Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

<p>Abstract</p> <p>Background</p> <p><it>Lyngbya majuscula </it>CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by <it>hupSL</it>) and a bidirectional enzyme (encoded by <it>hoxEFUYH</it>). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.</p> <p>Results</p> <p>In this work we demonstrated that, in <it>L. majuscula</it>, the structural genes encoding the bidirectional hydrogenase are cotranscribed, and that <it>hoxW </it>(the gene encoding its putative specific endopeptidase) is in the same chromosomal region but transcribed from a different promoter. The gene encoding the putative specific uptake hydrogenase endopeptidase, <it>hupW</it>, can be cotranscribed with the structural genes but it has its own promoter. <it>hoxH</it>, <it>hupL</it>, <it>hoxW </it>and <it>hupW </it>transcription was followed in <it>L. majuscula </it>cells grown under N₂-fixing and non-N₂-fixing conditions over a 12 h light/12 h dark cycle. The transcription of <it>hoxH</it>, <it>hoxW </it>and <it>hupW </it>did not vary remarkably in the conditions tested, while the <it>hupL </it>transcript levels are significantly higher under N₂-fixing conditions with a peak occurring in the transition between the light and the dark phase. Furthermore, the putative endopeptidases transcript levels, in particular <it>hoxW</it>, are lower than those of the respective hydrogenase structural genes.</p> <p>Conclusion</p> <p>The data presented here indicate that in <it>L. majuscula </it>the genes encoding the putative hydrogenases specific endopeptidases, <it>hoxW </it>and <it>hupW</it>, are transcribed from their own promoters. Their transcript levels do not vary notably in the conditions tested, suggesting that HoxW and HupW are probably constantly present and available in the cells. These results, together with the fact that the putative endopeptidases transcript levels, in particular for <it>hoxW</it>, are lower than those of the structural genes, imply that the activity of the hydrogenases is mainly correlated to the transcription levels of the structural genes. The analysis of the promoter regions indicates that <it>hupL </it>and <it>hupW </it>might be under the control of different transcription factor(s), while both <it>hoxH </it>and <it>xisH </it>(<it>hoxW</it>) promoters could be under the control of LexA.</p

Alkali and acid polysaccharides blend nanofibrous membranes prepared by electrospinning

Poster apresentado no "Smart and functional coatings conference", Torino, Italy, 2013Electrospinning allows the production of polymer fibres with diameters in the sub-micron size range, through the application of an external electric field, keeping intact the bulk properties of the polymers. Electrospun membranes possess some unique structural features, such as a high surface to volume ratio and very good mechanical performance, properties that are determinant to their use in several applications such as air and liquid filtration, tissue engineering, optical and chemical sensors [1]. In this work, alkali and acid biopolysaccharides blended with polyvinyl alcohol (PVA) were electrospinned into a polyvinylidene difluoride (PVDF) basal microfiltration membrane, with the goal of developing a mid-layer nanofibrous porous support for exploitable thin-film composite (TFC) membranes for water filtration. The alkali and acid biopolysaccharides chosen were, respectively, chitosan (CS), a cationic polyelectrolyte (in this case with deacetylation degree around 85), and cyanobacterial extracellular polymeric substances (EPS), an acidic polysaccharide isolated from Cyanothece sp.CCY 0110 [2]. The electrospun blended nanofibrous membranes were fully characterized in order to investigate their morphology, diameter, structure, mechanical and thermal properties. The results showed that these membranes have great potential for filtration purposes [3].This work was funded by FEDER funds through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Fundação para a Ciência e a Tecnologia under the projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011), FCOMP-01-0124-FEDER-009389 (PTDC/CTM/100627/2008) and FCOMP-01-0124-FEDER-009697 (PTDC/EBB-EBI/099662/2008), and the grants SFRH/BPD/37045/2007 and SFRH/BPD/72400/2010. The authors also thank to the project INVISIBLE NETWORK nº. 13857 * SI I&DT Mobilizador

Evaluation of the antioxidant activity of cell extracts from microalgae

A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA oxidative damage induced by copper(II)-ascorbic acid; and against decay in bacteriophage infection capacity induced by H2O2. Finally, performance of an Ames test revealed no mutagenic effects of the said extract.info:eu-repo/semantics/publishedVersio

Expanding the toolbox for Synechocystis sp. PCC 6803 : validation of replicative vectors and characterization of a novel set of promoters

Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference PrnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study

Internal Illumination to Overcome the Cell Density Limitation in the Scale-up of Whole-Cell Photobiocatalysis

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell‐density, which is a challenge for the scale‐up. Here, it was shown with the example of a light‐dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene‐reductase YqjM were used for the reduction of 2‐methylmaleimide to (R)‐2‐methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two‐fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h(−1) and specific activities of up to 65.5 U g(DCW) (−1) were obtained, allowing the reduction of 40 mm 2‐methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor

Cyanoflan: A cyanobacterial sulfated carbohydrate polymer with emulsifying properties

Abstract The extracellular polysaccharides produced by cyanobacteria have distinctive characteristics that make them promising for applications ranging from bioremediation to biomedicine. In this study, a sulfated polysaccharide produced by a marine cyanobacterial strain and named cyanoflan was characterized in terms of morphology, chemical composition, and rheological and emulsifying properties. Cyanoflan has a 71% carbohydrate content, with 11% of sulfated residues, while the protein account for 4% of dry weight. The glycosidic-substitution analysis revealed a highly branched complex chemical structure with a large number of sugar residues. The cyanoflan high molecular mass fractions (above 1 MDa) and entangled structure is consistent with its high apparent viscosity in aqueous solutions and high emulsifying activity. It showed to be a typical non-Newtonian fluid with pseudoplastic behavior. Altogether, these results confirm that cyanoflan is a versatile carbohydrate polymer that can be used in different biotechnological applications, such as emulsifying/thickening agent in food or cosmetic industries

A new cyanobacterial species with a protective effect on lettuce grown under salinity stress: envisaging sustainable agriculture practices

In this work, a new terrestrial cyanobacterial species, Oculatella lusitanica LEGE 161147, was isolated and characterized using a polyphasic approach. Morphologically, O. lusitanica shares characteristics with different Oculatella species (mainly with O. crustae-formantes), lacking distinctive features. However, the phylogeny based on the 16S rRNA gene sequence and the 16S-23S ITS secondary structures support the establishment of this isolate as a new species. O. lusitanica is placed within a clade mainly composed by other Oculatella terrestrial strains; however, it forms a separate lineage. In addition, our species differs from the other Oculatella described so far by lacking the V2 helix within the ITS region. Since cyanobacteria are known to release compounds that promote plant growth and/or increase their tolerance to stresses, the effect of this newly described cyanobacterial species on Lactuca sativa (lettuce) plants development and salinity stress resistance was evaluated. Our results showed that, although the cyanobacterium had no impact on plant growth under the conditions tested, it was able to mitigate the deleterious salinity stress effects on plant size, root and aerial part fresh weight, by eliciting the non-enzymatic antioxidant response system (proline, H2O2 and reduced glutathione). In addition, the microorganism was able to induce a priming effect on lettuce plants by stimulating defensive mechanisms under non-stress conditions, and enhances the activity of nitrogen metabolism-related enzymes glutamate dehydrogenase, glutamine synthetase and nitrate reductase. These results indicate that this native terrestrial cyanobacterial species could be employed as a tool in sustainable agricultural practices.This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the projects PCIF/RPG/0077/2017, UIDB/04293/2020, UIDP/04293/2020, UIDB/05748/2020 and UIDP/05748/2020. This work was also funded by the FCT grant SFRH/BPD/115571/2016 (to AB) and LTAUSA 18008 (to JK)info:eu-repo/semantics/publishedVersio

Thin-layer nanofiltration membranes using engineered biopolymers for seawater desalination pre-treatment processes

Nowadays water demand already exceeds supply and water scarcity is a global problem. So it is necessary to develop novel technologies to be able to use poorer quality source waters for drinking water production. Once considered as an expensive, ultimate solution for water supply, desalination is becoming affordable. The two most commonly used seawater desalination methods are Multi-stage Flash Distillation (MSF) and Seawater Reverse Osmosis (SWRO). SWRO is less energy demanding compared to MSF, which makes it economically attractive. However there is no backpulsing of the expensive and delicate reverse osmosis (RO) membranes with air or water, so they are susceptible to fouling, causing the loss of their performance. Therefore cleaning the feed water to the highest level possible by nanofiltration, before it reaches the RO membranes would highly increase the efficiency of the process. Nanofiltration (NF) as a feed pre-treatment step is a pressure driven membrane separation process that takes place on a selective layer formed by a semipermeable membrane with properties between RO and ultrafiltration. The objective of this project is the developement of highly efficient thin-film composite (TFC) membranes for SWRO pre-treatment processes based on low-fouling cyanobacterial extracellular polymeric substances (EPS). TFC membranes combine high flux and mechanical strenght, and they are expected to be the key components of any water purification technology in the future. Cyanobacterial EPS are complex heteropolysaccharides with putative antimicrobial and antiviral properties and a particular affinity to bind metal ions [1,2].Within this work, the unicellular N2-fixing marine cyanobacterium Cyanothece sp. CCY 0110 was chosen for RPS production, since it is among the most efficient released polysaccharide (RPS) producers and the polymer has been previously extensively characterised [3]. RPS was produced by growing Cyanothece CCY 0110 in 10L bioreactors, in conditions previously defined and the polymer was isolated following the standard methodology [3]. A polyvinyl alcohol (PVA) / cyanobacterial EPS blend nanofibrous membranes were fabricated by electrospinning using polyvinylidene fluoride (PVDF) as a basal membrane, in order to obtain thin-layer composite nanofiltration membranes. The production of the nanofibers using EPS and PVA as plasticizer in different ratios was produced in a NF-103 MECC Nanon electrospinning equipment with an applied electric field between 15 and 25 kV and a flow of 0,2 mL/h. Morphological, mechanical, chemical and thermal characterization of the electrospun fibers deposited on the basal membranes, were evaluated by atomic force microscopy (AFM), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), dynamical and mechanical analysis (DMA), thermogravimetry (TGA) and differential scanning calorimetry (DSC). The AFM and SEM results show the presence of fibers with dimensions between 54 and 121 nm with low bead formation. In the EDS analysis presence of sulfur elements was observed confirming the inclusion of EPS in the nanofibers. The morphology and diameter of the nanofibers were mainly affected by the concentration of the blend solution and the weight ratio of the blend, respectively. The best PVA/EPS nanofibers were achieved in a ratio of 12 % PVA and 0.4 % EPS. The solution conductivity was ranging 1500 to 3500 μS/cm with a viscosity of about 100 to 500 cP. The DMA results confirmed the miscibility of PVA/EPS blends. The elastic modulus of the nanocomposite mats increased significantly as a consequence of the reinforcing effect of EPS. Thermal and mechanical analysis demonstrated that there were strong intermolecular hydrogen bonds between the molecules EPS-PVA in the blends. The heat-treated electrospun blended membranes showed better tensile mechanical properties when compared with PVA alone, and resisted more against disintegration. A lab-scale nanofiltration was performed in a bench stainless steel Sterlitech tangential flow stirred cell (200 mL) connected to an air pressure system that allow pressure driven filtration up to 10 BAR. Bactericidal activity and biofilm formation were tested using Escherichia coli and Sthaphylococcus aureus as pathogenic microorganisms

Assessing the antitumor potential of variants of the extracellular carbohydrate polymer from synechocystis ΔsigF mutant

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.info:eu-repo/semantics/publishedVersio