Synechocystis PCC6803: the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 139 S. Ill., graph. Darst. 30 cm |
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| 245 | 1 | 0 | |a Synechocystis PCC6803 |b the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |c presented by Danika Maria Trautmann |
| 264 | 1 | |c 2013 | |
| 300 | |a 139 S. |b Ill., graph. Darst. |c 30 cm | ||
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| 502 | |a Freiburg (Breisgau), Univ., Diss., 2013 | ||
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| adam_text | IMAGE 1
TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 CYANOBACTERIA AS IMPORTANT MODEL ORGANISMS 1
1.1.1 EVIDENCES FOR MICROEVOLUTION IN CYANOBACTERIA 1
1.1.2 PHYCOBILISOMES - ADVANCED LIGHT-HARVESTING COMPLEXES IN
CYANOBACTERIA 2
1.2 OXIDATIVE STRESS RESPONSE IN CYANOBACTERIA 3
1.2.1 DEFENCE MECHANISMS 3
1.2.2 OXIDATIVE STRESS-INDUCED LIPID PEROXIDATION 4
1.2.3 OXIDATIVE STRESS LEADS TO FORMATION OF ALDEHYDES 4
1.2.4 CYANOBACTERIAL SUNSCREEN PIGMENTS 5
1.3 CAROTENOID METABOLISM IN CYANOBACTERIA 6
1.3.1 AN INTRODUCTION TO CAROTENOIDS-STRUCTURE AND RELEVANCE 6
1.3.2 BIOSYNTHESIS OF CAROTENOIDS 8
1.3.3 CAROTENOID CATABOLISM - ENZYMATIC CAROTENOID CLEAVAGE 10
1.4 ALKANES IN CYANOBACTERIA 13
1.5 ALDEHYDE DEHYDROGENASES (ALDHS) 14
1.5.1 STRUCTURE OF ALDHS 15
1.5.2 SIGNIFICANCE OF ALDHS 16
1.6 AIM OF THIS WORK 18
2 MATERIAL 19
2.1 INSTRUMENTS 19
2.2 CONSUMABLE MATERIALS 20
2.3 CHEMICALS 21
2.4 APOCAROTENOIDS AND FATTY ALDEHYDES 21
2.5 ENZYMES AND KITS 22
2.6 ANTIBODIES 22
2.7 GROWTH MEDIA 22
2.7.1 MEDIA FOR E. COLI 22
2.7.2 MEDIUM FOR CYANOBACTERIA (RIPPKA ETAL., 1979)..: 23
2.8 BIOLOGICAL MATERIAL 24
2.9 VECTORS 26
2.10 NUCLEIC ACIDS 29
2.10.1 GENOMIC DNA FROM SYNECHOCYSTIS 29
2.10.2 OLIGONUCLEOTIDES 30
2.10.2.1 PRIMERS FOR CLONING AND SEQUENCING 30
2.10.2.2 PRIMERS FOR SEQUENCE ANALYSIS OF SYNECHOCYSTIS SP. PCC6803
SUBSTRAINS: 30
2.10.2.3 PRIMERS AND PROBES FOR REAL-TIME-RT-PCR 32
2.10.2.4 PRIMERS FOR OPERON ANALYSIS 32
HTTP://D-NB.INFO/1036240940
IMAGE 2
3 METHODS 34
3.1 MOLECULAR BIOLOGICAL METHODS 34
3.1.1 ISOLATION OF NUCLEIC ACIDS 34
3.1.1.1 ISOLATION OF DNA 34
3.1.1.2 ISOLATION OF RNA FROM CYANOBACTERIA 34
3.1.1.3 ISOLATION OF GENOMIC DNAFROM CYANOBACTERIA 34
3.1.2 SEPARATION OF NUCLEIC ACIDS USING GEL ELECTROPHORESIS 35
3.1.2.1 AGAROSE GEL ELECTROPHORESIS FOR DNA 35
3.1.2.2 FORMALDEHYDE GELS FOR RNA 35
3.1.3 DNA MODIFICATIONS 36
3.1.3.1 DNA CLEAVAGE WITH RESTRICTION ENZYMES 36
3.1.3.2 DEPHOSPHORYLATION OF DNA USING ALKALINE PHOSPHATASE 36
3.1.3.3 CONVERSION OF STICKY ENDS TO BLUNT ENDS WITH T4-DNA-POLYMERASE
36
3.1.3.4 DNA LIGATION 36
3.1.4 SANGER DNA SEQUENCING 36
3.1.5 DEEP SEQUENCING 37
3.1.6 DNA SEQUENCE ALIGNMENTS 37
3.1.7 PCR (POLYMERASE CHAIN REACTION) 37
3.1.8 REAL-TIME-RT-PCR ANALYSIS 38
3.1.9 PRODUCTION OF COMPETENT E. COLI CELLS 39
3.1.10 TRANSFORMATION OF E. COLI CELLS 39
3.1.11 TRANSFORMATION OF SYNECHOCYSTIS CELLS 39
3.1.12 CLONING PROCEDURES OF SYNALHL CONSTRUCTS 39
3.2 PROTEIN CHEMICAL METHODS 45
3.2.1 BRADFORD PROTEIN ASSAY 45
3.2.2 SDS-PAGE 45
3.2.2.1 STANDARD SDS-GEL 45
3.2.2.2 UREA-SDS-GEL 46
3.2.3 COOMASSIE STAINING OF SDS GELS 46
3.2.4 WESTERN BLOT (IMMUNOBLOT) 46
3.2.5 EXPRESSION OF RECOMBINANT PROTEIN IN E. COLI 48
3.2.5.1 CRUDE PROTEIN PREPARATION 48
3.2.5.2 PROTEIN PURIFICATION WITH IMAC 48
3.2.5.3 PROTEIN PURIFICATION OF GST FUSION PROTEINS 49
3.2.5.4 PROTEIN PURIFICATION OF MBP-FUSION PROTEINS 50
3.2.5.5 REFOLDING OF HETEROLOGOUSLY EXPRESSED ALHL 50
3.2.5.6 ARTIFICIAL CHAPERONE-ASSISTED REFOLDING WITH P-CYCLODEXTRIN 53
3.2.6 BACULOVIRUS EXPRESSION IN SF9 INSECT CELLS 53
3.2.7 ISOLATION OF NATIVE CYANOBACTERIAL CYTOPLASM 54
3.3 METHODS OF PIGMENT OR LIPID EXTRACTION 54
3.3.1 CAROTINOID EXTRACTION FROM IN VITRO ASSAYS 54
3.3.2 CAROTINOID EXTRACTION FROM IN VIVO ASSAYS 54
3.3.3 PIGMENT EXTRACTION FROM CYANOBACTERIA 55
IMAGE 3
3.3.3.1 DETERMINATION OF TOTAL CHLOROPHYLL AND CAROTENOIDS 55
3.3.4 EXTRACTION OF THE UNKNOWN YELLOW PIGMENT FROM GT-VERMAAS
SUPERNATANT 55
3.3.5 TOCOPHEROL EXTRACTION FROM CYANOBACTERIA 56
3.3.6 EXTRACTION AND DERIVATIZATION OF FATTY ACIDS FROM CYANOBACTERIA 56
3.4 DERIVATIZATION OF FATTY ALCOHOLS TO FATTY ALDEHYDES 56
3.5 CHROMATOGRAPHIC METHODS 57
3.5.1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC OR UFLC) 57
3.5.1.1 PROGRAMS FOR THE WATERS HPLC SYSTEM 57
3.5.1.2 PROGRAMS FOR THE SHIMADZU UFLC SYSTEM 59
3.5.1.3 QUANTIFICATION OF ANALYTES WITH HPLC 60
3.5.2 THIN LAYER CHROMATOGRAPHY (TLC) 60
3.6 MASS SPECTROMETRY (MS) 61
3.6.1 LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) 61
3.6.2 GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) 6 1
3.7 DEHYDROGENASE ASSAYS 62
3.7.1 IN VITRO ASSAYS 62
3.7.2 IN VIVO ASSAYS 62
3.7.3 ASSAYS USING E. COLI CYTOPLASM 63
3.7.4 PHOTOMETRIC MEASUREMENT OF NADH-FORMATION 63
3.7.5 DETERMINATION OF CONVERSION RATES (PRODUCT FORMATION CURVES) 63
3.8 CULTURING AND GROWTH EXPERIMENTS WITH CYANOBACTERIA 64
3.8.1 STANDARD GROWTH CONDITIONS FOR SYNECHOCYSTIS SP. PCC6803 64
3.8.2 STRESS TREATMENTS 64
3.8.3 ALDEHYDE TOXICITY ASSAY 64
3.9 TRANSMISSION ELECTRON MICROSCOPY (TEM) 65
3.10 PHOTOSYNTHESIS RATE BY OXYGEN-EVOLUTION 65
3.11 HOMOLOGY ANALYSES 65
3.11.1 HOMOLOGY O F PROTEINS 65
3.11.2 PHYLOGENETIC TREE 65
4 RESULTS 66
4.1 THE SYNALHL GENE ENCODES A STRESS-INDUCED ALDH 66
4.1.1 SYNALHL IS PART OF A PUTATIVE STRESS-OPERON 66
4.1.2 SEQUENCE HOMOLOGY POINTS TO A DUAL FUNCTION OF SYNALHL 68
4.2 EXPRESSION ANALYSIS OF THE SYNALHL GENE 73
4.2.1 SYNALHL TRANSCRIPTION IS SIGNIFICANTLY UP-REGULATED IN HIGH-LIGHT
AND COLD TREATED CELLS.... 73
4.2.2 ALSO THE GENE ADJACENT TO SYNALHL, SLR0092, IS UP-REGULATED IN
HIGH-LIGHT 74
4.2.3 SYNALHL TRANSCRIPT INDUCTION UPON ROSE BENGAL AND PARAQUAT
TREATMENT 75
4.3 BIOCHEMICAL CHARACTERIZATION OF HETEROLOGOUSLY EXPRESSED SYNALHL 76
4.3.1 VARIOUS EXPRESSION AND PURIFICATION SYSTEMS WERE TESTED 76
4.3.1.1 EXPRESSION IN INSECT CELLS USING THE BACULOVIRUS SYSTEM 77
4.3.1.2 REFOLDING OF SYNALHL PURIFIED FROM INCLUSION BODIES 78
IMAGE 4
4.3.2 IN VITRO CONVERSION OF ALDEHYDES 79
4.3.2.1 LONG-CHAIN APOCAROTENALS ARE SUBSTRATES FOR SYNALHL 79
4.3.2.2 CONVERSION OF APOCAROTENOIC DI-ALDEHYDES UNDERLIES NO
CHAIN-LENGTH MINIMUM.... 8 1
4.3.2.3 ALSO ALKANALS ARE CONVERTED BY SYNALHL 82
4.3.3 COMPARISON OF VARIOUS SUBSTRATES 84
4.3.3.1 KINETICS MEASUREMENTS USING AN OXIDOREDUCTION DYE REPORTER ASSAY
85
4.3.3.2 PRODUCT FORMATION RATES 86
4.3.4 IN VIVO ACTIVITY OF SYNALHL IN CAROTENOID-ACCUMULATING E. COLI 87
4.4 SYNECHOCYSTIS SP. PCC6803 OCCURS IN DISTINCT SUBSTRAINS 90
4.4.1 DEEP-SEQUENCING THE GENOME OF SYNECHOCYSTIS SUBSTRAIN PCC-MOSCOW
9 1
4.4.2 THE GT-VERMAAS SUBSTRAIN 94
4.5 ANALYSIS OF ASYNALHL STRAINS 95
4.5.1 PIGMENT ANALYSIS 96
4.5.1.1 MYXOXANTHOPHYLL ACCUMULATES IN ASYNALHL GT-V BUT NOT IN ASYNALHL
PCC-M 96
4.5.1.2 THE CAROTENOID PHENOTYPE O F ASYNALHL IN THE GT-V BACKGROUND 98
4.5.1.3 THE PHOTOSYNTHETIC EFFICIENCY IS REDUCED IN ASYNALHL GT-V 99
4.5.1.4 LONG-TERM HIGH-LIGHT TREATMENT LEADS TO CHANGES IN THE PIGMENT
PATTERN
(BLEACHING) BUT REDUCES THE SYNALHL KNOCK-OUT PHENOTYPE 100
4.5.1.5 SYNALHL KNOCK-OUT IN GT-V LEADS TO HIGHER PIGMENT-LOSS DURING
HIGH-LIGHT
TREATMENT 101
4.5.2 A SYNALHL GT-V SECRETES LARGER AMOUNTS OF AN UNKNOWN YELLOW
PIGMENT 102
4.5.3 GROWTH CURVES 105
4.5.3.1 ASYNALHL GT-V SHOWS RETARDED GROWTH UNDER NORMAL CONDITIONS 105
4.5.3.2 GROWTH OF ASYNALHL IN THE PCC-M BUT NOT GT-V BACKGROUND IS
AFFECTED UNDER
HIGH-LIGHT CONDITIONS 106
4.5.4 FATTY ACID AND TOCOPHEROL PROFILES ARE NOT AFFECTED IN BSYNALHL
108
4.6 RESCUE MUTANT 109
4.6.1.1 COMPLEMENTATION MUTANT RESCUES THE PHOTOSYNTHESIS PHENOTYPE 110
4.6.1.2 SYNALHL RECONSTITUTION REDUCES PIGMENT DEGRADATION IN HIGH-LIGHT
I L L
4.7 ATTEMPTS TO FIND ACCUMULATED NATURAL SUBSTRATES OF SYNALHL IN THE
KNOCK-OUT MUTANT.... 112
5 DISCUSSION 114
5.1 SYNALHL IS THE FIRST ALDEHYDE DEHYDROGENASE SHOWN TO CONVERT BOTH
APOCAROTENALS AND FATTY ALDEHYDES 114
5.2 THE SYNALHL KNOCK-OUT MUTANT SHOWS A STRESS-RELATED PHENOTYPE 116
5.3 PUTATIVE BIOLOGICAL FUNCTION OF SYNALHL 119
5.4 MICROEVOLUTION IN CYANOBACTERIA 121
6 SUMMARY 123
7 ABBREVIATIONS 125
8 LITERATURE 127
9 ACKNOWLEDGEMENTS 139
|
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| author | Trautmann, Danika Maria |
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| author_facet | Trautmann, Danika Maria |
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| spelling | Trautmann, Danika Maria Verfasser (DE-588)1036240851 aut Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution presented by Danika Maria Trautmann 2013 139 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027029761&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Trautmann, Danika Maria Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |
| subject_GND | (DE-588)4113937-9 |
| title | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |
| title_auth | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |
| title_exact_search | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |
| title_full | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution presented by Danika Maria Trautmann |
| title_fullStr | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution presented by Danika Maria Trautmann |
| title_full_unstemmed | Synechocystis PCC6803 the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution presented by Danika Maria Trautmann |
| title_short | Synechocystis PCC6803 |
| title_sort | synechocystis pcc6803 the stress induced aldehyde dehydrogenase synalh1 and microevolution |
| title_sub | the stress-induced aldehyde dehydrogenase SynAlh1 and microevolution |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027029761&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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