Phormidium ambiguum and Leptolyngbya ohadii Exopolysaccharides under Low Water Availability.

Cyanobacteria can cope with various environmental stressors, due to the excretion of exopolysaccharides (EPS). However, little is known about how the composition of these polymers may change according to water availability. This work aimed at characterizing the EPS of Phormidium ambiguum (Oscillatoriales; Oscillatoriaceae) and Leptolyngbya ohadii (Pseudanabaenales; Leptolyngbyaceae), when grown as biocrusts and biofilms, subject to water deprivation. The following EPS fractions were quantified and characterized: soluble (loosely bound, LB) and condensed (tightly bound, TB) for biocrusts, released (RPS), and sheathed in P. ambiguum and glycocalyx (G-EPS) in L. ohadii for biofilms. For both cyanobacteria upon water deprivation, glucose was the main monosaccharide present and the amount of TB-EPS resulted was significantly higher, confirming its importance in these soil-based formations. Different profiles of monosaccharides composing the EPSs were observed, as for example the higher concentration of deoxysugars observed in biocrusts compared to biofilms, demonstrating the plasticity of the cells to modify EPS composition as a response to different stresses. For both cyanobacteria, both in biofilms and biocrusts, water deprivation induced the production of simpler carbohydrates, with an increased dominance index of the composing monosaccharides. The results obtained are useful in understanding how these very relevant cyanobacterial species are sensitively modifying the EPS secreted when subject to water deprivation and could lead to consider them as suitable inoculants in degraded soils.

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.

Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Strategies to Obtain Designer Polymers Based on Cyanobacterial Extracellular Polymeric Substances (EPS).

Biopolymers derived from polysaccharides are a sustainable and environmentally friendly alternative to the synthetic counterparts available in the market. Due to their distinctive properties, the cyanobacterial extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides, emerge as a valid alternative to address several biotechnological and biomedical challenges. Nevertheless, biotechnological/biomedical applications based on cyanobacterial EPS have only recently started to emerge. For the successful exploitation of cyanobacterial EPS, it is important to strategically design the polymers, either by genetic engineering of the producing strains or by chemical modification of the polymers. This requires a better understanding of the EPS biosynthetic pathways and their relationship with central metabolism, as well as to exploit the available polymer functionalization chemistries. Considering all this, we provide an overview of the characteristics and biological activities of cyanobacterial EPS, discuss the challenges and opportunities to improve the amount and/or characteristics of the polymers, and report the most relevant advances on the use of cyanobacterial EPS as scaffolds, coatings, and vehicles for drug delivery.

Characterization and antitumor activity of the extracellular carbohydrate polymer from the cyanobacterium Synechocystis ΔsigF mutant.

Cyanobacterial extracellular carbohydrate polymers are particularly attractive for biotechnological applications. Previously, we determined the monosaccharidic composition of the polymer of a Synechocystis ΔsigF overproducing mutant. Here, we further characterized this polymer, demonstrated that it is possible to recover it in high yields, and successfully use it for biomedical research. This amorphous polymer is formed by a mesh of fibrils/lamellar structures with high porosity, is constituted by high molecular mass fractions, is highly sulfated and displays low viscosity, even in highly concentrated aqueous solutions. FTIR analysis confirmed the presence of several functional groups. We demonstrated that the ΔsigF polymer has strong biological activity, decreasing the viability of melanoma, thyroid and ovary carcinoma cells by inducing high levels of apoptosis, through p53 and caspase-3 activation. Therefore, the ΔsigF Synechocystis mutant is a promising platform for the sustainable production of biological active carbohydrate polymer(s) with the desired characteristics for biomedical applications.

The role of the tyrosine kinase Wzc (Sll0923) and the phosphatase Wzb (Slr0328) in the production of extracellular polymeric substances (EPS) by Synechocystis PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803.

Cyanobacterial alternative sigma factors are crucial players in environmental adaptation processes, which may involve bacterial responses related to maintenance of cell envelope and control of secretion pathways. Here, we show that the Group 3 alternative sigma factor F (SigF) plays a pleiotropic role in Synechocystis sp. PCC 6803 physiology, with a major impact on growth and secretion mechanisms, such as the production of extracellular polysaccharides, vesiculation and protein secretion. Although ΔsigF growth was significantly impaired, the production of released polysaccharides (RPS) increased threefold to fourfold compared with the wild-type. ΔsigF exhibits also impairment in formation of outer-membrane vesicles (OMVs) and pili, as well as several other cell envelope alterations. Similarly, the exoproteome composition of ΔsigF differs from the wild-type both in amount and type of proteins identified. Quantitative proteomics (iTRAQ) and an in silico analysis of SigF binding motifs revealed possible targets/pathways under SigF control. Besides changes in protein levels involved in secretion mechanisms, our results indicated that photosynthesis, central carbon metabolism and protein folding/degradation mechanisms are altered in ΔsigF. Overall, this work provided new evidences about the role of SigF on Synechocystis physiology and associates this regulatory element with classical and non-classical secretion pathways.

Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

Phylum-wide analysis of genes/proteins related to the last steps of assembly and export of extracellular polymeric substances (EPS) in cyanobacteria.

Many cyanobacteria produce extracellular polymeric substances (EPS) with particular characteristics (e.g. anionic nature and presence of sulfate) that make them suitable for industrial processes such as bioremediation of heavy metals or thickening, suspending or emulsifying agents. Nevertheless, their biosynthetic pathway(s) are still largely unknown, limiting their utilization. In this work, a phylum-wide analysis of genes/proteins putatively involved in the assembly and export of EPS in cyanobacteria was performed. Our results demonstrated that most strains harbor genes encoding proteins related to the three main pathways: Wzy-, ABC transporter-, and Synthase-dependent, but often not the complete set defining one pathway. Multiple gene copies are mainly correlated to larger genomes, and the strains with reduced genomes (e.g. the clade of marine unicellular Synechococcus and Prochlorococcus), seem to have lost most of the EPS-related genes. Overall, the distribution of the different genes/proteins within the cyanobacteria phylum raises the hypothesis that cyanobacterial EPS production may not strictly follow one of the pathways previously characterized. Moreover, for the proteins involved in EPS polymerization, amino acid patterns were defined and validated constituting a novel and robust tool to identify proteins with similar functions and giving a first insight to which polymer biosynthesis they are related to.

Differential proteomes of the cyanobacterium Cyanothece sp. CCY 0110 upon exposure to heavy metals.

The proteomes of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110, grown in medium supplemented with an essential metal (Cu(2+)) or a non-essential metal (Cd(2+)),were compared using iTRAQ technology. The data were obtained within a larger study that evaluated the overall effects of different heavy metals on growth/survival, EPS production and ultrastructure of this cyanobacterium [1]. To allow a broader understanding of the strategies triggered to coupe with toxic effects of the metals, Cyanothece's proteomes were evaluated after chronic and acute exposure to Cu(2+) and Cd(2+) in two independent 8-plex iTRAQ studies. For the chronic exposure 0.1 mg/l of Cu(2+) or 5 mg/l of Cd(2+) were used for 10 and 20 days, while in the acute experiments the cells were exposed to 10× these concentrations for 24 h. 202 and 268 proteins were identified and quantified for studies 1 (Cu(2+)) and 2 (Cd(2+)), respectively. The majority of the proteins with significant fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism.

Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles.

The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. BIOLOGICAL SIGNIFICANCE: This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers.

Preparation and characterization of polysaccharides/PVA blend nanofibrous membranes by electrospinning method.

A series of polyvinyl alcohol (PVA), PVA/chitosan (CS) and PVA/cyanobacterial extracellular polymeric substances (EPS) blended nanofibrous membranes were produced by electrospinning using a microfiltration poly(vinylidene fluoride) (PVDF) basal membrane, for potential applications in water filtration. Nanofibres were obtained from solutions of 20% (w/w) PVA with 1% (w/w) CS or EPS, using a weight ratio of 60/40. Blended nanofibres have shown a smooth morphology, no beads formation and diameters between 50 and 130 nm. Thermo-mechanical analysis demonstrated that there were inter and/or intramolecular hydrogen bonds between the molecules of PVA/CS and PVA/EPS in the blends. The electrospun blended PVA/EPS membrane showed better tensile mechanical properties when compared with PVA and PVA/CS, and resisted more against disintegration in the temperature range between 10 and 50 °C. Finally, the blended membranes have shown an increase in chromium binding capacity of 5%. This is the first successful report of a blended membrane of electrospinned cyanobacterial polysaccharide with PVA.

Production and characterization of extracellular carbohydrate polymer from Cyanothece sp. CCY 0110.

Cyanobacterial extracellular polymeric substances (EPS) are heteropolysaccharides that possess characteristics suitable for industrial applications, notably a high number of different monomers, strong anionic nature and high hydrophobicity. However, systematic studies that unveil the conditions influencing EPS synthesis and/or its characteristics are mandatory. In this work, Cyanothece sp. CCY 0110 was used as model organism. Our results revealed that this strain is among the most efficient EPS producers, and that the amount of RPS (released polysaccharides) is mainly related to the number of cells, rather than to the amount produced by each cell. Light was the key parameter, with high light intensity enhancing significantly RPS production (reaching 1.8 g L(-1)), especially in the presence of combined nitrogen. The data showed that RPS are composed by nine different monosaccharides (including two uronic acids), the presence of sulfate groups and peptides, and that the polymer is remarkably thermostable and amorphous in nature.

iTRAQ-based quantitative proteomic analysis of Gloeothece sp. PCC 6909: Comparison with its sheathless mutant and adaptations to nitrate deficiency and sulfur limitation.

Gloeothece sp. PCC 6909 is a unicellular N(2)-fixing cyanobacterium with a well defined and highly developed sheath surrounding its cells. A sheathless mutant of this strain was previously obtained by chemical mutagenesis and, although lacking the sheath, it releases large amounts of polysaccharides into the culture medium. To provide a global understanding on the metabolic differences between the two phenotypes, the proteomes of the wild type and mutant were analyzed using a cross-species proteomics approach coupled with iTRAQ isobaric tagging technology, since their genome sequences are not yet available. Effects arising from the presence/absence of nitrate and sulfur are presented as two metabolically directed follow-up iTRAQ studies. These nutrients are believed to play a major role in Gloeothece's metabolism, including the production of extracellular polymeric substances - EPS. 454, 124, and 53 proteins were identified and reliably quantified using homology anchoring approaches for iTRAQ previously described. The results obtained strongly suggest that the chemical mutagenesis affected the regulation of a number of key cellular processes, as revealed by the significant fold changes observed for proteins covering a large spectrum of functional groups. Moreover, they provide new insights on the adaptations of Gloeothece cells to nitrate-deficiency and sulfur-limitation.