Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp

To investigate the abundance of active and inactive microcystin genotypes in populations of the filamentous cyanobacterium Planktothrix spp., individual filaments were grown as clonal strains in the laboratory and analysed for microcystin synthetase (mcy) genes and microcystin. Twenty-three green-pigmented strains of P. agardhii originating mostly from shallow water bodies fell into two groups, those possessing mcyA and those lacking mcyA. In contrast, all of the 49 strains that were assigned to the red-pigmented P. rubescens contained mcyA. One strain of P. agardhii and eight strains of P. rubescens contained the total microcystin synthetase gene cluster but were found inactive in microcystin synthesis. To investigate the natural abundance of inactive mcy genotypes in P. rubescens individual filaments sampled from Lake Irrsee and Lake Mondsee (Austria) were analysed directly for the presence of mcyA and microcystin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. All filaments assigned to P. rubescens contained mcyA. The proportion of inactive microcystin genotypes in populations with a low (Irrsee) or high density (Mondsee) of P. rubescens was 5% and 21%, each. The results of this study demonstrate that P. rubescens typically contain mcy genes whereas P. agardhii have a patchy distribution of mcy genes. In both species microcystin producers co-occur with non-microcystin producers due to the absence/inactivation of mcy genes.     

Allelopathic activity of Spirogyra sp.: stimulating bloom formation and toxin production by Oscillatoria agardhii in some irrigation canals, Egypt

It has often been observed that irrigation canals in Egypt thatcontain Spirogyra are covered with blooms of Oscillatoria duringthe summer season. These blooms do not occur in the canals notcontaining Spirogyra. Thus, two irrigation canals, one containingSpirogyra and another not containing Spirogyra, were chosento study the possible allelopathic activity of Spirogyra onthe growth of Oscillatoria agardhii. The growth of O. agardhiiand other associated cyanobacterial species was followed inthese two canals during the period May–September 2000.The results revealed that O. agardhii was dominant and formedblooms in the canals containing Spirogyra, while it had a moderate/rareoccurrence in those not containing Spirogyra. To confirm thestimulatory allelopathic activity of this green alga on thegrowth of and microcystin production by O. agardhii, a laboratoryexperiment was run by growing O. agardhii with different concentrationsof Spirogyra aqueous extract. The results showed that the growthof and microcystin production by O. agardhii increased withincreasing concentrations of Spirogyra aqueous extract. Thisfinding demonstrates the allelopathic activity of the greenalga Spirogyra stimulating the growth of and toxin productionby O. agardhii. Such a biotic factor should be taken into considerationwhen cyanobacterial blooms are monitored in freshwater bodies.     

Isolation and identification of eight microcystins from thirteen Oscillatoria agardhii strains and structure of a new microcystin.

Microcystins (cyclic heptapeptide hepatotoxins), isolated from 13 freshwater Oscillatoria agardhii strains from eight different Finnish lakes by high-performance liquid chromatography, were characterized by amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and tandem FABMS (FABMS/collisionary-induced dissociation/MS). All strains produced two to five different microcystins. In total, eight different compounds, of which five were known microcystins, were isolated. The known compounds identified were [D-Asp3]MCYST (microcystin)-LR, [Dha7]MCYST-LR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. This is the first time that isolation of these toxins from Oscillatoria spp., with the exception of [D-Asp3]MCYST-RR, has been reported. Three of the strains produced a new microcystin, and the structure was assigned as [D-Asp3,Mser7]MCYST-RR. The structures of two new microcystins, produced as minor components by one Oscillatoria strain, could not be determined because of the small amounts isolated from the cells. Four strains produced [Dha7]MCYST-RR as the main toxin, but [D-Asp3]MCYST-RR was clearly the most abundant and most frequently occurring toxin among these isolates of O. agardhii.     

Allelopathic growth inhibition by the toxic, bloom-forming cyanobacterium Planktothrix rubescens

Blooms of freshwater cyanobacteria are typically accompanied by an important decrease in phytoplankton biodiversity in the water bodies where they occur. This study examines the potential production of growth-inhibiting substances by the toxic, bloom-forming cyanobacterium Planktothrix rubescens, following the observation of physical segregation between this and another cyanobacterium during previously performed mixed-culture competition experiments. Inhibition assays examining the growth of target strains exposed to donor culture filtrates showed that the growth of Planktothrix agardhii TCC 83-2, P. agardhii PMC 75.02 and Mougeotia gracillima TCC 50-2 was significantly inhibited in the presence of culture filtrate from P. rubescens TCC 29-1, isolated from Lake Bourget, France. Filtrates from P. rubescens TCC 69-6 and P. rubescens TCC 69-7, isolated from Lakes Nantua and Paladru (France), respectively, did not, however, inhibit the growth of P. agardhii TCC 83-2. This brief exploration of the allelopathic activity of P. rubescens suggests that it may potentially inhibit coexisting competitors as well as phytoplankton isolated from other freshwater ecosystems, and that this capacity may vary among different strains of Planktothrix. The potential importance of this phenomenon in pelagic competition dynamics is discussed.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains.

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Regulation of growth and photosynthesis by Oscillatoria agardhii grown with a light/dark cycle

Abstract The cyanobacterium Oscillatoria agardhii was grown in turbidostat cultures with the light energy supply in either the continuous mode or in the pulsed mode (8/16 h light/dark (L/D) cycle). The light irradiance value used was sufficient to allow the maximal growth rate to be attained, when supplied continuously. Adaptation of O. agardhii to the L/D cycle was characterized by an increase in pigment content and photosynthetic performance, accompanied by a decrease in growth rate. This mode of adaptation resembled the adaptation of O. agardhii to continuous low light intensities. It is suggested that in this case the L/D cycle provokes this adaptation in order to allow the cells to accumulate carbohydrate rapidly during the light period. This was attributed to the storage of polyglucose, which served as a carbon and energy source for growth in the dark. The utilization of polyglucose in the dark was able to sustain the synthesis of all other cell components at the same rate as when cells were growing in the light. The growth yield in the dark, whilst metabolizing internally stored polyglucose, was 0.52 g cell C/g polyglucose C, or 0.62 g cell dry weight/g polyglucose. Although in the pulsed mode there is a 66% loss in light irradiance per 24 h when compared with a continuous light regime, the growth rate of the cyanobacteria grown in the pulsed mode was only 35% lower than the growth rate of a culture grown in continuous light. This can be explained by a high growth yield in the dark and by increased CO₂ fixation rates in the light of cells grown in the pulsed mode.     

Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii

Microcystins (MCs) are toxic heptapeptides that are produced by filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii via nonribosomal peptide synthesis. MCs share a common structure cyclo (-D-Alanine(1)-L-X(2)- D-erythro-beta-iso-aspartic acid(3)-L-Z(4)-Adda(5)-D-Glutamate(6)- N-methyl-dehydroalanine(7)) where X(2) and Z(2) are variable L-amino acids in positions 2, 4 of the molecule. Part of the mcyB gene (1,451 bp) that is involved in the activation of the X(2) amino acid during MC synthesis was sequenced in 49 strains containing different proportions of arginine, homotyrosine, and leucine in position 2 of the MC molecule. Twenty-five genotypes were found that consisted of eight genotype groups (A-H, comprising 2-11 strains) and 17 unique genotypes. P. rubescens and P. agardhii partly consisted of the same mcyB genotypes. The occurrence of numerous putative recombination events that affected all of the genotypes can explain the conflict between taxonomy and mcyB genotype distribution. Genotypes B (homotyrosine and leucine in X(2)) and C (arginine in X(2)) showed higher nonsynonymous/synonymous (d(N)/d(S)) substitution ratios implying a relaxation of selective constraints. In contrast, other genotypes (arginine, leucine, homotyrosine) showed lowest d(N)/d(S) ratios implying purifying selection. Restriction fragment length polymorphism (RFLP) revealed the unambiguous identification of mcyB genotypes, which are indicative of variable X(2) amino acids in eight populations of P. rubescens in the Alps (Austria, Germany, and Switzerland). The populations were found to differ significantly in the proportion of specific genotypes and the number of genotypes that occurred over several years. It is concluded that spatial isolation might favour the genetic divergence of microcystin synthesis in Planktothrix spp.     

Chemotaxonomy of planktonic cyanobacteria based on non-polar and 3-hydroxy fatty acid composition

Twenty-eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acids were detected in any strain, but 3-hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non-polar fatty acids. Qualitative and quantitative analyses of 3-hydroxy fatty acids showed no taxonomic value in these strains, while the type of non-polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon-Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological species criteria, suggesting that non-polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.     

A comparison of toxins isolated from the cyanobacteria Oscillatoria agardhii and Microcystis aeruginosa

1. A toxin isolated from a strain of Oscillatoria agardhii var. was compared to a peptide toxin isolated from Microcystis aeruginosa. 2. The Oscillatoria toxin possessed similar hepatotoxic properties on mice as the Microcystis toxin but had a higher LD50 than the latter; 320 micrograms/kg compared to 43 micrograms/kg (i.p. mouse), respectively. 3. Ultra-violet and infra-red spectra showed that the Oscillatoria toxin is a peptide which is not identical to the Microcystis toxin. 4. The spectra also indicated some structural similarities in these toxins.     

Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins

The role of zooplankton in the control of cyanobacterial bloomsand the transfer of cyanotoxins to higher trophic levels areof great importance to the management of water resources. Manystudies have focused on the cyanobacterium Microcystis, butfew have examined the interactions between zooplankton and filamentouscyanobacteria. In this study, we provide experimental evidencefor the potential grazing of two toxic strains of filamentouscyanobacteria, Planktothrix rubescens and P. agardhii, by Daphniapulicaria, and for transfer of toxins in the planktonic foodchain. We determined clearance rates (CRs) by adult and juvenileD. pulicaria of the two Planktothrix strains, Scenedesmus acutusand a mixture of S. acutus cells with P. rubescens culture filtrate.Filament lengths were analyzed, and microcystin (MCY) presencein Daphnia was assessed using the Protein Phosphatase-2A (PP-2A)Inhibition Assay. The two Planktothrix strains were equallygrazed by D. pulicaria, but at lower CRs than S. acutus. Potentialanti-grazer toxins in P. rubescens filtrate did not inhibitDaphnia grazing. Small P. rubescens (<100 µm) filamentswere preferentially grazed by adult D. pulicaria, suggestingtheir limited ability to control a Planktothrix population duringa bloom. Large quantities of MCYs were found in unstarved Daphniapreviously exposed to Planktothrix, whereas quantities weresignificantly smaller in individuals starved for 24 h beforepreservation. This indicated a potential for transfer of toxinsin the food chain by Daphnia, especially immediately after ingestionof toxic cyanobacteria.     

Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study

Abstract The incorporation of a very low concentration (0.015 μM) of an [³H]amino acid mixture was measured for a natural population of Oscillatoria rubescens DC in samples from a eutrophic lake, Lake Nantua (France).
The kinetics of amino acid incorporation in the size fraction ⩾12 μ m showed that uptake was fast and that the maximum was reached after 4 h.
Microautoradiography demonstrated that Oscillatoria rubescens is able to utilize for protein synthesis an external pool of amino acids whose [³H] label becomes distributed generally throughout the cell.     

Suboptimal light conditions negatively affect the heterotrophy of Planktothrix rubescens but are beneficial for accompanying Limnohabitans spp

We examined the effect of light on the heterotrophic activity of the filamentous cyanobacterium Planktothrix rubescens and on its relationship with the accompanying bacteria. In situ leucine uptake by bacteria and cyanobacteria was determined in a subalpine mesotrophic lake, and natural assemblages from the zone of maximal P. rubescens abundances were incubated for 2 days at contrasting light regimes (ambient, 100× increased, dark). Planktothrix rubescens from the photic zone of the lake incorporated substantially more leucine, but some heterotrophic activity was maintained in filaments from the hypolimnion. Exposure of cyanobacteria to increased irradiance or darkness resulted in significantly lower leucine incorporation than at ambient light conditions. Highest abundances and leucine uptake of Betaproteobacteria from the genus Limnohabitans were found in the accompanying microflora at suboptimal irradiance levels for P. rubescens or in dark incubations. Therefore, two Limnohabitans strains (representing different species) were co-cultured with axenic P. rubescens at different light conditions. The abundances and leucine incorporation rates of both strains most strongly increased at elevated irradiance levels, in parallel to a decrease of photosynthetic pigment fluorescence and the fragmentation of cyanobacterial filaments. Our results suggest that Limnohabitans spp. in lakes might profit from the presence of physiologically stressed P. rubescens.     

Anabaenopeptins G and H, potent carboxypeptidase A inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-595).

Anahaenopeptins G (1) and H (2) were isolated from the cultured cyanobacterium Oscillatoria agardhii (NIES-595) as potent carboxypeptidase A (CPA) inhibitors. The gross structure of 1 and 2 were established by spectroscopic analysis including the 2D NMR technique and the absolute configurations of 1 and 2 were determined by the spectral and chemical methods. 1 and 2 inhibited CPA with IC50's of 0.0018 and 3.4 microg/mL, respectively.     

Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii

The primary structure of a lectin, designated Oscillatoria agardhii agglutinin (OAA), isolated from the freshwater cyanobacterium O. agardhii NIES-204 was determined by the combination of Edman degradation and electron spray ionization-mass spectrometry. OAA is a polypeptide (Mr 13,925) consisting of two tandem repeats. Interestingly, each repeat sequence of OAA showed a high degree of similarity to those of a myxobacterium, Myxococcus xanthus hemagglutinin, and a marine red alga Eucheuma serra lectin. A systematic binding assay with pyridylaminated oligosaccharides revealed that OAA exclusively binds to high mannose (HM)-type N-glycans but not to other N-glycans, including complex types, hybrid types, and the pentasaccharide core or oligosaccharides from glycolipids. OAA did not interact with any of free mono- and oligomannoses that are constituents of the branched oligomannosides. These results suggest that the core disaccharide, GlcNAc-GlcNAc, is also essential for binding to OAA. The binding activity of OAA to HM type N-glycans was dramatically decreased when alpha1-2 Man was attached to alpha1-3 Man branched from the alpha1-6 Man of the pentasaccharide core. This specificity of OAA for HM-type oligosaccharides is distinct from other HM-binding lectins. Kinetic analysis with an HM heptasaccharide revealed that OAA possesses two carbohydrate binding sites per molecule, with an association constant of 2.41x10(8) m-1. Furthermore, OAA potently inhibits human immunodeficiency virus replication in MT-4 cells (EC50=44.5 nm). Thus, we have found a novel lectin family sharing similar structure and carbohydrate binding specificity among bacteria, cyanobacteria, and marine algae.     

Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii

In the survey of 14 species of laboratory-cultured cyanobacteria for hemagglutinins, we newly detected the activity in two species, Oscillatoria agardhii, strain NIES-204, and Phormidium foveolarum, strain NIES-503. From the extract of O. agardhii, which showed the highest activity with trypsin-treated erythrocytes of rabbit, a lectin was purified to homogeneity by the combination of precipitation with (NH4)2SO4, gel filtration, hydrophobic chromatography and reverse phase chromatography. The purified lectin, designated OAA, was a monomeric protein with an apparent molecular weight of 13,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 16,000 on gel filtration. The amino acid composition was rich in glycine and acidic amino acids. The hemagglutination activity was inhibited by glycoproteins such as yeast mannan, but not by any of the monosaccharides tested. The activity was stable over a wide range of pH (4-11) and at a high temperature of 80 degrees C, and independent on the presence of divalent cations. The features of OAA resembled those of many of lectins from marine macroalgae. The sequence of amino-terminal residues of OAA was determined as ALYNVENQWGGSSAPWNEGG, which was highly homologous to those of lectins from macroalgae of the genus Eucheuma and that of a myxobacterium Myxococcus xanthus hemagglutinin.     

Photosynthetic Activity of Dominant Algal Species in Eutrophic Shallow Lake (Großer Müggelsee,Berlin) Investigated by Microautoradiography

The photosynthetic activity of dominant phytoplankton in eutrophic shallow lake was investigated by the autoradiographic method in 1979 and 1980. It was shown by light and dark field microscopy that all species of Cyanophyta (Oscillatoria redekei, Oscillatoria agardhii, Aphanizomenon flos-aquae) were characterized by a continuously high uptake of NaH¹⁴CO₃. Similarly high photosynthetic activity was observed during the occurrence of Cryptomonas sp. and nanoplankton. Contrary to these observations, diatoms showed remarkably high portions of photosynthetically inactive biomass when their development was abundant. The reasons for this discrepancy between high biomass of diatoms and relatively low primary production (measured by the ¹⁴C-method and autoradiography) are discussed.     

Decomposition of aquatic biota and sediment formation: lipid components of two blue-green algal species and of detritus resulting from microbial attack

To simulate early stages in the diagenesis of algal material in lakes, microbial attack was allowed to proceed on natural populations of two blue-green algal species, Gloeotrichia echinulata (J. E. Smith) P. Richter and Oscillatoria agardhii var. isothrix Skuja. Changes in composition and abundance of the major lipid fractions were related to differences in the ease of microbial attack and to the effect of oxygen on the decay process. The loss of labile unsaturated compounds during diagenesis is consistent with the small amount of these compounds in most lake sediments.     

Toxicity Studies with the Blue-Green Alga Oscillatoria Agardhii from two Eutrophic Norwegian Lakes

Extracts of the blue-green alga Oscillatoria agardhii were tested for acute toxicity on laboratory mice and rats. Material originating from lake Gjersjøen proved to be toxic to the animals, samples from the nearby lake Årungen did not. Clinical symptoms culminated in the development of a fatal shock due to decrease in circulating blood volume. Pathological examination revealed heavy pooling of blood in the liver and severe damage to the organ. Blood analyses also indicated liver damage. Effects were the same with extracts from a laboratory clone culture as from a natural water bloom, but the toxin content was higher in the bloom material. Toxicity was not affected by heat, acid or alkali treatment.     

A comparison of chlorophyll a and carotenoid concentrations as indicators of algal volume

SUMMARY. 1. Carotenoid concentration, as measured hy absorbance at 480 nm. was a better indicator of algal volume than chlorophyll a when the results from two lakes and laboratory studies on Osciltaloria agardhii var. isothrix Skuja were compared. The correlation between algal volume and carotenoid in White Lough ( r =0.91) was significantly higher (0.001 P <0.01) than ihat between algal volume and chlorophyll a (r=0.77). The Lotigh Neagh correlation coefficient lor algal volume with carotenoid (r=0.89) was only marginally stronger than that with chlorophyll a (r=0.87).
2. The relatively weak correlation between algal volume and chlorophyll a in White Lough was a result of a summer decline in the chlorophyll a content of O. agardhii var. isothrix , which dominated the phytoplankton. The chlorophyll a content of the phytoplankton was depressed by high summer daily totals ol light hours received by the phytoplankton in White Lough of up to 14 h in comparison to a maximum value of 3.8 h in Lough Neagh. where no seasonal cycle of chlorophyll a content was evident.
3. Laboratory studies demonstrated that while chlorophyll a per unit algal volume of O. agardhii var. isothri.x declined with increasing light dose, carotenoid content did not. Nitrogen and phosphorus limitation depressed the carotenoid content but to a lesser degree than was observed for chlorophyll a.