The Pkn22 Ser/Thr kinase in Nostoc PCC 7120: role of FurA and NtcA regulators and transcript profiling under nitrogen starvation and oxidative stress

International audienceBackground: The filamentous cyanobacterium Nostoc sp. strain PCC 7120 can fix N2 when combined nitrogen is not available. Furthermore, it has to cope with reactive oxygen species generated as byproducts of photosynthesis and respiration. We have previously demonstrated the synthesis of Ser/Thr kinase Pkn22 as an important survival response of Nostoc to oxidative damage. In this study we wished to investigate the possible involvement of this kinase in signalling peroxide stress and nitrogen deprivation. Results: Quantitative RT-PCR experiments revealed that the pkn22 gene is induced in response to peroxide stress and to combined nitrogen starvation. Electrophoretic motility assays indicated that the pkn22 promoter is recognized by the global transcriptional regulators FurA and NtcA. Transcriptomic analysis comparing a pkn22-insertion mutant and the wild type strain indicated that this kinase regulates genes involved in important cellular functions such as photosynthesis, carbon metabolism and iron acquisition. Since metabolic changes may lead to oxidative stress, we investigated whether this is the case with nitrogen starvation. Our results rather invalidate this hypothesis thereby suggesting that the function of Pkn22 under nitrogen starvation is independent of its role in response to peroxide stress. Conclusions: Our analyses have permitted a more complete functional description of Ser/Thr kinase in Nostoc. We have decrypted the transcriptional regulation of the pkn22 gene, and analysed the whole set of genes under the control of this kinase in response to the two environmental changes often encountered by cyanobacteria in their natural habitat: oxidative stress and nitrogen deprivation

Evidence that the PatB (CnfR) factor acts as a direct transcriptional regulator to control heterocyst development and function in the cyanobacterium <i>Nostoc</i> PCC 7120

International audienceNumerous multicellular cyanobacteria are able to form specialized cells that acquire new properties to fulfill specific tasks: atmospheric nitrogen fixation for heterocysts, motility for hormogonia, and dormancy for akinetes. In addition, in the case of heterocysts, mutual interactions occur between vegetative and differentiated cells, making from these prokaryotes exciting models to investigate cell differentiation and its connection to multicellularity (Flores & Herrero, 2010). Like any differentiation, heterocyst formation follows a precise temporal dynamic in which each step is orchestrated through often interconnected transcriptional regulators. Since the early 1990s, the regulation of this process has been extensively studied in the model bacterium Nostoc (Anabaena) PCC7120 (hereafter Nostoc). When the combined nitrogen source (ammonium or nitrate) becomes limiting, this bacterium differentiates heterocyst with a semi-regular pattern along the filaments. Heterocyts are micro-oxic, non-dividing cells, that provide a suitable environmen

PCBs and DDTs in Stenella coeruleoalba dolphins from the French Mediterranean coastal environment (2007-2009): Current state of contamination

International audienceOrganochlorinated compounds including PolyChloroBiphenyles, Dichloro-DiphenylTrichloroethan and metabolites are determinated in Stenella coeruleoalba (n = 37) stranded on the french Mediterranean coasts from 2007 till 2009. Studies are carried out on lung, muscle, kidney, liver, and blubber. The sought-after compounds are all detected to variable levels in each tissue and organ. In general, total PCBs are the most abondant, followed by total DDTs. The concentration (in ng g(-1) of lipid weight) in blubber of S. coeruleoalba, varied from 2,052 to 158,992 for PCBs and from 1,120 to 45,779 for DDTs. The ratios DDE/tDDTs are higher than 80% in almost all samples. The overall results of this work, compared to previous studies concerning the Mediterranean Sea, seems to confirm the tendency to a decrease of the contamination by organics compounds for the cetaceans in the Western Mediterranean Sea. (C) 2012 Elsevier Ltd. All rights reserved

The CcdB toxin is an efficient selective marker for CRISPR-plasmids developed for genome editing in cyanobacteria

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HetL, HetR and PatS form a reaction-diffusion system to control pattern formation in the cyanobacterium Nostoc PCC 7120

International audienceLocal activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here, we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum

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

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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

Étude de la faisabilité de la désorption thermique pour les sols pollués par des molécules organiques

A better knowledge of the phenomena governing the thermal decontamination of soils allows to improve the conception of the thermal treatment processes: this study was realised in this aim. The first stage of this work was done by thermogravimetric analysis with small amounts of soils artificially polluted. The fundamental mechanisms controlling the release of typical organic pollutants from the soil matrix during the thermal treatment have been identified. Results have shown the importance of the inter-particle diffusive transport of the gaseous pollutants within the porous media and above the bed of soil. Furthermore, decontamination rate and decontamination temperature domains were easily obtained with this tool for several polluted soils. In a second time, the study of the thermal decontamination of soils was led on a laboratory-scale pilot : decontamination temperatures, residual contamination level and the toxicity of the gases released were studied. To validate the fundamental results obtained and to confirm the feasibility of the thermal desorption process at the semi-industrial scale, a thermal treatment unity (30kg/h) was developed. The influence of the main parameters (temperature, pressure, residence time and air flow-rate) was carried out for soils polluted with HAP. Treatment temperature and air flow-rate are the most important factors influencing the residual HAP level in the treated soils.Une meilleure connaissance des phénomènes régissant la décontamination thermique des sols permet d’améliorer la conception des procédés de traitement thermique et leur mise en œuvre. La première étape de ce travail a été menée par analyse thermogravimétrique sur des petites quantités de sols pollués artificiellement afin de déterminer les mécanismes fondamentaux mis en jeu lors du traitement thermique. L’analyse thermogravimétrique apparaît comme un outil rapide permettant d’estimer les températures et les vitesses de décontamination de sols pollués. L’étude de la décontamination thermique (températures de décontamination et contamination résiduelle après traitement) a ensuite été réalisée sur un pilote de laboratoire puis à l’échelle semi-industrielle. L’influence des principaux paramètres a pu être étudiée pour des sols pollués par des HAP : la température et le débit d’air de balayage sont les facteurs ayant une influence prépondérante sur la contamination résiduelle en HAP

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