................................... 56 4.1 Stoichiometry and robustness of 2CSs ......................................................................................... 58 4.1.1 Stoichiometry and robustness of other signal transduction pathways ................. 58 4.1.2 Stoichiometry and robustness of LiaFSR – revolutionary or only one of many? 61 4.2 Mechanistic insights of stimulus perception and signal transduction of LiaFSR‐like systems ...................................................................................................................................................... 62 4.2.1 Stimulus perception by LiaS and LiaF ................................................................................. 62 4.2.2 Catalytic activity of LiaS‐like HKs and phosphotransfer to LiaR .............................. 64 4.2.3 LiaS‐independent activation of LiaR .................................................................................... 65 4.3 Development of a new protein expression system for B. subtilis ...................................... 66 4.3.1 Protein expression in the Gram‐negative bacterium E. coli ....................................... 67 4.3.2 Protein expression in the Gram‐positive bacterium B. subtilis ................................. 68 4.3.3 The LIKE system – pros and cons .......................................................................................... 70 4.4 Conclusions/outlook ............................................................................................................................ 71 5 Supplemental Material – CHAPTER 2 ...................................................................................... 74 6 Supplemental Material – CHAPTER 4 ...................................................................................... 79 6.1 Protein expression systems for E. coli .......................................................................................... 79 6.2 Protein expression in Gram‐positive bacteria ........................................................................... 81 6.2.1 Protein expression systems for B. subtilis ......................................................................... 81 6.2.2 Protein expression systems for other Firmicutes bacteria ......................................... 84 References ................................................................................................................................................. 86 Acknowledgements ................................................................................................................................ 98 III Abbreviations Abbreviations 1CS one‐component system 2CS two‐component system ABC ATP binding cassette AckA acetate kinase ATP adenosine‐5’‐triphosphate cAMP cyclic adenosine‐3’,5’‐monophosphate CAMP cationic antimicrobial peptide CAP catabolite activator protein CESR cell envelope stress response CoA coenzyme A CRE catabolite‐responsive element CTD C‐terminal DNA‐binding domain DNA deoxyribonucleic acid ECF extracytoplasmic function e.g. for example GlcNAc N‐acetylglucosamin HK histidine kinase IPTG isopropyl‐β‐D‐thiogalactopyranoside MLS macrolide‐lincosamide‐streptogramin mRNA messenger RNA MurNAc N‐acetylmuramic acid OD optical density PCR polymerase chain reaction Pta phosphotransacetylase RNA ribonucleic acid RR response regulator TCA tricarboxylic acid UMP uridine monophosphate UDP uridine diphosphate UPP undecaprenol pyrophosphate X‐Gal 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐galactopyranoside IV Publications Originating from this Thesis CHAPTER 1 and 4 Schrecke, K.*, Staroń, A.*, Mascher, T. (2012). Two‐component systems in bacteria. Chapter 11: Two‐Component signaling in the Gram‐positive envelope stress response: intramembrane‐sensing histidine kinases and accessory membrane proteins. D. Beier and R. Gross (eds). Horizon Scientific Press, pp. 199‐229 (* shared first authorship) CHAPTER 2 Schrecke, K., Jordan, S., Mascher, T. (2013). Stoichiometry and perturbation studies of the LiaFSR system of Bacillus subtilis. Molecular Microbiology 87(4): 769‐788 CHAPTER 3 Toymentseva, A. A., Schrecke, K., Sharipova, M. R., Mascher, T. (2012). The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter. Microbial Cell Factories 11: 143 V Contributions to Publications CHAPTER 1 and 4 Schrecke, K.*, Staroń, A.*, Mascher, T. (2012). D. Beier and R. Gross (eds). Horizon Scientific Press, pp. 199‐229 (* shared first authorship) Karen Schrecke, Anna Staroń, and Thorsten Mascher performed the literature search and wrote the manuscript. CHAPTER 2 Schrecke, K., Jordan, S., Mascher, T. (2013). Molecular Microbiology 87(4): 769‐788 Karen Schrecke carried out all experiments, drew the figures and constructed the tables. Sina Jordan generated some strains. Karen Schrecke and Thorsten Mascher designed the experiments and wrote the manuscript. CHAPTER 3 Toymentseva, A. A., Schrecke, K., Sharipova,