Effect of nitrogen starvation on Synechocystis physiology and FNR accumulation 

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Synechocystis sp. strain PCC 6803

Our model organism is the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter referred to as Synechocystis). Synechocystis was originally isolated from a freshwater lake in California by R. Kunisawa(Stanier et al., 1971).It is the first photosynthetic organism and the second bacterium to have its genome fully sequenced (Kaneko et al., 1996). Synechocystis is the most popular laboratory strain serving as a model in the research fields of photosynthesis, stress response and metabolism (Ikeuchi and Tabata, 2001). There are two major reasonsfor its importance: it is naturally transformable by exogenous DNA (Grigorieva and Shestakov, 1982), and it can grow heterotrophically in the presence of glucose, which permits the selection of photosynthesis-deficient mutants(Rippka et al., 1979; Ikeuchi and Tabata, 2001).
Synechocystis is a unicellular coccoid or spherical cyanobacterium (Figure I.1-b andFigure I.2).Its circular genome is about 3.5 Mb,with an average GC content of 47.7%(Kaneko et al., 1996). A.Thin section showing intracellular structures present in Synechocystis sp. PCC 6803. Band C. Enlargements of the boxed areas in A, showing close proximity of thylakoid membrane (T) and plasma membrane (PM). g: glycogen granule; r ribosomes; OM: outer membrane; PD: peptidoglycan layer. From (Liberton et al., 2006).
As all gram-negative bacteria, Synechocystis has a cell envelope consisting of a plasma membrane, a peptidoglycan layer and an outer membrane (Figure I.2). In addition, as in all cyanobacteria exceptforGloeobacter violaceus, Synechocystis contains an internal membrane system,the thylakoid, where photosynthetic and respiratory electron-transfer reactions occur (Figure I.2). Components of both photosynthetic and respiratory systems, as well astheir electron transferpathwaysare introduced in the following section.

Linear photosynthetic electron transfer

LET involves two photosynthetic reaction centers called photosystem I (PSI) and photosystem II (PSII) (Figure I.3-A). It implicates water oxidation to molecular oxygen by PSII, as a first step, then the reduction of NADP+ to NADPH via PSI, as a final step. The membrane-protein complex called cytochrome b6f (cyt b6f) mediates electron transport between PSII and PSI in the thylakoid. During this process an electrochemical proton gradient is produced across the thylakoid membrane. This gradient is used, by ATP synthase, to produce ATP (Figure I.3-A). NADPH and ATP are then used to fix CO2 in the Calvin cycle, among other anabolic reactions.
In the following sections, we will introduce the four integral membrane-protein complexes, PSII, cyt b6f, PSI and ATP-synthasethat are involved in photosynthetic electron transfer and ATP synthesis. The photosystem IIreaction center (PSII) is composed mainly of two similar protein subunits referred to as D1 and D2. It carries out the first step of LET, which is the light-catalyzed oxidation of water. This reaction provides almost all earth’s atmospheric molecular oxygen.

Electron redirection and cyclic photosynthetic electron flow

Duringphotosynthetic LET, O2, ATP and reduced NADPH are produced. However, stress conditions, like high-light or low CO2, may lead to electron redirection towards other compounds. In these cases, electrons can be transferred from PSI to molecular oxygen, which resultsin the photoreduction of O2, via superoxideanion (O2-), to form H2O2 in plant chloroplastes(Mehler-reaction) (Mehler, 1951). This reaction produces reactive oxygen species (ROS), known to cause a significant damage to the cell. In photosynthetic organisms PSII is the preferential target of ROS resulting in photoinhibition (Hackenberg et al., 2009). ROS are quickly detoxified by the combined action of superoxide dismutase and peroxidases. O2 photoreduction in cyanobacteria is quite different from that in plants. InSynechocystis, O2 is reduced directly to water in a reaction mediated by A-type flavoproteins, which avoids ROS accumulation(Vicente et al., 2002).
The different complexes implicated in photosynthesis are depicted. The two major pathways of cyclic-electron transfer are indicated with black arrows. They involve the respiratory NDH-1 complex and/or the Fd. Respiratory complexes such as cytochrome oxidase, Cox and NAD(P)H dehydrogenase I, NDH-1 are represented in addition to the photosynthetic complexes.
Another pathway of electron redirection is called CET (Figure I.7). In this alternative electron pathway ATP is generated with no NADPH accumulation. Two pathways for CET have beenproposed; both involve PSI, cyt b6f, and the PQ pool. Several partners were proposed to catalyze the donation of electrons from the acceptor side of PSI (Fd, FNR, NADPH) back into the PQ pool, reduced PQ transfer electrons to cyt b6f, which inturn transferthem to PSI and so on (Shikanai, 2007). Onepathway involveselectron redirection from Fd to the PQ pool (Figure I.7-black arrow) while another pathway involvesits redirection from NADPH to the NDH-I complex (introduced in the next section) then to the PQ pool(Shikanai, 2007)(Figure I.7-black arrow).

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The Ferredoxin:NADP(H) oxidoreductase

Ferredoxin:NADP(H) oxidoreductase (FNR) is a flavoprotein that catalyzes the last step of linear electron transfer between Fd and NADPH. FNR acceptstwo electrons, one at time, from ferredoxinand carries out the two-electron reduction of NADP+ to NADPH (Blankenship, 2002). The overall reaction catalyzed by FNR is as follows: 2Fdred + NADP+ + H+ 2Fdox + NADPH.

Evolution of photosynthetic pigments

Absorption spectra of whole cells, collected at different time-points of nitrogen starvation, depict the evolution of different photosynthetic pigments in WT, MI6 and FS1.
The spectra in figures II.2-A, B and C show that PBP absorption (620 nm) decreased, due to a well-known phenomenon called bleaching (Section I.4.2), where PBP degradation provides amino acids for cell survival in the absence of external nitrogen (Allen and Smith, 1969). The bleaching rate was clearly different in the different strains.On the other hand carotenoidabsorptionincreases(350 nm to 500 nm), carotenoid accumulation being a typical response to stress, while chlorophyll a (440 nm, 620 nm and 680 nm) does not seem to be greatly affected, in agreement with the results described in the last section (Figure II.1).
The spectra show that the rate of PBPdecrease during nitrogen starvation is different between the WT and the mutants. Compared to WT, the remaining level of PBP at 48 h is higher in MI6 and lower in FS1 (dark blue trace in figure II.2 B and C, respectively).
The intensity of PBP-complexes absorption at 620 nm (A620nm) provides an indication of the concentration of biliproteins in the cells. However, the bilins are not the only pigments that absorb at 620 nm. Chlorophyllaabsorbs at the same wavelength and this is demonstrated,in figure II.2-D, in the absorption spectrum of PAL – a mutant totally devoid of PBP (Ajlani and Vernotte, 1998). In PAL, A620nm is about 20% of the absorption at 680 nm (A680nm), indicating that about 20% of A680nm contributes to the A620nmin a PBP-containing strain.
In order to better characterize PBP degradation rate, curves that represented the variation of A620nm/A680nm during nitrogenstarvation were plotted (Figure II.2-E).

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 


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