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The present study examined the regulatory mechanism of hydrogen sulfide (H 2 S) and nitric oxide (NO) in nickel (Ni) stressed cyanobacteria viz., Nostoc muscorum and Anabaena sp. by analyzing growth, photosynthetic pigments, biochemical components (protein and carbohydrate), exopolysaccharides (EPS), inorganic nitrogen content, and activity of enzymes comprised in nitrogen metabolism and Ni accumulation. The 1 µM Ni substantially diminished growth by 18% and 22% in N. muscorum and Anabaena sp. respectively, along with declining the pigment contents (Chl a/Car ratio and phycobiliproteins), and biochemical components. It also exerted negative impacts on inorganic uptake of nitrate and nitrite contents; nitrate reductase and nitrite reductase; and ammonium assimilating enzymes (glutamine synthetase, glutamate synthase, and glutamate dehydrogenase exhibited a reverse trend) activities. Nonetheless, the adverse impact of Ni can be mitigated through the exogenous supplementation of NaHS [sodium hydrosulfide (8 µM); H 2 S donor] and SNP [sodium nitroprusside (10 µM); NO donor] which showed substantial improvement on growth, pigments, nitrogen metabolism, and EPS layer and noticeably occurred as a consequence of a substantial reduction in Ni accumulation content which minimized the toxicity effects. The accumulation of Ni on both the cyanobacterial cell surface (EPS layer) are confirmed by the SEM-EDX analysis. Further, the addition of NO scavenger (PTIO; 20 µM) and inhibitor of NO (L-NAME; 100 µM); and H 2 S scavenger (HT; 20 µM) and H 2 S inhibitor (PAG; 50 µM) reversed the positive responses of H 2 S and NO and damages were more prominent under Ni stress thereby, suggesting the downstream signaling of H 2 S on NO-mediated alleviation. Thus, this study concludes the crosstalk mechanism of H 2 S and NO in the mitigation of Ni-induced toxicity in rice field cyanobacteria., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

The TonB-dependent transport of scarcely available substrates across the outer membrane is a conserved feature in Gram-negative bacteria. The plasma membrane-embedded TonB-ExbB-ExbD accomplishes complex functions as an energy transducer by physically interacting with TonB-dependent outer membrane transporters (TBDTs). TonB mediates structural rearrangements in the substrate-loaded TBDTs that are required for substrate translocation into the periplasm. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, four TonB-like proteins have been identified. Out of these TonB3 accomplishes the transport of ferric schizokinen, the siderophore which is secreted by Anabaena to scavenge iron. In contrast, TonB1 (SjdR) is exceptionally short and not involved in schizokinen transport. The proposed function of SjdR in peptidoglycan structuring eliminates the protein from the list of TonB proteins in Anabaena . Compared with the well-characterized properties of SjdR and TonB3, the functions of TonB2 and TonB4 are yet unknown. Here, we examined tonB2 and tonB4 mutants for siderophore transport capacities and other specific phenotypic features. Both mutants were not or only slightly affected in schizokinen transport, whereas they showed decreased nitrogenase activity in apparently normal heterocysts. Moreover, the cellular metal concentrations and pigment contents were altered in the mutants, most pronouncedly in the tonB2 mutant. This strain showed an altered susceptibility toward antibiotics and SDS and formed cell aggregates when grown in liquid culture, a phenotype associated with an elevated lipopolysaccharide (LPS) production. Thus, the TonB-like proteins in Anabaena appear to take over distinct functions, and the mutation of TonB2 strongly influences outer membrane integrity. IMPORTANCE The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified. In addition to a role in uptake of scarcely available nutrients, including iron complexes, TonB proteins are related to virulence, flagellum assembly, pilus localization, or envelope integrity, including antibiotic resistance. The knowledge about the function of TonB proteins in cyanobacteria is limited. Here, we compare the four TonB proteins of Anabaena sp. strain PCC 7120, providing evidence that their functions are in part distinct, since mutants of these proteins exhibit specific features but also show some common impairments.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 g PHB g biomass -1 d -1 ) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L -1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L -1 , and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena . Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR ( s eptal j unction d isk r egulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena . In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel s eptal j unction d isk r egulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole-system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near-complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834 , alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.

The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. strain PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. They belong to the family of the two-partner secretion system proteins which are characteristic of pathogenic bacteria. Because pathogenicity of Anabaena sp. strain PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659 , while TpsB2 is encoded by all5116 The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. strain PCC 7120 putatively coding for substrates of the TpsB system, suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes resulted in altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates, a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. strain PCC 7120 and predict a large pool of putative substrates. IMPORTANCE Cyanobacteria are important organisms for the ecosystem, considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacterial overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment, including other organisms, is required to define their impact on ecosystems. While two-partner secretion systems in pathogenic bacteria are well known, we provide a first description of the cyanobacterial two-partner secretion system., (Copyright © 2021 American Society for Microbiology.)

Background: Cyanobacteria are well known for their inherent ability to serve as atmospheric nitrogen fixers and as bio-fertilizers; however, increased contaminants in aquatic ecosystem significantly decline the growth and function of these microbes in paddy fields. Plant growth regulators play beneficial role in combating the negative effects induced by heavy metals in photoautotroph. Current study evaluates the potential role of indole acetic acid (IAA; 290 nm) and kinetin (KN; 10 nm) on growth, nitrogen metabolism and biochemical constituents of two paddy field cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 exposed to two concentrations of chromium (Cr VI ; 100 μM and 150 μM)., Results: Both the tested doses of Cr VI declined the growth, ratio of chlorophyll a to carotenoids (Chl a/Car), contents of phycobiliproteins; phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), protein and carbohydrate associated with decrease in the inorganic nitrogen (nitrate; NO 3 - and nitrite; NO 2 - ) uptake rate that results in the decrease in nitrate and ammonia assimilating enzymes; nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) except glutamate dehydrogenase (GDH). However, exogenous supplementation of IAA and KN exhibited alleviating effects on growth, nitrogen metabolism and exopolysaccharide (EPS) (first protective barrier against metal toxicity) contents in both the cyanobacteria, which probably occurred as a result of a substantial decrease in the Cr uptake that lowers the damaging effects., Conclusion: Overall result of the present study signifies affirmative role of the phytohormone in minimizing the toxic effects induced by chromium by stimulating the growth of cyanobacteria thereby enhancing its ability as bio-fertilizer that improved fertility and productivity of soil even in metal contaminated condition.

RNase E is an endoribonuclease and plays a central role in RNA metabolism. Cyanobacteria, as ancient oxygen-producing photosynthetic bacteria, also contain RNase E homologues. Here, we introduced mutations into the S1 subdomain (F53A), the 5'-sensor subdomain (R160A), and the DNase I subdomain (D296A) according to the key activity sites of Escherichia coli RNase E. The results of degradation assays demonstrated that Asp296 is important to RNase E activity in Anabaena sp. PCC 7120 (hereafter PCC 7120). The docking model of RNase E in PCC 7120 (AnaRne) and RNA suggested a possible recognition mechanism of AnaRne to RNA. Moreover, overexpression of AnaRne and its N-terminal catalytic domain (AnaRneN) in vivo led to the abnormal cell division and inhibited the growth of PCC 7120. The quantitative analysis showed a significant decrease of ftsZ transcription in the case of overexpression of AnaRne or AnaRneN and ftsZ mRNA could be directly degraded by AnaRne through degradation assays in vitro, indicating that AnaRne was related to the expression of ftsZ and eventually affected cell division. In essence, our studies expand the understanding of the structural and functional evolutionary basis of RNase E and lay a foundation for further analysis of RNA metabolism in cyanobacteria., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Background: Filamentous cyanobacteria represent model organisms for investigating multicellularity. For many species, nitrogen-fixing heterocysts are formed from photosynthetic vegetative cells under nitrogen limitation. Intracellular Ca 2+ has been implicated in the highly regulated process of heterocyst differentiation but its role remains unclear. Ca 2+ is known to operate more broadly in metabolic signalling in cyanobacteria, although the signalling mechanisms are virtually unknown. A Ca 2+ -binding protein called the Ca 2+ Sensor EF-hand (CSE) is found almost exclusively in filamentous cyanobacteria. Expression of asr1131 encoding the CSE protein in Anabaena sp. PCC 7120 was strongly induced by low CO 2 conditions, and rapidly downregulated during nitrogen step-down. A previous study suggests a role for CSE and Ca 2+ in regulation of photosynthetic activity in response to changes in carbon and nitrogen availability., Results: In the current study, a mutant Anabaena sp. PCC 7120 strain lacking asr1131 (Δcse) was highly prone to filament fragmentation, leading to a striking phenotype of very short filaments and poor growth under nitrogen-depleted conditions. Transcriptomics analysis under nitrogen-replete conditions revealed that genes involved in heterocyst differentiation and function were downregulated in Δcse, while heterocyst inhibitors were upregulated, compared to the wild-type., Conclusions: These results indicate that CSE is required for filament integrity and for proper differentiation and function of heterocysts upon changes in the cellular carbon/nitrogen balance. A role for CSE in transmitting Ca 2+ signals during the first response to changes in metabolic homeostasis is discussed.

Although cyanobacteria are a common group of microorganisms well-suited to utilization in photobioreactors (PBRs), studies of cyanobacteria fouling and its prevention are scarce. Using a cyanobacterium, Anabaena sp. PCC 7120, which had been genetically modified to enhance linalool production, the formation of conditioning films and the effects of these on the physico-chemical surface properties of various PBR materials during initial adhesion and biofilm formation were investigated. The adhesion assay revealed that the overall attachment of Anabaena was substratum dependent and no correlation between the hydrophobicity/roughness of clean material and cell attachment was found. Surface hydrophilicity/hydrophobicity of all the materials changed within 12   h due to formation of conditioning films. ATR-FTIR spectroscopy revealed that the fractional change in protein deposition between 12 to 96   h was consistent with Anabaena cell attachment but polysaccharide deposition was material specific and did not correlate with cell attachment on the PBR materials. Also, the delay in conditioning film proteins on PVC and PTFE indicated that components other than proteins may be responsible for the decrease in contact angles on these surfaces within 12   h. This indicates the important role of the chemical nature of adsorbed conditioning films in determining the initial attachment of Anabaena to PBR materials. The lower rate of attachment of Anabaena on the hydrophilic surfaces (glass and PMMA) between 72   h to 96   h (regime 3) showed that these surfaces could potentially have low fouling characteristics at extended time scales and should be considered for further research.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) β-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of β-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of β-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between β-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaenasp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here, we studied the function of the putative nucleoside-diphosphate-sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site-directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdAmutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP-tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP-galactose 4-epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst-specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ca 2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca 2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca 2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca 2+ -binding protein which was called the Ca 2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca 2+ , which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mutant strain of the diazotrophic cyanobacterium Anabaena doliolum able to tolerate high temperature was isolated by induced mutation techniques using ethyl methane sulphonate. This mutant strain exhibited higher temperature tolerance than the wild type. The wild type was able tolerate temperature up to 40 °C whereas the mutant was able to grow at an elevated temperature of 48 °C. This mutant exhibited higher growth rate, heterocyst frequency, and nitrogen fixation. Mutant strains exhibited comparable levels of chlorophyll, phycocyanin, PS II activity, and O 2 evolution as compared to unexposed control. Results also showed that the mutant accumulated low levels of peroxides and lipid peroxidation products with enhanced activity of antioxidant enzymes. The FAME analysis revealed quantitative and qualitative changes in the profile of fatty acids in the mutant strain. Maximum number of saturated fatty acids was observed in the mutant strain followed by control whereas the wild type exposed to elevated temperature showed least diversity of fatty acids. Enhanced level of antioxidant enzymes coupled with efficient modulation of fatty acid profile could therefore enhance the mutant to resist the high temperature stress. The results could be exploited further to decipher molecular mechanisms underlying the temperature tolerance and enhancing the utility of A. doliolum as efficient biofertilizer for rice paddy keeping in view of the future climatic change scenario., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.

Anabaena sp. strain PCC 7120 is a multicellular, filamentous, freshwater cyanobacterium that is capable of differentiating specialized heterocyst cells for nitrogen fixation. This unit includes protocols for the growth and maintenance of Anabaena appropriate for a research or teaching laboratory. Controlled induction and assessment of heterocyst development is also covered. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Globally, eutrophication and warming of aquatic ecosystems has increased the frequency and intensity of cyanobacterial blooms and their associated toxins, with the simultaneous detection of multiple cyanotoxins often occurring. Despite the co-occurrence of cyanotoxins such as microcystins and anatoxin-a (ATX) in water bodies, their effects on phytoplankton communities are poorly understood. The individual and combined effects of microcystin-LR (MC-LR) and ATX on the cyanobacteria Microcystis spp., and Anabaena variabilis (a.k.a. Trichormus variabilis ), and the chlorophyte, Selenastrum capricornutum were investigated in the present study. Cell density, chlorophyll-a content, and the maximum quantum efficiency of photosystem II (Fv/Fm) of Microcystis cells were generally lowered after exposure to ATX or MC-LR, while the combined treatment with MC-LR and ATX synergistically reduced the chlorophyll-a concentration of Microcystis strain LE-3. Intracellular levels of microcystin in Microcystis LE-3 significantly increased following exposure to MC-LR + ATX. The maximum quantum efficiency of photosystem II of Anabaena strain UTEX B377 declined during exposure to the cyanotoxins. Nitrogen fixation by Anabaena UTEX B377 was significantly inhibited by exposure to ATX, but was unaffected by MC-LR. In contrast, the combination of both cyanotoxins (MC-LR + ATX) caused a synergistic increase in the growth of S. capricornutum . While the toxins caused an increase in the activity of enzymes that scavenge reactive oxygen species in cyanobacteria, enzyme activity was unchanged or decreased in S. capricornutum . Collectively this study demonstrates that MC-LR and ATX can selectively promote and inhibit the growth and performance of green algae and cyanobacteria, respectively, and that the combined effect of these cyanotoxins was often more intense than their individual effects on some strains. This suggests that the release of multiple cyanotoxins in aquatic ecosystems, following the collapse of blooms, may influence the succession of plankton communities.

Heterocyst-forming cyanobacteria are filamentous organisms that perform oxygenic photosynthesis and CO 2 fixation in vegetative cells and nitrogen fixation in heterocysts, which are formed under deprivation of combined nitrogen. These organisms can acclimate to use different sources of nitrogen and respond to different levels of CO 2 . Following work mainly done with the best studied heterocyst-forming cyanobacterium, Anabaena, here we summarize the mechanisms of assimilation of ammonium, nitrate, urea and N 2 , the latter involving heterocyst differentiation, and describe aspects of CO 2 assimilation that involves a carbon concentration mechanism. These processes are subjected to regulation establishing a hierarchy in the assimilation of nitrogen sources -with preference for the most reduced nitrogen forms- and a dependence on sufficient carbon. This regulation largely takes place at the level of gene expression and is exerted by a variety of transcription factors, including global and pathway-specific transcriptional regulators. NtcA is a CRP-family protein that adjusts global gene expression in response to the C-to-N balance in the cells, and PacR is a LysR-family transcriptional regulator (LTTR) that extensively acclimates the cells to oxygenic phototrophy. A cyanobacterial-specific transcription factor, HetR, is involved in heterocyst differentiation, and other LTTR factors are specifically involved in nitrate and CO 2 assimilation., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Eutrophication is caused by the rapid growth of microalgae. Iron and manganese are important micronutrients for microalgae growth. However, the effect of the limitation of iron and manganese on microalgae growth in fresh water has not been well understood. In this study, natural mixed algae, Anabaena flosaquae and Scenedesmus quadricanda, were cultivated under different quotas of iron and manganese to reveal the effect of the limitation of iron and manganese on the growth of microalgae in fresh water. The results showed that the growth rate of algae is influenced more by iron than by manganese. However, the effect of manganese cannot be overlooked: when the initial manganese quota was replete, i.e. 0.6-0.8 mg l -1 , manganese was able to relieve the effect of iron limitation on microalgae growth in fresh water. We further found that the microalgae showed an uptake preference for iron over manganese. Iron had a competitive effect on manganese uptake, while manganese had less impact on iron uptake by microalgae. The information obtained in the current study is useful for the provision of water quality warnings and for the control of microalgae bloom in fresh water.

Thioredoxin (Trx) family proteins perform redox regulation in cells, and they are involved in several other biological processes (e.g. oxidative stress tolerance). In the filamentous cyanobacterium Anabaena sp. PCC7120 (A. 7120), eight Trx isoforms have been identified via genomic analysis. Among these Trx isoforms, the absence of Trx-m1 and TrxC appears to result in oxidative stress in A. 7120 together with alterations of the thylakoid membrane structure and phycobiliprotein composition. To analyze the physiological changes in these Trx disruptants thoroughly, quantitative proteomics was applied. Certainly, the mutants exhibited similar alterations in the proteome including decreased relative abundance of phycobiliproteins and an increased level of proteins involved in amino acid and carbohydrate metabolism. Nevertheless, the results also indicated that the mutants exhibited changes in the relative abundance of different sets of proteins participating in reactive oxygen species detoxification, such as Fe-SOD in Δtrx-m1 and PrxQ in ΔtrxC, suggesting distinct functions of Trx-m1 and TrxC.

Nitrogen (N) is one of the most important limiting factors in conventional rice (Oryza sativa) production, which heavily relies on synthetic fertilizers. In this study, we researched on the development and use of a vertical semi-closed airlift photobioreactor (PBR) for microalgal cultivation and subsequently determined the efficacy of microalgae-based fertilizers to rice plant growth. The PBR system was developed to produce two strains of N2-fixing cyanobacteria (Anabaena sp. UTEX 2576, Nostoc muscorum UTEX 2209S), and a polyculture of Chlorella vulgaris (UTEX 2714) and Scenedesmus dimorphus (UTEX 1237). When these biofertilizers were evaluated for rice under the greenhouse conditions, results showed that the rice plant heights treated with polyculture-based microalgal biomass were similar to or better than the urea treatment. The effects of the inoculation of the N2-fixing cyanobacterial inoculation on seedling growth was not statistically significant. In conclusion, the vertical semi-closed system PBR cultivation method developed in this study proved to be a simple and effective method for cultivating microalgae. Demonstration of the reliable production system for N2-fixing cyanobacteria and chlorophytes at a medium scale could potentially open the future application of microalgal biofertilizers in rice production., Competing Interests: The authors have declared that no competing interests exist.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N 2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena., (© 2018 John Wiley & Sons Ltd.)

Controlling blooms of toxigenic phytoplankton, including cyanobacteria, is a high priority for managers of aquatic systems that are used for drinking water, recreation, and aquaculture production. Although a variety of treatment approaches exist, hydrogen peroxide (H 2 O 2 ) has the potential to be an effective and ecofriendly algaecide given that this compound may select against cyanobacteria while not producing harmful residues. To broadly evaluate the effectiveness of H 2 O 2 on toxigenic phytoplankton, we tested multiple concentrations of H 2 O 2 on (1) four cyanobacterial cultures, including filamentous Anabaena, Cylindrospermopsis, and Planktothrix, and unicellular Microcystis, in a 5-day laboratory experiment and (2) a dense cyanobacterial bloom in a 7-day field experiment conducted in a nutrient-rich aquaculture pond. In the laboratory experiment, half-maximal effective concentrations (EC 50 ) were similar for Anabaena, Cylindrospermopsis, and Planktothrix (average EC 50  = 0.41 mg L -1 ) but were ∼10x lower than observed for Microcystis (EC 50  = 5.06 mg L -1 ). Results from a field experiment in an aquaculture pond showed that ≥1.3 and ≥ 6.7 mg L -1 of H 2 O 2 effectively eliminated Planktothrix and Microcystis, respectively. Moreover, 6.7 mg L -1 of H 2 O 2 reduced microcystin and enhanced phytoplankton diversity, while causing relatively small negative effects on zooplankton abundance. In contrast, 20 mg L -1 of H 2 O 2 showed the greatest negative effect on zooplankton. Our results demonstrate that H 2 O 2 can be an effective, rapid algaecide for controlling toxigenic cyanobacteria when properly dosed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study was carried out using indoor controlled experiments to study the arsenic (As) uptake, biotransformation, and release behaviors of freshwater algae under growth stress. Three freshwater algae, Microcystis aeruginosa, Anabaena flosaquae, and Chlorella sp., were chosen. Two types of inhibitors, e.g., Cu 2+ and isothiazolinone, were employed to inhibit the growth of the algae. The algae were cultivated to a logarithmic stage in growth media containing 0.1 mg/L P; then, 0.8 mg/L As in the form of arsenate (iAs V ) was added, while both inhibitors were simultaneously added at dosages of 0.1 and 0.3 mg/L, with no addition of inhibitors in the control. After 2 days of exposure, the average growth rate (μ 2d ) was measured to represent the growth rates of the algae cells; the extra- and intracellular As concentrations in various forms, i.e., arsenate, arsenite (iAs III ), monomethyl arsenic (MMA), and dimethyl arsenic (DMA), were also measured. Without inhibitors, the average growth rate followed the order of M. aeruginosa, Chlorella sp., and A. flosaquae, with the growth rate of M. aeruginosa significantly higher than that of the other two algae. However, when Cu 2+ was added as an external inhibitor, the order of the average growth rate for the three algae became partially reversed, suggesting differentiation of the algae in response to the inhibitor. This differentiation can be seen by the reduction in the average growth rate of M. aeruginosa, which was as high as 1730% at the 0.3-mg/L Cu 2+ dosage when compared with the control, while for the other two algae, much fewer changes were seen. The great reduction in M. aeruginosa growth rate was accompanied by increases in extracellular iAs V and iAs III and intracellular iAs V concentrations in the algae, indicating that As transformation is related to the growth of this algae. Much fewer or neglectable changes in growth were observed that were consistent with the few changes in the extra- and intracellular As speciation for the other two algae with Cu 2+ inhibition and all the three algae with isothiazolinone inhibition, corroborating the above hypothesis again. All the algae tested in this study demonstrated great abilities for As transformation and release, as seen by the much higher rates of 86.11-99.98% and 81.11-99.89% for transformation and release when compared to the control, respectively. When inhibitors were added, the transformation and release values of only A. flosaquae decreased remarkably down to 72.37-86.79% and 64.67-85.24%, respectively, while no changes were seen for these values in the other two algae, indicating that growth stress did not affect the As transformation and release of the other algae. The biological productivity of As by the three algae followed the order of M. aeruginosa, Chlorella sp., and A. flosaquae, which was generally consistent with the As transformation and release in conditions with and without inhibitors, suggesting that the As behavior in the algae that was related to growth stress largely differed among algae species.

A novel imaging-driven technique with an integrated fluorescence signature to enable automated enumeration of two species of cyanobacteria and an alga of somewhat similar morphology to one of the cyanobacteria is presented to demonstrate proof-of-concept that high accuracy, imaging-based, rapid water quality analysis can be with conventional equipment available in typical water quality laboratories-this is not currently available. The results presented herein demonstrate that the developed method identifies and enumerates cyanobacterial cells at a level equivalent to or better than that achieved using standard manual microscopic enumeration techniques, but in less time, and requiring significantly fewer resources. When compared with indirect measurement methods, the proposed method provides better accuracy at both low and high cell concentrations. It extends the detection range for cell enumeration while maintaining accuracy and increasing enumeration speed. The developed method not only accurately estimates cell concentrations, but it also reliably distinguishes between cells of Anabaena flos-aquae, Microcystis aeruginosa, and Ankistrodesmus in mixed cultures by taking advantage of additional contrast between the target cell and complex background gained under fluorescent light. Thus, the proposed image-driven approach offers promise as a robust and cost-effective tool for identifying and enumerating microscopic cells based on their unique morphological features.

Anabaena sp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication. hetC is a gene upregulated in developing cells during heterocyst formation in Anabaena sp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation of hetC in 3 substrains of Anabaena sp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of P hetZ - gfp in these hetC mutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function of hetC is influenced by the genomic background and epistasis and constitutes an example of evolution under way. IMPORTANCE Our knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N 2 fixation, is mostly based on studies with Anabaena sp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such as hetC This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism., (Copyright © 2018 American Society for Microbiology.)

Various species of cyanobacteria, diatoms and dinoflagellates are capable of synthesizing the non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA), which is known to be a causative agent of human neurodegeneration. Similar to most cyanotoxins, the biological and ecological functions of BMAA in cyanobacteria are unknown. In this study, we show for the first time that BMAA, in micromolar amounts, inhibits the formation of heterocysts (specialized nitrogen-fixing cells) in heterocystous, diazotrophic cyanobacteria [Anabaena sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), Nostoc sp. strain 8963] under conditions of nitrogen starvation. The inhibitory effect of BMAA is abolished by the addition of glutamate. To understand the genetic reason for the observed phenomenon, we used qPCR to study the expression of key genes involved in cell differentiation and nitrogen metabolism in the model cyanobacterium Anabaena sp. PCC 7120. We observed that in the presence of BMAA, Anabaena sp. PCC 7120 does not express two essential genes associated with heterocyst differentiation, namely, hetR and hepA. We also found that addition of BMAA to cyanobacterial cultures with mature heterocysts inhibits nifH gene expression and nitrogenase activity., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Under nitrogen deprivation, the one-dimensional cyanobacterial organism Anabaena sp. PCC 7120 develops patterns of single, nitrogen-fixing cells separated by nearly regular intervals of photosynthetic vegetative cells. We study a minimal, stochastic model of developmental patterns in Anabaena that includes a nondiffusing activator, two diffusing inhibitor morphogens, demographic fluctuations in the number of morphogen molecules, and filament growth. By tracking developing filaments, we provide experimental evidence for different spatiotemporal roles of the two inhibitors during pattern maintenance and for small molecular copy numbers, justifying a stochastic approach. In the deterministic limit, the model yields Turing patterns within a region of parameter space that shrinks markedly as the inhibitor diffusivities become equal. Transient, noise-driven, stochastic Turing patterns are produced outside this region, which can then be fixed by downstream genetic commitment pathways, dramatically enhancing the robustness of pattern formation, also in the biologically relevant situation in which the inhibitors' diffusivities may be comparable.

Soil salinity in nature is generally mixed type; however, most of the studies on salt toxicity are performed with NaCl and little is known about sulfur type of salinity (Na 2 SO 4 ). Present study discerns the physiologic mechanisms responsible for salt tolerance in salt-adapted Anabaena fertilissima, and responses of directly stressed parent cells to NaCl and NaCl+Na 2 SO 4 mixture. NaCl at 500 mM was lethal to the cyanobacterium, whereas salt-adapted cells grew luxuriantly. Salinity impaired gross photosynthesis, electron transport activities, and respiration in parent cells, but not in the salt-adapted cells, except a marginal increase in PSI activity. Despite higher Na + concentration in the salt mixture, equimolar NaCl appeared more inhibitive to growth. Sucrose and trehalose content and antioxidant activities were maximal in 250 mM NaCl-treated cells, followed by salt mixture and was almost identical in salt-adapted (exposed to 500 mm NaCl) and control cells, except a marginal increase in ascorbate peroxidase activity and an additional fourth superoxide dismutase isoform. Catalase isoform of 63 kDa was induced only in salt-stressed cells. Salinity increased the uptake of intracellular Na + and Ca 2+ and leakage of K + in parent cells, while cation level in salt-adapted cells was comparable to control. Though there was differential increase in intracellular Ca 2+ under different salt treatments, ratio of Ca 2+ /Na + remained the same. It is inferred that stepwise increment in the salt concentration enabled the cyanobacterium to undergo priming effect and acquire robust and efficient defense system involving the least energy.

Heterocyst-forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N 2 -fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi-regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell-cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC-type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N 2 as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell-cell communication by directly interacting with AmiC1., (© 2018 John Wiley & Sons Ltd.)

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO 2 from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 ( Anabaena 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though Anabaena G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from Anabaena 7120 and its activator protein OpcA, we found that m -type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys 393 and Cys 399 of OpcA are responsible for the redox regulation property of this protein. Moreover, in vivo analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The microalgae-based technology has been developed to reduce biogas slurry nutrients and upgrade biogas simultaneously. In this work, five microalgal strains named Chlorella vulgaris, Scenedesmus obliquus, Selenastrum capricornutum, Nitzschia palea, and Anabaena spiroides under mono- and co-cultivation were used for biogas upgrading. Optimum biogas slurry nutrient reduction could be achieved by co-cultivating microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Nitzschia palea) with fungi using the pelletization technology. In addition, the effects of different ratio of mixed LED light wavelengths applying mixed light-emitting diode during algae strains and fungi co-cultivation on CO 2 and biogas slurry nutrient removal efficiency were also investigated. The results showed that the COD (chemical oxygen demand), TN (total nitrogen), and TP (total phosphorus) removal efficiency were 85.82 ± 5.37%, 83.31 ± 4.72%, and 84.26 ± 5.58%, respectively at red: blue = 5:5 under the co-cultivation of S. obliquus and fungi. In terms of biogas upgrading, CH 4 contents were higher than 90% (v/v) for all strains, except the co-cultivation with S. obliquus and fungi at red: blue = 3:7. The results indicated that co-cultivation of microalgae with fungi under mixed light wavelengths treatments was most successful in nutrient removal from wastewater and biogas upgrading.

Rice straw is supposed to be an environment-friendly biomaterial for inhibiting the growth of harmful blooms of the cyanobacterium Microcystis aeruginosa . The effects of rice straw extract(RSE) on algal growth, morphologic parameters(cell size), and physiological parameters( in vivo Chl-a fluorescence) were investigated using flow cytometry. We examined the selective inhibitory potential of rice straw on four cyanobacterial strains(toxic and non-toxic Microcystis aeruginosa , toxic Anabaena flos-aquae , and Microcystis ichthyoblabe ), in comparison with inhibitory effects on three common freshwater green algae( Selenastrum capricornutum, Chlorella pyrenoidosa , and Scenedesmus obliqnus ). Concentrations from 2.0 to 10.0 g·L -1 of RSE were found to efficiently inhibit the growth of cyanobacteria in a dose-dependent manner, simultaneously modifying the in vivo Chl-a fluorescence and cell size. The 50% growth-inhibition concentration(7 d) of A. flos-aquae, M. ichthyoblabe, M. aeruginosa (toxic strain), M. aeruginosa (non-toxic strain) was 1.72, 2.21, 2.92 and 5.72 g·L -1 , respectively. Interestingly, the growth and cell size of C. pyrenoidosa and S. obliqnus increased with the addition of RSE and colony formation was observed. In the case of S. capricornutum , the inhibitory effect of RSE on growth and in vivo Chl- a fluorescence occurred at 1.0-4.0 g·L -1 , while RSE induced a stimulatory effect on algal growth at 8.0-10.0 g·L -1 . Taken together, the sensitivity of cyanobacteria to RSE was significantly higher than that of S. capricornutum, C. pyrenoidosa and S. obliqnus . The higher sensitivity of PSⅡ reaction center of cyanobacteria and the ability to form colonies of green algae may have important implications for the species-specific allelopathic antialgal activity of rice straw.

The present study provides data on the insertion of an extra copy of phytochelatin synthase (alr0975) in Anabaena sp. PCC 7120. The recombinant strain (AnFPN-pcs) compared to wild type showed approximately 22.3% increase in growth rate under UV-B, NaCl, heat, CuCl 2 , carbofuran, and CdCl 2 . It also registered 2.25-fold enhanced nitrogenase activity and 5-fold higher phytochelatin production. A comparison of the protein profile of wild type with the recombinant strain revealed that recombinant strain accumulated proteins belonging to the following categories: (i) detoxification (nutrient stress induced DNA binding protein, Mn-SOD, Alr0946 (CalA)), (ii) protein folding and modification (molecular chaperone DnaK, FKBP-type peptidyl-prolyl cis-trans isomerase), (iii) nucleotide and amino acid biosynthesis (dihydroorotase and Ketol-acid reductoisomerase), (iv) photosynthesis and respiration (coproporphyrinogen III oxidase, phycocyanin alpha chain, ferredoxin-NADP + reductase), and (v) transport (sugar transport ATP-binding protein). Thus, it can be concluded that, above category proteins with their respective role in scavenging reactive oxygen species, proper folding of unfolded proteins, and protection of protein from degradation, sustained carbon fixation and energy pool and active transport of sugar together conceivably help the recombinant cyanobacterium (AnFPN-pcs) to cope with abiotic stress employed in the present study. Such recombinant strains have potential for future use as biofertilizer.

Abandoned waste piles from ancient mining activities are potential hot spots for the pollution of the surrounding areas. A pot experiment was carried out to check the potential toxicity of the dumping material present in one of these scenarios, and several amendments were tested to attenuate the spread of the contamination events. The waste material had an acid pH and a large total concentration of As and Cu. A dose-response experiment was performed with this material following OCDE 208 test. A proportion 90:10 uncontaminated soil: dumping material (% w/w) was selected for the following experiment, in order to surpass the amount of dumping material that caused 50% reduction in plant growth. Pots were filled with the 90:10 mixture, planted with seeds of Brassica napus and amended with the following materials: three iron oxides of Bayoxide® E33 series, iron (II) sulphate in combination with de-inking paper sludge (Fe+PS), iron oxide-rich rolling mill scale (ROL) and iron oxide-rich cement waste (CEM). Amendment effectiveness evaluation was based on chemical and biological assays: extractable trace element concentration, soil enzymatic activities, inhibition of light emission of V. fischeri and Anabaena sp., B. napus L. fresh weight and screening test for emergence of B. napus L. seedlings. Amendments E33HCF and Fe+PS were the most effective in reducing extractable As and Zn concentration. B. napus weight and dehydrogenase and β-glucosidase activities were positively increased with the two above mentioned treatments but they triggered more toxic effects for V. fischeri luminescence. E33P treatment was the only in which the EC 50 was higher than in the control. Anabaena sp. was less sensitive than V. fischeri as its luminescence was not hampered by any treatment. Trace element concentration did not significantly affect the failure in seed emergence. E33HCF and Fe+PS could act as proper amendments as they decreased extractable As and Zn. Further, plant fresh weight, enzymatic activities and some of the bioassays identified the latter treatments as the best ones among those tested here to this type of multi-contaminated soil., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Anabaena PCC7120 has two annotated toxin-antitoxin systems: MazEF and HicAB. Overexpression of either of the toxins severely inhibited the growth of Escherichia coli BL21(plysS)(DE3). Of the two Anabaena toxins, MazF exhibited higher toxicity than HicA as evidenced by (i) 100-fold lower viability upon overexpression of MazF compared to HicA; (ii) complete loss of cell viability within 1 h of induction of MazF expression, as against >10 3 colony forming units mL -1 in case of HicA; (iii) inability to maintain the MazF overexpressing plasmid in E. coli cells; and (iv) neutralisation of the toxin was effective at the molar ratio of 1:1.9 for MazF:MazE and 13:1 for HicA:HicB, indicating higher antitoxin requirement for neutralisation of MazF. The growth inhibitory effect of MazF was found to be higher in lag phase cultures compared to mid-logarithmic phase cultures of E. coli, while the reverse was true for HicA. The results suggest possible distinct roles for MazEF and HicAB systems of Anabaena., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na(+) content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K(+) content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K(+)/Na(+) ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K(+)/Na(+). Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF-MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na(+) content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na(+) ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the halotolerant cyanobacterium Anabaena sp is grown under low and high salinity levels.

Phytoplankton (eutrophication, biogeochemical) models are important tools for ecosystem research and management, but they generally have not been updated to include modern biology. Here, we present a dynamic, mechanistic, molecular-level (i.e. gene, transcript, protein, metabolite) model of Anabaena - nitrogen interaction. The model was developed using the pattern-oriented approach to model definition and parameterization of complex agent-based models. It simulates individual filaments, each with individual cells, each with genes that are expressed to yield transcripts and proteins. Cells metabolize various forms of N, grow and divide, and differentiate heterocysts when fixed N is depleted. The model is informed by observations from 269 laboratory experiments from 55 papers published from 1942 to 2014. Within this database, we identified 331 emerging patterns, and, excluding inconsistencies in observations, the model reproduces 94% of them. To explore a practical application, we used the model to simulate nutrient reduction scenarios for a hypothetical lake. For a 50% N only loading reduction, the model predicts that N fixation increases, but this fixed N does not compensate for the loading reduction, and the chlorophyll a concentration decreases substantially (by 33%). When N is reduced along with P, the model predicts an additional 8% reduction (compared to P only)., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The use of Rhizobium inoculants in chickpea is well established; however, meagre efforts have been directed towards the use of other microbial supplements for improving nutrient uptake and yields. A set of novel cyanobacterial and biofilmed inoculants were evaluated in chickpea under field conditions. A significant two-fold enhancement in leghaemoglobin content of nodules and plant biomass was recorded with Anabaena laxa treatment. The inoculants - Anabaena laxa and Anabaena - Rhizobium biofilmed formulation proved to be the top-ranking treatments. Soil chlorophyll, nitrogen-fixation and available N possessed high positive direct effects on grain yield through positive - correlations and - high direct effects and also had high positive indirect effects through other component traits. The cumulative effect of improved plant growth and nutrient uptake exhibited a positive correlation with microbiological activity, especially nitrogen fixation, soil chlorophyll and soil available nitrogen. This may account for the significantly higher yield parameters in the A. laxa treatment, which recorded 50% higher grain yield (1724kgha(-1)) as compared to control (847kgha(-1))., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Surviving of crews during future missions to Mars will depend on reliable and adequate supplies of essential life support materials, i.e. oxygen, food, clean water, and fuel. The most economical and sustainable (and in long term, the only viable) way to provide these supplies on Martian bases is via bio-regenerative systems, by using local resources to drive oxygenic photosynthesis. Selected cyanobacteria, grown in adequately protective containment could serve as pioneer species to produce life sustaining substrates for higher organisms. The very high (95.3 %) CO2 content in Martian atmosphere would provide an abundant carbon source for photo-assimilation, but nitrogen would be a strongly limiting substrate for bio-assimilation in this environment, and would need to be supplemented by nitrogen fertilizing. The very high supply of carbon, with rate-limiting supply of nitrogen strongly affects the growth and the metabolic pathways of the photosynthetic organisms. Here we show that modified, Martian-like atmospheric composition (nearly 100 % CO2) under various low pressure conditions (starting from 50 mbar to maintain liquid water, up to 200 mbars) supports strong cellular growth. Under high CO2 / low N2 ratio the filamentous cyanobacteria produce significant amount of H2 during light due to differentiation of high amount of heterocysts.

In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

Bioflocculant exopolysaccharide (EPS) production by 40 cyanobacterial strains during their photoautotrophic growth was investigated. Highest levels of EPS were produced by Nostoc sp. BTA97 and Anabaena sp. BTA990. EPS production was maximum during stationary growth phase, when nitrogenase activity was very low. Maximum EPS production occurred at pH 8.0 in the absence of any combined nitrogen source. The cyanobacterial EPS consisted of soluble protein and polysaccharide that included substantial amounts of neutral sugars and uronic acid. The EPS isolated from Anabaena sp. BTA990 and Nostoc sp. BTA97 demonstrated high flocculation capacity. There was a positive correlation between uronic acid content and flocculation activity. The flocculant bound a cationic dye, Alcian Blue, indicating it to be polyanionic. The 16S rRNA gene sequences for Nostoc sp. BTA97 and Anabaena sp. BTA990 were deposited at NCBI GenBank, and accession numbers were obtained as KJ830951 and KJ830948, respectively. The results of these experiments indicate that strains Anabaena sp. BTA990 and Nostoc sp. BTA97 are good candidates for the commercial production of EPS and might be utilized in industrial applications as an alternative to synthetic and abiotic flocculants.

In the forthcoming decades, it is widely believed that the dominance of colonial and filamentous bloom-forming cyanobacteria (e.g. Microcystis, Planktothrix, Anabaena and Cylindrospermopsis) will increase in freshwater systems as a combined result of anthropogenic nutrient input into freshwater bodies and climate change. While the physicochemical parameters controlling bloom dynamics are well known, the role of biotic factors remains comparatively poorly studied. Morphology and toxicity often - but not always - limit the availability of cyanobacteria to filter feeding zooplankton (e.g. cladocerans). Filamentous and colonial cyanobacteria are widely regarded as trophic dead-ends mostly inedible for zooplankton, but substantial evidence shows that some grazers (e.g. copepods) can bypass this size constraint by breaking down filaments, making the bloom biomass available to other zooplankton species. A wide range of algicidal bacteria (mostly from the Alcaligenes, Flavobacterium/Cytophaga group and Pseudomonas) and viruses (Podoviridae, Siphoviridae and Myoviridae) may also contribute to bloom control, via their lytic activity underpinned by a diverse array of mechanisms. Fungal parasitism by the Chytridiomycota remains the least studied. While each of these biotic factors has traditionally been studied in isolation, emerging research consistently point to complex interwoven interactions between biotic and environmental factors., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)