Brendan P. Burns, Martin L. Saker, Michelle C. Moffitt, and Brett A. Neilan
Cyanobacteria are ubiquitous in the freshwater environment. Their success as a group in a wide range of aquatic habitats has been attributed to their unique physiological characteristics and their high adaptive ability over a wide range of environmental conditions. They are capable of reaching very high biomass levels, often dominating the other aquatic biota, and under some circumstances can accumulate near the water surface, producing scums. Such cyanobacterial "blooms" are of particular concern in reservoirs used to supply potable water. Dense aggregations of cyanobacterial cells may block water filters, and many species produce compounds that affect the taste and odor of water supplies. Of greatest concern, however, is the potential of many bloom-forming cyanobacteria to produce a wide range of toxic substances. These natural compounds, known as cyanotoxins, are chemically diverse and are usually either neuro- or hepatotoxic in pathology.
1.1. Chemical and Toxicological Diversity of Microcystins, Nodularin, and Cylindrospermopsin
Among the most common hepatotoxins encountered in the freshwater environment are the cyclic heptapeptide toxins (containing seven peptide-linked amino acids) known as microcystins (MCYSTs). Over 60 variants of MCYSTs have been found to date, most with median lethal dose (LD50) values (ip mouse bioassay) from 50 to 500 ^g/ kg and molecular weights ranging from 800 to 1100 (1). The general structure of MCYST is given in Fig. 1A. Most of the structural variants of MCYST are formed by the substitution of l-amino acids at positions 2 or 4, or by the demethylation of amino acids at positions 3 and/or 7 (Fig. 1A) (1). The MCYSTs are produced by several
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
genera of cyanobacteria including Microcystis, Anabaena, Planktothrix, Nostoc, Hepalosiphon, and Anabaenopsis (1) and have caused death in humans and other animals (2-5). The MCYSTs have also been shown to be potent tumor promotors in rats and have been linked to liver cancer in humans (6,7).
A compound with similar chemical structure and also similar toxicological properties has been identified from the brackish water cyanobacterium Nodularia. The compound termed nodularin is a pentapeptide (containing five peptide-linked amino acids).
To date, only two toxic variants of nodularin have been reported, one with a demethylated amino acid at position 1 and the other with a demethylated amino acid at position 3 (Fig. 1B) (1). Nodularin has been implicated in the death of domestic animals (8).
Another cyanotoxin of increasing concern throughout the world is cylindrospermopsin (Fig. 1C). This hepatotoxic compound is a cyclic guanidine alkaloid with a molecular weight of 415 and LD50 (ip mouse bioassay) of 200 ^g/kg. Only one toxic structural variant of cylindrospermopsin (named 7-epicylindrospermopsin) has been reported (9). This compound differs from cylindrospermopsin by the lack of the 7-hydroxy function and also by differences in the uracil nucleus. Cylindrospermopsin has been implicated in an outbreak of human gastroenteritis (10,11) and mortality in cattle (12) and has been shown to display carcinogenic activity (13). Cylindrospermopsin is produced by several genera of cyanobacteria including Anabaena, Aphanizomenon, Cylindrospermopsis, Umezakia, and Raphidiopsis (14-16).
1.2. Variation in Toxicity of Cyanobacterial Isolates at the Subspecies Level
The production of MCYST, nodularin, and cylindrospermopsin by potentially toxic strains of cyanobacteria is complicated by the fact that toxin production not only differs spatially and temporally within a bloom population, but also between morphologically indistinct strains within a single population (1). Until very recently evaluation of strain toxigenicity was not possible since microscopic examination of a bloom sample does not provide information on toxigenicity or the toxic bloom-forming potential of a particular strain present. Recent developments, using molecular techniques, have resulted in the identification of the genes responsible for the production of some of the most commonly occurring freshwater cyanotoxins, namely, microcystins (17,18), nodularin (19), and cylindrospermopsin (16). These methods, based on poly-merase chain reaction (PCR), are of rapidly increasing use in the identification of potentially toxic cyanobacterial bloom populations and will in the future be important tools for the management of water quality.
In this chapter we detail methods for cyanobacterial DNA extraction followed by PCR methods used for the molecular detection of microcystin, nodularin, and cylindrospermopsin toxigenicity from laboratory-cultured cyanobacterial cells. It should be noted that the following techniques are also applicable to natural bloom samples (consisting of multiple genotypes) and can also be adapted for the use of single cyanobacterial colonies/trichomes/filaments as the DNA template for PCR.
Caution: All cyanobacterial culture material should be treated with care because of the potential risk of serious adverse acute and long-term effects resulting from inhalation or skin exposure. Wear gloves at all times, and when dealing with lyophilized material, manipulation should be carried out under ventilation.
For the following procedures, all chemicals should be of analytical grade quality.
Characteristics of Oligonucleotide Primers Used
Characteristics of Oligonucleotide Primers Used
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