Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization.

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.

Gas vesicle genes in Planktothrix spp. from Nordic lakes: strains with weak gas vesicles possess a longer variant of gvpC.

In cyanobacteria of the genus Planktothrix:, there are three length variants of gvpC, the gene that encodes the outer protein of the gas vesicle. Sequence analyses indicated that the three allelic variants of gvpC differ principally in the presence or absence of a 99 nt and a 213 nt section. Strains with the new variant, gvpC(28), which encodes a 28 kDa form of GvpC, produce gas vesicles that collapse at the relatively low critical pressure (p(c)) of 0.61-0.75 MPa. The authors have identified 12 classes of gvp genotypes that differ in the number and arrangement of alternating gvpA-gvpC genes and in the presence of OmegaC, a fragment of gvpC. The gvpC(28) gene was found to be the most common variant of gvpC amongst 71 strains of Planktothrix: isolated from Nordic lakes: 34 strains contained only gvpC(28); 22 strains, which possessed only the shorter gvpC(20) gene, produced gas vesicles with a higher p(c) of 0.76-0.91 MPa; and 15 strains, which possessed both gvpC(20) and gvpC(28), also produced the stronger gas vesicles. Genotypes with only the gvpC(28) genes were more common amongst green Planktothrix: strains (33 out of 38) than red strains (one out of 33). It is suggested that there is competition between the strains producing the two types of gas vesicles, with the stronger forms favoured in lakes deeper than 60 m, in which the combination of cell turgor pressure and hydrostatic pressure can collapse the weaker gas vesicles. The fact that none of the Nordic lakes are deeper than 67 m would explain the absence of the gvpC(16)-containing strains that produce even narrower gas vesicles of p(c) 1.0-1.2 MPa, which are common in the much deeper Lake Zürich.

Antibacterial properties of extracts from selected planktonic freshwater cyanobacteria--a comparative study of bacterial bioassays.

Aqueous and methanol extracts from five selected cyanobacteria were examined for antibacterial properties in six different bacterial bioassays. All five cyanobacteria revealed antibacterial properties. Methanol extracts made from Tychonema bourrellyi, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii showed the most pronounced inhibitory effects, Aqueous extracts made from Microcystis aeruginosa and T. bourrellyi possessed evident antibacterial properties. The bacterial bioassays were based on agar diffusion tests and included pour-plate methods commonly used to detect residues of antibacterial substances in food. In addition, a pourplate bioassay with Aeromonas hydrophila was developed and described. Antibacterial effects were observed in five of the six bacterial bioassays. No antibacterial effect was observed in the Micrococcus luteus bioassay. Bioassays based on Aer. hydrophila, Bacillus cereus and B. subtilis grown in Antibiotic Medium 8, pH5.85, seemed to be sensitive and suitable. The MIC value of diluted MeOH extracts made from C. raciborskii and T. bourrellyi against Aer. hydrophila corresponded to 38 mg freeze-dried cyanobacteria. Bacillus subtilis was more sensitive when grown in a culture medium with pH 5.85 than 7.9. The antibacterial properties of extracts from the cyanobacteria examined differed from defined cyanotoxins and antibacterial substances. The pattern of inhibition in the bacterial bioassays indicated that various antibacterial substances are involved.

Quantification of toxic cyanobacteria in water by use of competitive PCR followed by sequence-specific labeling of oligonucleotide probes.

A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genus Microcystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

Evolution of cyanobacteria by exchange of genetic material among phyletically related strains.

The cyanobacterial radiation consists of several lineages of phyletically (morphologically and genetically) related organisms. Several of these organisms show a striking resemblance to fossil counterparts. To investigate the molecular mechanisms responsible for stabilizing or homogenizing cyanobacterial characters, we compared the evolutionary rates and phylogenetic origins of the small-subunit rRNA-encoding DNA (16S rDNA), the conserved gene rbcL (encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit), and the less conserved gene rbcX. This survey includes four categories of phyletically related organisms: 16 strains of Microcystis, 6 strains of Tychonema, 10 strains of Planktothrix, and 12 strains of Nostoc. Both rbcL and rbcX can be regarded as neutrally evolving genes, with 95 to 100% and 50 to 80% synonymous nucleotide substitutions, respectively. There is generally low sequence divergence within the Microcystis, Tychonema, and Planktothrix categories both for rbcLX and 16S rDNA. The Nostoc category, on the other hand, consists of three genetically clustered lineages for these loci. The 16S rDNA and rbcLX phylogenies are not congruent for strains within the clustered groups. Furthermore, analysis of the phyletic structure for rbcLX indicates recombinational events between the informative sites within this locus. Thus, our results are best explained by a model involving both intergenic and intragenic recombinations. This evolutionary model explains the DNA sequence clustering for the modern species as a result of sequence homogenization (concerted evolution) caused by exchange of genetic material for neutrally evolving genes. The morphological clustering, on the other hand, is explained by structural and functional stability of these characters. We also suggest that exchange of genetic material for neutrally evolving genes may explain the apparent stability of cyanobacterial morphological characters, perhaps over billions of years.

Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection.

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.

The effect of water soluble cyanotoxin(s) produced by two species of Anabaena on the release of acetylcholine from the peripheral cholinergic nervous system of the rat airway.

A water extract of the lyophilised fresh-water alga Anabaena flos-aquae enhanced substantially the release of [(3)H]acetylcholine ([(3)H]acetylcholine and [(3)H]choline) from cholinergic nerves of rat bronchi. Parallel experiments performed with the related species Anabaena lemmermannii did not demonstrate this effect. The effect on the release of [(3)H]acetylcholine by A. flos-aquae extract was concentration dependent. The A. flos-aquae induced [(3)H]acetylcholine release was not reduced by exposure to a low concentration of Ca(2+), but ω-conotoxin GVIA (1.0 µM), a blocker of N-type Ca(2+) channels reduced the release of [(3)H]acetylcholine induced by the A. flos-aquae extract. Addition of verapamil in a concentration (1.0 µM) specific for inhibition of L-type Ca(2+) channels had no effect on the neurotransmitter release. A reduction in the release was, moreover, observed with the intracellular Ca(2+) chelator BAPTA/AM (30 µM) and with the Na(+) channel blocker tetrodotoxin (3.0 µM). During patch-clamp studies of GH(4)C(1) neuronal cells, which have L- and T-type Ca(2+) channels, but no Na(+) channels, it was shown that a water extract of A. flos-aquae depolarised these cells and reduced, rather than enhanced, the influx of Ca(2+). Such an effect was not seen following exposure of GH(4)C(1) cells to water extracts of A. lemmermannii. In addition to its presynaptic activity, the water extract of A. flos-aquae showed an antimuscarinic effect by displacing [(3)H]QNB binding from muscarinic receptors in homogenates of rat bronchi. A similar but more potent effect was observed during experiments with water extract of A. lemmermannii. None of the respective water extracts showed any effects on cholinesterase activities in rat bronchial smooth muscle. The present observations suggest, therefore, that water extracts of A. flos-aquae may depolarise cells by activation of mono and divalent cation channels in cholinergic nerve cells. These channels are probably Na(+) channels and N-type, but not L- or T-type Ca(2+) channels. L- and T-type Ca(2+) channels were blocked in experiments with GH(4)C(1) cells and high concentrations of Ca(2+) channel blockers were necessary to reduce the effects of A. flos-aquae extract in cholinergic nerves in the airways. Furthermore, A. flos-aquae extract may also mobilise Ca(2+) from intracellular compartments. A. lemmermannii, on the other hand, does not contain components which alter mono and divalent cation-fluxes across cell membranes, but may rather have substances with more potent antagonistic effects on muscarinic cholinergic receptors than what is observed in experiments with A. flos-aquae.

Effects of a homoanatoxin-a-containing extract from Oscillatoria formosa (Cyanophyceae/cyanobacteria) on neuromuscular transmission.

Experimental investigations were carried out with cultured and lyophilized material of the toxigenic strain Oscillatoria NIVA-CYA 92. This organism is classified as Phormidium formosum (Boryex Gom.) Anagnet kom. Aqueous extracts of the algal material, containing the bioactive secondary amine alkaloid 2-(propan-1-oxo-1-yl)-9-azabicyclo(4,2,1)non-2ene (homoanatoxin-a) in an amount of 2.57 micrograms/mg lyophilized material, were tested for acute in vivo toxicity in mice, and for toxicity on neuromuscular transmission by means of electrophysiological methods on the isolated phrenic-nerve hemidiaphragm from rat and in the frog rectus abdominis assay. Acute toxic effects in mice were observed by i.p. and oral (by gavage) administration. Lethal doses were in the range 112-225 and 1125-2250 mg of freeze-dried algal material per kg body weight (i.e. 288-578 and 2890-5780 micrograms homoanatoxin-a/ kg body weight), respectively. The nerve-initiated muscle contractions in the rat diaphragm were blocked by about 0.125 mg cyanophyte material per ml bath solution (i.e. 0.32 microgram homoanatoxin-a/ml or 1.8 microM), but muscle contractions, although slightly reduced, could still be elicited by direct electrical stimulation of the muscle. The compound action potentials recorded from the main phrenic-nerve trunk were not affected. An additive blocking effect on partly curarized preparations was observed and cholinesterase inhibition by physostigmine (eserine) transiently augmented the muscle twitch contraction in preparations partly blocked by the extract. Intracellular recordings from single muscle fibers of homoanatoxin-a-treated rat hemidiaphragm disclosed a partial depolarization and a decrease in the endplate potential to subthreshold level simultaneously with a decrease and then complete disappearance of the miniature endplate potentials. The neuromuscular transmission block was reversed by washing. The extract produced muscle contractions in the frog rectus abdominis assay. Homoanatoxin-a in the algal material was readily absorbed from the gastrointestinal tract in mice. Blockade of the neuromuscular transmission of the respiratory muscle may partly explain the acute toxic effects observed in mice. Thus, the main target of the homoanatoxin-a action at the mammalian neuromuscular junction was traced to the postsynaptic nicotinic acetylcholine receptor channel complex, where it reduced the sensitivity to the transmitter substance.

Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8.

A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.

Enhancement of acetylcholine release by homoanatoxin-a from Oscillatoria formosa.

The strain NIVA-CYA 92 of Oscillatoria formosa Bory ex Gormont produces phycotoxins with neurotoxic properties. Chemical analysis by gas chromatography/mass spectrometry of a water extract of lyophilized material of the organism showed the presence of only homoanatoxin-a. The mechanism of action of homoanatoxin-a on peripheral cholinergic nerves is so far not known. The neurotoxicity of O. formosa containing homoanatoxin-a was investigated in rat bronchi, rat brain synaptosomes and in GH(4)C(1) cells. The water extract of lyophilized material of the organism produced a concentration-dependent reversible increase in the release of [(3)H]acetylcholine from both K(+) (51 mM) depolarised and non-depolarised cholinergic nerves of the rat bronchial smooth muscle. The K(+)-evoked release of [(3)H]acetylcholine was enhanced by about 75% by a water extract from 15-20 mg/ml of lyophilized algal material. The enhanced release of [(3)H]acetylcholine was substantially reduced by the L-type Ca(2+)-channel blocker verapamil (100 µM) and not by the N-type Ca(2+)-channel blocker ω-conotoxin GVIA (1.0 µM) or the P-type Ca(2+)-channel blocker ω-agatoxin IV-A (0.2 µM). Chelation of intra-cellular Ca(2+) by 1,2-bis-(aminofenoxi)etan-N,N,N',N'-tetraacidic acid/acetoxymethyl (BAPTA/AM) (30 µM) had no effect on the phycotoxin-induced release of [(3)H]acetylcholine, indicating that an extracellular pool of Ca(2+) was important for the action of the phycotoxin on the release of [(3)H]acetylcholine from peripheral cholinergic nerves. In rat brain synaptosomes the algal extract enhanced the influx of (45)Ca(2+) in a tetrodotoxin (1.0 µM) and ω-conotoxin MVIIC (blocker of N-, P- and Q-type Ca(2+) channels) (1.0 µM) insensitive manner. Patch-clamp studies showed that the phycotoxin opened endogenous voltage dependent L-type Ca(2+) channels in neuronal GH(4)C(1) cells. These Ca(2+) channels and the effect of the toxin on the channels were blocked by the L-type Ca(2+)-channel antagonist gallopamil (200 µM). The present results suggest, therefore, that the investigated strain of O. formosa contains homoanatoxin-a, which enhances the release of acetylcholine from peripheral cholinergic nerves through opening of endogenous voltage dependent neuronal L-type Ca(2-) channels.

Two methyl ester derivatives of microcystins, cyclic heptapeptide hepatotoxins, isolated from Anabaena flos-aquae strain CYA 83/1.

Cultured cells of Anabaena flos-aquae strain CYA 83/1, isolated from Lake Edlandsvatn, Norway, produced two microcystin mono-methyl ester derivatives (1 and 2) at the D-Glu unit in addition to microcystin-LR (3), [D-Asp3]microcystin-LR (4), microcystin-RR (5), and [D-Asp3]microcystin-RR (6). Structures of these compounds were assigned based on their amino acid analysis with a Waters Pico Tag HPLC system plus fast atom bombardment mass spectrometry (FABMS), including tandem FABMS, analysis on the two new microcystins, [D-Glu(OCH3)6]microcystin-LR (1) and [D-Asp3, D-Glu(OCH3)6]microcystin-LR (2). Toxicity data were not obtained for 1 and 2 because of the small amounts isolated from the cells.

7-Desmethyl-microcystin-RR, a hepatotoxin from a waterbloom of Microcystis aeruginosa.

A peptide toxin was isolated from a waterbloom of Microcystis aeruginosa from Lake Frøylandsvatn in Norway. The isolation procedure included liquid and solid phase extraction and reversed phase high performance liquid chromatography. Amino acid analysis yielded D-glutamic acid, D-erythro-beta-methylaspartic acid and D-alanine in equimolar and L-arginine in twofold molar ratios. The presence of dehydroalanine was confirmed by hydrogenation and subsequent amino acid analysis with combined gas liquid chromatography/mass spectrometry. Investigation of the toxin with fast atom bombardment mass spectrometry showed a nominal relative molecular mass of 1023. 3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyl- 4,6-decadienoic acid (Adda) was identified by 1H NMR and 1H, 1H COSY spectroscopy. The structure of the toxin was elucidated as 7-desmethyl-microcystin-RR.

Structural characterization of toxic cyclic peptides from blue-green algae by tandem mass spectrometry.

Combined use of chemical degradation, derivatization, and tandem mass spectrometry for rapid structural characterization of toxic cyclic peptides from blue-green algae at the nanomole level is described. Previously, all blue-green algal toxins were thought to belong to a family of seven-residue cyclic peptides, having the general structure cyclo-D-Ala-L-Xaa-erythro-beta-methyl-D-isoaspartic acid-L-Yaa-Adda-D-isoglutamic acid-N-methyldehydroalanine, where Xaa and Yaa represent variable amino acids of the L configuration and Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid. Structural characterization of two additional toxins indicates that further variability can exist within this family of naturally occurring toxic cyclic peptides. Isoaspartic acid and dehydroalanine can substitute for beta-methylisoaspartic acid and N-methyldehydroalanine, respectively.

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.

Effect of Photon Fluence Rate and Specific Growth Rate on Geosmin Production of the Cyanobacterium Oscillatoria brevis (Kütz.) Gom.

The production of geosmin from the cyanobacterium Oscillatoria brevis was studied as a function of the photon fluence rate and appears to be related to the chlorophyll content.