Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

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

Under nitrogen-limiting conditions, the filamentous cyanobacterium Nostoc PCC7120 differentiates nitrogen-fixing heterocysts at semi-regular intervals along filaments generating a periodic pattern of two distinct cell types. Heterocysts are micro-oxic cells that host the oxygen-sensitive nitrogenase allowing two antagonistic activities to take place simultaneously. Although several factors required to control the differentiation process are known, the molecular mechanisms engaged have only been elucidated for a few of them. The patB (cnfR) gene has been shown to be essential for heterocyst formation and nitrogen fixation in this cyanobacterium, but its function remains to be clarified. Here, we show that PatB acts as a direct transcriptional regulator of genes required for nitrogenase production and activity. The DNA-binding activity of PatB does not depend on micro-oxia as it interacts with its target promoters under aerobic conditions both in vitro and in vivo. The absence of the DNA-binding domain of PatB can be rescued in the heterocyst but not in the vegetative cell. Furthermore, the putative ferredoxin domain of PatB is not essential to its interaction with DNA. The patB gene is widely conserved in cyanobacterial genomes and its function can be pleiotropic since it is not limited to nitrogen fixation control.

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

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.

Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do. In addition, some strains are able to 'fix' the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life. However, both processes cannot happen in a given cell at the same time. A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells. Under certain conditions, one in every ten cells stops drawing its energy from the sun, and starts fixing atmospheric nitrogen instead. Exactly how the bacteria are able to 'count to ten' and organize themselves in such a precise pattern is still unclear. Cells can communicate and establish patterns by exchanging molecular signals that switch on and off certain cell programs. For instance, a protein called HetR turns on the genetic program that allows cyanobacteria to fix nitrogen; on the other hand, a signal known as PatS binds to HetR and turns it off. Cells starting to specialise in fixing nitrogen produce both HetR and PatS, with the latter diffusing in surrounding cells and preventing them from extracting nitrogen. However, it remained unclear how the nitrogen-fixing cell could ignore its own PatS signal and keep its HetR signal active. HetL ­ another protein produced by the future nitrogen-fixing cell ­ could potentially play this role, but how it acts was unknown. Here, Xu et al. show that HetL cannot diffuse from one cell to the other, and that it binds to HetR at the same place than PatS does. When both PatS and HetL are present, they compete to attach to HetR, which stops PatS from turning off HetR and deactivating the nitrogen-fixing program. Understanding how cyanobacteria fix nitrogen could help to develop new types of natural fertiliser. More generally, dissecting how these simple organisms can create patterns could help to grasp how patterning emerges in more complex creatures.

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

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.

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.

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.

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

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 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.

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

BACKGROUND: 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.

Exploring the size limit of protein diffusion through the periplasm in cyanobacterium Anabaena sp. PCC 7120 using the 13 kDa iLOV fluorescent protein.

In the filamentous heterocyst-forming cyanobacterium Anabaena PCC 7120, vegetative cells and heterocysts are interdependent on each other and engaged in exchanges of metabolites for survival when grown under diazotrophic conditions. In this organism, the periplasm appears to be continuous along each filament, with a shared outer membrane; however, barriers exist preventing free diffusion of the fluorescent protein GFP (27 kDa) targeted into the periplasmic space. Here we expressed a smaller fluorescent protein iLOV (≈ 13 kDa) fused to the All3333 (a putative homologue of NrtA) signal sequence corresponding to those recognized by the TAT protein translocation system, which exports iLOV to the periplasm of either heterocysts or vegetative cells. Fluorescence microscopy and immunoblot analysis indicated that the iLOV protein is translocated into the periplasm of the producing cell and properly processed, but does not diffuse to neighboring cells via the periplasm. Thus, periplasmic barriers appear to block diffusion of molecules with a size of 13 kDa, the minimum size tested thus far. Assuming that the physical barrier is the peptidoglycan sacculus, its pores might allow diffusion of molecules within the size range between the PatS pentapeptide and iLOV, thus between 0.53 kDa and 13 kDa.

Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in Anabaena sp. PCC 7120.

The cyanobacterium Anabaena (Nostoc) sp. PCC 7120 is a model for cyanobacterial cell differentiation studies. pDU1, an endogenous plasmid in Nostoc sp. PCC 7524, is used as the only cyanobacterial replicon for Anabaena (Nostoc) studies. However, the relative copy numbers of pDU1-based plasmids in Anabaena (Nostoc) sp. PCC 7120 are not well studied. We found that the relative plasmid copy number of one such vector, pRL25T, varied widely, especially when the vector carried a recombinant insert, under different conditions, ranging from 0.53 to 1812 per chromosome in different recombinant strains tested, either in independent clones of the same strain or in the same clone under different growth conditions. The phenotypes caused by pRL25T-driven expression of green fluorescent protein or the GAF domain of Pkn41 or Pkn42 varied depending on the independent clones analyzed. This phenotypic variation correlated with the relative plasmid copy number present in cells.

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

Organochlorinated 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 abundant, 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.

Effects of temperature and soil components on emissions from pyrolysis of pyrene-contaminated soil.

Effects of temperature and soil on yields and identities of light gases (H2, CH4, C2H2, C2H4, C2H6, CO, and CO2) and polycyclic aromatic hydrocarbons (PAH) from thermal treatment of a pyrene-contaminated (5 wt%) soil in the absence of oxygen were determined for a U.S. EPA synthetic soil matrix prepared to proxy U.S. Superfund soils. Shallow piles (140-170 mg) of contaminated soil particles and as controls, neat (non-contaminated) soil (140-160 mg), neat pyrene (10-15 mg), neat sand (230 mg), and pyrene-contaminated sand (160 mg), were heated in a ceramic boat inside a 1.65 cm i.d. pyrex tube at temperatures from 500 to 1100 degrees C under an axial flow of helium. Volatile products spent 0.2-0.4s at temperature before cooling. Light gases, PAH and a dichloromethane extract of the residue in the ceramic boat, were analyzed by gas chromatography or high pressure liquid chromatography (HPLC). Over 99% pyrene removal was observed when heating for a few tens of seconds in all investigated cases, i.e., at 500, 650, 750, 1000, and 1100 degrees C for soil, and 750 and 1000 degrees C for sand. However, each of these experiments gave significant yields (0.2-16 wt% of the initial pyrene) of other PAH, e.g., cyclopenta[cd]pyrene (CPP), which mutates bacterial cells and human cells in vitro. Heating pyrene-polluted soil gave pyrene conversions and yields of acetylene, CPP, and other PAH exceeding those predicted from similar, but separate heating of neat soil and neat pyrene. Up to 750 degrees C, recovered pyrene, other PAH, and light gases accounted for all or most of the initial pyrene whereas at 1000 and 1100 degrees C conversion to soot was significant. A kinetic analysis disentangled effects of soil-pyrene interactions and vapor phase pyrolysis of pyrene. Increase of residence time was found to be the main reason for the enhanced conversion of pyrene in the case of the presence of a solid soil or sand matrix. Light gas species released due to the thermal treatment, such as acetylene and methane, lead the formation of other, pyrene-derived PAH, e.g., methylpyrenes, cyclopenta[cd]pyrene, and benzo[a]pyrene. Implications of these findings for the chemistry of soil thermal decontamination and for diagnosing potential defects in soil thermal cleaning, e.g., incomplete elimination of targeted pollutants and formation of adverse by-products, are discussed.