Functional analysis of the leader peptidases in cyanobacterium Synechocystis sp. PCC 6803 [Elektronische Ressource] / von Maria Zhbanko

Mascha

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 28 Mo

Extrait

Functional analysis of the leader peptidases
in cyanobacterium Synechocystis sp. PCC 6803.




Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat)




vorgelegt der

Matematisch-Naurwissenschaftlich-Technischen Fakultät
(matematisch-naturwissenschaftlicher Bereich)
der Martin-Luther-Universität Halle-Wittenberg



von Frau Maria Zhbanko

geb. am: 10. April 1974 in: Moskau





Gutachter:

1. Prof. Dr. R. B. Klösgen

2. Prof. Dr. U. Johanningmeier

3. Prof. Dr. M. Rögner


Halle (Saale), 28 Februar 2006

urn:nbn:de:gbv:3-000009972
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000009972]
Table of contents
Abbreviations............................................................................................................................4
1. Introduction ..........................................................................................................................7
1.1. Specific features of cyanobacteria important for this study ......................................... 7
1.2. Translocation of proteins and biogenesis of thylakoid membrane............................. 10
1.3. Role of the signal peptidases for the protein transport processes............................... 15
1.3.1. Types of signal peptidases in bacteria ................................................................. 15
1.3.2. Specific features and role of different signal peptides ........................................ 15
1.3.3. Structural and functional similarities of leader peptidases from bacteria and
thylakoid processing peptidase from higher plants. ...................................................... 17
1.4. Aims of this work ....................................................................................................... 19
2. Materials and Methods ......................................................................................................20
2.1. Chemicals and enzymes.............................................................................................. 20
2.2. Bacterial strains and plasmids .................................................................................... 20
2.3. Oligonucleotides......................................................................................................... 22
2.4. Molecular weight markers for gel electrophoresis ..................................................... 23
2.5. Cultivation of Escherichia coli cells .......................................................................... 23
2.6. Cultivation of Synechocystis sp. PCC6803 cells ........................................................ 24
2.7. Transformation of E. coli cells ................................................................................... 25
2.8. Transformation and conjugation of Synechocystis 6803 cells.................................... 25
2.9. Harvesting of Synechocystis 6803 cells...................................................................... 26
2.10. Preparation of stock cultures .................................................................................... 26
2.11. Synechocystis 6803 growth curves .......................................................................... 27
2.12. Molecular biology methods...................................................................................... 27
2.12.1. Standard methods .............................................................................................. 27
2.12.2. Polymerase chain reaction................................................................................. 27
2.12.3. Isolation of genomic DNA from Synechocystis 6803 cells ............................... 28
2.12.4. Isolation of plasmid DNA from Synechocystis 6803 cells ................................ 28
2.12.5. Construction of recombinant plasmids.............................................................. 29
2.13. Biochemical methods ............................................................................................... 30
2.13.1. Determination of protein concentration............................................................. 30
2.13.2. Protein precipitation .......................................................................................... 31
2.13.3. Isolation of expressed protein from E. coli ....................................................... 31
2.13.4. SDS-polyacrylamide gel electrophoresis (SDS-PAGE).................................... 32
1
2.13.5. Staining of polyacrylamid gels.......................................................................... 32
2.13.6. Staining of heme-containing proteins................................................................ 33
2.13.7. Western Blot Analysis....................................................................................... 33
2.13.8. Isolation of membranes from Synechocystis 6803 ............................................ 35
2.13.9. Blue native PAGE ............................................................................................. 35
2.13.10. Determination of chlorophyll content.............................................................. 37
2.13.11. Pigment analysis by HPLC.............................................................................. 37
2.13.12. Determination of the cell densities .................................................................. 38
2.14. Proteomic methods ................................................................................................... 38
2.14.1. Two-dimensional gel electrophoresis................................................................ 38
2.14.2. Peptide mass fingerprinting (performed by Dr. Angelika Schierhorn) ............. 40
2.15. Physiological methods.............................................................................................. 42
2.15.1. Measurements of the absorption spectra ........................................................... 42
2.15.2. Low temperature fluorescence emission spectra............................................... 42
2.15.3. Measurements of the photosynthetic activity with Clark-electrode .................. 42
2.16. Electron-microscopy of the Synechocystis 6803 cells (performed by Dr. Gerd
Hause)................................................................................................................................ 43
2.17. Computer analysis of polypeptides .......................................................................... 43
2.17.1. The search of Synechocystis 6803 proteins containing N-terminal signal
peptides with the Signal-P3.0 program ......................................................................... 43
2.17.2. Blast and ClustalW analysis .............................................................................. 44
3. Results..................................................................................................................................45
3.1. Analysis of the protein translocases and signal peptidases of Synechocystis 6803.... 45
3.2. The strategy of the targeted gene inactivation............................................................ 46
3.3. Functional analysis of the two genes for type I signal peptidases of Synechocystis
6803. .................................................................................................................................. 48
3.3.1. Analysis of amino acid sequences of signal peptidases I. ................................... 48
3.3.2. Inactivation of the genes encoding LepB1 and LepB2 proteins.......................... 54
3.3.3. Analysis of LepB1 antigen and production of antiserum.................................... 56
R
3.4. Phenotypic features of lepB1::Km mutant................................................................ 58
R3.4.1. Homozygous lepB1::Km cells are sensitive to high light intensities................. 58
R3.4.2. The alterations in thylakoid membrane structure revealed in lepB1::Km mutant
by electron microscopy of the Synechocystis 6803 cells............................................... 58
R3.5. The complementation of the leader peptidase function in the lepB1::Km mutant. .. 60
2
R3.6. Characterization of the lepB1::Km mutant strain of Synechocystis 6803 ................ 62
R3.6.1. The lepB1::Km mutant strain is incapable of photoautotrophic growth............ 62
3.6.2. The mutant cells show the altered pigment composition and PSI/PSII ratio ...... 63
R
3.6.3. The photosynthetic electron transport in lepB1::Km is inhibited by strong light
....................................................................................................................................... 67
3.6.4. The assembly of the core proteins of photosystems is not significantly affected in
the mutant. ..................................................................................................................... 70
3.6.4.1. Analysis of thylakoid membrane proteins using SDS-PAGE ...................... 70
3.6.4.2. Analysis of membrane protein complexes by blue-native PAGE. ............... 71
3.6.4.3. Analysis of cytochrome b f complex by specific staining........................... 75 6
3.6.4.4. Im