Study of alginate-encapsulated phycoerythrin in promoting the biological activity of synbiotic ice cream with Lactobacillus casei.

This study examines the effect of phycoerythrin (PE) from a cyanobacterial Nostoc strain encapsulated with alginate as a potential prebiotic to produce synbiotic ice cream products with Lactobacillus casei. It was found that the addition of the encapsulated PE affected, mostly favourably, the physicochemical properties, antioxidant activity, probiotic survival, volatile compound contents, and sensory acceptability of the synbiotic ice cream samples before and after aging at the freezing periods of one day to eight weeks. Thus, it confirms the prebiotic potential of PE for synbiotic ice creams with L. casei.

Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae.

BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production. RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells. CONCLUSION: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

The crane fly glycosylated triketide δ-lactone cornicinine elicits akinete differentiation of the cyanobiont in aquatic Azolla fern symbioses.

The restriction of plant-symbiont dinitrogen fixation by an insect semiochemical had not been previously described. Here we report on a glycosylated triketide δ-lactone from Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla. Only the glycosylated trans-A form of chemically synthesized cornicinine was active: 500 nM cornicinine in the growth medium turned all cyanobacterial filaments from Nostoc azollae inside the host leaf-cavities into akinetes typically secreting CTB-bacteriocins. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not impact development of the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc but affected the fern host without cyanobiont. Fern-host mRNA sequencing from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. After cornicinine treatment, it revealed activation of Cullin-RING ubiquitin-ligase-pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype, and increased JA-oxidase, sulfate transport and exosome formation. The work begins to uncover molecular mechanisms of cyanobiont differentiation in a seed-free plant symbiosis important for wetland ecology or circular crop-production today, that once caused massive CO2 draw-down during the Eocene geological past.

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.

UV-A radiation increases biomass yield by enhancing energy flow and carbon assimilation in the edible cyanobacterium Nostoc sphaeroides.

Ultraviolet (UV) A radiation (315-400 nm) is the predominant component of solar UV radiation that reaches the Earth's surface. However, the underlying mechanisms of the positive effects of UV-A on photosynthetic organisms have not yet been elucidated. In this study, we investigated the effects of UV-A radiation on the growth, photosynthetic ability, and metabolome of the edible cyanobacterium Nostoc sphaeroides. Exposures to 5-15 W m-2 (15-46 µmol photons m-2 s-1) UV-A and 4.35 W m-2 (20 µmol photons m-2 s-1) visible light for 16 days significantly increased the growth rate and biomass production of N. sphaeroides cells by 18%-30% and 15%-56%, respectively, compared to the non-UV-A-acclimated cells. Additionally, the UV-A-acclimated cells exhibited a 1.8-fold increase in the cellular nicotinamide adenine dinucleotide phosphate (NADP) pool with an increase in photosynthetic capacity (58%), photosynthetic efficiency (24%), QA re-oxidation, photosystem I abundance, and cyclic electron flow (87%), which further led to an increase in light-induced NADPH generation (31%) and ATP content (83%). Moreover, the UV-A-acclimated cells showed a 2.3-fold increase in ribulose-1,5-bisphosphate carboxylase/oxygenase activity, indicating an increase in their carbon-fixing capacity. Gas chromatography-mass spectrometry-based metabolomics further revealed that UV-A radiation upregulated the energy-storing carbon metabolism, as evidenced by the enhanced accumulation of sugars, fatty acids, and citrate in the UV-A-acclimated cells. Therefore, our results demonstrate that UV-A radiation enhances energy flow and carbon assimilation in the cyanobacterium N. sphaeroides.IMPORTANCEUltraviolet (UV) radiation exerts harmful effects on photo-autotrophs; however, several studies demonstrated the positive effects of UV radiation, especially UV-A radiation (315-400 nm), on primary productivity. Therefore, understanding the underlying mechanisms associated with the promotive effects of UV-A radiation on primary productivity can facilitate the application of UV-A for CO2 sequestration and lead to the advancement of photobiological sciences. In this study, we used the cyanobacterium Nostoc sphaeroides, which has an over 1,700-year history of human use as food and medicine, to explore its photosynthetic acclimation response to UV-A radiation. As per our knowledge, this is the first study to demonstrate that UV-A radiation increases the biomass yield of N. sphaeroides by enhancing energy flow and carbon assimilation. Our findings provide novel insights into UV-A-mediated photosynthetic acclimation and provide a scientific basis for the application of UV-A radiation for optimizing light absorption capacity and enhancing CO2 sequestration in the frame of a future CO2 neutral, circular, and sustainable bioeconomy.

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia, Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India.

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S-23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S-23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.

Tradeoffs between phage resistance and nitrogen fixation drive the evolution of genes essential for cyanobacterial heterocyst functionality.

Harmful blooms caused by diazotrophic (nitrogen-fixing) Cyanobacteria are becoming increasingly frequent and negatively impact aquatic environments worldwide. Cyanophages (viruses infecting Cyanobacteria) can potentially regulate cyanobacterial blooms, yet Cyanobacteria can rapidly acquire mutations that provide protection against phage infection. Here, we provide novel insights into cyanophage:Cyanobacteria interactions by characterizing the resistance to phages in two species of diazotrophic Cyanobacteria: Nostoc sp. and Cylindrospermopsis raciborskii. Our results demonstrate that phage resistance is associated with a fitness tradeoff by which resistant Cyanobacteria have reduced ability to fix nitrogen and/or to survive nitrogen starvation. Furthermore, we use whole-genome sequence analysis of 58 Nostoc-resistant strains to identify several mutations associated with phage resistance, including in cell surface-related genes and regulatory genes involved in the development and function of heterocysts (cells specialized in nitrogen fixation). Finally, we employ phylogenetic analyses to show that most of these resistance genes are accessory genes whose evolution is impacted by lateral gene transfer events. Together, these results further our understanding of the interplay between diazotrophic Cyanobacteria and their phages and suggest that a tradeoff between phage resistance and nitrogen fixation affects the evolution of cell surface-related genes and of genes involved in heterocyst differentiation and nitrogen fixation.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

INTRODUCTION: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. OBJECTIVES: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. METHODS: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. RESULTS: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. CONCLUSION: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

Effects of ultraviolet and photosynthetically active radiation on morphogenesis, antioxidants and photoprotective defense mechanism in a hot-spring cyanobacterium Nostoc sp. strain VKB02.

The continuous increase in global temperature and ultraviolet radiation (UVR) causes profound impacts on the growth and physiology of photosynthetic microorganisms. The hot-spring cyanobacteria have a wide range of mitigation mechanisms to cope up against current unsustainable environmental conditions. In the present investigation, we have explored the indispensable mitigation strategies of an isolated hot-spring cyanobacterium Nostoc sp. strain VKB02 under simulated ultraviolet (UV-A, UV-B) and photosynthetically active radiation (PAR). The adaptive morphological changes were more significantly observed under PAB (PAR, UV-A, and UV-B) exposure as compared to P and PA (PAR and UV-A) irradiations. PAB exposure also exhibited a marked decline in pigment composition and photosynthetic efficiency by multi-fold increment of free radicals. To counteract the oxidative stress, enzymatic and non-enzymatic antioxidants defense were significantly enhanced many folds under PAB exposure as compared to the control. In addition, the cyanobacterium has also produced shinorine as a strong free radicals scavenger and excellent UV absorber for effective photoprotection against UV radiation. Therefore, the hot-spring cyanobacterium Nostoc sp. strain VKB02 has unique defense strategies for survival under prolonged lethal UVR conditions. This study will help in the understanding of environment-induced defense strategies and production of highly value-added green photo-protectants for commercial applications.

Arsenite S-Adenosylmethionine Methyltransferase Is Responsible for Antimony Biomethylation in Nostoc sp. PCC7120.

Antimony (Sb) biomethylation is an important but uninformed process in Sb biogeochemical cycling. Methylated Sb species have been widely detected in the environment, but the gene and enzyme for Sb methylation remain unknown. Here, we found that arsenite S-adenosylmethionine methyltransferase (ArsM) is able to catalyze Sb(III) methylation. The stepwise methylation by ArsM forms mono-, di-, and trimethylated Sb species. Sb(III) is readily coordinated with glutathione, forming the preferred ArsM substrate which is anchored on three conserved cysteines. Overexpressing arsM in Escherichia coli AW3110 conferred resistance to Sb(III) by converting intracellular Sb(III) into gaseous methylated species, serving as a detoxification process. Methylated Sb species were detected in paddy soil cultures, and phylogenetic analysis of ArsM showed its great diversity in ecosystems, suggesting a high metabolic potential for Sb(III) methylation in the environment. This study shows an undiscovered microbial process methylating aqueous Sb(III) into the gaseous phase, mobilizing Sb on a regional and even global scale as a re-emerging contaminant.

Studying the impact of phycoerythrin on antioxidant and antimicrobial activity of the fresh rainbow trout fillets.

Marine cyanobacteria present a significant potential source of new bioactive compounds with vast structural diversity and relevant antimicrobial and antioxidant activities. Phycobiliproteins (PBPs) like phycocyanin (PC), phycoerythrin (PE), and water-soluble cyanobacterial photosynthetic pigments, have exhibited strong pharmacological activities and been used as natural food additives. In this study, phycoerythrin (PE) isolated from a marine strain of cyanobacterium Nostoc sp. Ft salt, was applied for the first time as a natural antimicrobial as well as an antioxidant to increase the shelf life of fresh rainbow trout i.e., (Oncorhynchus mykiss) fillets. Fresh trout fillets were marinated in analytical grade PE (3.9 µg/mL) prepared in citric acid (4 mg/mL), and stored at 4 °C and 8 °C for 21 days. Microbiological analysis, antioxidant activity and organoleptic evaluation of both control and treated fish fillets were then statistically compared. The results demonstrated noticeable (P < 0.05) differences in the microbial counts, antioxidant activity, and organoleptic characteristic values between PE-treated and non-treated groups. In addition, we observed that treating fresh fish fillets with a PE solution leads to a significant increase in shelf life by at least 14 days. Consequently, PE could be an alternative to synthetic chemical additives since it does not contain the potentially dangerous residues of the synthetic chemical additives and is thus healthier to the consumers.

Sulfur speciation in Sphagnum peat moss modified by mutualistic interactions with cyanobacteria.

Peat moss (Sphagnum spp.) develops mutualistic interactions with cyanobacteria by providing carbohydrates and S compounds in exchange for N-rich compounds, potentially facilitating N inputs into peatlands. Here, we evaluate how colonization of Sphagnum angustifolium hyaline cells by Nostoc muscorum modifies S abundance and speciation at the scales of individual cells and across whole leaves. For the first time, S K-edge X-ray Absorption Spectroscopy was used to identify bulk and micron-scale S speciation across isolated cyanobacteria colonies, and in colonized and uncolonized leaves. Uncolonized leaves contained primarily reduced organic S and oxidized sulfonate- and sulfate-containing compounds. Increasing Nostoc colonization resulted in an enrichment of S and changes in speciation, with increases in sulfate relative to reduced S and sulfonate. At the scale of individual hyaline cells, colonized cells exhibited localized enrichment of reduced S surrounded by diffuse sulfonate, similar to observations of cyanobacteria colonies cultured in the absence of leaves. We infer that colonization stimulates plant S uptake and the production of sulfate-containing metabolites that are concentrated in stem tissues. Sulfate compounds that are produced in response to colonization become depleted in colonized cells where they may be converted into reduced S metabolites by cyanobacteria.

Biological nitrogen fixation, diversity and community structure of diazotrophs in two mosses in 25 temperate forests.

Many moss species are associated with nitrogen (N)-fixing bacteria (diazotrophs) that support the N supply of mosses. Our knowledge relates primarily to pristine ecosystems with low atmospheric N input, but knowledge of biological N fixation (BNF) and diazotrophic communities in mosses in temperate forests with high N deposition is limited. We measured BNF rates using the direct stable isotope method and studied the total and potentially active diazotrophic communities in two abundant mosses, Brachythecium rutabulum and Hypnum cupressiforme, both growing on lying deadwood trunks in 25 temperate forest sites. BNF rates in both mosses were similar to those observed in moss species of pristine ecosystems. H. cupressiforme fixed three times more N2 and exhibited lower diazotrophic richness than B. rutabulum. Frankia was the most prominent diazotroph followed by cyanobacteria Nostoc. Manganese, iron, and molybdenum contents in mosses were positively correlated with BNF and diazotrophic communities. Frankia maintained high BNF rates in H. cupressiforme and B. rutabulum even under high chronic N deposition in Central European forests. Moss N concentration and 15 N abundance indicate a rather minor contribution of BNF to the N nutrition of these mosses.

Identification of a morphogene required for tapered filament termini in filamentous cyanobacteria.

Although the photosynthetic cyanobacteria are monophyletic, they exhibit substantial morphological diversity across species, and even within an individual species due to phenotypic plasticity in response to life cycles and environmental signals. This is particularly prominent among the multicellular filamentous cyanobacteria. One example of this is the appearance of tapering at the filament termini. However, the morphogenes controlling this phenotype and the adaptive function of this morphology are not well defined. Here, using the model filamentous cyanobacterium Nostoc punctiforme ATCC29133 (PCC73102), we identify tftA, a morphogene required for the development of tapered filament termini. The tftA gene is specifically expressed in developing hormogonia, motile trichomes where the tapered filament morphology is observed, and encodes a protein containing putative amidase_3 and glucosaminidase domains, implying a function in peptidoglycan hydrolysis. Deletion of tftA abolished filament tapering inidcating that TftA plays a role in remodelling the cell wall to produce tapered filaments. Genomic conservation of tftA specifically in filamentous cyanobacteria indicates this is likely to be a conserved mechanism among these organisms. Finally, motility assays indicate that filaments with tapered termini migrate more efficiently through dense substratum, providing a plausible biological role for this morphology.

Structural Diversity and Biological Activity of Cyanopeptolins Produced by Nostoc edaphicum CCNP1411.

Cyanopeptolins (CPs) are one of the most commonly occurring class of cyanobacterial nonribosomal peptides. For the majority of these compounds, protease inhibition has been reported. In the current work, the structural diversity of cyanopeptolins produced by Nostoc edaphicum CCNP1411 was explored. As a result, 93 CPs, including 79 new variants, were detected and structurally characterized based on their mass fragmentation spectra. CPs isolated in higher amounts were additionally characterized by NMR. To the best of our knowledge, this is the highest number of cyanopeptides found in one strain. The biological assays performed with the 34 isolated CPs confirmed the significance of the amino acid located between Thr and the unique 3-amino-6-hydroxy-2-piperidone (Ahp) on the activity of the compounds against serine protease and HeLa cancer cells.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Anti-SARS-CoV-2 activity of cyanopeptolins produced by Nostoc edaphicum CCNP1411.

Despite the advances in contemporary medicine and availability of numerous innovative therapies, effective treatment and prevention of SARS-CoV-2 infections pose a challenge. In the search for new anti-SARS-CoV-2 drug candidates, natural products are frequently explored. Here, fifteen cyanopeptolins (CPs) were isolated from the Baltic cyanobacterium Nostoc edaphicum and tested against SARS-CoV-2. Of these depsipeptides, the Arg-containing structural variants showed the strongest inhibition of the Delta SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells. The functional assays indicated a direct interaction of the Arg-containing CP978 with the virions. CP978 also induced a significant decline in virus replication in the primary human airway epithelial cells (HAE). Of the four tested SARS-CoV-2 variants, Wuhan, Alpha, Omicron and Delta, only Wuhan was not affected by CP978. Finally, the analyses with application of confocal microscopy and with the SARS-CoV-2 pseudoviruses showed that CP978-mediated inhibition of viral infection results from the direct binding of the cyanopeptolin with the coronaviral S protein. Considering the potency of viral inhibition and the mode of action of CP978, the significance of the peptide as antiviral drug candidate should be further explored.

Description of two new species of Nostoc (Nostocales, Cyanobacteria) from central Mexico, using morphological, ecological, and molecular attributes.

The present study describes two new Nostoc species, N. montejanii and N. tlalocii, based on a polyphasic approach that combines morphological, ecological, and genetic characteristics. The five investigated populations, including those from newly collected material from central Mexico, were observed to possess morphological features characteristic of the Nostoc genus. Results showed that both new species are strictly associated with running water, and they show clear differences in their habitat preferences. The 16S rRNA gene sequences of the five strains displayed between 98% and 99% similarity to the genus Nostoc sensu stricto. The 16S rRNA gene phylogenetic analyses inferred using Bayesian inference, maximum likelihood, and parsimony methods, placed these five strains in two separate clades distinct from other Nostoc species. The secondary structures of the 16S-23S internal transcribed spacer rRNA region in the two new species showed >10.5% dissimilarities in the operons when compared with other Nostoc species. In addition, clear morphological differences were observed between the two Mexican species, including the color of the colonies (black in N. montejanii and green in N. tlalocii), the size of the cells (greater in N. montejanii), and the number of polyphosphate granules present in the cells (one in N. montejanii and up to four in N. tlalocii).

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins.

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 µM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.