PKS, polyketide synthetase; PS, peptide synthetase.

PKS, polyketide synthetase; PS, peptide synthetase.

2.1. Cyanobacterial DNA Extraction

1. TNE buffer: 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, and 50 mM NaCl.

2. 50 mg/mL Lysozyme.

3. 10 mg/mL Proteinase K.

4. 10% Sodium dodecyl sulfate.

5. Phenol/chloroform/isoamylalcohol (25:24:1).

6. 4 M Ammonium acetate.

7. Isopropanol.

9. Agarose and gel electrophoresis equipment.

2.2. Polymerase Chain Reaction

1. GeneAmp® PCR System 2400 (Perkin Elmer).

2. PCR reaction buffer: 67 mM Tris-HCl, pH 7.6, 16 mM (NH4)SO4, 0.45% Triton X-100, 0.2% gelatin.

4. 200 ^M Deoxyribonucleotide triphosphates.

5. Forward/reverse primers (Table 1).

6. Taq DNA polymerase.

7. Sterile deionized H2O.

8. Agarose and gel electrophoresis equipment.

3. Methods

3.1. Cyanobacterial DNA Extraction

Total genomic DNA can be extracted from lyophilized (or fresh) cyanobacteria using a modification of a technique for purification of DNA from Gram-negative bacteria (20).

1. Suspend 5-10 mg of lyophilized cyanobacterial cells (or fresh culture equivalent) in 500 ^L of TNE buffer. Vortex for 10 s and then add 10 ^L of lysozyme (50 mg/mL). Incubate the solution at 55°C for 30 min.

2. Add 10 ^L proteinase K (10 mg/mL) and 20 ^L of 10% sodium dodecyl sulfate. Incubate at 55°C for 10 min, or until the solution has cleared (complete cell lysis; see Note 1).

3. Chill the solution on ice for 10 min and extract twice with an equal volume of phenol/ chloroform/isoamylalcohol (25:24:1). Vortex the solution for 10 s and centrifuge at 12,000g for 6 min.

4. Add the supernatant to an equal volume of 4 M ammonium acetate.

5. Precipitate total genomic DNA by addition of 2 vol of isopropanol followed by centrifu-gation (12,000g) for 12 min at room temperature. Remove the supernatant, taking care not to make contact with the DNA pellet, and air-dry. Then resuspend the genomic DNA in 40 ^L of sterile H2O.

6. Test the purity and concentration of the DNA by gel electrophoresis using a 1.5% gel and following standard gel electrophoresis protocols (21). Maintain the DNA (which can be diluted to the appropriate concentration with sterile deionized H2O) under refrigeration for the PCR analyses described below.

3.2. Polymerase Chain Extraction

The following is the standard PCR protocol used for cyanobacterial toxin analyses, with the only variations being the specific primers used and their respective annealing temperatures, which are listed in Table 1. Small-volume PCR is performed for rapid and highly specific generation of DNA molecules.

1. PCR reactions are performed in a 20-^L reaction volume, and thermal cycling is conducted using a GeneAmp PCR System 2400. The reaction mix consists of reaction buffer (67 mM Tris-HCl, 16 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 % gelatin), 2.5 mM MgCl2, 200 ^M deoxyribonucleotide triphosphates, ~1 ng chromosomal DNA template (see Subheading 3.1.), 1 pmol forward and reverse primers and 0.5 U Taq DNA polymerase. The final reaction volume is adjusted to 20 ^L with sterile deionized H2O.

2. Initial denaturation of template DNA is achieved by incubation at 95°C for 2 min, and standard reactions are then subjected to 30 cycles of 95°C for 1.5 min, primer annealing between 49 and 60°C (Table 1), and extension at 72°C for 50 s. A further 7-min extension is carried out at the end of the 30 cycles for each PCR.

3. As a positive control, cyanobacterial-specific 16S rRNA primers are used for each sample amplifying the region 27-809 (E. coli designation) (see Notes 2-5).

3.3. Indentificaiton of Toxin Genes and Interpretation of Results 3.3.1. Microcystis and Microcystins

The recent identification of the locus responsible for microcystin synthesis in Microcystis aeruginosa (17,18) has shown that microcystin synthetase is a member of the non-ribosomal peptide synthetase family (17,22). Microcystin was the first cyanobacterial peptide for which a non-ribosomal synthesis by the thio-template mechanism was discovered (23). Insertional mutagenesis of one of these putative microcystin synthetase genes from the Microcystis strain PCC 7806 led to the complete loss of all microcystin variants, thus providing evidence for a function of this gene in microcystin biosynthesis (17). Subsequently, the entire microcystin synthetase


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