Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.

Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa, K-139.

Two toxic heptapeptides were isolated from an axenic Microcystis aeruginosa strain, K-139. Using mainly a non-destructive NMR method, we determined the structure of the major toxin to be 7-desmethylmicrocystin LR which lacks an N-methyl group of the dehydroalanine moiety of microcystin LR. The minor toxin was deduced to be 3,7-didesmethylmicrocystin LR. The chromatographic and NMR analyses of 7-desmethylmicrocystin LR were compared with those of 3-desmethylmicrocystin LR.

Cell wall constituents of Microcystis sp. PCC 7806.

Cell walls of Microcystis sp. PCC 7806 were purified from cell homogenates by sucrose density centrifugation and Triton X-100 extraction. The outer membrane contained carotenoids, two major peptidoglycan-associated proteins (Mr 49,000 and 52,000), and lipopolysaccharide (LPS) as indicated by the presence of 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0), 4-oxo-18:0 fatty acid, and GlcN as lipid A components in addition to rare O-methyl sugars (2-O-methyl-6-deoxyhexoses I and II). The peptidoglycan (A1 gamma-type) was found to be covalently linked to a wall polysaccharide composed of GlcN, ManN, Man, Glc, and phosphate.

Purification of an acidic plastocyanin from Microcystis aeruginosa.

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806.

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-beta-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with Mr 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a Mr of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcystis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.

Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China.

Waterbloom samples of the colonial cyanobacterium Microcystis aeruginosa, collected in fish ponds at the Hydrobiological Institute, Wuhan, People's Republic of China, were hepatotoxic to mice. Lyophilized cells had an LD50 (i.p. mouse; 40 mg/kg) and signs of poisoning similar to that reported for other cyanobacterial hepatotoxic peptides. Two toxins, with an LD50 (i.p. mouse) of 40 and 150 micrograms/kg, were isolated using gel filtration and high performance liquid chromatography. The amino acid composition and mol. wt (994) of the 40 micrograms/kg toxin was the same as that for microcystin-LR, while the 150 micrograms/kg toxin had an amino acid composition and mol. wt (1048) different from any of the reported cyanobacteria heptapeptide toxins reported to date.

Toxins contained in Microcystis species of cyanobacteria (blue-green algae).

Cyclic peptide toxins were analyzed for three Microcystis species (M. aeruginosa, M. viridis and M. wesenbergii) using an ODS-silica gel cartridge and high performance liquid chromatography with ODS-silica gel. On strain of M. aeruginosa contained a high amount of microcystin (cyanoginosin) YR and a lesser amount of LR. Three toxins, microcystin-RR, -YR and -LR, were detected in two strains of M. aeruginosa and four of M. viridis. The main component of the toxins of these strains was microcystin-RR. M. wesenbergii showed no peak in the area where the three toxins were obtained in other Microcytisis species by HPLC analysis. LD50 values of the purified toxins of YR and LR were similar, while a lower toxicity was estimated for RR. This explains the relatively weak toxicity of M. viridis whose main component is microcystin-RR.

Properties of two toxins isolated from the blue-green alga Microcystis aeruginosa.

Attempts were made to characterize the two toxins (P-1 and P-2) isolated from the blue-green alga Microcystis aeruginosa, by amino acid analysis, mass spectrometry, 1H- and 13C-NMR. P-2, the major toxin, had a molecular weight of 1044, and consisted of one molecule each of beta-methylaspartic acid, D-Glu, D-Ala, L-Arg, L-Tyr, N-methyldehydroalanine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). P-1, with a molecular weight of 994, appeared to have almost the same composition except that it contained L-Leu instead of L-Tyr in P-2. Mass spectrometric studies, along with a negative ninhydrin reaction, indicated that each toxin was a cyclic peptide. P-2 showed an LD99 of 70 micrograms/kg mice when injected i.p. and [alpha]D24 of -72.42 degrees (c = 0.095 in methanol), and was decomposed at around 218 degrees C.

Injury to hepatocytes induced by a peptide toxin from the cyanobacterium Microcystis aeruginosa.

The freshwater, bloom forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin which causes death accompanied by liver necrosis. We show here that the time and dose-dependent blebbing of isolated hepatocytes is accompanied by the activation of phosphorylase a, with no changes in cyclic AMP levels, and by glutathione (acid-soluble thiols) depletion. These results suggest that the disruption of cytoskeletal structures is accompanied by disturbances in cellular calcium homeostasis and by decreased protection against oxidative damage to the cells.

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.

Toxic substance from a natural bloom of Microcystis aeruginosa.

A toxic substance contained in the blue-green alga Microcystis aeruginosa was purified and partially characterized. Toxic algal cells were collected from a highly eutrophic lake in Japan, and the toxin was purified by homogenization, ultrafiltration, gel filtration, and ion-exchange chromatography. The final preparation gave a single peak on high-performance liquid chromatography. The toxicity was somewhat less than that reported for other toxins from this alga. The water extract of 6.7 mg (dry weight) of cells and 72 microgram of the purified protein was required to kill a mouse (1 mouse unit). The main amino acids of the toxin were glutamic acid, asparatic acid, alanine, glycine, arginine, and leucine. The molecular weight of the toxin was 2,950 as determined by high-performance liquid chromatography.

Configuration assignments of the amino acid residues and the presence of N-methyldehydroalanine in toxins from the blue-green alga, Microcystis aeruginosa.

The configuration assignment of the alpha-carbon atom of amino acid residues in four toxin variants from Microcystis aeruginosa have been made by stereospecific enzymic transformations. The relative conformation assignment of the beta-carbon atom of beta-CH3-aspartic acid could be made by comparison of the electrophoretic mobility with literature values reported for the authentic compound. The presence of an N-methyldehydroalanine residue, which, due to elimination of methylamine under hydrolytic conditions, previously escaped detection by conventional means, has been confirmed by identification of N-methylalanine in the hydrolysate after reduction of toxin with sodium borohydride.

Isolation and characterization of four toxins from the blue-green alga, Microcystis aeruginosa.

Two alternative procedures for the isolation of toxins from the blue-green alga, Microcystis aeruginosa forma aeruginosa, are described. A novel approach is reported, whereby contaminating impurities are succinylated, exploiting the absence of free amino groups in toxin variants. All toxin variants comprise a hydrocarbon blocking group, five amino acid residues detectable by conventional means, while methylamine is liberated upon acid hydrolysis. Possible structural features are discussed relating to the observed chemical and physical properties of the toxins.

Isolation, characterization and pathology of the toxin from a Microcystis aeruginosa (= Anacystis cyanea) bloom.

The nature of the toxicity of a bloom of blue-green alga, M. aeruginosa (= Anacystis cyanea), that occurred in a man-made lake was investigated. Crude algal bloom extracts were toxic to laboratory mice when injected intraperitoneally. The lethal dose (LD100) of these extracts was 15-30 mg of lyophilized algal bloom per kilogram body weight. The toxin was purified by a procedure that included ammonium sulphate fractionation, solvent extraction, acid precipitation, Sephadex G25 and DEAE-Sephadex chromatography, and high-voltage electrophoresis at pH 6.5. The preparation gave a single spot on high-voltage electrophoresis at pH 9.0, had no free amino group, and was characterized by a simple amino acid composition of equimolar quantities of L-methionine, L-tyrosine, D-alanine, D-glutamic acid, erythro beta-methyl aspartic acid and methylamine. The LD50 for the purified toxin was estimated to be 0.056 mg/kg of mice, and the approximate LD100 is 0.070 mg/kg, based on the total material found from amino acid analysis. Parenteral administration of the purified toxin to mice produced extensive liver lobular haemorrhage and death within 1-3 h. Repeated inoculation of sublethal doses daily over some weeks produced progressive hepatocyte degeneration and necrosis and the development of fine hepatic fibrosis.

Diarrhea toxin obtained from a waterbloom-producing species, Microcystis aeruginosA Kützing.

A diarrhea-producing toxin from a blue-green alga, Microcystis aeruginosa Kützing, was obtained from standing laboratory cultures. The non-dialyzable fraction of the lysate from whole cells produced fluid accumulation in the ligated small intestinal loops in guinea pigs.