The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 is a model species fordevelopment of sustainable production methods of numerous compounds. One of its uniquefeatures is the anaerobic environment of the strains nitrogen fixing heterocyst cells. To be ableto properly utilize this environment, more knowledge regarding what regulates cell specificexpression is required. In this study, three motifs of the NsiR I promoter of Anabaena sp.PCC 7120 was studied in this system utilizing YFP-fluorescence as a reporter to determinetheir impact on spatial expression pattern. Investigations were performed on immobilizedcells with the use of confocal microscopy and results point towards sigma factor regulation.

Cyanobacteria belong to the oldest organisms of our planet. They use photosynthesis to produce ATP and gain biomass from carbon dioxide. The cyanobacteria Nostoc punctiforme is a filamentous bacterium that consists of two different types of cells, vegetative cells and heterocysts. The type of cell it differentiates into depends on the media they grow in. In an ammonium-rich medium, the N.punctiforme consists of vegetative cells that differentiate into heterocysts when in the medium is changed to a low-concentration ammonium medium. The ammonium-binding nitrogenase in the heterocysts does not work in an oxidative environment. During oxidative stress, N.punctiforme produces Dps (DNA binding protein from starved cells) which protects DNA. In the heterocysts the nitrogenase produces hydrogen as a side product. The hypothesis is that Dps is cell specific. In order to study this protein, a fusion of the promotor of Dps and GFP (Green Flourescent Protein) was constructed. To detect GFP, optimization of a Western Blot (WB) for GFP was performed. Protein samples were analyzed in strains of N.punctiforme. In strain 12A, the production of GFP was visualized but the band was not specific. Several attempts of optimization of the WB procedure were performed, but none of them showed clear specific protein detection in the N.punctiforme strains. Further optimization of the WB protocol is needed.

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The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function to accommodate the oxygen-sensitive process of nitrogen fixation. This dissertation focuses on my research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation. We using microarray experiments we found that conR (all0187) gene is necessary for normal septum-formation of vegetative cells, diazotrophic grow, and heterocyst morphogenesis. In our studies we characterized the expression of sigma factors genes in Anabaena PCC 7120 during heterocyst differentiation, and we found that the expression of sigC, sigG and sigE is localized primarily in heterocysts. Expression studies using sigE mutant showed that nifH is under the control of this specific sigma factor.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20604号 / 理博第4319号 / 新制||理||1620(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 熊﨑 茂一, 教授 林 重彦, 教授 寺嶋 正秀 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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Les métabolismes du carbone et de l’azote sont étroitement coordonnés chez tous les organismes vivants en raison de l’importance de ces deux éléments dans les différents mécanismes physiologiques. Le 2-oxoglutarate (2-OG) est une molécule signal conservée chez tous les organismes et est impliqué dans la balance carbone / azote. Malgré son importance, il n’existe pas d’outil permettant de mesurer la concentration de 2-OG à l’échelle cellulaire. Pour combler cette carence, nous avons utilisé Anabaena sp. PCC 7120 pour construire un système de quantification du 2-OG in vitro et in vivo. Cette bactérie appartient au groupe des cyanobactéries qui contribuent aussi bien au cycle du carbone via la photosynthèse qu’au cycle de l’azote via leur métabolisme.Au cours de ma thèse, j’ai construit différents types de biosenseurs au 2-OG en utilisant les techniques de FRET (Fluorescence Resonance Energy Transfer). Ces biosenseurs sont capables de mesurer le 2-OG in vitro et permettent de le détecter in vivo en utilisant l’analyse à l’échelle de la cellule unique et la microscopie en temps réel. Nous avons découvert l’existence de variations dynamiques du 2-OG au niveau des filaments et le profil formé semble correspondre au futur profil de développent cellulaire. En raison de la conservation du rôle régulateur du 2-OG dans de nombreuses activités cellulaires, le biosenseur développé durant ma thèse pourrait être appliqué à une grande variété d’organismes dont les bactéries, les plantes, les animaux et les humains. / Carbon and nitrogen metabolisms are tightly coordinated in all living organisms because of the importance of the two elements in the physiology. 2-oxoglutarate (2-OG) is a signal conserved in all living organisms involved in the balancing between carbon and nitrogen metabolisms; however, despite its importance, it is currently impossible to measure 2-OG in each cells under different conditions when 2-OG is subject to variations Cyanobacteria contribute to global carbon cycle by oxygenic photosynthesis as well as to global nitrogen cycle through their nitrogen metabolism and in this study, we used the cyanobacterium Anabaena PCC 7120 as a model, to construct a system for the quantification of 2-OG in vitro and in vivo.. During my thesis, I took the advantages of the FRET (Fluorescence Resonance Energy Transfer) techniques and constructed several 2-OG biosensors that can adequately detect 2-OG levels in vitro in a quantitative manner. I tested their performance for the detection of 2-OG in vivo by using single-cell analysis and time-lapse microscopy. We found that 2-OG display dynamic changes at single-cell basis, and these variations are strongly correlated to cell differentiation activities. The 2-OG biosensors developed during my thesis can be applied in a wide range of organisms, including other bacteria, plants, animals, and human, because of the conserved roles of 2-OG in regulating a variety of cellular activities.

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