A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Structural and functional investigation of AerF, a NADPH-dependent alkenal double bond reductase participating in the biosynthesis of Choi moiety of aeruginosin.

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is an essential residue for the antithrombotic activities of aeruginosins, which are a class of cyanobacterial derived bioactive linear tetrapeptides. Biosynthetic pathway of Choi is still elusive. AerF was suggested to be involved in the biosynthesis of Choi, and can be assigned to the short-chain dehydrogenase/reductase (SDR) superfamily. However, both the exact role and the catalytic mechanism of AerF have not been elucidated. In this study, functional and mechanistic analyses of AerF from Microcystis aeruginosa were performed. Observation of enzymatic assay demonstrates that AerF is a NADPH-dependent alkenal double bond reductase that catalyzes the reduction of dihydro-4-hydroxyphenylpyruvate (H2HPP) to generate tetrahydro-4-hydroxyphenylpyruvate (H4HPP), which is the third step of the biosynthetic pathway from prephenate to Choi. Comparative structural analysis indicates that ligand binding-induced conformational change of AerF is different from that of the other SDR superfamily reductase using H2HPP as a substrate. Analyses of NADPH and substrate analogue binding sites combined with the results of mutagenesis analyses suggest that a particular serine residue mainly involves in the initiation of the proton transfer between the substrate and the residues of AerF, which is an uncommon feature in SDR superfamily reductase. Furthermore, based on the observations of structural and mutagenesis analyses, the catalytic mechanism of AerF is proposed and a proton transfer pathway in AerF is deduced.

Physiological and thylakoid ultrastructural changes in cyanobacteria in response to toxic manganese concentrations.

In this study, two cyanobacterial strains (morphologically identified as Microcystis novacekii BA005 and Nostoc paludosum BA033) were exposed to different Mn concentrations: 7.0, 10.5, 15.7, 23.6 and 35.4 mg L-1 for BA005; and 15.0, 22.5, 33.7, 50.6, and 76.0 mg L-1 for BA033. Manganese toxicity was assessed by growth rate inhibition (EC50), chlorophyll a content, quantification of Mn accumulation in biomass and monitoring morphological and ultrastructural effects. The Mn EC50 values were 16 mg L-1 for BA005 and 39 mg L-1 for BA033, respectively. Reduction of chlorophyll a contents and ultrastructural changes were observed in cells exposed to Mn concentrations greater than 23.6 and 33.7 mg L-1 for BA005 and BA033. Damage to intrathylakoid spaces, increased amounts of polyphosphate granules and an increased number of carboxysomes were observed in both strains. In the context of the potential application of these strains in bioremediation approaches, BA005 was able to remove Mn almost completely from aqueous medium after 96 h exposure to an initial concentration of 10.5 mg L-1, and BA033 was capable of removing 38% when exposed to initial Mn concentration of 22.5 mg L-1. Our data shed light on how these cyanobacterial strains respond to Mn stress, as well as supporting their utility as organisms for monitoring Mn toxicity in industrial wastes and potential bioremediation application.

Extracellular polysaccharide synthesis in a bloom-forming strain of Microcystis aeruginosa: implications for colonization and buoyancy.

Microcystis, the dominant species among cyanobacterial blooms, normally forms colonies under natural conditions but exists as single cells or paired cells in axenic laboratory cultures after long-term cultivation. Here, a bloom-forming Microcystis aeruginosa strain CHAOHU 1326 was studied because it presents a colonial morphology and grows on the water surface during axenic laboratory culturing. We first examined the morphological features of strain CHAOHU 1326 and three other unicellular M. aeruginosa strains FACHB-925, FACHB-940, and FACHB-975 cultured under the same conditions by scanning and transmission electron microscopy. Then, we compared the extracellular polysaccharide (EPS)-producing ability of colonial strain CHAOHU 1326 to that of the three unicellular M. aeruginosa strains, and found that strain CHAOHU 1326 produced a higher amount of EPS than the other strains during growth. Moreover, based on genome sequencing, multiple gene clusters implicated in EPS biosynthesis and a cluster of 12 genes predicted to be involved in gas vesicle synthesis in strain CHAOHU 1326 were detected. These predicted genes were all functional and expressed in M. aeruginosa CHAOHU 1326 as determined by reverse transcription PCR. These findings provide a physiological and genetic basis to better understand colony formation and buoyancy control during M. aeruginosa blooming.

Environmental risks of ZnO nanoparticle exposure on Microcystis aeruginosa: Toxic effects and environmental feedback.

The vast majority of studies measure the toxic effect of organisms exposed to nanoparticles (NPs) while there is still a lack of knowledge about the influence of NPs on the aquatic environment. It is unknown whether or not the interaction between NPs and algae will result in the variation of algal organic matter (AOM) and stimulate the production of more algal toxins. In this study, zinc oxide nanoparticles (nano-ZnO) as a typical representative of metal oxide NPs were used to evaluate the toxic effects and environmental feedback of Microcystis aeruginosa. Reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to explain the toxicity mechanism. Changes of AOM, including the production of toxins, the molecular weight distribution and the excitation-emission matrices of algal solution were also studied as environmental feedback indicators after nano-ZnO destroyed the algae. As the nano-ZnO exceeded the comparable critical concentration (1.0 mg/L), the algae were destroyed and intracellular organic matters were released into the aquatic environment, which stimulated the generation of microcystin-LR (MC-LR). However, it is worth noting that the concentration of nano-ZnO would need to be high (at mg/L range) to stimulate more MC-LR production. These findings are expected to be beneficial in interpreting the toxicity and risks of the releasing of NPs through the feedback between algae and the aquatic environment.

Adsorptive removal of harmful algal species Microcystis aeruginosa directly from aqueous solution using polyethylenimine coated polysulfone-biomass composite fiber.

In recent times, the treatment of harmful algal blooms (HABs) became an important environmental issue to preserve and remediate water resources globally. In the present study, the adsorptive removal of harmful algal species Microcystis aeruginosa directly from an aqueous medium was attempted. Waste biomass (Escherichia coli) was immobilized using polysulfone and coated using the cationic polymer polyethylenimine (PEI) to generate PEI-coated polysulfone-biomass composite fiber (PEI-PSBF). The density of M. aeruginosa in an aqueous medium (BG11) was significantly decreased by treatment with PEI-PSBF. additionally, analysis using FE-SEM, confirmed that the removal of M. aeruginosa algal cells by PEI-PSBF was caused by the adsorption mechanism. According to the profiles of phosphorus for the algal cell growth in M. aeruginosa cultivating samples, we found that the adsorbed M. aeruginosa onto the PEI-PSBF lost their biological activity compared to the non-treated M. aeruginosa cells.

Influences of small-scale oscillations on growth inhibition and ultrastructural changes of Microcystis cells.

We investigated the effects of small-scale oscillation (SSO) on toxic Microcystis cells. The oscillating device was made of silicon with two axes that had a diameter of ∼40 mm, and a frequency of 2.5 Hz was observed at 150 rpm. The SSO was effective in inhibiting Microcystis growth. Microcystin release was not observed, whereas cell density barely increased in the oscillating group. Cell size and morphology of the oscillating group were no different from the control group. However, cell quotas of chl.a and microcystin in the oscillating group were half the level of the control group. Crucially, a number of large-sized holes were observed and layered long linear thylakoids were rarely observed in the oscillating group. Therefore, SSO was found to be very effective in Microcystis growth inhibition, and it caused ultrastructural changes without damage to the cell membrane and subsequent microcystin release.

Vitamin C modulates Microcystis aeruginosa death and toxin release by induced Fenton reaction.

Cyanobacterial blooms and their associated toxins pose a great threat to human beings. The situation is even worse for those whose drinking water source is a cyanotoxin-polluted water body. Therefore, efficient and safe treatments urgently need to be developed. The present study verified the application of vitamin C on the inhibition of toxic Microcystis aeruginosa. Our results showed that vitamin C drove the Fenton reaction and significantly sterilized cultures of M. aeruginosa. The algicidal activity of vitamin C was dependent on its involvement in iron (Fe) metabolism. Vitamin C enhanced iron absorption leading to high ferrous ion levels. The ferrous ion increased production of reactive oxygen species (ROS) by Fenton reaction, which play a crucial role in the killing process. Interestingly, vitamin C also dramatically decreased the release of microcystins. This study highlights the possible benefits of using a vitamin C-induced Fenton reaction to remove M. aeruginosa and microcystins from drinking water sources.

pH-dependent gas vesicle formation in Microcystis.

In lakes with seasonal cyanobacterial blooms, the pH fluctuates from slightly above 7 to around 10. In this study, we found that the abundance of gas vesicles in Microcystis species, in parallel to the buoyancy of cells, increased in response to elevation of the extracellular pH. Within 48 h after pH upshift, gas vesicle protein genes (gvp) were upregulated at both mRNA and protein levels due to reduced decay of gvp transcripts. The effect of pH on GvpC level was basically unaffected by inorganic carbon availability. This is the first report that long-term pH range plays a role in controlling gas vesicle formation in certain Microcystis species.

Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide.

Growth inhibition and possible mechanism of oleamide against the toxin-producing cyanobacterium Microcystis aeruginosa NIES-843.

Oleamide, a fatty acid derivative, shows inhibitory effect against the bloom-forming cyanobacterium Microcystis aeruginosa. The EC50 of oleamide on the growth of M. aeruginosa NIES-843 was 8.60 ± 1.20 mg/L. In order to elucidate the possible mechanism of toxicity of oleamide against M. aeruginosa, chlorophyll fluorescence transient, cellular ultrastructure, fatty acids composition and the transcription of the mcyB gene involved in microcystins synthesis were studied. The results of chlorophyll fluorescence transient showed that oleamide could destruct the electron accepting side of the photosystem II of M. aeruginosa NIES-843. Cellular ultrastructure examination indicated that the destruction of fatty acid constituents, the distortion of thylakoid membrane and the loss of integrity of cell membrane were associated with oleamide treatment and concentration. The damage of cellular membrane increased the release of microcystins from intact cells into the medium. Results presented in this study provide new information on the possible mechanisms involved and potential utilization of oleamide as an algicide in cyanobacterial bloom control.

Inquisition of Microcystis aeruginosa and Synechocystis nanowires: characterization and modelling.

Identification of extracellular conductive pilus-like structures (PLS) i.e. microbial nanowires has spurred great interest among scientists due to their potential applications in the fields of biogeochemistry, bioelectronics, bioremediation etc. Using conductive atomic force microscopy, we identified microbial nanowires in Microcystis aeruginosa PCC 7806 which is an aerobic, photosynthetic microorganism. We also confirmed the earlier finding that Synechocystis sp. PCC 6803 produces microbial nanowires. In contrast to the use of highly instrumented continuous flow reactors for Synechocystis reported earlier, we identified simple and optimum culture conditions which allow increased production of nanowires in both test cyanobacteria. Production of these nanowires in Synechocystis and Microcystis were found to be sensitive to the availability of carbon source and light intensity. These structures seem to be proteinaceous in nature and their diameter was found to be 4.5-7 and 8.5-11 nm in Synechocystis and M. aeruginosa, respectively. Characterization of Synechocystis nanowires by transmission electron microscopy and biochemical techniques confirmed that they are type IV pili (TFP) while nanowires in M. aeruginosa were found to be similar to an unnamed protein (GenBank : CAO90693.1). Modelling studies of the Synechocystis TFP subunit i.e. PilA1 indicated that strategically placed aromatic amino acids may be involved in electron transfer through these nanowires. This study identifies PLS from Microcystis which can act as nanowires and supports the earlier hypothesis that microbial nanowires are widespread in nature and play diverse roles.

The combined effects of UV-C radiation and H2O2 on Microcystis aeruginosa, a bloom-forming cyanobacterium.

In order to get insight into the impacts of UVC/H2O2 on Microcystis aeruginosa, physiological and morphological changes as well as toxicity were detected under different UVC/H2O2 treatments. In the presence of sole UVC or H2O2, the net oxygen evolution rate decreased significantly (p<0.05) since activity of photosystem II (PSII) was inhibited. Meanwhile, increase of intracellular reactive oxygen species (ROS), degradation of microcystin (MC) and ultrastructure destructions were observed. Under sole UVC treatment, no changes happened in the activity of photosystem I (PSI), but the degradation of D1 protein was observed. Under sole H2O2 treatment, an increase of malondialdehyde, aggregation of D1 protein and deformation of the thylakoid membrane were observed. ROS content under H2O2 treatment was about 5 times than that under UVC treatment. Combined use of UVC and H2O2, as well as 20mJcm(-2) UVC and 60µM H2O2, showed high synergetic effects. Obvious damage to membrane systems, the marked degradation of MC and inhibition of the photosystems were observed. It could be deduced that UVC worked on intracellular membrane components directly and the damaged oxygen-evolving complex, which was followed by the D1 protein degradation. H2O2 oxidised the membrane lipids via an ROS-mediated process, with thylakoid injury and the aggregation of D1 protein being the lethal mechanisms, and both PSII and PSI being the attacking targets. With regard towards the effective inactivation of M. aeruginosa and high removal of MC, UVC/H2O2 proposed a novel practical method in controlling cyanobacterial blooms.

The performance of chitosan/montmorillonite nanocomposite during the flocculation and floc storage processes of Microcystis aeruginosa cells.

This study aimed to investigate the performance of chitosan-modified nano-sized montmorillonite (CTS/NMMT) during the flocculation of Microcystis aeruginosa (MA). The release of intracellular microcystins (MCs) caused by the damage of intact MA cells during the flocculation and floc storage processes was also comprehensively evaluated through scanning electron microscopy (SEM) and measurement of K(+) and Mg(2+) release. With the application of the Box-Behnken experimental design combined with response surface methodology, the quadratic statistical model was established to predict and optimize the interactive effects of content of CTS/NMMT, weight ratio of NMMT to CTS, and agitation time on the removal efficiency of MA cells. A maximum removal of 94.7 % MA cells was observed with content of CTS/NMMT 300-320 mg L(-1), weight ratio of NMMT to CTS 14-16, and agitation time 16-50 min. During the flocculation process, CTS/NMMT aggregated MA cells as flocs and served as a protection shield for cells. The extracellular and intracellular microcystin-leucine-arginine (MC-LR) decreased remarkably and the yield of intracellular MC-LR showed a decreasing trend during the flocculation. The cell integrity was slightly damaged by the mechanical actions rather than by the flocculant. During the floc storage process, cell lysis and membrane damage were remarkably aggravated. The noticeable increase of K(+) and Mg(2+) release indicated that CTS/NMMT damaged the integrity of most MA cells in the flocs and liberated the intracellular MC-LR. Meanwhile, NMMT and CTS polymers assisted the adsorptive removal of extracellular MC-LR released to water. The flocs should be timely treated within 12 h to prevent the leakage of MCs.

The fate of Microcystis aeruginosa cells during the ferric chloride coagulation and flocs storage processes.

Microcystis blooms could cause severe problems for drinking water supplies with their associated microcystins (MCs). As the majority of MCs are retained inside the cells, the effective removal of the intact Microcystis cells to avoid the release of additional MCs plays an important role in drinking water treatment. This study evaluated the effect of ferric chloride (FeCl3) coagulation and the flocs storage process on the integrity of Microcystis aeruginosa cells and the intracellular MCs release (and possible degradation) in both processes. Multiple analysis techniques including scanning electron microscopy and chlorophyll fluorescence were used to assess the integrity of M. aeruginosa. In the coagulation process, the coagulant dosage and mechanical actions caused no cell damage, and all the cells remained intact. Furthermore, 100 mg/L FeCl3 was effective in removing the extracellular MCs. In the flocs storage process, a number of intracellular MCs were released into the supernatant, but the cells remained viable up to 10 d.

Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa.

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8-14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF from Microcystis aeruginosa PCC 7806 is reported at 2.7 Šresolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+ß class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

Aquatic environmental safety assessment and inhibition mechanism of chemicals for targeting Microcystis aeruginosa.

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, fungi, bacteria and cell lines. Recently the main methods of controlling cyanobacteria are using chemicals, medicinal plants and microorganism but fewer involved the safety research in hydrophytic ecosystems. In search of an environmentally safe compound, 53 chemicals were screened against the developed heavy cyanobacteria bloom Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that 9 chemicals inhibited M. aeruginosa effectively at 20 mg L(-1) after 7 days of exposure. Among them dimethomorph, propineb, and paraquat were identified that they are safe for Chlorella vulgaris, Scenedesmus obliquus, Carassius auratus (Goldfish) and Bacillus subtilis within half maximal effective concentration (EC50) values 5.2, 4.2 and 0.06 mg L(-1) after 7 days, respectively. Paraquat as the positive control observed to be more efficient than the other compounds with the inhibitory rate (IR) of 92% at 0.5 mg L(-1). For the potential inhibition mechanism, the chemicals could destroy the cell ultrastructure in different speed. The safety assay proved dimethomorph, propineb and paraquat as harmless formulations or products having potential value in M. aeruginosa controlling, with the advantage of its cell morphology degrading ability.

Removal of Microcystis aeruginosa using hydrodynamic cavitation: performance and mechanisms.

Algal blooms are a seasonal problem in eutrophic water bodies, and novel approaches to algal removal are required. The effect of hydrodynamic cavitation (HC) on the removal of Microcystis aeruginosa was investigated using a laboratory scale device. Samples treated by HC were subsequently grown under illuminated culture conditions. The results demonstrated that a short treatment with HC could effectively settle naturally growing M. aeruginosa without breaking cells. Algal cell density and chlorophyll-a of a sample treated for 10 min were significantly decreased by 88% andv 94%, respectively, after 3 days culture. Various HC operating parameters were investigated, showing that inhibition of M. aeruginosa growth mainly depended on treatment time and pump pressure. Electron microscopy confirmed that sedimentation of algae was attributable to the disruption of intracellular gas vesicles. Damage to the photosynthetic apparatus also contributed to the inhibition of algal growth. Free radicals produced by the cavitation process could be as an indirect indicator of the intensity of HC treatment, although they inflicted minimal damage on the algae. In conclusion, we suggest that HC represents a potentially highly effective and sustainable approach to the removal of algae from water systems.

Effects of limonene stress on the growth of and microcystin release by the freshwater cyanobacterium Microcystis aeruginosa FACHB-905.

The effects of limonene exposure on the growth of Microcystisaeruginosa and the release of toxic intracellular microcystin (MCY) were tested by evaluating the results obtained from the batch culture experiments with M. aeruginosa FACHB-905. The time series of cell as well as intracellular and extracellular MCY concentrations were evaluated during 5d of the incubation. After exposure to limonene, the number of cells gradually diminished; the net log cell reduction after 5d of the exposure was 3.0, 3.6, and 3.8log when the initial cell densities were set at 1.6×10(7), 1.1×10(6) and 4.1×10(5)cell/mL, respectively. Limonene was found to significantly influence the production and release of MCY. As the limonene exposure could inhibit the increase in the number of cells, the increase in the total MCY concentration in the medium was also inhibited. In the presence of limonene, the intracellular MCY was gradually released into the medium through a gradual reduction in the number of cells. The extracellular MCY concentration in the medium was significantly higher in the limonene-exposed samples than in the control samples, which confirmed that limonene cannot decompose the extracellular MCY.

Inactivation of Microcystis aeruginosa by DC glow discharge plasma: Impacts on cell integrity, pigment contents and microcystins degradation.

We proposed a method to inactivate M. aeruginosa by using discharge plasma taking at the gas-solution interface supplied by DC power. Multiple analysis techniques including fluorescence excitation-emission matrix (EEM) and flow cytometry (FCM) were used to reveal the inactivation mechanism of M. aeruginosa. The photosynthetic pigment contents including phycocyanin, chlorophyll and metabolites were examined quantitatively. The DC glow discharge plasma caused an increased level of reactive oxygen species (ROS), and the damage of M. aeruginosa cells are mainly attributed to the oxidative stress including OH attack and H2O2 oxidation. Our findings demonstrate that plasma oxidation is a promising technology for inactivation of M. aeruginosa cells with simultaneous removal of microcystins and so it may lead us to a new route to efficient treatment of cyanobacterial blooms.