Analysis of protein-protein interactions linked to the formation of a bacterial cytoskeleton in Mycoplasma pneumoniae [Elektronische Ressource] / presented by Atcha Boonmee

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences























presented by

Diplom-Biologin Atcha Boonmee
born in: Ubon Ratchathani, Thailand
Oral-examination: 12.07.2005




Analysis of protein-protein interactions
linked to the formation of a bacterial
Cytoskeleton in Mycoplasma pneumoniae

























Referees: Prof. Dr. Richard Herrmann
Prof. Dr. Claus Hobe Schröder Acknowledgments

This thesis has been accomplished at the Zentrum für Molekulare Biologie Heidelberg (ZMBH),
Ruperto-Carola University, Heidelberg, under the supervision of Prof. Richard Herrmann.

First of all, I would like to especially express my sincere gratitude to Prof. Richard
Herrmann for giving me the opportunity to work in his research group and for his
supervision throughout my PhD years, as well as for his interest, thoughtful discussions,
patience and solicitude.

Furthermore, I would like to thank:
- Prof. Claus Hobe Schröder for his worthful comments as the second referee;
- Dr. Diana Hofmann for assistance with the 2-D gel electrophoresis and warmhearted
advice during the thesis writing process;
- Elsbeth Pirkl for help in solving many problems in the daily laboratory work;
- Katrin Tagscherer for providing very useful data concerning the synthesis of recombinant
HMW2-s;
- Armin Bosserhoff and Dr. Thomas Ruppert for mass spectrometric analyses of HMW2-s
and components of the subfractions from TAP-tag purification;
- Dr. Carl-Ulrich Zimmerman for useful suggestions in many experimental approaches;
- Dr. Jan Hegermann for collaboration in eletron microscopy of M. pneumoniae;
- Dr. Manfred Koegl for introduction into the two-hybrid system and important comments;
- Werner Schaller for colaboration in two-hybrid screens;
- Dr. Jörg Regula, Dr. Barbara Ueberle , Dr. January Weiner III and Dr. Ina Catrein, for
valuable discussions;
- Michael Schulz, Peter Pechel and all lab members for providing a good working atmosphere.

I am grateful to all the people of the ZMBH who helped me during my thesis work.

Finally, I am thankful to my family and friends for their incessant support and encouragement.
Index
Index
Summary ........................................................................................................................1
Zusammenfassung ................................................................................................2
Abbreviations ........................................................................................................................3
1 Introduction ............................................................................................................6
1.1 Mycoplasma pneumoniae : general information .................................................6
1.2 Cytoskeleton-like structures in prokaryotes and cytoskeletal elements in
M. pneumoniae ................................................................................................9
1.3 Methods for detection and analysis of protein-protein interactions ............15
1.4 “The Yeast Two Hybrid System” .......................................................................19
1.5 “The Tandem Affinity Purification (TAP) Method”: A general procedure of
protein complex purification .......................................................................23
1.6 The outline for the experimental approach ...........................................................25
2 Materials ......................................................................................................................26
2.1 Bacteria strains ...........................................................................................................26
2.2 Yeast strains ...........................................................................................................27
2.3 Culture media
2.3.1 Supplementary ingredients and culture media ...........................................................27
2.3.2 Antibiotics
2.3.3 Culture medium for Escherichia coli .......................................................................27
2.3.4 Agar plates for E. coli ...............................................................................................28
2.3.5 Culture medium for M. pneumoniae .......................................................................28
2.3.6 Agar plates for ...................................................................................29
2.3.7 Culture medium and agar plates for Saccharomyces cerevisiae ........................29
2.4 Nucleic acids ...........................................................................................................30
2.4.1 Cosmids ......................................................................................................................30
2.4.2 Plasmids......................................................................................................................30
2.4.3 Oligonucleotides ...............................................................................................31
2.4.4 DNA Ladders and markers for DNA gel electrophoresis ....................................33
2.5 Proteins ......................................................................................................................34
2.5.1 Restriction endonucleases ...................................................................................34
2.5.2 Polymerases ...........................................................................................................34
2.5.3 DNA modifying enzymes and other enzymes ...........................................................34
iIndex
2.5.4 Antibodies ...........................................................................................................35
2.5.5 Protein ladders and markers for SDS-PAGE ...........................................................36
2.5.6 Other standard protein ...............................................................................................36
2.5.7 Peptides ......................................................................................................................36
2.6 Buffers and solutions
2.6.1 Reaction solutions for enzymes .......................................................................36
2.6.2 Solutions for plasmid isolation ...................................................................................36
2.6.3 Solutions for Colony transfer
2.6.4 Solution for Southern blot transfer .......................................................................37
2.6.5 Buffers for hybridization ...................................................................................37
2.6.6 Buffers for DIG-detection ...................................................................................37
2.6.7 Buffer for DNA gel electrophoresis .......................................................................38
2.6.8 Buffers for protein gel electrophoresis
2.6.9 Buffers for 2-D gel electrophoresis
2.6.10 Western blot transfer buffer ...................................................................................39
2.6.11 Buffers and solutions for Immunoblot analysis ...............................................39
2.6.12 Staining solution for polyacrylamide protein gels and protein blots ........................40
2.6.13 Buffers for His-tagged protein purification ...........................................................41
2.6.14 Buffers for TAP-tagged protein purification
2.6.15 Buffer for M. pneumoniae transformation ...........................................................42
2.6.16 Solutions for S. cerevisiae transformation
2.6.17 Other buffers and solutions ...................................................................................43
2.7 Chemical compounds ...............................................................................................43
2.7.1 Special chemical compounds ...................................................................................43
2.7.2 Reagents for biochemical and molecular biological methods ....................................46
2.7.3 Isotopes ......................................................................................................................46
2.8 Laboratory articles/ consumer items .......................................................................46
2.9 Apparatuses ...........................................................................................................47
2.9.1 Gel electrophoresis ...............................................................................................47
2.9.2 Chromatography
2.9.3 Other apparatuses
2.10 Computer Software ...............................................................................................48
iiIndex
3 Methods ..............................................................................................................