An analysis of two-component regulatory systems in Myxococcus xanthus [Elektronische Ressource] / vorgelegt von Xingqi Shi

Shi

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

DECKBLATT







An analysis of two-component regulatory
systems in Myxococcus xanthus


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









dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von

Xingqi Shi
aus Anhui, China



Marburg / Lahn, Oktober 2008 Die Untersuchungen zur vorliegenden Arbeit wurden von Oktober 2005 bis
Oktober 2008 am Max-Planck-Institut für terrestrische Mikrobiologie unter der
Leitung von Prof. Dr. MD Lotte Sogaard-Andersen durchgeführt.















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


Erstgutachter: Prof. Dr. MD Lotte Sogaard-Andersen
Zweitgutachter: Prof. Dr. Erhard Bremer


Tag der mündlichen Prüfung am:

The first part of results in this thesis has been published:

Shi, X., S. Wegener-Feldbrugge, S. Huntley, N. Hamann, R. Hedderich & L.
Sogaard-Andersen, (2008) Bioinformatics and experimental analysis of
proteins of two-component systems in Myxococcus xanthus. J Bacteriol 190:
613-624.
List of contents
Abbreviations ..................................................................................................... 1
1 Abstract.................................................................................................... 2
Zusammenfassung ............................................................................................ 4
2 Introduction ............................................................................................. 7
2.1 Two-component regulatory system.................................................................7
2.1.1 General introduction ...................................................................................................... 7
2.1.2 Domains of HPK............................................................................................................ 8
2.1.3 Domains of RR............................................................................................................ 12
2.1.4 Architecture of TCS pathways ..................................................................................... 14
2.1.5 Recognition specificity of TCS proteins ....................................................................... 15
2.2 Development of Myxococcus xanthus..........................................................19
2.2.1 Life cycles of M. xanthus ............................................................................................. 19
2.2.2 Intercellular signal transduction pathways during development................................... 20
2.2.3 Motility of M. xanthus................................................................................................... 25
2.3 TCS in M. xanthus ...........................................................................................27
3 Results ................................................................................................... 29
3.1 An analysis of two-component regulatory systems in M. xanthus ............29
3.1.1 Identification of TCS genes in M. xanthus ................................................................... 29
3.1.2 Genetic organization of TCS genes in M. xanthus....................................................... 30
3.1.3 Histidine protein kinases ............................................................................................. 31
3.1.4 Histidine protein kinase-like proteins ........................................................................... 32
3.1.5 Response regulators and output domains ................................................................... 33
3.1.6 Transcriptional regulation of TCS genes during development ..................................... 34
3.1.7 Genetic analysis of transcriptionally up-regulated, orphan hpk genes ......................... 38
3.2 In search of the FruA kinase using a candidate approach..........................43
3.2.1 A candidate approach to identify for the FruA kinase .................................................. 43
3.2.2 Y2H analyses to test the interactions between FruA and 25 developmentally up-
regulated orphan HPKs................................................................................................ 43
3.2.3 Analyses of the FruA kinase candidates...................................................................... 47
3.2.4 Epistasis analyses of fruA and fruA kinase candidates................................................ 49
3.2.5 FrzCD methylation and FruA accumulation in the mutants of FruA kinase candidates 51
3.2.6 Developmental gene expression in the mutants of FruA kinase candidates ................ 53
3.2.7 Purification of FruA and the FruA kinase candidates................................................... 57
3.2.8 Autophosphorylation assay of Hpk8 ............................................................................ 59
3.2.9 Chemical stability of phosphorylated Hpk8.................................................................. 60
3.2.10 Preliminary data of phosphotransfer from Hpk8 to FruA.............................................. 64
3.2.11 Accumulation of Hpk8 during development ................................................................. 65 3.2.12 Preliminary data of autophosphorylation of SdeK and Hpk12...................................... 66
3.2.13 Preliminary analyses of the redundant FruA kinase candidates .................................. 67
3.3 Characterization of Hpk37..............................................................................70
3.3.1 Analysis of Hpk37........................................................................................................ 70
3.3.2 Motility assay of ∆hpk37.............................................................................................. 73
3.3.3 FrzCD methylation and FruA accumulation in ∆hpk37................................................. 74
3.3.4 Developmental gene expression in ∆hpk37................................................................. 75
3.3.5 Purification of Hpk37 ........................................................................................ 76 1148-1967
3.4 In search of the FruA kinase with bioinformatics method ..........................78
3.5 Miscellaneous hpk mutants ...........................................................................79
4 Discussion ............................................................................................. 81
4.1 Two-component regulatory systems in M. xanthus.....................................81
4.2 In search of the FruA kinase......................85
4.3 Hpk37................................................................................................................92
5 Material and methods ........................................................................... 94
5.1 Reagents, Enzymes and Kits .........................................................................94
5.2 Microbiological methods..94
5.2.1 Escherichia coli strains................................................................................................ 94
5.2.2 Saccharomyces cerevisiae strain ................................................................................ 95
5.2.3 Myxococcus xanthus stains......................................................................................... 95
5.2.4 Cultivation of E. coli..................................................................................................... 97
5.2.5 Cultivation of S. cerevisiae .......................................................................................... 97
5.2.6 Cultivation of M. xanthus ............................................................................................. 97
5.2.7 Development assay and spore assay of M. xanthus.................................................... 98
5.2.8 Motility assay of M. xanthus ........................................................................................ 98
5.3 Molecular biological methods........................................................................99
5.3.1 Oligonucleautide and plasmids.................................................................................... 99
5.3.2 Preparation of chromosomal DNA from M. xanthus................................................... 111
5.3.3 PCR reaction , digestion and ligation......................................................................... 111
5.3.4 Transformation of E. coli............................................................................................ 112
5.3.5 Electroporation of M. xanthus.................................................................................... 112
5.3.6 Co-transformation of S. cerevisiae ............................................................................ 113
5.3.7 RNA preparation from M. xanthus ............................................................................. 113
5.3.8 RNA clean up, cDNA synthesis and qRT-PCR....................................................