Analysis of the interaction of the LuxR-type transcription factors BglJ and RcsB and antagonism of H-NS mediated silencing in Escherichia coli [Elektronische Ressource] / vorgelegt von Raja Venkatesh Ganesan

Sabari

Sujets

• Biologie

Informations

• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Analysis of the interaction of the LuxR-type
transcription factors BglJ and RcsB and antagonism of
H-NS mediated silencing in Escherichia coli




I n a u g u r a l - D i s s e r t a t i o n

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Universität zu Köln










vorgelegt von

Raja Venkatesh Ganesan

aus Tamil Nadu, Indien








Köln, März 2010


































Berichterstatter/in: Prof. Dr. Karin Schnetz
Prof. Dr. Jürgen Dohmen




Tag der letzten mündlichen Prüfung: 26 April 2010
Acknowledgements

I take this opportunity to thank all the people who have helped, encouraged and
inspired me during my graduation and made this thesis possible. First and foremost, I
would like to express my deepest gratitude to my PhD supervisor Prof. Karin Schnetz
for providing me the opportunity to work in her group and sharing her knowledge for
the betterment of my life. I am very thankful for her valuable advice, support and
teaching me the word “organization”. Thanks for the pushes and constructive
criticism which helped me to make this happen.

I would like to thank Prof. Dr. Siegfried Roth and Prof. Dr. Jürgen Dohmen for being
part of my thesis committee. I thank Dr. Kevin Johnson for agreeing to be part of my
thesis committee.

I am deeply grateful to Dr. Sabari and Dr. Palani for their immeasurable help and
support professionally and personally. I thank Dr. Jain Nayar for his wise words
during tougher times. I also thank him for the lighter moments and great movie
sessions.

Many thanks to Dr. Antonio Jose Marque for his help with FPLC for the protein
purification and also for the invitation for volleyball games.

I feel extremely fortunate having enjoyed my time with wonderful colleagues. I thank
all my past lab members Vel, Madhu, Andreas, Kathleen, Birgit, Idriss for their help. I
thank my present lab members, Kostya, Thomas, Lilia and Katrin for being very
helpful and very supportive. I enjoyed our movie sessions, funny jokes, visit to the
zoo, football talks, parties at Lilia`s house. Everything was really great and fun. It was
pleasure working with you all.

I am very thankful to Brigitte Wilcken-Bergmann for her countless helps from IKEA
to AOK and for taking care of us like her own child. I also thank Isabell Witt for all
her help and time during our presentations and retreats. Thanks to Inge Götz-Krichi
for making the administrative work easier.

My graduate school coordinators deserve a loud mention: Jo and Kathy, You two are
awesome. I bet we would not see many better people than you two. I thank you both
for all your help.

My special thanks to Prof. Dohmen`s group members Palani, Leo, Daniela, Toze,
Marion, Maria, Annie, Ganapathy, Kerstin for treating me like one of them and
inviting me for all occasions.

Special thanks to Daniela for being my Deutsch-Englisches Wörterbuch and helping
me translating many things with patience. Thanks for being supportive and sharing
“The Good, the bad and the ugly” things in the PhD life.

I like to thank all my friends Bhagi, Vivek, Sam, Venky, Rajesh, Charles, Chenna,
Ravi, Jain, Sudhir, Racchappa, Karthik, Ram, Vinodh, Zahid, Johnny, Numan, Giri,
Arun, Saba, Naga, Andrews, Devender, for all their help and making Sunday more
Acknowledgements

fun filled with cricket and arguments. I am also thankful for the delicious dishes
provided by Viji, Kayal and Preeti during special occasions.

I thank my wonderful lab neighbors Dr. Mirka, Nils, Eva, Sabrina, Dr. Martina, Dr.
Renjith, Dr, Sanjita, Dr. Gritt, Katarina, Julia, and Cornelia, for being nice and
helpful. I also thank them for effectively engaging in Friday beer sessions and grilling
in summer and extending fun beyond science.

I am lucky enough to come across many good people during this time like Ines,
Fabian, Florian, Osmon, Carsten, Tanja, and Anne. I would like to thank Sascha
Dargazanli and Nina Leuschen for their help in getting the chemicals with a smiling
face even at their busiest times. Big thanks to our lovely kitchen staffs Elfie Hansen
and Karin Küllenberg for the friendly smiles and help. I also thank my institute
Hausmeister Ramdane and Ute for their help and funny chit chats.

I should also thank the people for building this Institute closer to life saving places
like Uni Mensa, Backwerk, and Happy Döner. I thank Frau. Gotzmann for her
extended help in filling the application form.

I thank Leo and Luis for helping me to adjust to the new life in Köln in the early days
and special thanks to Jinu for her hospitality and treating me like a family member.

I thank my first German friend and roommate Sebastian, for saving my life in the very
first days by cooking Fischstäbchen. I am indebted to him for introducing me to the
great “Kölsch”. I also thank my Deutsch lehrerin Frau.Petra for her efforts to teach me
some Deutsch.

I thank my soccer team members Tobi, Benny, Fabiano, Florian, Sascha, Max, Malou,
Richie, Frank, Yinka, Marcel, Anni, Heike and Michael for the fun times on and off
the ground.

Verena deserves a special mention for her unconditional care and support all the time.
I am thankful to my other family Alex, Jenny, Mara, Lina, Thomas, Wiebke, Arne,
Kurt, Edith, Renate, Wolfgang, Michaela, Marion and Romina for their love and care.

My deepest gratitude goes to my father and mother for their support and love. This
stage would have been simply impossible without them. I also thank my brother for
being very supportive and helpful.

Finally my appreciation also goes to the city Köln, which has an extraordinary
diversity of culture and life style. This city teaches you how to cherish the good things
in your life. Everything is fun, from Zülpicher to Schildergasse, Live Music Hall to
Rhein-Energie-Stadion and Carnival to Christopher Street Day. Ich liebe Köln!

Da simmer dabei, dat is prima, VIVA COLONIA!




Acknowledgements

Contents

Abbreviations

I Zusammenfassung 01

I Summary 03
II Introduction 04

1. H-NS, a nucleoid associated protein 04
2. H-NS mediated repression 05
3. Anti-silencing of H-NS repression 06
4. Bgl operon 07
5. Rcs two-component system 09
6. yjjQ-bglJ operon 10
7. Aim of the thesis 11

III Results 12
1. Interaction of RcsB with BglJ and YjjQ 12
1.1 Expression of epitope tagged proteins 12
1.2 Phenotype analysis 16
1.3 Interaction of RcsB with BglJ 17
1.4 RcsB interaction with YjjQ 20

2. RcsB-BglJ heterodimer binding to the bgl regulatory region 23
2.1 Linker insertion mutants of a putative RcsB/BglJ site in 23
bgl regulatory region
2.2 Bacterial one hybrid system for DNA-binding specificity 25
2.2.1 Testing bacterial one-hybrid system 27
2.2.2 Analysis of putative binding site 28
2.3 Protein purification and Electrophoretic Mobility Shift Assay (EMSA) 32
2.3.1 Expression of epitope tagged proteins 33
2.3.2 Strep-tagged protein purification 37
2.3.4 Electrophoretic Mobility Shift Assay (EMSA) 40


Contents i3. BglJ, a protease target in E. coli 46
3.1 BglJ protein stability 46

4. Microarray 48
IV Discussion 52

1. RcsB interaction with BglJ and YjjQ 52
2. De-repression of bgl operon by BglJ/RcsB might require a co-factor 54
3. Lon protease targets BglJ 56
4. Model 57

V Materials and methods 58
1. Media and agar plates 58
2. Antibiotics, sugars and amino acids 60
3. Buffers 60
4. Standard molecular biology techniques 61
5. Bacterial strains 62
6. Plasmids 62
7. DNA sequencing 65
8. Deletion of genes according to Datsenko and Wanner 65
9. Transduction with phage T4GT7 66
10. Integration of plasmids into the attB site of the E. coli chromosome 67
11. β-galactosidase assay 67
12. Preparation of competent cells and transformation 68
13. SDS-PAGE and Immunoblotting 69
14. Co-immunoprecipitation 69
15. Strep-tag protein purification 71
16. BglJ protein purification 72
17. Electrophoretic Mobility Shift Assay (EMSA) 73

VI Bibliography 74
Erklärung 85
Lebenslauf 86
Curriculum Vitae 87

Contents i