Filamentous Nitrogen-fixing Cyanobacterium Research Articles

Filamentous nitrogen-fixing cyanobacteria: contributing to filling nitrogen and water gaps in a context of climate change

Filamentous nitrogen-fixing cyanobacteria: contributing to filling nitrogen and water gaps in a context of climate change

Tipping points of nitrogen use efficiency in freshwater phytoplankton along trophic state gradient

Eutrophication and harmful algal blooms have severe effects on water quality and biodiversity in lakes and reservoirs. Ecological regime shifts of phytoplankton blooms are generally thought to be driven by the rapidly rising nutrient use efficiency of bloom-forming species over short periods, and often exhibit nonlinear dynamics. Regime shifts of trophic state, eutrophication, stratification, and clear or turbid waters are well-studied topics in aquatic ecology. However, information on the prevalence of regime shifts in relationships between trophic states and phytoplankton resource transfer efficiencies in ecosystems is still lacking. Here, we provided a first insight into regime shifts in nitrogen use efficiency of phytoplankton along the trophic state gradient. We explored the regime shifts of phytoplankton resource use efficiency and detected the tipping points by combining four temporal or spatial datasets from tropical to temperate zones in Asia and Europe. We first observed significant abrupt transitions (abruptness > 1) in phytoplankton nitrogen use efficiency along the trophic state gradient. The tipping point values were lower in subtropical/tropical waterbodies (mesotrophic states; TSIc: around 50) than those in temperate zones (eutrophic states; TSIc: 60–70). The regime shifts significantly reduced the primary production transfer efficiency via zooplankton (from 0.15 ± 0.03 to 0.03 ± 0.01; mean ± standard error) in the aquatic food web. Nitrogen-fixing filamentous cyanobacteria can drive eutrophication under mesotrophic state. Our findings imply that the time-window of opportunity for harmful algae prevention and control in lakes and reservoirs is earlier in subtropical/tropical regions.

Identification and toxicity towards aquatic primary producers of the smallest fractions released from hydrolytic degradation of polycaprolactone microplastics.

Bioplastics are thought as a safe substitute of non-biodegradable polymers. However, once released in the environment, biodegradation may be very slow, and they also suffer abiotic fragmentation processes, which may give rise to different fractions of polymer sizes. We present novel data on abiotic hydrolytic degradation of polycaprolactone (PCL), tracking the presence of by-products during 132 days by combining different physicochemical techniques. During the study a considerable amount of two small size plastic fractions were found (up to∼6mg of PCL by-product/g of PCL beads after 132 days of degradation); and classified as submicron-plastics (sMPs) from 1μm to 100nm and nanoplastics (NPs, <100nm) as well as oligomers. The potential toxicity of the smallest fractions, PCL by-products < 100nm (PCL-NPs+PCL oligomers) and the PCL oligomers single fraction, was tested on two ecologically relevant aquatic primary producers: the heterocystous filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and the unicellular cyanobacterium Synechococcus sp. PCC 7942. Upon exposure to both, single and combined fractions, Reactive Oxygen Species (ROS) overproduction, intracellular pH and metabolic activity alterations were observed in both organisms, whilst membrane potential and morphological damages were only observed upon PCL-NPs+PCL oligomers exposure. Notably both PCL by-products fractions inhibited nitrogen fixation in Anabaena, which may be clearly detrimental for the aquatic trophic chain. As conclusion, fragmentation of bioplastics may render a continuous production of secondary nanoplastics as well as oligomers that might be toxic to the surrounding biota; both PCL-NPs and PCL oligomers, but largely the nanoparticulate fraction, were harmful for the two aquatic primary producers. Efforts should be made to thoroughly understand the fragmentation of bioplastics and the toxicity of the smallest fractions resulting from that degradation.

Draft genome sequence of the cyanobacterium Sphaerospermopsis aphanizomenoides BCCUSP55 from the Brazilian semiarid region reveals potential for anti-cancer applications.

Sphaerospermopsis aphanizomenoides is a filamentous nitrogen-fixing and bloom-forming cyanobacterium, which biomass can fertilize natural water with nutrients, especially through nitrogen fixation. The Sphaerospermopsis aphanizomenoides strain BCCUSP55 was previously isolated from a water supply reservoir in the Brazilian semiarid region, and its draft genome assembly coupled with the gene contents are reported here. The obtained BCCUSP55 draft genome comprised 254 scaffolds with a genome size estimated of 6,096,273bp. In addition, it has 5250 predicted coding sequences (CDS) and the G + C content is 38.5%. Further, the BCCUSP55 draft genome presented the putative nocuolin A gene complete cluster, a natural oxadiazine that triggers apoptosis in human cancer cells. Thus, our results contribute to extend the knowledge on the genus Sphaerospermopsis and reveal its biotechnological potential.

Genomic and transcriptomic insights into the habitat adaptation of the diazotrophic paddy-field cyanobacterium Nostoc sphaeroides.

Nitrogen-fixing cyanobacteria are common in paddy fields, one of the most productive wetland ecosystems. Here, we present the complete genome of Nostoc sphaeroides, a paddy-field diazotroph used for food and medicine for more than 1700 years and deciphered the transcriptional regulation during the developmental transition from hormogonia to vegetative filaments with heterocysts. The genome of N. sphaeroides consists of one circular chromosome (6.48 Mb), one of the largest ever reported megaplasmids (2.34 Mb), and seven plasmids. Multiple gene families involved in the adaption to high solar radiation and water fluctuation conditions were found expanded, while genes involved in anoxic adaptation and phosphonate utilization are located on the megaplasmid, suggesting its indispensable role in environmental adaptation. Distinct gene expression patterns were observed during the light-intensity-regulated transition from hormogonia to vegetative filaments, specifically, genes encoding proteins involved in photosynthetic light reaction, carbon fixation, nitrogen metabolism and heterocyst differentiation were significantly upregulated, whereas genes related to cell motility were down-regulated. Our results provide genomic and transcriptomic insights into the adaptation of a filamentous nitrogen-fixing cyanobacterium to the highly dynamic paddy-field habitat, suggesting N. sphaeroides as an excellent system to understand the transition from aquatic to terrestrial habitats and to support sustainable rice production.

Biogenic synthesis of silver-nanoparticles with the brackish water cyanobacterium Nostoc sphaeroides and assessment of antibacterial activity against urinary tract infecting bacteria

The aim of this study was to isolate a filamentous nitrogen-fixing cyanobacterium Nostoc sphaeroides (NS) from brackish water of Chilika Lake, Odisha coast, and exploration of its use in the biosynthesis of silver-nanoparticles (AgNPs) against the urinary tract–infecting bacterial strains. The synthesized NS-AgNPs were structurally characterized by analytical techniques such as UV-visible-spectrophotometry, FTIR, XRD, SEM and SEM-EDX. The antibacterial efficacy of synthesized AgNPs had remarkable in vitro activities with the MIC value of 50 mg/ml. Moreover, Nostocine A, the inherent bioactive compound of Nostoc sp. was further investigated for molecular docking studies against the targeted bacterial DNA gyrase (Escherichia coli) and the obtained docking score was −7.9 kCal/mol as compared with the standard drug, Ciprofloxacin of a docking score −7.8 kCal/mol. The concordance between molecular docking and experimental results is excellent. Thus, the biogenic-synthesized “N. sphaeroides-AgNPs” would be used as an antibacterial agent in future.

Genome-Wide Assessment of Putative Superoxide Dismutases in Unicellular and Filamentous Cyanobacteria

Abstract Cyanobacteria are photoautotrophic prokaryotes capable to grow in diverse ecological habitats, originated 2.5-3.5 billion years ago and were first to produce oxygen. Since then superoxide dismutases (SOD) acquired great significance due to their ability to catalyze detoxification of byproducts of oxygenic photosynthesis i.e. superoxide radicals. In the present study, we extracted information regarding SODs from species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. 144 putative SOD homologs were identified. Unlike other protein families (ex. serine-threonine kinases) SODs are present in all cyanobacterial species reflecting their significant role in survival. However, their distribution varies fewer (0.01%-0.09%) found in unicellular marine strains whereas abundant (0.02%-0.07%) in filamentous nitrogen-fixing cyanobacteria. They were classified into three major subfamilies according to their domain structures: Fe/MnSOD, Cu/ZnSOD and NiSOD. Interestingly, they lack additional domains as found in proteins of other families however motifs and invariant amino acids typical in eukaryotic SODs were conserved well in these proteins indicating similar catalytic mechanism as eukaryotic SODs. Phylogenetic relationships correspond well with phylogenies based on 16S rRNA and clustering occurs on the basis of structural characteristics such as domain organization. Gene gain-and-loss is insignificant during SOD evolution as evidenced by the absence of additional domain. This study has not only examined an overall background of sequence-structure-function interactions for the SOD gene family but also revealed variation among SOD distribution based on ecophysiological and morphological characters.

Effect of crude extracts from Nodularia spumigena on round goby (Neogobius melanostomus)

Nodularia spumigena is a nitrogen-fixing filamentous cyanobacteria in the Baltic Sea. Nodularin (NOD), the hepatotoxic peptide produced by this cyanobacterium, accumulates in the organisms from different trophic levels. In this paper, the effects of N. spumigena cell extract on the round goby (Neogobius melanostomus) was investigated under laboratory conditions. This benthic fish species feed on mussels in which nodularin accumulation was well documented.In current study a sharp increase in the NOD concentration in analyzed organs was observed after 24 h (PPIA) after 72 h of exposure (LC/MS). To determine the direction and strength of the changes induced in the fish by the toxin, several biochemical markers of exposure such as concentration of glutathione and activities of catalase, guaiacol peroxidase and glutathione S-transferase were used. In analyzed organs (liver, gills and muscle) of the round goby, the activity of these enzymes were suppressed. Higher GSH/protein amount and CAT and POD activity in gills than in liver reflects the importance of gills in NOD entering into analyzed fish body when exposed to toxin. The results indicate that the round goby (Neogobius melanostomus) exposed to extracts from N. spumigena cells triggered a defense system in a time-dependent manner. The obtained results contribute to a better understanding of fish response to the presence of compounds produced by N. spumigena.

Newly isolated Nodularia phage influences cyanobacterial community dynamics.

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community-level impact due to the release of nutrients by phage-induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS-2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage-induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%-27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven-fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS-2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.

Functional Significance of NADPH-Thioredoxin Reductase C in the Antioxidant Defense System of Cyanobacterium Anabaena sp. PCC 7120.

The redox regulation system is widely accepted as a crucial mechanism for controlling the activities of various metabolic enzymes. In addition to thioredoxin reductase/thioredoxin cascades, NADPH-thioredoxin reductase C (NTRC), a hybrid protein formed by an NADPH-thioredoxin reductase domain and a thioredoxin (Trx) domain, is present in chloroplasts and in most cyanobacteria species. Although several target proteins and physiological functions of NTRC in chloroplasts have been characterized, little is known about NTRC functions in cyanobacteria. Therefore, we investigated the molecular basis and physiological significance of NTRC-dependent redox regulation in the filamentous heterocyst-forming nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120). Initially, we identified six candidate NTRC targets in Anabaena 7120 using NTRC affinity chromatography. Subsequently, we compared the efficiency of reducing-equivalent transfer from NTRC and Trx-m1 to the NTRC target protein 2-Cys peroxiredoxin. Biochemical analyses revealed that compared with Trx-m1, NTRC more efficiently transfers reducing equivalents to 2-Cys peroxiredoxin. Subsequently, we constructed and analyzed an ntrC knockout strain in Anabaena 7120. The mutant showed impaired growth under oxidative stress conditions and lower concentrations of reduced 2-Cys peroxiredoxin in cells. Taken together, the present in vitro and in vivo results indicate that NTRC is a significant electron donor for 2-Cys peroxiredoxin and plays a pivotal role in antioxidant defense systems in Anabaena 7120 cells.

A Comprehensively Curated Genome-Scale Two-Cell Model for the Heterocystous Cyanobacterium Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a nitrogen-fixing filamentous cyanobacterium. Under nitrogen-limiting conditions, a fraction of the vegetative cells in each filament terminally differentiate to nongrowing heterocysts. Heterocysts are metabolically and structurally specialized to enable O2-sensitive nitrogen fixation. The functionality of the filament, as an association of vegetative cells and heterocysts, is postulated to depend on metabolic exchange of electrons, carbon, and fixed nitrogen. In this study, we compile and evaluate a comprehensive curated stoichiometric model of this two-cell system, with the objective function based on the growth of the filament under diazotrophic conditions. The predicted growth rate under nitrogen-replete and -deplete conditions, as well as the effect of external carbon and nitrogen sources, was thereafter verified. Furthermore, the model was utilized to comprehensively evaluate the optimality of putative metabolic exchange reactions between heterocysts and vegetative cells. The model suggested that optimal growth requires at least four exchange metabolites. Several combinations of exchange metabolites resulted in predicted growth rates that are higher than growth rates achieved by only considering exchange of metabolites previously suggested in the literature. The curated model of the metabolic network of Anabaena sp. PCC 7120 enhances our ability to understand the metabolic organization of multicellular cyanobacteria and provides a platform for further study and engineering of their metabolism.

Down-Regulation of the Alternative Sigma Factor SigJ Confers a Photoprotective Phenotype to Anabaena PCC 7120.

Alternative sigma factors belonging to Group 3 are thought to play an important role in the adaptation of cyanobacteria to environmental challenges by altering expression of genes needed for coping with such stresses. In this study, the role of an alternative sigma factor, SigJ, was analyzed in the filamentous nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120 by knocking down the expression of the sigJ gene (alr0277) employing an antisense RNA-mediated approach. In the absence of any stress, the knock-down (KD0277) or the wild-type strain both grew similarly. Upon exposure to high-intensity light, KD0277 showed substantially reduced bleaching of its pigments, higher photosynthetic activity and consequently better survival than the wild type. KD0277 also showed an enhanced accumulation of two carotenoids, which were identified as myxoxanthophyll and keto-myxoxanthophyll. Further, KD0277 was more tolerant to ammonium-triggered photodamage than the wild type. Moreover, PSII was better protected against photodamage in KD0277 than in the wild type. Down-regulation of sigJ in Anabaena PCC 7120, however, reduced its ability to cope with desiccation. This study demonstrates that down-regulation of the sigJ gene in Anabaena PCC 7120 differentially affects its ability to tolerate two environmentally relevant stresses, i.e. high-intensity light and desiccation.

Designing Synthetic Flexible Gene Regulation Networks Using RNA Devices in Cyanobacteria.

In recent years, studies on the development of gene regulation tools in cyanobacteria have been extensively conducted toward efficient production of valuable chemicals. However, there is considerable scope for improving the economic feasibility of production. To improve a recently reported gene induction system using anhydrotetracycline (aTc)-TetR and an endogenous gene repression system using small antisense RNA in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop, in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter. GFP expression in this improved system was higher and longer than the system lacking tetR repression. In addition, by using TetR aptamer and a riboswitch, we succeeded in achieving a superior and longer induction of GFP expression even under high-light conditions. Hence, efficient gene induction systems were established in Anabaena by designing a gene regulation network using RNA-based tools.

A Proposal on the Restoration of Nostoc flagelliforme for Sustainable Improvement in the Ecology of Arid Steppes in China

Nostoc flagelliforme, a filamentous nitrogen-fixing cyanobacterium, is widely distributed in arid steppes of the west and northwestern parts of China. However, as a food delicacy this species has been overexploited from 1970 to 2000. Moreover, overgrazing, land reclamation and the removal of medicinal herbs have caused severely reduced vegetation coverage there. In this communication, a badly damaged but slowly rehabilitating N. flagelliforme-inhibiting steppe is described, and the rehabilitation of desertified steppes by the renewed growth of N. flagelliforme is proposed. The restoration of this dominant nitrogen supplier would be an ecologically sustainable solution for supplementing current measures already taken in the desertified regions. In addition, a goal of 50%–60% vegetation coverage is proposed by the N. flagelliforme restoration.

Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01.

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB, or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4 and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8% reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 and 81.3% after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor.

Missing nitrogen fixation in the Benguela region

Opposing opinions on the importance of nitrogen fixation in the northern Benguela upwelling region provoked us to investigate the magnitude of nitrogen fixation in front of northern Namibia and southern Angola. Measurements of nitrogen fixation rates using the 15N method at 66 stations during seven cruises from 2008 to 2014 showed that, in general, the 15N content in the biomass did not increase after tracer incubation with 15N2, indicating that no nitrogen fixation occurred. Correspondingly, the filamentous nitrogen-fixing cyanobacterium Trichodesmium was almost not present. The abundant picocyanobacteria did obviously not perform nitrogen fixation to a significant degree. The artificial improvement of conditions for nitrogen fixation in mesocosm experiments, including phosphate and iron additions and a warmer temperature, failed to induce nitrogen fixation. A plausible explanation of these findings is a lack of conditioned cells for nitrogen fixation in the Benguela region.

Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

Carbon dioxide and poultry waste utilization for production of polyhydroxyalkanoate biopolymers by Nostoc muscorum Agardh: a sustainable approach

The new paradigm is to view wastes as resources for sustainable development. In this regard, the feasibility of poultry waste and CO2 utilization for cultivation of a filamentous nitrogen-fixing cyanobacterium, Nostoc muscorum Agardh, was investigated for production polyhydroxyalkanoates, the biodegradable polymers. This cyanobacterium showed profound rise in biomass yield with up to 10 % CO2 supply in airstream with an aeration rate of 0.1 vvm. Maximum biomass yield of 1.12 g L−1 was recorded for 8 days incubation period, thus demonstrating a CO2 biofixation rate of 0.263 g L−1 day−1 at 10 % (v/v) CO2-enriched air. Poultry litter (PL) supplementation also had a positive impact on the biomass yield. The nutrient removal efficiency of N. muscorum was reflected in the significant reduction in nutrient load of PL over the experimental period. A maximum poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] copolymer yield of 774 mg L−1 (65 % of dry cell wt.), the value almost 11-fold higher than the control, was recorded in 10 g L−1 PL-supplemented cultures with 10 % CO2 supply under the optimized condition, thus demonstrating that N. muscorum has good potential for CO2 biomitigation and poultry waste remediation while simultaneously producing eco-friendly polymers.

Draft Genome Sequence of Tolypothrix boutellei Strain VB521301.

We report here the draft genome sequence of the filamentous nitrogen-fixing cyanobacterium Tolypothrix boutellei strain VB521301. The organism is lipid rich and hydrophobic and produces polyunsaturated fatty acids which can be harnessed for industrial purpose. The draft genome sequence assembled into 11,572,263 bp with 70 scaffolds and 7,777 protein coding genes.

Visualization of channels connecting cells in filamentous nitrogen‐fixing cyanobacteria

Cyanobacteria, formerly called blue-green algae, are abundant bacteria that carry out green plant photosynthesis, fixing CO2 and generating O2. Many species can also fix N2 when reduced nitrogen sources are scarce. Many studies imply the existence of intracellular communicating channels in filamentous cyanobacteria, in particular, the nitrogen-fixing species. In a species such as Anabaena, growth in nitrogen-depleted medium, in which ∼10% of the cells differentiate into anaerobic factories for nitrogen fixation (heterocysts), requires the transport of amino acids from heterocysts to vegetative cells, and reciprocally, the transport of sugar from vegetative cells to heterocysts. Convincing physical evidence for such channels has been slim. Using improved preservation of structure by high-pressure rapid freezing of samples for electron microscopy, coupled with high-resolution 3D tomography, it has been possible to visualize and measure the dimensions of channels that breach the peptidoglycan between vegetative cells and between heterocysts and vegetative cells. The channels appear to be straight tubes, 21 nm long and 14 nm in diameter for the latter and 12 nm long and 12 nm in diameter for the former.-Omairi-Nasser, A., Haselkorn, R., Austin, J. II. Visualization of channels connecting cells in filamentous nitrogen-fixing cyanobacteria.