Characterization of CorS, a histidine protein kinase involved in temperature-dependent synthesis of the phytotoxin coronatine in Pseudomonas syringae [Elektronische Ressource] / vorgelegt von Angela Vladimirovna Smirnova

Angela Vladimirovna Smirnova

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2001
• Nombre de lectures: 29
• Poids de l'ouvrage: 1 Mo

Extrait

Characterization of CorS, a histidine protein kinase
involved in temperature-dependent synthesis of the
phytotoxin coronatine in Pseudomonas syringae
Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)
dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von
Angela Vladimirovna Smirnova
aus
Odessa/Ukraine
Marburg/Lahn 2001Vom Fachbereich Biologie der Philipps-Universität Marburg
Als Dissertation am 13.06.2001 angenommen.
Erstgutachter: PD Dr. M.S. Ullrich
Zweitgutachter: Prof. Dr. R.K. Thauer
Die öffentliche Disputation der Dissertation erfolgte am 17.07.2001Meinen Eltern und Lehrern
Моим родит е лям и учи т елямIm Zusammenhang mit der Thematik der vorliegenden Dissertation wurde folgende
Publikation erstellt:
Smirnova, A., Wang, L., Rohde, B., Budde, I., Weingart, H., and Ullrich, M. (2001). Control
of temperature-responsive synthesis of the phytotoxin in Pseudomonas syringae by the two-
component system CorRPS. J. Mol. Microbiol. Biotechnol., in press. Contents
CONTENTS
1 SUMMARY .................................................................................................................................... 1
ZUSAMMENFASSUNG.................................................................................................................2
2 INTRODUCTION ........................................................................................................................... 3
2.1 Bacterial thermoregulation ................................................................................................................... 3
2.1.1 Temperature as a global environmental factor influencing cellular function
at the molecular level.................................................................................................................... 3
2.1.2 Influence of temperature on bacterial pathogenicity ...................................................................... 4
2.1.3 Molecular mechanisms for thermoregulation of bacterial gene expression ................................... 5
2.2 Signal transduction by two-component regulatory systems............................................................... 7
2.2.1 Bacterial signal transduction .......................................................................................................... 7
2.2.2 Structure and function of histidine protein kinases ........................................................................8
2.2.3 Structural determinants for signal perception by class I histidine protein kinases ....................... 11
2.3 The plant pathogenic bacterium Pseudomonas syringae .................................................................. 14
2.3.1 General characteristics of Pseudomonas syringae 14
2.3.2 Pseudomonas syringae pv. glycinea 16
2.3.3 Phytotoxin coronatine................................................................................................................... 17
2.4 Aim of this work................................................................................................................................... 22
3 MATERIAL .................................................................................................................................. 24
3.1 Equipment...................................................................................................................................... 24
3.2 Chemicals, antibiotics and enzymes............................................................................................. 25
3.3 Kits............................................................................................................................................... 25
3.4 Antibodies .................................................................................................................................... 26
3.5 Additional materials ..................................................................................................................... 26
3.6 Media ........................................................................................................................................... 26
3.6.1 Complex medium for Escherichia coli.................................................................................... 27
3.6.2 Complex medium for Pseudomonas syringae......................................................................... 27
3.6.3 Minimal media for Pseudomonas syringae ............................................................................. 27
3.7 Oligonucleotides........................................................................................................................... 28
3.8 Software ....................................................................................................................................... 30
3.9 Microorganisms............................................................................................................................ 30
3.10 Plasmids ....................................................................................................................................... 31
4 METHODS 34
4.1 Bacterial growth............. 34
4.1.1 Escherichia coli............................................................................................................................ 34
4.1.2 Pseudomonas syringae................................................................................................................. 34
4.1.3 Storage of bacterial strains ........................................................................................................... 34
4.2 Molecular biology methods........................................................................................................ 34
4.2.1 Plasmid DNA isolation................................................................................................................. 34
4.2.1.1 1-2-3 Isolation of plasmid DNA.............................................................................................. 34
4.2.1.2 Midi and Maxi plasmid DNA isolation by Qiagen procedure................................................. 35
4.2.1.3 Pseudomonas plasmid isolation according to Kado and Liu ................................................... 36
4.2.2 DNA separation by gel electrophoresis........................................................................................ 36
4.2.3 Polymerase chain reaction (PCR)................................................................................................. 37
4.2.4 Cloning techniques....................................................................................................................... 39
4.2.4.1 Digestion of plasmid DNA with endonucleases...................................................................... 39
4.2.4.2 Desphosphorylation of digested DNA 39
4.2.4.3 Converting 5’-overhanging sticky ends to blunt ends ............................................................. 39
4.2.4.4. Conv 5’-overhanging sticky endst ends 40
4.2.4.5 Precipitation of DNA by ethanol ............................................................................................. 40 Contents
4.2.4.6 QIAEX II agarose gel extraction procedure ............................................................................ 41
4.2.4.7 QIAquick PCR purification kit................................................................................................ 41
4.2.4.8 Estimation of DNA concentration ........................................................................................... 41
4.2.4.9 Ligation of DNA...................................................................................................................... 41
4.2.4.10 Preparation of competent E. coli cells using calcium chloride................................................ 42
4.2.4.11 Transformation of DNA into E. coli cells by heat shock......................................................... 43
4.2.5 Introduction of recombinant DNA into Pseudomonas syringae .................................................. 43
4.2.5.1 Preparation of P. syringae electrocompetent cells................................................................... 43
4.2.5.2 Electroporation of P. syringae cells 43
4.2.5.3 Conjugation by triparental mating........................................................................................... 43
4.2.6 Pentapeptide scanning mutagenesis... 44
4.3 Biochemical and analytical methods.......................................................................................... 45
4.3.1 Estimation of enzymatic activities................................................................................................ 45
4.3.1.1 Quantitative estimation of specific ß-glucuronidase activity ................................................... 45
4.3.1.2 Quantitative estimation offic alkaline phosphatase (PhoA) activity............................... 46
4.3.1.3 Quantitative estimation of specific ß-galactosidase (LacZ) activity ........................................ 47
4.3.1.4 Qualitative determination of GUS, PhoA and LacZ