Outer membrane proteins of Anabaena sp. strain PCC 7120 [Elektronische Ressource] / vorgelegt von Sunčana Moslavac

Suncana Moslavac

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Outer membrane proteins of Anabaena sp.
strain PCC 7120





Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München



vorgelegt von
Sun čana Moslavac





München, 2007























Dissertation eingereicht am: 18. 06. 2007

Datum der mündlichen Prüfung: 20. 07. 2007



Erstgutachter: PD Dr. Enrico Schleiff
Zweitgutachter: Prof. Dr. Jörg Nickelsen



Ehrenwörtliche Versicherung

Die vorliegende Dissertation wurde von Sun čana Moslavac selbständig und ohne
unerlaubte Hilfe angefertigt. Die Verfasserin hat zuvor nicht versucht, anderweitig eine
Dissertation einzureichen oder sich einer Doktorprüfung zu unterziehen. Die Dissertation
wurde keiner weiteren Prüfungskommission weder in Teilen noch als Ganzes vorgelegt.





München, 15.06.2007 _______________________________
Sunčana Moslavac




Table of Contents

1. Abstract ........................................................................................................................1
2. Zusammenfassung ....................................................................................................... 2
3. Introduction ................................................................................................................. 3
3.1. Filamentous nitrogen fixing cyanobacterium Anabaena sp. PCC 7120................ 3
3.2. Anabaena sp. PCC 7120 – a model organism for plastid evolution...................... 6
3.3. β-barrel proteins of the outer membrane ............................................................... 7
3.3.1 TolC-dependent protein secretion.................................................................9
3.3.2 TonB-dependent transporters and iron uptake............................................. 11
3.4. An objective.........................................................................................................16
4. Materials..................................................................................................................... 17
4.1. Chemicals............................................................................................................17
4.2. Enzymes and kits................................................................................................. 17
4.3. Primers.................................................................................................................18
4.4. Vectors19
4.4.1 Cyanobacterial and bacterial strains............................................................ 20
4.5. Antibodies20
4.6. Glass beads, TLC plates and membranes 20
4.7. System for the growth of Anabaena sp. PCC 7120 cultures ............................... 21
5. Methods ...................................................................................................................... 22
5.1. Molecular biological methods ............................................................................. 22
5.1.1 General molecular biological methods........................................................ 22
5.1.2 Polymerase chain reaction (PCR)................................................................ 22
5.1.3 Cloning strategies........................................................................................22
5.1.3.1 Generation of deletion strains.................................................................. 22
5.1.3.2 Generation of over-expression strains ..................................................... 23
5.1.3.3 Generation of GFP-protein fusion strains............................................... 23
5.1.3.4 Generation of GFP-promoter fusion strains ............................................ 24
5.1.4 RT-PCR.......................................................................................................25
5.1.5 Southern blotting.........................................................................................26
5.2. Biochemical methods..........................................................................................26
5.2.1 Determination of protein concentration....................................................... 26
I5.2.2 SDS-Polyacrylamid Gel Electrophoresis (SDS-PAGE) and Blue-Native
electrophoresis (BN-PAGE) ........................................................................ 26
5.2.3 Western blotting..........................................................................................27
5.2.4 Protein extraction.........................................................................................27
5.2.5 Digestion of Anabaena sp. outer membrane proteins and mass spectrometry
.....................................................................................................................27
5.3. Chromatography methods...................................................................................28
5.3.1 Thin Layer Chromatography (TLC) of lipids.............................................. 28
5.4. Methods related to Anabaena sp. PCC 7120....................................................... 28
5.4.1 Media and growth conditions ...................................................................... 28
5.4.2 Determination of the chlorophyll a concentration....................................... 29
5.4.3 Growth curve...............................................................................................29
5.4.4 DNA isolation out of Anabaena sp. PCC 7120........................................... 30
5.4.5 RNA isolation out of 30
5.4.6 Conjugation.................................................................................................31
5.4.7 Heterocyst isolation.....................................................................................32
5.4.8 Membrane fractionation of vegetative cells ................................................ 32
5.4.9 Membrane fractionation of isolated heterocysts.......................................... 33
5.4.10 Thermoluminescence measurements...........................................................34
5.4.11 Determination of metal uptake .................................................................... 34
5.4.12 Amino acid transport assay ......................................................................... 34
5.4.13 Spectrometric GFP fluorescence measurement........................................... 35
5.4.14 Measurements of the chlorophyll fluorescence by PAM ............................ 35
5.4.15 ents of nitrogenase activity ......................................................... 36
5.4.16 Analysis of in the medium secreted Anabaena sp. proteins........................ 36
5.5. Microscopy..........................................................................................................37
5.5.1 Light microscopy.........................................................................................37
5.5.2 Electron microscopy....................................................................................37
5.6. Protein modelling and bioinformatic analyses .................................................... 38
6. Results.........................................................................................................................39
6.1. Proteomic analysis of the outer membrane of Anabaena sp. PCC 7120............. 39
6.1.1 New method for the membrane fractionation of vegetative cells of
Anabaena sp. PCC 7120 .............................................................................. 39
6.1.2 General characterisation of identified proteins............................................ 41
II6.1.3 Classification of identified proteins............................................................. 43
6.1.4 Cytoplasmic proteins...................................................................................46
6.1.5 Inner membrane and thylakoid membrane proteins .................................... 46
6.1.6 Periplasmic proteins and exoproteins.......................................................... 47
6.1.7 Outer membrane proteins ............................................................................ 48
6.1.8 Complex analysis.........................................................................................49
6.2. Proteomic analyses of the heterocyst outer membrane of Anabaena sp. PCC 7120
.............................................................................................................................52
6.2.1 Alr2269 protein as an outer membrane / cell wall marker .......................... 52
6.2.2 New method for the outer membrane / cell wall isolation from heterocyst of
Anabaena sp. PCC 7120 .............................................................................. 54
6.2.3 Proteome analysis of the isolated heterocyst outer membrane / cell wall
fraction.........................................................................................................56
6.3. Alr2887 – the TolC-like transporter of Anabaena sp. PCC 7120 ....................... 59
6.3.1 alr2887 gene