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Deliverables
Cylindrospermopsis raciborskii (Wolosz.) Seenaya and Subba Raju strain (ACT-9502, Algal Culture Tihany) was isolated from Lake Balaton and maintained in modified BG-11 medium. The sodium nitrate was omitted and the copper content of medium was decreased tenfold because C. raciborskii could not grow at the original copper concentration.
The chemostat experiments were run at 25 degrees Celsius. The cultures were illuminated by cool-white fluorescent tubes with an irradiance of 200mmol m-2 s-1. To investigate the growth dependence of C. raciborskii on the N sources the concentration mineral nitrogen sources of the inflowing medium varied between 0mg/l and 6mg/l. The dilution rate was 0,36/day. When the growth of C. raciborskii was studied as the function of the dilution rate, there was no NO3-N in the medium and the dilution rate ranged between 0,36 and 0,9 day-1.
Our results demonstrated that this cyanobacterium was able to utilise atmospheric nitrogen besides the NO3-N and NH4-N. In our experiments fixation of atmospheric nitrogen decreased when the inorganic nitrogen concentration increased in the inflowing medium. However even at the highest NO3-N concentration the contribution of nitrogen fixation to the total nitrogen uptake was approximately 30%. The Thamnotoxkit FTM was evaluated for detecting cyanobacterial toxins compared with the rat hepatocyte test and mouse test.
This test kit is a 24-hour bioassay using larvae of the freshwater anostracan crustacean Thamnocephalus platyurus hatched from cysts. Thirteen freeze-dried cyanobacterial samples from freshwaters in Hungary, Germany and Brazil, and strains from the Culture Collection of University of Helsinki were tested and analysed for microcystins and anatoxin a by high performance liquid chromatography (HPLC) and in some cases ELISA test. The samples were examined by a double blind approach. The Thamnotoxkit test reacts to pure microcystins, neurotoxins and crude extracts of cyanobacteria. It can be concluded that the Thamnotox test is an alternative simple, cost-effective method that may replace the mouse bioassay used previously for determination of cyanobacterial toxicity.
Within CYANOTOX we focused our effort on obtaining the complete sequences of genes involved in microcystin production (mcy genes) of a Planktothrix strain. We could use our experience obtained by isolating and sequencing the mcy genes of Microcystis aeruginosa strain PCC 7806 (Tillett et al., 2000).
We performed PCR with the primer pairs MTF2 and MTR2 (Neilan et al., 1999) using total genomic DNA of the microcystin producing strain P. agardhii CYA 126/8 as template. The resulting amplicon was cloned in the pGEM-T vector and 20 clones were randomly sequenced. Four different peptide synthetase gene fragments were obtained, one of which showed 75% homology on amino acid level to McyA of M. aeruginosa.
This fragment was used to screen a genomic library of P. agardhii CYA 126/8. The initial phagemid clone encoded a protein with homology to a larger part of McyA from M. aeruginosa, including the very characteristic condensation domain. Subsequently 60kb were sequenced from overlapping phagemid clones spanning the putative microcystin biosynthesis gene cluster and flanking regions The sequence has been deposited in the database (EMBL).
Sequence analysis of the mcy region revealed a cluster of 9 genes that are, according to the results of gene mutation involved in microcystin biosynthesis in P. agardhii. Eight of these genes (mcyA, B, C, D, E, G, H, J) show significant similarity to mcy genes from M. aeruginosa encoding peptide synthetases, polyketide synthases and modifying enzymes.
At the 5'- end of the gene cluster and transcribed in the opposite direction, an additional ORF was found showing homology to genes and gene domains, respectively, encoding thioesterases. The ORF was designated mcyT. It is not contained in the mcy gene cluster of Microcystis. In contrast, two ORFs, present in Microcystis, are missing: the racemase gene mcyF and mcyI, an ORF, having a similarity to D-3-phosphoglycerate dehydrogenases genes.
The arrangement of the mcy genes clearly differs from that in Microcystis. In Microcystis, mcyA-C and McyD-J form two operons that are transcribed bi-directionally from an internal promoter region (Kaebernick et al. 2002). In contrast, the mcy genes of Planktothrix (except mcyT) are all on the same strand and might be transcribed into one polycistronic mRNA.
Based on the sequences of mcy genes obtained from different cyanobacterial genera by the CYANOTOX partners at Humboldt University and the University of Helsinki, primers for PCR could be designed that amplify specifically genes formicrocystin biosynthesis from these species (Neilan et al. 1999, Hisbergues et al. 2002).
For early warning it would be particularly useful to have primers that allow for the sensitive detection of mcy genes, i.e. for the discrimination by PCR between microcystin¿producing strains from non-producing-ones in any relevant genus. We found that PCR with primers designed to amplify part of the condensation domain of mcyA gene is a powerful means to detect microcystin-producing strains in the 3 major producer genera: Microcystis, Anabaena, Planktothrix but also in one microcystin-containing Nostoc strain. Moreover, RFLP analysis of the PCR products and sequencing permit undoubtedly the determination of the genus of the toxic cyanobacteria. The applicability of these primers was tested on natural samples.
Cyanotoxin concentrations (microcystin-LR and total microcystin) in the cyanobacterial water blooms samples (47 in total) that were developed in the Czech Republic during 1993 - 1998 were summarised and compared to the previously reported concentrations from the world literature. More than 87% of all samples were found to be toxic (containing microcystins). The concentrations of microcystins ranged from 0.7 to 5804µg per gram of biomass dry weight (g/g d.w.).
During 1999, 142 samples from 108 localities were collected. Using HPLC, microcystins were analysed in 81 samples from 56 water reservoirs (in which cyanobacterial water blooms developed). 67 samples (83%) were found to be toxic. At least one of three dominant variants of microcystins were detected in the analysed samples (microcystin-RR was detected in 60 samples, -YR in 38 samples and -LR in 66 samples). The total microcystin concentrations ranged from 7-to 6171 g/g d.w. The median of % content of microcystin-LR was 50% (ranging from 16 to 100%). The content of minor microcystin variants was lower than 10% in all samples.
In order to address the objectives of our section of the CYANOTOX project we have investigated some aspects of the toxicity of cyanobacterial blooms in a number of Portuguese water bodies. Our work has involved the routine monitoring of phytoplankton populations, nutrient concentrations and physical conditions in some important water resources to gain a better understanding of the factors influencing the development of potentially toxic cyanobacterial populations and the factors affecting cyanobacterial species succession.
Our work has also aimed at improving our knowledge of the peptides and other toxic compounds within Portuguese potable and recreational water bodies. This aspect of the work has relied on the isolation of strains into pure culture and subsequent toxicity screening by mouse bioassay, HPLC, ELISA and other techniques. The primary focus of the research has been with water bodies affected by blooms of Microcystis, and analysis of isolated Microcystis strains. However, the high diversity and incidence of other cyanobacteria including Aphanizomenon, Anabaena, Lyngbia, Oscillatoria and Cylindrospermopsis has also prompted investigations into the toxicity of these other genera.
Cylindrospermopsin:
Cultures of Cylindrospermopsis raciborskii (6 Australian and 1 Hungarian) were established in the laboratory and cylindrospermopsin was identified in 5 of the Australian strains. An HPLC system, with PDA detection, was established for cylindrospermopsis analysis and one Australian strain, which contained the greatest amount of cylindrospermopsin according to HPLC-PDA, was mass-cultured.
Further analysis of actively growing cultures of Cylindrospermopsis revealed over 50% of the cylindrospermopsin was present in the extracellular, spent medium. To recover this toxin, solid phase extraction methods were successfully developed using C18 and graphitised carbon solid phase extraction cartridges arranged in series. This has provided a simple and effective purification step for cylindrospermopsin, which is not commercially available. Hitherto, the mouse bioassay has been used for cylindrospermopsin toxicity assessment and alternative toxicity assays were explored. The brine shrimp Artemia salina was found to be susceptible to this toxin and permitted statistically relevant toxicity assessment (Metcalf et al. 2002b). This procedure is advocated for the bioassay of Cylindrospermopsis blooms in European waters.
Neosaxitoxin:
Neosaxitoxin was purified from a neosaxitoxin-producing cyanobacterial strain of Aphanizomenon flos-aquae. Polyclonal antibodies were produced against the toxin. Prior addition of the antibodies to neosaxitoxin from Aphanizomenon in vitro protected mice from the action of this toxin in bioassay. An ELISA was developed and this was applied to the analysis of saxitoxins from environmental samples of cyanobacteria. In addition, bacterial isolates from the neosaxitoxin-producing cyanobacterial strain were analysed for saxitoxins and some isolates were positive according to ELISA.
The cyanobacterium Planktothrix agardhii is often found in shallow lakes and can be toxic. In this study, the effect of light intensity on the production of toxins (microcystin RR and LR) of P. agardhii was investigated.
Much of the research on the effects of environmental factors on toxin production has been performed in batch cultures. This complicates the results as many factors are changed during the growth of the cyanobacteria in the cultures. To investigate only one factor continuous cultures have to be used.
The Continuous cultures used were specially designed for light limited growth, i.e. the vessels are flat which facilitates precise measurement of the average light intensity (Huisman et al., in press). The turbidostat technique was used: the pump was adjusted to keep the optical density (at 750nm) constant at about 0.01.
The light:dark period was 12:12. During a steady state, samples were taken at five days at the same time (about one hour after the light has been switched on). A good relation was found between the light conditions and the production of toxins. Maximal toxin productions were found at a light intensity of 60µEm-2s-1.
The effects of light on the regulation of microcystin production can be detected on an earlier level than the microcystin content by detection of RNA transcripts. We found that both the transcript levels and the microcystin production are increasing with increasing photon irradiance. Only the highest growth irradiance showed a low transcript level while the production was very high. This is probably due to the fact that the growth rate in this high light culture was overestimated. From the results it can be concluded that light affects the transcript of the mcy gene directly.
A MALDI-TOF MS method for the quantification of microcystins was developed. With the respective internal standard this could be accomplished, however, culture studies showed that recovery decreases with increasing cell density, probably due to quenching effects. This problem can be solved by substantial dilution of the samples, but in combination with the envisaged simple extraction procedure of drying 1 ml lake water, this resulted in a detection limit of 3 µg/l.
This must be considered as too high for a MS method and does not fully exploit the sensitivity of MALDI-TOF MS. Extraction of enriched cells on filters resolved this problem, but miniaturisation of the sample preparation should be examined as well. The method developed so far was successfully applied on field material from Lake Wannsee, 1999.
Comparison with high-performance liquid chromatography coupled with photodiode array detection (HPLC-PDA) analysis revealed a good agreement between the two methods, except for some samples where concentrations obtained with HPLC were twice compared to MALDI-TOF MS. This might be explained by low peak purity and thus overestimation of concentrations by HPLC-PDA. Furthermore, MALDI-TOF MS has been successfully applied in the analysis of other cyanotoxins such cylindrospermopsin and anatoxin-a.
Development of new immuno-methods for microcystin detection and analysis and characterization of existing ELISA methods. New antibodies to microcystin-LR were produced after conjugating the toxin to Keyhole Limpet haemocyanin. Immunisation of this conjugate resulted in the production of polyclonal antibodies against microcystin-LR and an enzyme-linked immunosorbent assay (ELISA) was subsequently developed. The ELISA used was sensitive enough to detect microcystin-LR at a concentration of below 1 mg l-1 in accordance with the provisional guideline value for drinking water of the World Health Organisation.
The cross-reactivity of the antibodies used in the ELISA was performed by comparison of reactivity of microcystin-LR with 5 other microcystins (-LA, -LY, -LW, -LF, -RR) and the related cyanobacterial pentapeptide toxin, nodularin. All purified toxins used were found to cross-react with the antibodies to varying degrees, the maximum difference in the 50% binding concentration being three-fold. The ELISA procedure was further extended to the analysis of microcystin-containing cyanobacterial strains and environmental samples of cyanobacteria. The analysis of the strains and environmental samples for microcystins by ELISA was compared to analysis by HPLC and showed good correlation (r = 0.71; n = 22). This procedure contributed to the analysis of microcystin pools in the laboraory investigations of the Amsterdam and Oslo partners and throughout the joint 2001 Wannsee Workshop.
The genes for the synthesis of anabaenopeptilides and microcystins have been fully sequenced and characterised. Anabaenopeptilide synthetase work has been published (Rouhiainen et al., 2000) and the manuscript of microcystins synthetase in Anabaena 90 is in preparation.
Approximately one half of the anabaenopeptin genes are sequenced. The anabaenopeptilide synthetase operon was about 30kb and consisted of six ORFs (apdA-F) and seven amino activating domains corresponding to the seven amino acids contained by anabaenopeptilides. The operon contained two specific features formyl transferase coding region and halogenase gene (Rouhiainen et al., 2000).
The microcystin synthetase of Anabaena 90 contains two sets of genes, which are transcribed in the opposite directions. One code for the peptide synthetases (genes mcyA, mcyB and mcyC), the other code for the polyketide synthase (mcyD) or the combined polyketide synthase/peptide synthetase (mcyE and mcyG), the modifications (mcyF, mcyI, mcyJ) and the putative transporter (mcyH).
The sequence similarity to the genes of Microcystis PCC7806 is from 70 % to 80 %. The order of the genes was first determined for Microcystis PCC7806 to be from mcyA to mcyC for the first set of genes and from mcyD to mcyI for the second set. In Anabaena 90, the order of the second set is different: mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI and mcyH.
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