Olav M. Skulberg

NAMING OF CYCLIC HEPTAPEPTIDE TOXINS OF CYANOBACTERIA (BLUE-GREEN-ALGAE)

UNIV ILLINOIS,COLL VET MED,URBANA,IL 61801; USA,MED RES INST INFECT DIS,DIV PATHOPHYSIOL,FREDERICK,MD 21701; UNIV NEW ENGLAND,DEPT BIOCHEM MICROBIOL & NUTR,ARMIDALE,NSW 2351,AUSTRALIA; UNIV ALBERTA,DEPT BOT,EDMONTON T6G 2E1,ALBERTA,CANADA; MEIJO UNIV,FAC PHARM,TEMPA KU,NAGOYA,AICHI 468,JAPAN; CHEM RES & DEV CTR,ABERDEEN,MD 21701; NATL BOT GARDENS,CLAREMENT,SOUTH AFRICA; ACAD SINICA,INST HYDROBIOL,WUHAN,PEOPLES R CHINA; NORWEGIAN INST WATER RES,OSLO 3,NORWAY; UNIV HAWAII MANOA,DEPT CHEM,HONOLULU,HI 96822; UNIV ILLINOIS,SCH CHEM SCI,URBANA,IL 61801; UNIV CALIF LOS ANGELES,DEPT MICROBIOL,LOS ANGELES,CA 90024; TOKYO METROPOLITAN RES LAB PUBL HLTH,SHINJUKU KU,TOKYO 160,JAPAN

Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.

Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization

ABSTRACT 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.

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.

Microalgae as a source of bioactive molecules : experience from cyanophyte research

Detrimental and beneficial properties of prokaryoticand eukaryotic microalgae may be qualifications givingthem posibillities for their biotechnologicalutilization. Phycotoxins and related products mayserve as material for useful drugs. Cyanotoxins arean example. Bioassay screening methods are used todetect specific biological activity (e.g. antibioticeffects). A study demonstrating antibacterialproperties among cyanophytes showed that these effectswere caused by substances produced by both toxigenicand non-toxigenic strains. Differences inbiosynthetic activity are influenced by the type ofclones and by the culture conditions (genetic andbiochemical variation). Experience of recentscreening (1997–1998) of compounds from cyanophytes –including 25 species from 14 genera – display thediscovery of more than 30 different antibioticsubstances. The further isolation of newpharmaceuticals from natural resources is becomingdifficult, if not uncommon species of producerorganisms are taken into consideration.

Optical purity of (3S,3'S)-astaxanthin from Haematococcus pluvialis

Abstract Haematococcus pluvialis cultivated in a N-deficient medium produced astaxanthin (1 % of total carotenoids), the monoester (76 %) and diester (7 %) of astaxanthin, β,β-carotene (1 %), an adonirubin ester (3 %), (3 R ,3′ R ,6′ R )-lutein (7 %), violaxanthin (2 %) and neoxanthin (1 %). The CD values of the mono- and diesters of astaxanthin, the HPLC properties of astaxanthin monoester further esterified with (−)-camphanic acid and the optical purity of astaxanthin [determined by HPLC analysis of the diester of(−)-camphanic acid] produced by saponification of the natural mono- and diesters of astaxanthin in the absence of oxygen showed that this green alga synthesizes pure (3 S ,3′ S )-astaxanthin esters.

Algal Problems Related to the Eutrophication of European Water Supplies, and a Bio-Assay Method to Assess Fertilizing Influences of Pollution on Inland Waters

It has been said that Europe is almost as complicated physically as it is politically. Although the European continent is the smallest principal division of the land surface of the globe distinguished by this term, the area still amounts to more than 10 million km2. Situated between 71 °N (Norway) and 36°N (Spain) and from 9°W (Portugal) to 66°E (Ural), deeply penetrated by the sea and with several great mountain ranges, the consequence is an outstanding variation in physical features and climatic contrasts. The hydrographic relations are correspondingly varied. The openness of Europe to oceanic influences and the topography give the landscape a multiplicity of rivers and lakes. But there are also great areas with small amounts of annual rainfall and with surface waters of sparse occurrence. Generalizations are misleading, but it is not wrong to state that almost all types of aquatic biotopes are represented, giving support to the most varied types of algal communities.

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.

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn, Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 µg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).

The first identification of the rare cyanobacterial toxin, homoanatoxin-a, in Ireland

The first identification of the rare cyanobacterial neurotoxin, homoanatoxin-a, in Ireland is reported. A sensitive fluorimetric liquid chromatographic (LC) method was applied to the analysis of homoanatoxin-a in the low microg/l range. The analysis of the anatoxins in water samples required weak cation exchange solid phase extraction, fluorimetric derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and isocratic reversed-phase LC. Confirmation of toxin identity was made using LC with electrospray mass spectrometry (MS) of the NBD-derivatised homoanatoxin-a as well as LC-MS/MS of the free toxin. Application of the fluorimetric LC protocol to examine cyanotoxins in 20 Irish lakes resulted in the detection of homoanatoxin-a at four locations, Lough Sillan (24 microg/l), Inniscarra Reservoir (34 microg/l), Lough Key (12 microg/l), Caragh Lake (1.4 microg/l). An improved procedure for the isolation of homoanatoxin-a from cyanobacteria was also developed and confirmation of homoanatoxin-a was achieved by chromatographic and mass spectral comparison with authentic toxin isolated from a laboratory clone culture of Planktothrix (formerly Oscillatoria) formosa.