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.

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.

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.

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.

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.

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).

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.

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.

PatU3 plays a central role in coordinating cell division and differentiation in pattern formation of filamentous cyanobacterium Nostoc sp. PCC 7120.

Spatial periodic signal for cell differentiation in some multicellular organisms is generated according to Turing's principle for pattern formation. How a dividing cell responds to the signal of differentiation is addressed with the filamentous cyanobacterium Nostoc sp. PCC 7120, which forms the patterned distribution of heterocysts. We show that differentiation of a dividing cell was delayed until its division was completed and only one daughter cell became heterocyst. A mutant of patU3, which encodes an inhibitor of heterocyst formation, showed no such delay and formed heterocyst pairs from the daughter cells of cell division or dumbbell-shaped heterocysts from the cells undergoing cytokinesis. The patA mutant, which forms heterocysts only at the filament ends, restored intercalary heterocysts by a single nucleotide mutation of patU3, and double mutants of patU3/patA and patU3/hetF had the phenotypes of the patU3 mutant. We provide evidence that HetF, which can degrade PatU3, is recruited to cell divisome through its C-terminal domain. A HetF mutant with its N-terminal peptidase domain but lacking the C-terminal domain could not prevent the formation of heterocyst pairs, suggesting that the divisome recruitment of HetF is needed to sequester HetF for the delay of differentiation in dividing cells. Our study demonstrates that PatU3 plays a key role in cell-division coupled control of differentiation.

Differential effects of nitrate and ammonium on the growth of algae and microcystin production by nitrogen-fixing Nostoc sp. and non-nitrogen-fixing Microcystis aeruginosa.

Cyanotoxins produced by cyanobacteria are a significant threat to human health. However, their responses to nitrogen (N) supplies could differ between N-fixing and non-N-fixing species, which has been poorly understood. This study aimed to compare the responses of the non-N-fixing Microcystis aeruginosa and N-fixing Nostoc sp. to varying concentrations of nitrate and ammonium. This comparison had been conducted by analyzing chlorophyll-a contents, maximum quantum efficiencies of photosystem II, microcystin production, and related gene expressions. Our findings revealed that nitrate substantially stimulated the growth of both M. aeruginosa and Nostoc sp. with biomass increase by 366.2 ± 56.5 and 93.0 ± 14.0%, respectively, at 16 mg-N/L. In contrast, high ammonium concentrations suppressed their growth. Furthermore, the intracellular concentration of microcystins produced by M. aeruginosa was higher under high nitrate. Extracellular microcystins showed an opposite trend to increases in nitrate and ammonium. Ammonium increases the production and releases microcystin from Nostoc sp. N metabolism genes showed a similar trend with toxin formation genes, which were up-regulated under the high N treatments. This study provides valuable insights into the impacts of N supplies on growths of N- and non-N-fixing cyanobacteria, as well as microcystin production, which helps to develop effective strategies for managing cyanobacterial blooms.

Sulfur is in the Air: Cyanolichen Marriages and Pollution.

Cyanolichens are symbiotic organisms involving cyanobacteria and fungi (bipartite) or with the addition of an algal partner (tripartite). Cyanolichens are known for their heightened susceptibility to environmental pollution. We focus here on the impacts on cyanolichens due to rising air pollution; we are especially interested in the role of sulfur dioxide on cyanolichen biology. Cyanolichens due to air pollution including sulfur dioxide exposure, show symptomatic changes including degradation of chlorophyll, lipid membrane peroxidation, decrease in ATP production, changes in respiration rate, and alteration of endogenous auxins and ethylene production, although symptoms are known to vary with species and genotype. Sulfur dioxide has been shown to be damaging to photosynthesis but is relatively benign on nitrogen fixation which proposes as a hypothesis that the algal partner may be more in harm's way than the cyanobiont. In fact, the Nostoc cyanobiont of sulfur dioxide-susceptible Lobaria pulmonaria carries a magnified set of sulfur (alkane sulfonate) metabolism genes capable of alkane sulfonate transport and assimilation, which were only unraveled by genome sequencing, a technology unavailable in the 1950-2000 epoch, where most physiology- based studies were performed. There is worldwide a growing corpus of evidence that sulfur has an important role to play in biological symbioses including rhizobia-legumes, mycorrhizae-roots and cyanobacteria-host plants. Furthermore, the fungal and algal partners of L. pulmonaria appear not to have the sulfonate transporter genes again providing the roles of ambient-sulfur (alkanesulfonate metabolism etc.) mediated functions primarily to the cyanobacterial partner. In conclusion, we have addressed here the role of the atmospheric pollutant sulfur dioxide to tripartite cyanolichen viability and suggest that the weaker link is likely to be the photosynthetic algal (chlorophyte) partner and not the nitrogen-fixing cyanobiont.

Interlink between ExoD (Alr2882), exopolysaccharide synthesis and metal tolerance in Nostoc sp. strain PCC 7120: Insight into its role, paralogs and evolution.

Exopolysaccharides (EPS) produced by bacterial species are an important component of bacteria's survival strategy. Synthesis of EPS, principal component of extracellular polymeric substance, occurs through multiple pathways involving multitude of genes. While stress-induced concomitant increase in exoD transcript levels and EPS content have been shown earlier, experimental evidence for direct correlation is lacking. In the present study, role of ExoD in Nostoc sp. strain PCC 7120 was evaluated by generating a recombinant Nostoc strain AnexoD+, wherein the ExoD (Alr2882) protein was constitutively overexpressed. AnexoD+ exhibited higher EPS production, propensity for formation of biofilms and tolerance to Cd stress compared to vector control AnpAM cells. Both Alr2882 and its paralog All1787 exhibited 5 transmembrane domains, with only All1787 predicted to interact with several proteins in polysaccharide synthesis. Phylogenetic analysis of orthologs of these proteins across cyanobacteria indicated that the two paralogs Alr2882 and All1787 and their corresponding orthologs arose divergently during evolution, and could have distinct roles to perform in the biosynthesis of EPS. This study has thrown open the possibility of engineering overproduction of EPS and inducing biofilm formation through genetic manipulation of EPS biosynthesis genes in cyanobacteria, thus building a cost-effective green platform for large scale production of EPS.

Functional specialization of expanded orange carotenoid protein paralogs in subaerial Nostoc species.

Orange carotenoid protein (OCP) is a photoactive protein that participates in the photoprotection of cyanobacteria. There are 2 full-length OCP proteins, 4 N-terminal paralogs (helical carotenoid protein [HCP]), and 1 C-terminal domain-like carotenoid protein (CCP) found in Nostoc flagelliforme, a desert cyanobacterium. All HCPs (HCP1 to 3 and HCP6) from N. flagelliforme demonstrated their excellent singlet oxygen quenching activities, in which HCP2 was the strongest singlet oxygen quencher compared with others. Two OCPs, OCPx1 and OCPx2, were not involved in singlet oxygen scavenging; instead, they functioned as phycobilisome fluorescence quenchers. The fast-acting OCPx1 showed more effective photoactivation and stronger phycobilisome fluorescence quenching compared with OCPx2, which behaved differently from all reported OCP paralogs. The resolved crystal structure and mutant analysis revealed that Trp111 and Met125 play essential roles in OCPx2, which is dominant and long acting. The resolved crystal structure of OCPx2 is maintained in a monomer state and showed more flexible regulation in energy quenching activities compared with the packed oligomer of OCPx1. The recombinant apo-CCP obtained the carotenoid pigment from holo-HCPs and holo-OCPx1 of N. flagelliforme. No such carotenoid transferring processes were observed between apo-CCP and holo-OCPx2. The close phylogenetic relationship of OCP paralogs from subaerial Nostoc species indicates an adaptive evolution toward development of photoprotection: protecting cellular metabolism against singlet oxygen damage using HCPs and against excess energy captured by active phycobilisomes using 2 different working modes of OCPx.

Identification of novel smORFs and microprotein acting in response to rehydration of Nostoc flagelliforme.

Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in N. flagelliforme remains unknown. Here we first constructed a smORF database in N. flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms.

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.

NtcA, LexA and heptamer repeats involved in the multifaceted regulation of DNA repair genes recF, recO and recR in the cyanobacterium Nostoc PCC7120.

Regulation of DNA repair genes in cyanobacteria is an unexplored field despite some of them exhibiting high radio-resistance. With RecF pathway speculated to be the major double strand break repair pathway in Nostoc sp. strain PCC7120, regulation of recF, recO and recR genes was investigated. Bioinformatic approach-based identification of promoter and regulatory elements was validated using qRT-PCR analysis, reporter gene and DNA binding assays. Different deletion constructs of the upstream regulatory regions of these genes were analysed in host Nostoc as well as heterologous system Escherichia coli. Studies revealed: (1) Positive regulation of all three genes by NtcA, (2) Negative regulation by LexA, (3) Involvement of contiguous heptamer repeats with/without its yet to be identified interacting partner in regulating (i) binding of NtcA and LexA to recO promoter and its translation, (ii) transcription or translation of recF, (4) Translational regulation of recF and recO through non-canonical and distant S.D. sequence and of recR through a rare initiation codon. Presence of NtcA either precludes binding of LexA to AnLexA-Box or negates its repressive action resulting in higher expression of these genes under nitrogen-fixing conditions in Nostoc. Thus, in Nostoc, expression of recF, recO and recR genes is intricately regulated through multiple regulatory elements/proteins. Contiguous heptamer repeats present across the Nostoc genome in the vicinity of start codon or promoter is likely to have a global regulatory role. This is the first report detailing regulation of DSB repair genes in any algae.

SepN is a septal junction component required for gated cell-cell communication in the filamentous cyanobacterium Nostoc.

Multicellular organisms require controlled intercellular communication for their survival. Strains of the filamentous cyanobacterium Nostoc regulate cell-cell communication between sister cells via a conformational change in septal junctions. These multi-protein cell junctions consist of a septum spanning tube with a membrane-embedded plug at both ends, and a cap covering the plug on the cytoplasmic side. The identities of septal junction components are unknown, with exception of the protein FraD. Here, we identify and characterize a FraD-interacting protein, SepN, as the second component of septal junctions in Nostoc. We use cryo-electron tomography of cryo-focused ion beam-thinned cyanobacterial filaments to show that septal junctions in a sepN mutant lack a plug module and display an aberrant cap. The sepN mutant exhibits highly reduced cell-cell communication rates, as shown by fluorescence recovery after photobleaching experiments. Furthermore, the mutant is unable to gate molecule exchange through septal junctions and displays reduced filament survival after stress. Our data demonstrate the importance of controlling molecular diffusion between cells to ensure the survival of a multicellular organism.

Anabaenopeptins from Nostoc edaphicum CCNP1411.

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.

Phycological exploration of the global biodiversity hotspots of Northeast India: discovery of a new species of soil-dwelling cyanobacteria, Desikacharya kailashaharensis sp. nov.

A soil-dwelling cyanobacterial strain (KLS-BP-3A_PS), has been isolated from the biodiversity rich Northeast region of India and characterized using a polyphasic approach. The strain was collected from a field covered with grass, near a stream from the Unakoti district of Tripura. Upon culturing in the laboratory, initial studies indicated the strain to be showing typical Nostoc or Nostoc-like morphology. Subsequently, 16S rRNA gene phylogenetic analyses using Neighbour joining, Maximum-likelihood, and Bayesian inference methods gave a distinct and stable positioning of the strain inside the genus Desikacharya. Upon recovery of the full-length operon of the 16S-23S ITS region with both tRNAs (tRNAIle and tRNAAla), the folded secondary structures revealed unique patterns of the D1-D1', V2, Box-B, and V3 regions of the strain KLS-BP-3A_PS as compared to phylogenetically related species of the genus Desikacharya. The total evidence approach indicated conclusively that the strain under investigation is a new species of the genus Desikacharya, which we describe as Desikacharya kailashaharensis in accordance with the International Code of Nomenclature for algae, fungi, and plants. Further, 16S rRNA gene phylogeny and evaluation of the 16S-23S ITS operons along with implying a re-examination of the family level affiliation of Desikacharya as well its generic limits may be in order. Notably, this study brings into focus the very less explored Northeast region of India which shares two global biodiversity hotspots in the world.

Phylogeny and fatty acid profiles of Aliinostoc vietnamicum sp. nov. (cyanobacteria) from the soils of Vietnam.

A new cyanobacterial species of Aliinostoc, A. vietnamicum sp. nov., is recorded in the tropical forest soil from the Cát Tiên National Park, Vietnam. The analysis is based on morphological characters, 16S rDNA phylogeny, ITS secondary structure, and fatty acid composition analysis. Aliinostoc vietnamicum differed from the other species of the genus by the size and shape of vegetative cells, size of akinetes and heterocytes, and presence of granular polyphosphate inclusions in vegetative cells. The evolutionary distance matrix based on the 16S rRNA gene shared 96.2-98.2% similarities with other Aliinostoc sequences. The phylogeny inferred by maximum likelihood and Bayesian inference placed A. vietnamicum in the Aliinostoc clade, within the Nostocaceae. For the first time, fatty acid composition analysis was obtained for a member of the genus Aliinostoc with cultivation time experiments. α-linolenic (27.54-37.75%), palmitic (13.87-22.65%), and stearic (10.08-20.27%) acids were the dominant fatty acids when cultured during the exponential growth phase, as well as during stationary. This is the first finding of a strain with such a high content of stearic acid among cyanobacteria with Nostoc-like morphology.