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Dissertation Cross-talk between two-component systems in Escherichia coli Miriam Fischer May 2016 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biologin Miriam Fischer born in Frankfurt am Main Oral examination: August 2016 Cross-talk between two-component systems in Escherichia coli Referees: Prof. Dr. Victor Sourjik PD Dr. Axel Mogk Acknowledgements I would like to thank Prof. Dr. Victor Sourjik for his supervision and support during the last years. I also want to thank my thesis advisory committee members PD Dr. Axel Mogk and Prof. Dr Rüdiger Hell for valuable advice and input. I would to thank the present and former members of the Sourjik lab, who are always very helpful and willing to share their experience and an occasional banter with me. I am grateful for the encouragement by my friends and family. I especially want to thank my parents for their consistent support and love. Contents 1 Introduction 17 1.1 Signaling in bacteria 17 1.2 Two-component systems 18 1.3 TCS structure and signaling mechanisms 21 1.3.1 Histidine kinases 21 1.3.2 Response regulators 25 1.3.3 Hybrid HKs and phosphorelays 26 1.3.4 Pathway specificity and pathway variants 27 1.3.5 Cross-talk among two-component systems 30 1.4 Copper homeostasis and sensing 32 1.4.1 Copper homeostasis in bacterial cells 32 1.4.2 Copper sensing in E. coli 34 1.5 Aims of this work 36 2 Materials and Methods 39 2.1 Chemicals and consumables 39 2.1.1 Media and plates 39 2.1.2 Buffers and stock solutions 40 2.1.3 Reaction Kits 40 2.2 Strains 41 2.2.1 Gene deletion strains derived from Keio collection 41 2.2.2 Deletion strains derived from genomic integration of PCR products 41 2.3 Plasmids 42 2.4 Cloning strategies 42 2.4.1 Primers 42 2.4.2 Polymerase Chain Reaction (PCR) 42 2.4.3 Mutation of conserved protein sites 43 9 Contents 2.4.4 Introduction of mutations using overlap PCR 44 2.4.5 Introduction of mutations into plasmid DNA 44 2.4.6 Restriction Digest 45 2.4.7 Phosphatase treatment 45 2.4.8 Ligation 45 2.5 Competent cells 46 2.5.1 Preparation of chemical competent cells 46 2.5.2 Preparation of competent cells for electroporation 46 2.5.3 Transformation of chemical competent cells 46 2.5.4 Transformation of electro competent cells for genomic integration 47 2.6 Cultures 47 2.7 Acceptor photobleaching experiments 47 2.8 Promoter activation 48 2.9 RNA Isolation and Deep Sequencing 49 2.10 Membrane protein pull-down 50 2.11 Data Analysis 51 2.11.1 Promoter activation and FRET data 51 2.11.2 RNA sequencing data 51 2.12 Software 52 3 Results 53 3.1 In vivo interactions among TCSs 53 3.1.1 FRET measurements of interaction of cognate TCS components 53 3.1.2 FRET measurements of TCS cross-talk 55 3.1.3 Cross-talk among the CusS/ CusR, YedV/ YedW and BaeS/ BaeR TCSs 56 3.1.4 Pull-down of membrane-integral HKs 59 3.2 Promoter activation experiments 62 3.2.1 Promoter activation by CusS/ CusR and YedV/ YedW TCSs 62 3.2.2 Complementation of CusS deletion 65 10 Contents 3.2.3 Effect of YedV on gene expression 66 3.2.4 Screen for YedV/ YedW activating stimulus 70 3.3 Transcriptomic analysis of TCS 70 3.3.1 Gene regulation by the CusS/ CusR TCS 72 3.3.2 Gene regulation by the BaeS/ BaeR TCS 82 3.3.3 Gene regulation by the YedV/ YedW TCS 90 4 Discussion 109 4.1 Protein interactions and cross-talk 109 4.2 Effects of interactions on gene expression 112 4.2.1 Effect on expression of promoter-GFP reporter 112 4.2.2 Effect on transcriptomic data 114 5 Conclusion and Outlook 121 6 Literature 123 7 Appendix 143 7.1 Tables 143 7.2 Figures 183 8 List of Figures and Tables 186 11 Contents 12 Zusammenfassung Mikroorganismen sind einer Vielzahl von Umweltbedingungen ausgesetzt. Sie nutzen Zweikomponenten-Signalsysteme um diese zu erfassen und ihren Stoffwechsel anzupassen. Zweikomponentensysteme kommen in Genomen von Bakterien, Archaeen und niederen Eukaryoten sehr häufig vor. Sie bestehen aus einer Sensor-Histidinkinase und einem 'Response Regulator', der als Transkriptionsfaktor die Genexpression reguliert. Eine Anregung der Signalübertragung durch Zweikomponentensysteme führt zur Autophosphorylierung der Histidinkinase mit nachfolgender Phosphoryl- gruppenübertragung auf den 'Response Regulator'. Ähnlichkeiten sowohl in der Struktur als auch der Proteinsequenzen verschiedener Komponenten können eine Signalverknüpfung zwischen verschiedenen Zweikomponentensystemen in unter- schiedlichen Abschnitten des Signalübertragungswegs herbeiführen. Während dieser 'cross-talk' zwischen nicht-verwandten Komponenten der Zweikomponentensysteme in vitro untersucht wurde, ist über das in vivo Verhalten wenig bekannt. In dieser Arbeit haben wir in vivo Wechselwirkungen der Zweikomponentensysteme in dem Enterobakterium Escherichia coli mit Hilfe von FRET Mikroskopie identifiziert, und die physiologisch bedeutsamen Wechselwirkungen der nicht-verwandten Komponenten des CusS/ CusR and YedV/ YedW Zweikomponentensystems beschrieben. Unsere Studien mit 'promoter-reportern', sowie Transkriptionsanalysen haben zwei Ebenen der Signalkopplung nachgewiesen, eine zwischen den Histidinkinasen und den nicht- verwandten 'response regulatoren', eine weitere auf dem Genexpressions- induktionsniveau. Unsere Ergebnisse vermitteln somit ein genaues Verständnis der gegenseitigen Regulation der Zielgene und des 'cross-talks' zwischen den CusS/ CusR and YedV/ YedW Zweikomponentensystemen. 13 14 Summary Microorganisms are faced with a huge variety of environmental conditions. They use two-component signaling systems to sense and adjust their metabolism accordingly. Such two-component systems are highly abundant in genomes of bacteria, archaea and lower eukaryotes. They are comprised of a sensor histidine kinase and a response regulator which serves as a transcription factor regulating gene expression. Induction of two-component system signaling leads to autophosphorylation of the histidine kinase and the subsequent transfer of the phosphoryl-group to the response regulator. Both modularity and protein sequence similarities can give rise to signal integration between two-component systems at different levels of their signaling pathways. While cross-talk between non-cognate components of two-component systems has been assessed in vitro, not much is yet known about cross-talk in vivo. In this work, we identified in vivo interactions of two-component systems in the enterobacterium Escherichia coli using FRET microscopy. We have also characterized physiological relevant interactions of non-cognate components of the CusS/ CusR and YedV/ YedW two-component systems. In addition, our studies with promoter reporters and transcriptomic analysis showed two interconnection mechanisms, one between the histidine kinases and the non-cognate response regulators, the other on the level of induction of gene expression. Our data provide a detailed understanding of this cooperative regulation of target genes and cross-talk between the CusS/ CusR and YedV/ YedW two-component systems. 15 16 1 Introduction This work is focused on studying signal transduction in two-component systems (TCSs) of the Gram-negative model microorganism Escherichia coli [1]. We are interested in how the activation of signal transduction of the 30 E. coli TCSs in response to environmental stimuli gets integrated into a particular cell response [2]. Here we focus on cross-talk among different TCSs. The cross-talk among TCSs associated with the response to an extracellular copper stimulus exemplifies how cells regulate the integration of signals in order to respond to pressing conditions. 1.1 Signaling in bacteria Prokaryotes are ubiquitous in a wide range of environments on earth which challenge them for instance with fluctuations in temperature, oxygen and nutrient availability. To alter their metabolism, lifestyle or response to stress and external signals, bacteria regulate differential gene expression via signal transduction. External signals can be small molecules which enter the cell and function as effectors, but in many cases the signal is detected by a sensor which transmits to the regulatory machinery of the cell [3]. One-component systems have a sensor input and an output domain in one protein. They make up a vast part of intracellular prokaryotic signaling systems [4]. In eukaryotes signal transduction is predominantly mediated by Ser/Thr/Tyr kinases. They are less abundant in prokaryotes where the second most important signal transduction pathway are two-component systems (TCSs). Both systems rely on phosphoryl-transfer as a means of signal transduction [5], [2]. Other intracellular signaling systems utilize small molecules like cyclic-di-GMP, guanosine tetraphosphate (ppGpp) or cyclic adenosine monophosphate (cAMP). It has also been shown that the uptake and phosphorylation of sugars by the phosphotransferase systems (PTS) impacts global regulators as well as small regulatory RNAs, which are for instance involved in bacterial quorum sensing and can play a role in the regulation of gene expression in response to stimuli [6],[7]. The unique feature of TCSs among the described signaling systems is the extracellular or 17 1 Introduction periplasmic sensing whereas the others require the stimulus to enter the cell before detection. 1.2 Two-component systems The wide range of signal recognition and regulation of physiological processes, cell division and virulence by TCSs makes them an interesting subject when studying signaling networks in bacteria. After one-component systems TCSs are the second most predominant signaling system in bacteria [2]. They are also involved in the signaling of archaea and eukaryotes with the exception of the animal kingdom where no genes encoding for TCSs have been found yet [8]. The number of TCSs genes in bacterial genomes can vary greatly and is correlated with the lifestyle and the environmental conditions the cell faces.
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