Dokument: Network architecture and heme-responsive gene regulation of the two-component systems HrrSA and ChrSA
| Titel: | Network architecture and heme-responsive gene regulation of the two-component systems HrrSA and ChrSA | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=48343 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20190131-085537-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Keppel, Marc [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Frunzke, Julia [Gutachter] Prof. Dr. Groth, Georg [Gutachter] | |||||||
| Stichwörter: | Heme, Corynebacterium glutamicum, Two-component signalling, transient heme sensing, HrrSA, ChrSA, | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Heme (iron bound protoporphyrin IX) is a versatile molecule that serves as the prosthetic group of various proteins, including hemoglobins, hydroxylases, catalases, peroxidases, and cytochromes. Because of its essential role in many cellular processes, such as electron transfer, respiration and oxygen metabolism, heme is synthesized and used by virtually all aerobic eukaryotic and prokaryotic species. As hemoglobin represents the most abundant iron reservoir in mammalian hosts and heme proteins are ubiquitously found in organic material, heme represents an important alternative source of iron for pathogenic and nonpathogenic bacteria alike. Despite is necessity for nearly all cellular life, elevated, intracellular heme levels are extremely toxic. To cope with this Janus-faced nature of heme, sophisticated regulatory systems have evolved to tightly balance its uptake, synthesis and utilization.
Remarkably, in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, two paralogous, heme-responsive two-component systems (TCSs), HrrSA and ChrSA, are dedicated to the coordinated control of heme homeostasis. Previous studies showed that, while HrrSA is required for utilization of heme as an alternative iron source by activating expression of a heme oxygenase (hmuO), ChrSA drives detoxification by induction of the hrtBA operon, which encodes a heme export system. Additionally, the histidine kinases (HKs) of both systems are able to phosphorylate both response regulators (RRs). This cross-talk is proofread by a specific phosphatase activity of the HKs. In the framework of this PhD thesis, the signal perception, the temporal dynamics of heme-induced target gene activation and the constitution of the HrrSA regulon have been studied in detail. A comparative analysis of the membrane topology and the heme-binding characteristics of the HKs HrrS and ChrS revealed that while N-terminal sensing parts share only minor sequence similarity, both proteins are embedded into the cytoplasmic membrane via six α-helices and bind heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain of HrrS revealed three aromatic residues (Y112, F115, F118), which apparently contribute to heme binding and suggest an intra-membrane sensing mechanism of this HK. Exchange of the corresponding residues in ChrS and the resulting red shift of the soret band of the heme-protein complex indicated, that in this HK, an altered set of ligands might contribute to binding in the triple mutant. To understand how the particular regulatory network architecture of HrrSA and ChrSA shapes the dynamic response to heme, experimental reporter profiling was combined with a quantitative mathematical model. We found, that both HKs contribute to the fast onset of the detoxification response (hrtBA) upon stimulus perception and that the instant deactivation of the hrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of the detoxification response is uncoupled from further factors, heme utilization (hmuO) is additionally governed by the global iron regulator DtxR, which integrates information on iron availability into the regulatory network. Time-resolved and genome-wide monitoring of in-vivo promoter occupancy of HrrA revealed binding to more than 250 different genomic targets, which encode proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. Additionally, we found that in heme-rich environments, HrrA represses sigC, which encodes an activator of the cydABCD operon. Thereby, HrrA prioritizes the expression of genes encoding the cytochrome bc1 aa3 supercomplex for the constitution of the respiratory chain. In conclusion, this thesis provides comprehensive insights into the regulatory interplay of the HrrSA and ChrSA TCS shaping a systemic response to the versatile signaling molecule heme. Furthermore, for the first time, a time-resolved dataset of stimulus-dependent regulator binding and the resulting transcriptional changes grant global insight into heme-regulated gene expression in bacterial cells. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 31.01.2019 | |||||||
| Dateien geändert am: | 31.01.2019 | |||||||
| Promotionsantrag am: | 30.10.2018 | |||||||
| Datum der Promotion: | 15.01.2019 |
