Benthic cyanobacteria (Oscillatoriaceae) that produce microcystin-LR, isolated from four reservoirs in southern California.

Cyanobacteria that produce the toxin microcystin have been isolated from many parts of the world. Most of these organisms are planktonic; however, we report on several microcystin-producing benthic filamentous cyanobacterial isolates from four drinking-water reservoirs in southern California (USA): Lake Mathews, Lake Skinner, Diamond Valley Lake (DVL), and Lake Perris. Some samples of benthic material from these reservoirs tested positive for microcystin by an ELISA tube assay, and all the positive samples had in common a green filamentous cyanobacterium 10-15microm in diameter. Seventeen unialgal strains of the organism were isolated and tested positive by ELISA, and 11 cultures of these strains were found to contain high concentrations of microcystin-LR (90-432microgL(-1)). The cultures were analyzed by protein phosphatase inhibition assay (PPIA) and HPLC with photodiode array detector (PDA) or liquid chromatography/mass spectrometry (LC/MS). Microcystin per unit carbon was determined for six cultures and ranged from 1.15 to 4.15microgmg(-1) C. Phylogenetic analysis of four cultures from Lake Skinner and DVL using cyanobacterial-specific PCR and sequencing of the partial 16S rRNA gene suggested the highest similarity to an unidentified cyanobacterium in the oscillatoriales, and to a Phormidium sp. Morphologically, some of the isolates were similar to Oscillatoria, and others resembled Lyngbya. The significance of these organisms lies in the relative scarcity of known toxin producers among freshwater benthic cyanobacteria, and also as a source of cell-bound microcystin in these reservoirs.

Characterization of microcystin production in an Antarctic cyanobacterial mat community.

Cyanobacteria are well known for their production of non-ribosomal cyclic peptide toxins, including microcystin, in temperate and tropical regions, however, the production of these compounds in extremely cold environments is still largely unexplored. Therefore, we investigated the production of protein phosphatase inhibiting microcystins by Antarctic cyanobacteria. We have identified microcystin-LR and for the first time [D-Asp3] microcystin-LR by mass spectrometric analysis in Antarctic cyanobacteria. The microcystins were extracted from a benthic microbial community that was sampled from a meltwater pond (Fresh Pond, McMurdo Ice Shelf, Antarctica). The extracted cyanobacterial cyclic peptides were equivalent to 11.4 ng MC-LR per mg dry weight by semi-quantitative analyses using HPLC-DAD and the protein phosphatase inhibition assay. Furthermore, we were able to identify the presence of cyanobacterial non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes in total DNA extracts from the mat community.

Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria.

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain.

Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica.

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds--Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.

Genetic potential for secondary metabolite production in stromatolite communities.

The cyanobacterial communities associated with stromatolites surviving in extreme habitats are a potentially rich source of bioactive secondary metabolites. We screened for the potential for production of bioactive metabolites in diverse species of cyanobacteria isolated from stromatolites in Hamelin Pool, Shark Bay, Australia. Using degenerate primer sets, putative peptide synthetase and polyketide synthase genes were detected from strains of Symploca, Leptolyngybya, Microcoleus, Pleuorocapsa, and Plectonema sp. Sequence analysis indicates the enzymes encoded by these genes may be responsible for the production of different secondary metabolites, such as hepatotoxins and antibiotics. Computer modelling was also conducted to predict the putative amino acid recognised by the unknown adenylation domain in the NRPS sequences. Mass spectral analysis also allowed the putative identification of the cyclic peptides cyanopeptolin S and 21-bromo-oscillatoxin A in two of the isolates. This is the first time evidence of secondary metabolite production has been shown in stromatolite-associated microorganisms.