Assembly, stability and regulation of flagellar motor in Escherichia coli [Elektronische Ressource] / presented by Hui Li

-

English
128 Pages
Read an excerpt
Gain access to the library to view online
Learn more

Description

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 MSc. Hui LI born in: Hebei, China Oral-examination: November 2010 Assembly, stability and regulation of flagellar motor in Escherichia coli First referee: Prof. Dr. Victor Sourjik Second referee: PD Dr. Matthias P. Mayer To my parents Acknowledgements First and foremost, I would like to express my sincere gratitude to Prof. Dr. Victor Sourjik for his supervision, encouragement and support throughout the entire course of my PhD study. Also, I am very grateful to PD Dr. Matthias Mayer for his kind guidance, helpful discussion and valuable advice. This work would not have been done without the financial support by the “Heinz Goetze Memorial Fellowship Program” of the Athenaeum Foundation. Many thanks go to Dr. Dietlind Wünsche for her all-round help and assistance when I was just starting in Heidelberg. Part of this work has been done by collaborating with Prof. Dr. Urs Jenal and Dr. Alex Boehm in Biocenter, University of Basel, Switzerland. I wish to thank them for the pleasant collaboration. I am grateful as well to all the former and present colleagues in the lab for giving me indispensible help, creating a wonderful and fun working environment.

Subjects

Informations

Published by
Published 01 January 2010
Reads 24
Language English
Document size 6 MB
Report a problem


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

MSc. Hui LI
born in: Hebei, China
Oral-examination: November 2010



Assembly, stability and regulation
of flagellar motor in Escherichia coli











First referee: Prof. Dr. Victor Sourjik
Second referee: PD Dr. Matthias P. Mayer











To my parents

Acknowledgements
First and foremost, I would like to express my sincere gratitude to Prof. Dr. Victor
Sourjik for his supervision, encouragement and support throughout the entire course of
my PhD study.
Also, I am very grateful to PD Dr. Matthias Mayer for his kind guidance, helpful
discussion and valuable advice.
This work would not have been done without the financial support by the “Heinz Goetze
Memorial Fellowship Program” of the Athenaeum Foundation. Many thanks go to Dr.
Dietlind Wünsche for her all-round help and assistance when I was just starting in
Heidelberg.
Part of this work has been done by collaborating with Prof. Dr. Urs Jenal and Dr. Alex
Boehm in Biocenter, University of Basel, Switzerland. I wish to thank them for the
pleasant collaboration.
I am grateful as well to all the former and present colleagues in the lab for giving me
indispensible help, creating a wonderful and fun working environment. Especially, I
would like to thank, Linda Løvdok for her patience when I was new to the lab, David
Kentner for providing many plasmids and helpful discussions about my project, Silke
Neumann for helping me to translate the summary of my dissertation into German and
also sharing a laugh during movie nights, Abiola Pollard and Alvaro Banderas for their
proof-reading of my dissertation manuscript and very helpful suggestions.
Despite the distance, my parents have always been there for me. My deepest gratitude
goes to them for their endless support and constant inspiration through years.
Lastly, I would like to thank my beloved Qiang for sharing his memories and experiences
with me, for his constant encouragement and understanding, and for always being willing
to help me.
Contents

Contents
Zusammenfassung ····································································································· III
Summary ···················· V
1 Introduction ············· 1
1.1 Bacterial chemotaxis sensory system ············································································ 1
1.1.1 The biased random walk ··············································· 1
1.1.2 Chemotaxis signalling pathway ···································································· 2
1.2 Bacterial flagellar motor ······························· 4
1.2.1 Structure and mechanism of flagellar motor ················································································ 4
1.2.2 The flagellar type III export apparatus ·························· 7
1.3 Regulation of flagellar operon expression ····· 9
1.4 The morphogenetic pathway of motor assembly ························································· 10
1.5 Additional proteins controlling flagellar motor function and assembly ························ 13
1.5.1 Proteins reported to interact with flagellar motor ····································· 13
1.5.2 The Hsp70/Hsp90 multichaperone machinery ··········································· 14
1.6 Aims of the current work ···························································· 16
2 Materials and methods ·························································· 19
2.1 Chemicals ·················································································· 19
2.2 Enzymes····················································· 20
2.3 Antibodies ················· 20
2.4 Media and buffers ······································································································ 20
2.4.1 Growth media ····························· 20
2.4.2 Tethering buffer ·························· 21
2.4.3 Buffers for DNA gel electrophoresis ··························································································· 21
2.4.4 Buffers and solutions for immunoblot ························ 21
2.5 Bacterial strains ········································································· 23
2.6 Primers and plasmids ································· 24
2.7 Experimental methods ······························································· 28
2.7.1 Construction of Fluorescent protein fusions ·············································· 28
2.7.2 Strains and their growth ············································· 29
2.7.3 Tethering assay ··························································································· 30
2.7.4 Fluorescence imaging ················· 30
2.7.5 Quantification of protein expression ·························································· 30
2.7.6 Immunoblot analyses ················································································· 31
I Contents
2.7.7 Mass spectrometry assay ············································································································ 31
2.7.8 Studying protein binding kinetics by FRAP ·················· 32
2.7.9 Studying protein interactions by FRET························································································· 34
3 Results ··················································································· 39
3.1 Fluorescent protein fusions to flagellar motor and export apparatus proteins ·············· 39
3.1.1 Construction and functionality of fusions ··················································································· 39
3.1.2 Background-dependent localization of fusions ··········· 42
3.2 Assembly of FliF oligomers is conditional ····································· 46
3.2.1 Assembly of FliF oligomers depends on the expression level ····················· 46
3.2.2 FliF oligomerization is promoted by FlhA and FliG ······································ 46
3.3 FlhA stoichiometry at the motor ················································· 49
3.4 Complex formation enhances stability of FliF and FlhA in vivo ······································ 51
3.5 Measurements of proteins exchange at the functional motor ······ 54
3.6 FRET mapping of protein interactions at the motor ······················································ 56
3.6.1 Interactions of motor proteins in vivo ························································· 56
3.6.2 HtpG interaction with flagellar motor and chemotaxis proteins ················································ 61
3.6.3 YcgR interaction with flagellar motor ·························· 68
4 Discussion ·············································································································· 73
4.1 Order of the early steps in motor assembly ································· 73
4.2 Cooperativity of motor assembly ················ 74
4.3 Additional protein interactions at the motor ······························································· 74
4.4 Structure and stability of the motor ············································ 75
4.5 Regulation of protein stability by the complex formation ············ 76
4.6 Involvement of DnaK/HtpG machinery in assembly of flagellar motor
and chemosensory complexes ····················································································· 77
4.7 Cyclic di-GMP regulates bacterial flagellar motor ························· 78
5 References ············································· 81
6 Publications ··········································································· 93


II