The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H. As a result, their H 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 H production activities, but their sensitivity to O inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O 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 O amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 μmol-H 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 H production by an O-sensitive hydrogenase.

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Hanks-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.

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In this paper we report yields, identities, and mutagenicities of products from heating a polycyclic aromatic hydrocarbon (PAH)-contaminated, Superfund-related synthetic soil matrix without exogenous oxygen. We heated batch samples of soil pretreated with 5.08 wt% (by weight) pyrene in a tubular furnace under a constant flow of helium gas at 250, 500, 750, and 1,000 +/- 20 degrees C. Dichloromethane (DCM) extracts of cooled residues of heated soil and of volatiles condensed on a cold finger after 1 sec residence time at furnace temperature were assayed gravimetrically and analyzed for PAH by HPLC, HPLC coupled to mass spectrometry, and gas chromatography coupled to mass spectrometry. All four temperatures volatilized pyrene and generated other PAHs, including alkylated pyrenes. We detected bioactive PAHs in the product volatiles: cyclopenta[cd]pyrene (CPP) at 750 and 1,000 degrees C and benzo[a]pyrene (BaP) at 1,000 degrees C. We found a clean soil residue, i.e., no pyrene or other DCM extracts, only at 750 degrees C. Control experiments with uncontaminated soil, pyrene, and Ottawa sand plus 4.89 wt% pyrene revealed no CPP or BaP production from soil itself, but these experiments imply that pyrene interactions with soil, e.g., soil-bound silica, stimulate CPP and BaP production. We detected mutagenicity to human diploid lymphoblasts (in vitro) in volatiles from 1,000 degrees C heating of soil plus pyrene and sand plus pyrene, and in the residue from 500 degrees C heating of soil plus pyrene. Three plausible pathways for pyrene conversion to other PAHs are a) a reaction with light gas species, e.g., soil- or pyrene-derived acetylene; b) loss of C(2)-units followed by reaction with a PAH; and c) dimerization with further molecular weight growth via cyclodehydrogenation. This study shows that thermal treatment of PAH-polluted soil may generate toxic by-products that require further cleanup by oxidation or other measures.ImagesFigure 2

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Local 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.