Functional analysis of peptidases from the cyanobacterium Synechocystis sp. PCC 6803 [Elektronische Ressource] / vorgelegt von Elena Pojidaeva

ludwig-maximilians-universitat_munchen - Pojidaeva , Elena

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Biologie

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Publié par	ludwig-maximilians-universitat_munchen
Publié le	01 janvier 2004
Nombre de lectures	14
Langue	English
Poids de l'ouvrage	3 Mo

Extrait

Functional analysis of peptidases from the cyanobacterium
Synechocystis sp. PCC 6803

Dissertation
zur Erlangung des Doktorgrades der Fakult t f r Biologie
der Ludwig-Maximilians-Universit t M nchen

vorgelegt von
Elena Pojidaeva
aus Moskau, Russland

M nchen
30.Juni 2004

1. Gutachter: Prof. Dr. R. G. Herrmann
2. Gutachter: Prof. Dr. H. Scheer

Tag der m ndlichen Pr fung: 16.09.2004

To my family

CONTENT I
CONTENT

Abbreviations ????????????????????????????????? V

1. INTRODUCTION ???????????????????????????? . 1
1.1 Proteolytic enzymes and their role in cell homeostasis . ... 1
1.2 Characterization of the protease complement of cyanobacterial cells .. 7
1.2.1 The Clp peptidase family . 8
1.2.2 The Deg peptidase family ?? ???? ?? ???? ?? ???? ?? . 9
1.2.3 The FtsH peptidase family ???? ?? ???? ?? ???? ?? ?? 10
1.2.4 The Ctp family of carboxypeptidases ... 11
1.2.5 The SppA peptidase family ???? ?? ???? ?? ???? ?? ??? 12

1.3 Cyanobacteria as models for the analysis of the photosynthetic machinery ? 13
1.3.1 Structure of cyanobacterial photosynthetic complexes ????????? . 13
1.3.2 Adaptation of cyanobacteria to environmental changes ?? ? ?? ?? 17
1.3.2.1 Light acclimation ? ???? ?? ???? ?? ???? ?? ???? .. 17
1.3.2.2 Heat stress . 19
1.3.2.3 Nutrient stress ?????????????? . ?????? .. 20

2. MATERIALS AND METHODS ???? ? ???? ?? ???? .. ???? ?? . 22
2.1 Materials ?? ?? ???? ?? ???? ?? ???? ?? ???? ?? .. ??? . 22
2.1.1 Chemicals and enzymes ??? ?? ???? ?? ???? ?? ???? ?? . 22
2.1.2 Molecular weight markers .. 22
2.1.3 Vectors and strains ???????????????????????? . 23
2.1.4 Antibodies ??????????? .. 23
2.1.5 Oligonucleotides ???? ?? ???? ?? ???? ?? ???? ?? ?? 23
2.1.5.1 Primers for Escherichia coli strains ???? ?? ???? ?? ???? ... 24 1.5.2 Synechocystis gene-specific primers ???? ?? ???? . ???? . 24

2.1.6 General buffers and solutions . 25
2.1.7 Medium for bacterial growth ??? ???? ?? ???? ?? .. ???? ?? 26
2.1.8 Transfer membranes ? ???? ?? ???? ?? ???? ?? ???? ?? ?? 27
2.1.9 Plant material ????????????????????????? .. 28

2.2 Methods .. 28
2.2.1 Sequence analysis ???? ?? ???? ?? ???? ?? ???? ?? ?? 28
2.2.2 Strains and growth conditions for Synechocystis ???? ?? ???? ?? ??? 28
2.2.3 Construction of knock-out mutants in Synechocystis .. . 29
2.2.3.1 Construction of recombinant plasmids ??? ? ???? ?? ???? . 29

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??CONTENT II
2.2.3.2 Transformation of Synechocystis ???? ?? ???? ?? ???? .. 30
2.2.3.3 Conjugal transfer of plasmids into cyanobacterial cells . .. 31

2.2.4 DNA and RNA analysis ?? ???? ?? ???? ?? ???? ?? . 31
2.2.4.1 DNA isolation from Synechocystis ???? ?? ???? ?? ???? ? 31
2.2.4.2 PCR analysis . 32
2.2.4.3 Isolation of plasmid DNA from E. coli ???????????????. 32 2.4.4 RNA isolation from Synechocystis .. . 32
2.2.4.5 Analysis of geneexpression ?. ????????????????. 33
2.2.4.5.1 Northern analysis ?????????????????? . 33
2.2.4.5.2 RT-PCR ??? ? ?? ?? ?? 34

2.2.5 Overexpression of proteins ?????????????????????? . 34
2.2.5.1 Overexpression of proteins in E. coli cells . 34
2.2.5.2 Overexon of proteins in E. coli lysates . 35

2.2.6 Generation of protein-specific antisera .. .. 35
2.2.6.1 Preparation of probes for rabbit immunization ? ???? ?? ???? .. 35
2.2.6.2. Injection of rabbits and antibody preparation . .. 35
2.2.6.3 Purification of antibodies .. . 36
2.2.6.3.1 Purification of antibodies with overexpressed
protein on the membrane ????? ?? ???? ?? ???? 36
2.2.9.3.2 Purificatiofantibodies
with Protein A-Sepharose .. .. 36

2.2.7 Pigment analysis of Synechocystis cells ??????????????? .. 37
2.2.7.1 Determination of chlorophyll a concentration ?? ???? ?? ???? ... 37
2.2.7.2 Determinatiof carotenoid concentration ??? ???? ?? ???? ? 37
2.2.7.3 Determination of C-phycocyanin concentration . 37

2.2.8 Determination of cell densities 38
2.2.9 Protein analysis ???? ?? ???? ?? ???? ?? ???? ?? ??? 38
2.2.9.1 Determination of protein concentrations ? ? ? ???? . .. 38
2.2.9.2 Protein gel electrophoresis . 39
2.2.9.2.1 Gel electrophoresis according to Laemmli (1970) . .. 39
2.2.8.2.2 electrophoresis according to Kashino (2001) ........... . 39
2.2.9.3 Staining of PAA gels .. 40
2.2.9.3.1 Coomassie Brilliant Blue staining . 40
2.2.9.3.2. Silver staining ? ???? ?? ???? ?? . ? ? . 41
2.2.9.3.3. Imidazol staining .. 42

2.2.9.4 Isolation and fractionation of thylakoid membranes???? ?? ???? ? 42
2.2.9.4.1 Isolation of total cellular and membrane proteins
from Synechocystis ???? ?? ???? ?? ???? 42
2.2.9.4.2 Extraction of peripherally associated proteins .. 43
2.2.9.4.3 Preparation of phycobilisomes .. 43
2.2.9.4.4 Isolation of photosynthetic complexes from Synechocystis
cells by sucrose gradient centrifugation ? ???? .. ???? ... 44
2.2.9.4.5 Isolation of photosynthetic complexes from Sy
by non-denaturing gel electrophoresis 44

2.2.9.5 Immunological detection of proteins .. ??? ???? ?? ???? ? 46
2.2.9.5.1 Transfer of proteins onto nitrocellulose membranes ?? ?? ? ? . 46

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????CONTENT III
2.2.9.5.2 Staining blots with Ponceau S ?? ???? ?? ???? ?? . 47
2.2.9.5.3 Western analysis using horseradish
peroxidase-conjgated antibodies ?????????? .. 47
1252.9.5.4 Western analysis using I labeled Protein A antibody ??? . 48

2.2.10 Proteolysis assay ? ???? . ?? . ?? ???? ?? ??? 48
35 2.2.11 Protein pulse-labeling with S-methionine ??? ???? ?? ???? ?? ?? 49
2.2.12 Measurements of oxygen evolution by the Clark electrode . 49
2.2.13 Low temperature (77K) fluorescence analysis ???? ?? ???? ?? .. . 49

3. RESULTS .. 51
3.1 Characterization of peptidases in Synechocystis . . 51
3.1.1 Physiological analysis of peptidase knock-out mutant strains ? ???? ?? . 52
3.1.1.1 Acclimation of cyanobacterial cells to different light intensities . 53
3.1.1.2 Heat stress ??? ???? ?? ???? ?? ???? ?? . 53
3.1.1.3 Nutrient deprivation ................................................................................ . 55

3.2 Functional analysis of the SppA peptidase family in Synechocystis ??? ?? ???? ?? . 57
3.2.1 Functional analysis of SppA1 protease ??? ?? ???? ?? ???? ?? . . 57
3.2.1.1 Analysis of SppA1 protein sequence ?? ???? ?? ???? ?? . 57
3.2.1.2 Construction of ∆sppA1 and complement pVZsppA1 mutant strains??? .. 59
3.2.1.3 Analysis of sppA1 gene ?? ???? ?? ???? ?? ???? ?? .. 61
3.2.1.4 Expression of the sppA1 gene under different light intensities . . 63
3.2.1.5 Phenotypical characterization of the ∆sppA1 ??? ???? ?? . 63
3.2.1.6 Spectroscopic characterization of the ∆sppA1 ??? ?? ???? ?? 67
3.2.1.7 Analysis of content of photosynthetic proteins .. 69
3.2.1.8 Genes and expression analysis of phycobiliproteins .. 70
3.2.1.9 In vivo labeling of the wild-type and ∆sppA1 ??? ?? ???? ?? . 72
3.2.1.10 Kinetics of phycobiliproteins degradation .. 73
3.2.1.11 Analysis of phycobiliproteins .. 74
3.2.1.11.1 Characterization and isolation of phycobilisomes .. . 74
3.2.1.11.2 SppA1 is involved in the light-dependent cleavage
of rod linkers .. 76

3.2.2 Functional analysis of SppA2 protease ??? ?? ???? ?? ???? ?? . . 78
3.2.2.1 Analysis of SppA2 amino acid sequence ??? ? ???? ?? . ? . 78
3.2.2.2 Overexpression of SppA2 protein ????????????? 79
3.2.2.3 Analysis of proteolytic activity of SppA2 81
3.2.2.4 Localization of the SppA2 protein 82
3.2.2.4.1 Intracellular localization of the SppA2 protein ? ???? ?? 82