Morphology,phylogeny, growth rate and nodularin production of Nodularia spumigena from Brazil

Blooms of the toxic cyanobacterium Nodularia spumigena occur in various locations worldwide, but have not been observed in Brazil until recently. Three Nodularia strains were isolated from summer blooms in experimental shrimp production ponds of Penaeus vannamei in Rio Grande, in southern Brazil; these strains were characterized by morphology, phylogeny, growth rate and toxicity. The strains were identified as N. spumigena based on the size of vegetative cells, heterocytes and akinetes under a light microscope and based on the number of gas vesicles per μm² under a transmission electron microscope. The 16S rRNA gene sequences of the three strains showed high identity (> 99%) with N. spumigena sequences available on the NCBI database but were grouped closer in the phylogenetic tree with N. spumigena strains from Australia and USA than those from the Baltic Sea. The growth rate in batch culture varied between 0.2 and 0.6 μ?d^?1 based on cell density, optical density and chlorophyll-a content. The three strains produced the hepatotoxin nodularin (ELISA plate kit) with similar toxicity values (4.8–4.9?µg?l^?1). We conclude that the three isolated strains are N. spumigena with similar rates of growth and nodularin production. The presence of N. spumigena now represents a potential problem in aquaculture and estuarine environments in Brazil.     

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.     

The cyanobacterium Synechocystis sp. PCC 6803 is able to express an active [FeFe]-hydrogenase without additional maturation proteins

[FeFe]-hydrogenases have been claimed as the most promising catalysts of hydrogen bioproduction and several efforts have been accomplished to express and purify them. However, previous attemps to obtain a functional recombinant [FeFe]-hydrogenase in heterologous systems such as Escherichia coli failed due to the lack of the specific maturation proteins driving the assembly of its complex active site. The unique exception is that of [FeFe]-hydrogenase from Clostridium pasteurianum that has been expressed in active form in the cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase with a well characterized maturation system, suggesting that the latter is flexible enough to drive the synthesis of a [FeFe]-enzyme. However, the capability of cyanobacteria to correctly fold a [FeFe]-hydrogenase in the absence of its auxiliary maturation proteins is a debated question. In this work, we expressed the [FeFe]-hydrogenase from Chlamydomonas reinhardtii as an active enzyme in the cyanobacterium Synechocystis sp. PCC 6803. Our results, using a different experimental system, confirm that cyanobacteria are able to express a functional [FeFe]-hydrogenase even in the absence of additional chaperones.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

CGFS-type monothiol glutaredoxins from the cyanobacterium Synechocystis PCC6803 and other evolutionary distant model organisms possess a glutathione-ligated [2Fe-2S] cluster

When produced in Escherichia coli, the CGFS-type monothiol Grxs from this organism (EcGrx4p) and the model cyanobacterium Synechocystis (SyGrx3p) exist as a dimeric iron-sulfur containing holoprotein or as a monomeric apoprotein in solution. Spectroscopic and site-directed mutagenesis analyses show that the SyGrx3 holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteine located in the CGFS motif of each monomer and the cysteines of two molecules of glutathione. The biochemical characterization of several monothiol Grxs from the cyanobacteria Gloeobacter violaceus (GvGrx3p) and Thermosynechococcus elongatus (TeGrx3p), the yeast Saccharomyces cerevisiae (ScGrx3p, ScGrx4p, and ScGrx5p), the plant Arabidopsis thaliana (AtGrx5p), and human (HsGrx5p) indicate that the incorporation of a GSH-ligated [2Fe-2S] center is a common feature of prokaryotic and eukaryotic CGFS-active site monothiol Grxs. In light of these results, the involvement of these enzymes in the sensing of iron and/or the biogenesis and transfer of Fe-S cluster is discussed.     

Refined X-ray structures of the oxidized, at 1.3 A, and reduced, at 1.17 A, [2Fe-2S] ferredoxin from the cyanobacterium Anabaena PCC7119 show redox-linked conformational changes

The chemical sequence of the [2Fe-2S] ferredoxin from the cyanobacterium AnabaenaPCC7119 (Fd7119) and its high-resolution X-ray structures in the oxidized and reduced states have been determined. The Fd7119 sequence is identical to that of the ferredoxin from the PCC7120 strain (Fd7120). X-ray diffraction data were collected at 100 K with an oxidized trigonal Fd7119 crystal, at 1.3 A resolution, and with an orthorhombic crystal, previously reduced with dithionite and flash frozen under anaerobic conditions, at 1.17 A resolution. The two molecular models were determined by molecular replacement with the [2Fe-2S] ferredoxin from the strain PCC7120 (Rypniewski, W. R., Breiter, D. R., Benning, M. M., Wesenberg, G., Oh, B.-H., Markley, J. L., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 4126-4131.) The final R-factors are 0. 140 (for the reduced crystal) and 0.138 (for the oxidized crystal). The [2Fe-2S] cluster appears as a significantly distorted lozenge in the reduced and oxidized redox states. The major conformational difference between the two redox forms concerns the peptide bond linking Cys46 and Ser47 which points its carbonyl oxygen away from the [2Fe-2S] cluster ("CO out") in the reduced molecule and toward it ("CO in") in the oxidized one. The "CO out" conformation could be the signature of the reduction of the iron atom Fe1, which is close to the molecular surface. Superposition of the three crystallographically independent molecules shows that the putative recognition site with the physiological partner (FNR) involves charged, hydrophobic residues and invariant water molecules.     

Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES-2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes

The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.     

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803

The two operons atp1 and atp2, encoding the subunits of the F_OF₁ ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F_OF₁ ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5 end of atp1, coding for a putative gene product similar to uncI in Escherichia coli.A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.     

Complete nucleotide sequence of the freshwater unicellular cyanobacterium Synechococcus elongatus PCC 6301 chromosome: gene content and organization

The entire genome of the unicellular cyanobacterium Synechococcus elongatus PCC 6301 (formerly Anacystis nidulans Berkeley strain 6301) was sequenced. The genome consisted of a circular chromosome 2,696,255 bp long. A total of 2,525 potential protein-coding genes, two sets of rRNA genes, 45 tRNA genes representing 42 tRNA species, and several genes for small stable RNAs were assigned to the chromosome by similarity searches and computer predictions. The translated products of 56% of the potential protein-coding genes showed sequence similarities to experimentally identified and predicted proteins of known function, and the products of 35% of the genes showed sequence similarities to the translated products of hypothetical genes. The remaining 9% of genes lacked significant similarities to genes for predicted proteins in the public DNA databases. Some 139 genes coding for photosynthesis-related components were identified. Thirty-seven genes for two-component signal transduction systems were also identified. This is the smallest number of such genes identified in cyanobacteria, except for marine cyanobacteria, suggesting that only simple signal transduction systems are found in this strain. The gene arrangement and nucleotide sequence of Synechococcus elongatus PCC 6301 were nearly identical to those of a closely related strain Synechococcus elongatus PCC 7942, except for the presence of a 188.6 kb inversion. The sequences as well as the gene information shown in this paper are available in the Web database, CYORF (http://www.cyano.genome.jp/).     

Reaction mechanism of phosphoribulokinase from a cyanobacterium,Synechococcus PCC7942

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg²⁺ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The K_m values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. K_i values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants K_i1 and K_i2 for 6-phosphogluconate were 9.3 and 0.49 mM. K_ia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min^–1 mg^–1 chl.     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena . We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.     

A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803

A vector-host system for testing promoters in the cyanobacterium Synechocystis PCC6803 has been constructed. It relies on a small Escherichia coli promoter-probe plasmid, pFF11, which has four unique restriction sites in a polylinker upstream from the cat reporter gene. This plasmid is able to obtain a cyanobacterial origin of replication by homologous recombination with the resident plasmid of the recipient host, generating a new E. coli-Synechocystis PCC6803 shuttle vector. This plasmid does not confer any detectable chloramphenicol acetyl transferase activity to this cyanobacterium in the absence of a promoter insert. Several heterologous promoters were tested in Synechocystis PCC6803 using this system. Results obtained with the lambda pR promoter and the repressor-encoding cI857 gene demonstrate that these elements can be used for high-level and tightly regulated gene expression in Synechocystis PCC6803.     

Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110.

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

The pleiotropic effects of ftn2 and ftn6 mutations in cyanobacterium Synechococcus sp. PCC 7942

Two cell division mutants (Ftn2 and Ftn6) of the cyanobacterium Synechococcus sp. PCC 7942 were studied using scanning electron microscopy and transmission electron microscopy methods. This included negative staining and ultrathin section analysis. Different morphological and ultrastructural features of mutant cells were identified. Ftn2 and Ftn6 mutants exhibited particularly elongated cells characterized by significantly changed shape in comparison with the wild type. There was irregular bending, curving, spiralization, and bulges as well as cell branching. Elongated mutant cells were able to initiate cytokinesis simultaneously in several division sites which were localized irregularly along the cell. Damaged rigidity of the cell wall was typical of many cells for both mutants. Thylakoids of mutants showed modified arrangement and ultrastructural organization. Carboxysome-like structures without a shell and/or without accurate polyhedral packing protein particles were often detected in the mutants. However, in the case of Ftn2 and Ftn6, the average number of carboxysomes per section was less than in the wild type by a factor of 4 and 2, respectively. These multiple morphological and ultrastructural changes in mutant cells evinced pleiotropic responses which were induced by mutations in cell division genes ftn2 and ftn6. Ultrastructural abnormalities of Ftn2 and Ftn6 mutants were consistent with differences in their proteomes. These results could support the significance of FTN2 and FTN6 proteins for both cyanobacterial cell division and cellular physiology.     

Fermentation metabolism of the unicellular cyanobacterium Cyanothece PCC 7822

The hydrogenase-catalyzed hydrogen production exhibited by the unicellular cyanobacterium Cyanothece 7822 during anoxic incubation in the dark is a result of the fermentative degradation of carbon reserves. Simultaneously with hydrogen production, evolution of carbon dioxide was detected, and excretion of ethanol, lactate, formate and acetate was demonstrated. The fermentation balance indicates that carbohydrates are fermented via a branched pathway, in which both the pentose phosphate pathway and glycolysis appear to be involved. It is proposed that the physiological function of hydrogen production is the introduction of protons as terminal electron acceptors. This removal of reducing equivalents might give rise to continuation of the pyruvate decarboxylation and consequently of the acetate formation, thereby increasing the efficiency of fermentative energy generation.