DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Markus Wehland-von Trebra :
The Rcs-Systems and the RcsAB-Box
Identification of a new, essential operator for the regulation of capsule biosynthesis in enterobacteria
Das Rcs-System und die RcsAB-Box

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Abstract

The ability to form capsules is a widespread feature between various bacterial species. The capsules consist of long, modular polysaccharides and represent the interface for the interaction of the bacteria with their environment. Furthermore, the exopolysaccharide (EPS) of pathogenic organisms serves as a protection against host-defense mechanisms and is also an important virulence determinant. The enterobacteria Escherichia coli, Erwinia amylovora, and Pantoea stewartii subsp. stewartii synthesize the EPS colanic acid, amylovoran, and stewartan, respectively. All these capsules belong to the group IA of bacterial polysaccharides. Their biosynthetic genes are organized in operon-like clusters (the wca-, ams-, and cps-cluster) and are regulated by the Rcs (regulation of capsule synthesis) system, consisting of the three proteins RcsA, RcsB, whose activity can be modulated by phosphorylation, and RcsC, a membrane located sensor kinase. Activation of transcription is achieved by binding of an RcsA/RcsB heterodimer (RcsAB) to suitable promoter sequences. In this work, the RcsAB/DNA interaction was further characterized. The apparent equilibrium constant KD = 100 nM of the RcsAB/ams-promoter complex has been determined using the bandshift technique, while the KD = 77 nM of the RcsAB/wza-promoter complex was calculated using the surface plasmon resonance (SPR) technique. An in vitro selection of the ams-promoter made it possible to formulate a first consensus motif for RcsAB binding (Wehland et al., .1999), which allowed to find 13 more RcsAB binding sites in the EPS- and rcsA- promoters from E. coli, Ew. amylovora, P. stewartii, Salmonella typhi, and Klebsiella pneumoniae as well as in the bvgA- and fha-promoters from Bordetella pertussis and B. parapertussis by data bank search. A compilation of these sequences resulted in the finding of a common RcsAB binding motif, the RcsAB box : TaAGaatatTCctA (Wehland et al., 2000). The essential role of the RcsAB box in EPS regulation was additionally confirmed by in vivo experiments Besides the RcsAB binding sites in the EPS main promoters, internal, weaker binding sites inside the ams- and wca-operons were identified, but their biological function remained unclear. The RcsAB binding to various rcsA promoters has been shown for the first time. In vitro and in vivo experiments confirmed the central role of the RcsAB box for RcsAB/DNA interaction. Expression of an RcsB protein bearing a mutation in its phophorylation motif (RcsB(11D-A)) led to constitutive, RcsA independent EPS synthesis in E. coli. SPR analyses showed a tenfold increased DNA affinity of RcsB(11D-A) compared to the wildtype protein, which cannot be suppressed by phosphorylation. Furthermore, SPR and in vivo studies provided evidence for the involvement of the RcsAB box in RcsB/DNA interaction as a deletion of the RcsAB box abolished RcsB binding completely. Additionally, the first experimental proof together with the kinetic data for an RcsA/RcsB interaction in solution is presented.

Table of Contents

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Titelblatt und Inhaltsverzeichnis
Zusammenfassung 5
Abstract 6
1. Einleitung 7
1.1. Bakterielle Polysaccharide 7
1.2. Funktionen bakterieller Kapseln 8
1.3. Die industrielle Nutzung bakterieller Exopolysaccharide 9
1.4. Typisierung von Kapselpolysacchariden 10
1.5. Die Kapseln von Ew. amylovora, P. stewartii subsp. stewartii und E. coli 12
1.6. Aufgabenstellung 21
2. Materialien 22
2.1. Benutzte Geräte 22
2.2. Bakterienstämme und Plasmide 23
2.3. Medien, Puffer und Lösungen 24
2.4. Primer für die PCR bzw. zur Rekonstitution von Promotorfragmenten 29
3. Methoden 33
3.1. Herstellung elektrokompetenter Bakterienzellen 33
3.2. Herstellung Calcium-kompetenter E. coli Zellen 33
3.3. Transformation elektrokompetenter Bakterienzellen (Elektroporation) 33
3.4. Transformation Calcium-kompetenter Bakterienzellen 34
3.5. Analytische Präparation bakterieller Plasmid-DNA durch alkalische Lyse 34
3.6. Plasmid-DNA-Gewinnung im präparativen Maßstab 35
3.7. DNA-Amplifikation duch die Polymerase-Kettenreaktion (polymerase chain reaction, PCR) 35
3.8. Sequenzspezifische DNA-Spaltung durch Restriktionsendonukleasen 36
3.9. Ligation von DNA-Fragmenten 37
3.10. Agarose Gelelektrophorese zur Größentrennung von DNA-Fragmenten 37
3.11. DNA Elution aus Agarose-Gelen 38
3.12. Photometrische DNA-Konzentrationsbestimmung 39
3.13. Beta-Galaktosidase-Reportergen-Assay (ONPG-Test) 39
3.14. Digoxigenin-Markierung von Oligonukleotid-Sonden für den Southern-Blot 41
3.15. Southern Blot 41
3.16. Überexpression von Proteinen in E. coli 43
3.17. Zellaufschluß durch Druck in der French Press 45
3.18. Gelperfusionschromatographie 46
3.20. Dialyse 47
3.21. Proteinkonzentrationsbestimmung nach Bradford 48
3.22. Diskontinuierliche SDS-Polyacrylamidgelelektrophorese (SDS-PAGE) 48
3.23. Färbung von SDS-Polyacrylamid-Gelen mit Coomassie Brilliant Blue 49
3.24. In-vitro-Phosphorylierung von Proteinen 50
3.25. EPS-Präparation 50
3.26. Anthron-Test zur Bestimmung der Glucose-Konzentration 51
3.27. Hybridisierung von Oligonukleotiden 51
3.28. Radioaktive Markierung von Oligonukleotiden mittels Fill-in 51
3.29. Elektrophoretischer Mobilitäts Shift Assay (EMSA) 52
3.30. Szintillationsmessung 53
3.31. In-vitro-Selektion (SELEX) 54
3.32. DNA-Elution aus Polyacrylamidgelen 54
3.33. DNA-Sequenzierung nach Sanger 55
3.34. Biomolekül-Interaktions-Studien mit der Oberflächen Plasmon Resonanz (surface plasmon resonance, SPR) 56
3.35. In-vivo-Mutagenese der bakteriellen chromosomalen DNA durch homologe Rekombination 60
3.36. Isolation bakterieller chromosomaler DNA 63
4. Ergebnisse 64
4.1. Charakterisierung des RcsAB-DNA-Komplexes am Ew. amylovora amsG-Promotor 64
4.2. Bestimmung eines RcsA/RcsB Bindungsmotivs im amsG-Promotor 67
4.3. Identifikation einer RcsA/RcsB-Bindungsstelle im P. stewartii cpsA-Promotor 70
4.4. Lokalisierung einer RcsA/RcsB-Bindungsstelle im E. coli wza-Promotor 73
4.5. RcsA und RcsB binden an die rcsA-Promotoren von E. coli, K. pneumoniae, S. typhi und Ew. amylovora 82
4.6. Identifikation einer RcsAB-Box in den Gen-Clustern für die K2-Antigen-Expression in K. pneumoniae und für die Vi-Anitgen-Expression in S. typhi 88
4.7. Das RcsAB-Heterodimer und BvgA, ein transkriptioneller Regulator aus Bordetella pertussis, erkennen gleiche DNA-Sequenzen. 89
4.8. Identifikation von RcsAB-Bindungsstellen an intergenische Regionen innerhalb des wza-Operons zur Colansäure-Biosynthese von E. coli 91
4.9. Identifikation von RcsAB-Bindungsstellen in intergenischen Regionen innerhalb des ams-Operons zur Amylovoran-Biosynthese von Ew. amylovora 95
4.10. Konstruktion dreier Mutanten-RcsB-Proteine im Phosphorylierungsmotiv 99
5. Diskussion 111
6. Literatur 120
7. Anhang 130
Lebenslauf 130
Veröffentlichungen 131
Danksagung 133

More Information:

Online available: http://www.diss.fu-berlin.de/2001/241/indexe.html
Language of PhDThesis: german
Keywords: Rcs-system, capsule biosynthesis, RcsAB-Box, enterobacteria
DNB-Sachgruppe: 30 Chemie
Date of disputation: 12-Nov-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Wolfram Saenger
Second Referee: Prof. Dr. Volker A. Erdmann
Contact (Author): markus.wehlandvontrebra@medizin.fu-berlin.de
Contact (Advisor): saenger@chemie.fu-berlin.de
Date created:29-Nov-2001
Date available:29-Nov-2001

 

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