Evolution of photosynthetic pigments

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Table of contents

I. Introduction 
I.1. Cyanobacteria
I.2. Synechocystis sp. strain PCC 6803
I.3. Bioenergetics
I.3.1. Linear photosynthetic electron transfer
I.3.2. Electron redirection and cyclic photosynthetic electron flow
I.3.3. Respiratory electron transfer
I.4. The phycobilisome
I.4.1. Phycobilisome structure
I.4.2. Phycobilisome function
I.5. The Ferredoxin:NADP(H) oxidoreductase
I.5.1. Structure and function
I.5.2. Isoforms
I.6. Gene regulation
I.6.1. Transcriptional regulation
I.6.2. Post-transcriptional regulation
II. Results and discussion 
II.1. Effect of nitrogen starvation on Synechocystis physiology and FNR accumulation
II.1.1. Culture turbidity and chlorophyll content
II.1.2. Evolution of photosynthetic pigments
II.1.3. Total-protein and FNR-accumulation pattern
II.2. Ectopic expression of the FNRL orf
II.2.1. petH 5’UTR is required for FNRS accumulation
II.2.2. Ectopic expression resulted in a gene dosage effect
II.3. A promoter responsible for FNRS accumulation
II.3.1. Deletions within the petH 5’-noncoding region
II.3.2. Genetic mapping of petH promoters
II.4. A specific promoter for each FNR isoform
II.4.1. petH transcription-start sites
II.4.2. Genetic confirmation of promoters locations
II.4.3. Transcriptional regulation of the large mRNA
II.5. Translation regulation retained in E. coli
II.6. Involvement of RNA secondary structures
II.6.1. petH mRNA 5’-end secondary structure prediction
II.6.2. Deletions within the petH 5’UTR encoding sequence
II.6.3. Additional characterization of the mRNA fold
II.7. 5’UTR affects ribosome binding in vitro
III. Conclusions and perspectives 
III.1. Conclusions
III.2. Perspectives
IV. Experimental procedures 
IV.1. Strains and growth conditions
IV.1.1. Synechocystis sp. PCC 6803
IV.1.2. Escherichia coli
IV.2. Genetic transformation of Synechocystis sp. PCC 6803
IV.3. DNA isolation from Synechocystis sp. PCC 6803
IV.4. Cloning, mutagenesis and plasmid constructions
IV.4.1. Construction of the cargo plasmids
IV.4.2. Insertions in the petH 5′-noncoding region
IV.4.3. Point mutation in the long transcript’s 5’-end
IV.4.4. Deletions in the long transcript’s 5′-end
IV.4.5. NtcA-binding site mutagenesis
IV.5. Expression of petH in E. coli
IV.6. Total-cell extracts and western blots
IV.6.1. Cell extracts preparation
IV.6.2. Chlorophyll a quantification
IV.6.3. Gel electrophoresis and immunoblotting
IV.7. Phycobilisomes analysis
IV.7.1. PBS purification
IV.7.2. SDS-PAGE and FNRL quantification
IV.8. Transcriptional-start sites mapping
IV.9. In vitro probingof the translation initiation complex
IV.9.1. Plasmid preparation
IV.9.2. In vitro transcription
IV.9.3. Toeprinting assays
IV.9.4. Sequence ladders
V. References 
VI. Annexe