Transcriptional activation and sensing properties of DegS-DegU [Elektronische Ressource] : a two-component system involved in the osmotic regulation of Bacillus subtilis / vorgelegt von Teodora Hadzhieva

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Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Transcriptional activation and sensing properties
of DegS-DegU: a two-component system involved
in the osmotic regulation of Bacillus subtilis









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









dem Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von





Teodora Hadzhieva
aus Varna (Bulgarien)



Marburg/Lahn 2007

Die Untersuchungen zu der vorliegender Arbeit wurden in der Zeit von Mai 2004 bis August
2007 im Laboratorium für Mikrobiologie der Philipps-Universität Marburg unter der Leitung
von Prof. Dr. Erhard Bremer dirchgeführt.
































Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation

am..............................................angenomen



Erstgutachter: Prof. Dr. Erhard Bremer

Zweitgutachter: Prof. Dr. Wolfgang Buckel



Tag der mündlichen Prüfung am:






The voyage of discovery is not in seeking new landscapes but in having new eyes

- Marcel Proust CONTENT 1

Content

I. Summary ............................................................................................................................. ..7

II. Introduction....................................................................................................................... ..9
1. Bacillus subtilis........................................................................................................... ..9
2. Significans of water for living cells............................................................................ ..9
3. Osmosensing and osmoregulation in bacteria with focus on B. subtilis..................... 11
3.1. Osmoregulation in response to hypoosmotic conditions .................................... 11
3.2. Osmoregulation in response to hyperosm ................................... 12
3.2.1. The initial stress response 13
3.2.2. Osmoadaptation via biosynthesis of compatible solutes............................. 14
3.2.3. Transport of compatible solutes for osmoprotective purposes.................... 18
3.3. General stress response ....................................................................................... 20
4. Two-component regulatory systems........................................................................... 21
4.1. System architecture.............................................................................................22 4.2. Structure of histidine kinases .............................................................................. 23 4.3. Structure of response regulators.......................................................................... 25
5. The DegS-DegU two-component system from Bacillus subtilis................................ 27
6. Aim of the work.......................................................................................................... 32

III. Materials and methods.................................................................................................... 33
1. Chemicals and reagents .............................................................................................. 33
2. Bacterial strains and plasmids..................................................................................... 33
2.1. Bacterial strains................................................................................................... 33
2.2. Plasmids .............................................................................................................. 35
3. Oligonucleotides.........................................................................................................36
4. Growth media and cultivation conditions................................................................... 39
4.1. Complex media39
4.2. Minimal media4.3. Transformation medium......................................................................................40
4.4. Compatible solutes and antibiotics .....................................................................
4.5. Sterilization41
4.6. Determination of the osmolarity ......................................................................... 41
4.7. Cultivation conditions ......................................................................................... 41
5. Molecular biology approaches.................................................................................... 42
5.1. Agarose gel electrophoresis ................................................................................ 42
5.2. DNA techniques.................................................................................................. 43
5.2.1. Isolation of genomic DNA .......................................................................... 43
5.2.2. Isolation of plasmid DNA ........................................................................... 43
5.2.3. Polymerase chain reaction (PCR)................................................................43
5.2.4. Determination of DNA concentration ......................................................... 43
5.2.5. Digestion and ligation of DNA ................................................................... 43
5.2.6. DNA sequencing.........................................................................................44
5.3. RNA techniques 44
5.3.1. Isolation of total RNA from B.subtilis ........................................................ 44
5.3.2. Determination of RNA concentration and purity........................................ 45
5.3.3. Synthesis of digoxigenin-labelled RNA probes .......................................... 46
5.3.4. Northern blot analysis ................................................................................. 46
5.3.5. Dot blot........................................................................................................47 CONTENT 2

5.3.6. Primer extension analysis............................................................................ 47
5.4. Bacterial transformation...................................................................................... 48
5.4.1. Preparation of competent B. subtilis cells and transformation.................... 48
5.4.2. Preparation of competent E. coliation ......................... 48
5.5. Construction of plasmids and bacterial strains ................................................... 49
5.5.1. Plasmid construction...................................................................................49
5.5.2. Strain .......................................................................................51
6. Biochemical approaches ............................................................................................. 52
6.1. SDS Polyacrylamide Gel Electrophoresis (PAGE) ............................................ 52
6.2. Determination of protein concentration .............................................................. 53
6.3. Heterologous overexpression and purification of DegS and DegU proteins
from B. subtilis .......................................................................................................... 53
6.3.1. Heterologous expression of DegS ............................................................... 53
6.3.2. Heterologous expression of DegU 54
6.3.3. Purification of DegS and DegU................................................................... 54
6.4. Phosphorylation assay......................................................................................... 55
6.4.1. Autophosphorylation of DegS sensor kinase .............................................. 55
6.4.2. Phosphotransfer from the DegS sensor kinase to the DegU regulator........ 55
6.5. HPLC..................................................................................................................56
6.5.1. Extraction according to Bligh and Dyer...................................................... 56
6.5.2. Precolumn derivatisation with FMOC ........................................................ 57
6.5.3. HPLC analysis.............................................................................................57
6.6. Determination of TreA activity........................................................................... 58
6.7. Amylase test........................................................................................................ 59
6.8. Protease assay ..................................................................................................... 59

IV. Results ............................................................................................................................... 60
1. High levels of DegU~P lead to the overproduction of degradative enzymes............. 60
2. The degSU deletion mutant shows no phenotype concerning growth