Différenciation cellulaire chez la cyanobactérie Nostoc PPC 7120 : relation entre le régulateur transcriptionnel HetR et la Sérine. Thréonine kinase Pkn22, et exploitation des hétérocystes pour la production de biohydrogène

Les cyanobactéries sont des microorganismes photosynthétiques de différentes tailles et morphologies. En cas de carence en azote combiné, certaines cyanobactéries filamenteuses comme Nostoc sp. PCC 7120 sont capables de former des cellules spécialisées dans la fixation du diazote atmosphérique, appelées hétérocystes retrouvées selon un motif semi-régulier toutes les 10-15 cellules végétatives. Ils fournissent un environnement favorable au fonctionnement de la nitrogénase une enzyme sensible à l’oxygène produisant de l’hydrogène comme sous-produit de réaction.Ma thèse s’articule autour de deux objectifs : l’étude du régulateur central de la différenciation HetR en suivant la piste d’une régulation post-traductionnelle, et sur l’utilisation des hétérocystes comme centres de production d’hydrogène.L’étude de HetR nous a permis de montrer que cette protéine est phosphorylée et interagit avec une Ser/Thr kinase, Pkn22. Un mutant de cette dernière présente un retard de différenciation suggérant un rôle dans les étapes précoces. Nous avons identifié des résidus importants dans l’interaction avec la kinase, et nous avons montré que la substitution de certaines serine impacte la différenciation et la capacité de se fixer à l’ADN.Les hétérocystes se sont révélés être un lieu propice au fonctionnement de l’hydrogénase HydA de la bactérie anaérobie Clostridium acetobutylicum. En couplant la production de l’hydrogénase et la diminution du taux d’oxygène par la cyanoglobine GlbN de Nostoc commune, ou de la flavoprotéine Flv3 nous avons pu augmenter drastiquement la production d’hydrogène.Cyanobacteria are photosynthetic microorganisms with various shapes and morphologies. In case of combined nitrogen starvation, some filamentous cyanobacteria such as Nostoc sp. PCC 7120 are capable to form cells specialized in atmospheric nitrogen fixation, called heterocysts which form a semi-regular pattern of one each 10-15 vegetative cells. They provide a microoxic environment favorable to nitrogenase extremely sensitive to oxygen to function which produces hydrogen as byproduct. My thesis discusses two topics: study of HetR, the master regulator of differentiation based on post translational modification, and the use of heterocyst as hydrogen manufactories.We demonstrate that HetR is a phosphoprotein interacting with a Ser/Thr kinase, Pkn22. A pkn22 mutant displays a delay in the differentiation process suggesting an involvement in the early steps of differentiation. We highlight residues important for the interaction with the kinase and showed that substitution of serine residues in alanine impacts differentiation and DNA-binding. We showed that heterocysts are favorable to the function of HydA hydrogenase from the anaerobic bacteria Clostridium acetobutylicum. Coupling HydA production to the decrease of oxygen amount via GlbN a cyanoglobin from Nostoc commune or the flavodiiron protein Flv3 permit to increase hydrogen evolution. The discovery of a new level of regulation of the differentiation by Pkn22 through the phosphorylation of HetR and the demonstration of new means to increase hydrogen evolution arouse new questions and could permit in fine to develop new strains optimized for hydrogen evolution at high scale

Tilted rotating annular bioreactors

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The Pkn22 Kinase of Nostoc PCC 7120 Is Required for Cell Differentiation via the Phosphorylation of HetR on a Residue Highly Conserved in Genomes of Heterocyst-Forming Cyanobacteria

International audienceHanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation

Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120

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Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120

International audienceThe conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 μmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase