The Myxococcus xanthus Red two-component signal transduction system: a novel four component signaling mechanism [Elektronische Ressource] / vorgelegt von Sakthimala Jagadeesan

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DECKBLATT The Myxococcus xanthus Red two-component signal transduction system: a novel “four-component” signaling mechanism Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Sakthimala Jagadeesan aus Coimbatore, India Marburg (Lahn), Oktober 2008 Die Untersuchungen zur vorliegenden Arbeit wurden von September 2005 bis Oktober 2008 am Max-Planck-Institut für Terrestrische Mikrobiologie unter der Leitung von Dr. Penelope I. Higgs durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: Erstgutachter: Prof. Dr. Lotte Sogaard-Andersen Zweitgutachter: Prof. Dr. Hans-Ulrich Mösch Tag der mündlichen Prüfung am: The following paper is in preparation by the date of submission of the present thesis: Jagadeesan S. and Higgs P.I.

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DECKBLATT


The Myxococcus xanthus Red two-component
signal transduction system: a novel “four-
component” signaling mechanism






Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)





dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von







Sakthimala Jagadeesan
aus Coimbatore, India









Marburg (Lahn), Oktober 2008
Die Untersuchungen zur vorliegenden Arbeit wurden von September 2005 bis
Oktober 2008 am Max-Planck-Institut für Terrestrische Mikrobiologie unter der
Leitung von Dr. Penelope I. Higgs durchgeführt.





























Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation
angenommen am:



Erstgutachter: Prof. Dr. Lotte Sogaard-Andersen
Zweitgutachter: Prof. Dr. Hans-Ulrich Mösch



Tag der mündlichen Prüfung am:








The following paper is in preparation by the date of submission of the present
thesis:

Jagadeesan S. and Higgs P.I. Red proteins in Myococcus xanthus constitute a
novel four-component Histidine-Aspartate phosphorelay system
















The publication that is not included in this thesis which was performed during
my PhD:

Higgs PI, Jagadeesan S, Mann P, Zusman DR (2008) EspA, an orphan hybrid
histidine protein kinase, regulates the timing of expression of key developmental
proteins of Myxococcus xanthus. J Bacteriol 190(13): 4416-4426
































Dedicated to my parents



TABLE OF CONTENTS

TABLE OF CONTENTS................................................................................................6
ABBREVIATIONS..........................................................9
1 SUMMARY...............................................................10
ZUSAMMENFASSUNG..............................................11
2 INTRODUCTION.......................................................13
2.1 Two-component signal transduction system in bacteria.......................................13
2.2 Domain architecture and function of histidine kinases..........................................15
2.2.1 Sensors.......................................................................................................................................... 15
2.2.2 Transmitters.................................................................................................................................. 16
2.2.3 Hybrid Kinases ............................................................................................................................. 16
2.3 Domain architecture and function of response regulators....................................17
2.4 Regulatory mechanisms.............................................................................................19
2.5 Myxococcus xanthus...................................................................................................21
2.5.1 Regulation of M. xanthus development program ................................................................... 22
2.5.2 Regulation of developmental progression by the TCS systems ......................................... 23
2.5.3 M. xanthus TCS system.............................................................................................................. 24
2.6 The Red two-component signal transduction system in M. xanthus...................26
3 RESULTS..........................................................................................................30
3.1 Biochemical characterization of Red signal transduction proteins......................30
3.1.1 Heterologous overexpression and purification of putative histidine ................................... 30
kinases, RedC and RedE.......................................................................................................................... 30
3.1.2 RedC-T but not RedE autophosphorylates on conserved histidine.................................... 34
3.1.3 Heterologous overexpression and purification of putative response ................................. 36
regulators, RedD and RedF...................................................................................................................... 36
3.1.4 RedD and RedF both can be autophosphorylated by acetyl-.............................................. 40
phosphate .................................................................................................................................................... 40
3.2 Expression of Red signal transduction proteins in M. xanthus.............................42
3.3 Analysis of signal flow in Red TCS system.............................................................44
3.3.1 The phosphorylated form of RedF represses developmental progression....................... 44
3.3.2 RedE acts as a phosphatase on RedF-P ................................................................................ 48
3.3.3 In vitro stability of phosphorylated RedF ................................................................................. 49
3.3.4 RedC might act as kinase on RedF.......................................................................................... 50
3.3.5 RedD is necessary to induce development............................................................................. 53
3.3.6 RedC acts as a kinase and a phosphatase on RedD ........................................................... 56
3.3.7 RedE is epistatic to RedD .......................................................................................................... 58
3.3.8 RedE receives phosphoryl group from RedD......................................................................... 59 4 DISCUSSION....................................................................................................61
5 MATERIALS AND METHODS.........71
5.1 Chemicals and Materials............................................................................................71
5.2 Microbiology methods.................................................................................................72
5.2.1 Culture media, conditions and storage .................................................................................... 72
5.2.2 Bacterial strains............................................................................................................................ 74
5.2.3 Analysis of M. xanthus developmental phenotypes .............................................................. 74
5.3 Molecular biology methods........................................................................................75
5.3.1 Plasmids ........................................................................................................................................ 75
5.3.2 Oligonucletides............................................................................................................................. 76
5.3.3 Construction of plasmids ............................................................................................................ 77
5.3.4 Construction of in-frame deletion in M. xanthus..................................................................... 81
5.3.5 Construction of in vivo non-functional point mutants in M. xanthus ................................... 81
5.4 DNA techniques...........................................................................................................82
5.4.1 Agarose gel electrophoresis ...................................................................................................... 82
5.4.2 Isolation of genomic DNA from M. xanthus............................................................................. 82
5.4.3 Isolation of plasmid DNA from E. coli....................................................................................... 83
5.4.4 Polymerase chain reaction (PCR) ............................................................................................ 83
5.4.5 Determination of DNA concentration........................................................................................ 84
5.4.6 Digestion and ligation of DNA.................................................................................................... 84
5.4.7 Preparation and transformation of electro competent E. coli cells ..................................... 84
5.4.8 Preparn of chemical competent E. coli cells ................................. 85
5.4.9 Preparation and transformation of electro competent M. xanthus cells............................. 85
5.4.10 DNA sequencing ..................................................................................................................... 86
5.5 Biochemical methods..................................................................................................87
5.5.1 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) ...................................................... 87
5.5.2 Tricine SDS Polyacrylamide Gel Electrophoresis (Tricine–SDS-PAGE) ........................... 88
5.5.3 Determination of protein concentration.................................................................................... 89
5.6 Heterologous overexpression and purification of Red proteins in.......................90
E. coli..........................................................................................................................................90
5.6.1 Heterologous expression of RedC............................................................................................ 90
5.6.2 Heterologous expression of RedD............................................................................................ 90
5.6.3 Heterologous expression of RedE............................................................................................ 90
5.6.4 Heterologous expression of RedF ............................................................................................ 91
5.6.5 Purification of Red proteins........................................................................................................ 91
5.7 Phosphorylation assays.............................................................................................92
5.7.1 Autophosphorylation of RedC and RedE kinases ................................................................. 92
5.7.2 Phosphotransfer from the kinase to the response regulators.............................................. 92
5.7.3 Autophosphorylation of RedD and RedF response regulators............................................ 93 5.7.4 Dephosphorylation assays......................................................................................................... 93
5.8 Immunoblot analysis...................................................................................................93
5.8.1 Antibody generation for Red proteins ...................................................................................... 93
5.8.2 Antibody purification.................................................................................................................... 94
5.8.3 Immunoblot analysis ................................................................................................................... 95
6 REFERENCES.................................................................................................96
CURRICULUM VITAE......................102
ACKNOWLEDGEMENTS.........................................................................................103
Erklärung.............................................................................................104

ABBREVIATIONS 9
ABBREVIATIONS
APS Ammonium persulfate
CF agar clone fruitingagar
CYE medium casitone yeast extract medium
daH O demineralized and autoclaved water 2
DTT Dithiothreitol
EDTA ethylene diamine tetra-acetic acid
FPLC Fast performance liquid chromatography
IPTG Isopropyl-1-thio-D-galactopyranoside
kDa Kilo Dalton
LB medium Luria-Bertani medium
NaOAc sodium acetate
OD optical density
rpm rounds per minute
SDS-PAG sodium dodecyl sulfate polyacrylamide gel
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TAE Tris-acetate-EDTA
Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol
TE buffer Tris EDTA buffer
TEMED N,N,N’,N’- Tetramethylethylendiamin