Draft genome database construction from four strains (NIES-298, FCY- 26, -27, and -28) of the cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa is a cyanobacterium that can form harmful algal blooms (HABs) producing toxic secondary metabolites. We provide here draft genome information of four strains of this freshwater cyanobacterium that was obtained by the Next Generation Sequencing approach to provide a better understanding of molecular mechanisms at the physiological and ecological levels. After gene assembly, genes of each strain were identified and annotated, and a genome database and G-browser of M. aeruginosa were subsequently constructed. Such genome information resources will enable us to obtain useful information for molecular ecological studies with a better understanding of modulating mechanisms of environmental factors associated with blooming.     

Heat shock and iron limitation modulate the metabolic profile of the cyanobacterium Microcystis aeruginosa NIES-88

The freshwater cyanobacterium Microcystis aeruginosa NIES-88, which can produce microcystins, micropeptins, and argicyclamides, was subjected to a one strain many compounds (OSMAC) analysis. We report its response to two environmental stressors, temperature and iron limitation, by means of untargeted and targeted metabolomics. The results demonstrated a slower specific growth rate of 0.20 per day and 0.16 per day in adverse conditions of 37°C and iron limitation, respectively. The metabolic signature of M. aeruginosa was highly dependent on incubation temperatures. Production of microcystins LR and RR was severely downregulated while that of argicyclamide B was significantly upregulated, with a highest 10-fold increase on day 14 of heat shock treatment. M. aeruginosa NIES-88 was found to produce a new compound, argicyclamide D (1), in iron limited medium, which has the same macrocyclic structure as the previously reported analogs. Hence, it is proposed that acclimation of M. aeruginosa to environmental stressors might be mediated by a change in the metabolic pathways as well as modulation of the levels of their expressed metabolites.     

Hydrogenase from the unicellular cyanobacterium,Microcystis aeruginosa

A hydrogenase was isolated from a unicellular and non-nitrogen-fixing cyanobacterium, Microcystis aeruginosa strain NIES 44. The enzyme was easily solubilized and was capable of evolving hydrogen gas in the presence of reduced methyl viologen and benzyl viologen. The enzyme was stimulated by divalent ions and showed a pH optimum around 6.8. The M_r of the enzyme, estimated by gel filtration, was 50 000.     

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.     

Photoinhibition of photosynthesis in the cyanobacterium Microcystis aeruginosa

We have examined characteristics of the photoinhibition of photosynthesis which occur in the unicellular cyanobacterium Microcystis aeruginosa, following exposure to photon fluence rates in excess of those required for growth. Photoinhibition occurs following exposure of cells to a photon fluence rate of 1,000 μmol m^-2 s^-1, which is manifested as a decrease in either light-limited CO₂ fixation or light-saturated CO₂-dependent O₂ evolution. The extent and rapidity of this photoinhibition is greatly enhanced under CO₂-depleted conditions. Experiments in which cultures were sparged with different gases indicate that photoinhibition is not an obvious consequence of elevated O₂ tensions, unlike the photooxidative bleaching of photosynthetic pigments. Comparative studies on the photoinactivation of CO₂-dependent O₂ evolution and of the methyl viologen-dependent Mehler reaction, in whole cells, indicate that a primary site of light damage is within the photosynthetic electron-transport reactions and that carbon fixation is initially unaffected.     

Complete Genomic Structure of the Bloom-forming Toxic Cyanobacterium Microcystis aeruginosa NIES-843

The nucleotide sequence of the complete genome of a cyanobacterium,Microcystis aeruginosa NIES-843, was determined. The genomeof M. aeruginosa is a single, circular chromosome of 5 842 795base pairs (bp) in length, with an average GC content of 42.3%.The chromosome comprises 6312 putative protein-encoding genes,two sets of rRNA genes, 42 tRNA genes representing 41 tRNA species,and genes for tmRNA, the B subunit of RNase P, SRP RNA, and6Sa RNA. Forty-five percent of the putative protein-encodingsequences showed sequence similarity to genes of known function,32% were similar to hypothetical genes, and the remaining 23%had no apparent similarity to reported genes. A total of 688kb of the genome, equivalent to 11.8% of the entire genome,were composed of both insertion sequences and miniature inverted-repeattransposable elements. This is indicative of a plasticity ofthe M. aeruginosa genome, through a mechanism that involveshomologous recombination mediated by repetitive DNA elements.In addition to known gene clusters related to the synthesisof microcystin and cyanopeptolin, novel gene clusters that maybe involved in the synthesis and modification of toxic smallpolypeptides were identified. Compared with other cyanobacteria,a relatively small number of genes for two component systemsand a large number of genes for restriction-modification systemswere notable characteristics of the M. aeruginosa genome.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Simulation of water-bloom formation in the cyanobacterium Microcystis aeruginosa

A mechanism for buoyancy increases in the cyanobacterium Microcystisaeruginosa and the associated formation of surface water-bloomsis presented. The mechanism is based on considering a responsetime in the rate of carbohydrate accumulation. When irradianceincreases, the Microcystis cells may require time to increasetheir rate of carbohydrate accumulation. If irradiance decreasesbefore adjustment, the maximum rate of carbohydrate accumulationis not reached. Colony buoyancy increases during mixing whenthe time scales of the light fluctuations are shorter than theresponse time. To examine the mechanism, a model of Microcystisbuoyancy that incorporates the response time has been coupledwith a hydrodynamics model that simulates mixing. The modelwas applied to a shallow lake to show that a prolonged episodeof intense mixing caused the simulated Microcystis coloniesto become excessively buoyant. Once the mixing subsided, thecolonies accumulated at the surface. Decreases in carbohydratewere reduced in large colonies as their size afforded buoyancyforces that could readily overcome the entraining forces ofthe mixing.     

Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

Toxicity and extrachromosomal DNA in strains of the cyanobacterium Microcystis aeruginosa

Abstract Correlations were sought between toxicity and the presence of plasmids in toxic and non-toxic strains of Microcystis aeruginosa . Plasmids were present in toxic and non-toxic cultures. Cultivation of the toxic Microcystis PCC7820 in the presence of novobiocin did not influence toxicity, although extrachromosomal DNA was no longer detected after novobiocin treatment. The data indicate that it is unlikely that plasmids are involved in the toxicity of Microcystis PCC7820.     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO₂ in the dark reactions of photosynthesis. The importance of these findings is discussed.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - PSI photosystem I - PSH photosystem II     

Intraspecific variation in growth and morphology of the bloom-forming cyanobacterium Microcystis aeruginosa

In the laboratory, we documented large variation in the morphology, toxicity, and maximum population growth rates for 32 Microcystis aeruginosa strains isolated from 12 lakes. Growth rates and mean colony sizes varied significantly across strains and were positively correlated. However, growth rates were unrelated to toxin production.     

Photoinactivation in vivo of superoxide dismutase and catalase in the cyanobacterium Microcystis aeruginosa

Abstract The planktonic cyanobacterium Microcystis aeruginosa is particularly sensitive to photoinhibition by visible light, Photosystem II and ribulose 1,5-bisphosphate (RuBP) carboxylase activities being affected. Although the organism contains superoxide dismutase (SOD) and catalase, these protective enzymes are also photoinactivated during the illumination of whole cells by visible light.     

Characteristics of the freshwater cyanobacterium Microcystis aeruginosa grown in iron-limited continuous culture

A continuous culturing system (chemostat) made of metal-free materials was successfully developed and used to maintain Fe-limited cultures of Microcystis aeruginosa PCC7806 at nanomolar iron (Fe) concentrations (20 to 50 nM total Fe). EDTA was used to maintain Fe in solution, with bioavailable Fe controlled by absorption of light by the ferric EDTA complex and resultant reduction of Fe(III) to Fe(II). A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe (i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of the ferric EDTA complex. Prediction by chemostat theory modified to account for the light-mediated formation of bioavailable Fe rather than total Fe was in good agreement with growth characteristics of M. aeruginosa under Fe limitation. The cellular Fe quota increased with increasing dilution rates in a manner consistent with the Droop theory. Short-term Fe uptake assays using cells maintained at steady state indicated that M. aeruginosa cells vary their maximum Fe uptake rate (ρ(max)) depending on the degree of Fe stress. The rate of Fe uptake was lower for cells grown under conditions of lower Fe availability (i.e., lower dilution rate), suggesting that cells in the continuous cultures adjusted to Fe limitation by decreasing ρ(max) while maintaining a constant affinity for Fe.     

Inhibition of Chlorella growth by the lipids of cyanobacterium Microcystis aeruginosa

The total lipids of the cyanobacterium Microcystis aeruginosa have been isolated and fractionated into its components. Of these lipid components, only the fatty acid-containing fraction inhibited the growth of the green alga Chlorella pyrenoidosa. The inhibitory activity appears to be due to linoleic and linolenic acids, which are both present in significants quantities. These acids may be the substances responsible for the reported toxicity of Microcystis aeruginosa to Chlorella.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Comparative protein expression in different strains of the bloom-forming cyanobacterium Microcystis aeruginosa

Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported.     

Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.