T Nt T T T T T Nt

Fig. 2. PCR amplification of the N-methyl transferase region of microcystin synthetase from an environmental bloom (22). Samples that proved toxic (T) or nontoxic (NT) by the phosphatase inhibition assay are indicated.

(mcy) gene cluster of the strain PCC 7806 has been sequenced. It comprises 10 genes, encoding peptide synthetases, polyketide synthases, and tailoring functions. Two large bidirectionally transcribed operons (A-C and D-J) span a region of 55 kb. Further knockout mutations have confirmed the involvement of other mcy genes in microcystin biosynthesis (15) (see Fig. 2 for sample results).

3.3.2. Nodularia and Nodularins

Nodularin is a peptide thought to be produced via a large multienzyme complex made up of peptide synthetase and polyketide synthase enzymes, similar to the biosynthesis of microcystin. Recent studies by Moffit and Neilan (19) have identified peptide-synthetase (PS) and polyketide synthetase (PKS)-like gene regions in Nodularia. The corresponding PCR using this toxic Nodularia-specific primer identified all nodularin-producing N. spumigena strains within the toxic bloom-forming cluster and was unable to amplify nontoxic Nodularia strains and toxic strains from other genera (Fig. 3).

Specific primers have also been designed to detect the presence of nodularin syn-thetase genes in environmental samples, hence directly detecting the presence of the nodularin biosynthetic pathway (19).

3.3.3. Cylindrospermopsis and Cylindrospermopsin

Biochemical studies and structural analysis have suggested a hybrid PS-PKS system for the synthesis of cylindrospermopsin by C. raciborskii (22). Schembri et al. (16) screened 17 toxic and nontoxic cyanobacterial strains for the presence of PKS and PS genes by hybridization of specifically designed radiolabeled probes. The

Fig. 3. Analysis of 16S rRNA gene regions using cyanobacterial- and Nodularia-specific oligonucleotide primers (Table 1). The cyanobacterial specific 16S rDNA PCR amplified a 400-bp fragment from all strains analyzed (CYAN). The Nodularia genus-specific 16S rDNA PCR amplified a 780-bp fragment from all Nodularia strains analyzed (NOD). The toxic N. spumigena-specific 16S rDNA PCR amplified a 200-bp fragment from all toxic planktic bloom-forming Nodularia (TOX).

Fig. 3. Analysis of 16S rRNA gene regions using cyanobacterial- and Nodularia-specific oligonucleotide primers (Table 1). The cyanobacterial specific 16S rDNA PCR amplified a 400-bp fragment from all strains analyzed (CYAN). The Nodularia genus-specific 16S rDNA PCR amplified a 780-bp fragment from all Nodularia strains analyzed (NOD). The toxic N. spumigena-specific 16S rDNA PCR amplified a 200-bp fragment from all toxic planktic bloom-forming Nodularia (TOX).

hybridization studies revealed that the PKS and PS genes were found only in the CYL-producing strains examined, C. raciborskii and Anabaena bergi. No PKS or PS genes were detected in non-CYL-producing strains, and the PKS and PS genes appeared to be linked in that either both were detected or both were absent in any one strain.

DNA sequence analysis and subsequent amino acid sequence determination of the PKS and PS gene fragments from C. raciborskii revealed a high identity to genes encoding similar functions in other bacteria. The similarity in sequence and function of the genes investigated by Schembri et al. (16) to other PKS and PS genes, combined with the finding that these genes are found only in strains that produced CYL, suggests a hybrid PS-PKS system in the production of CYL.

Using the PCR protocol described in Subheading 3.2. and the primers in Table 1, the PCR resulted in the amplification of a 650-bp fragment of the PKS gene and an approx 500-bp fragment of the PS gene (Fig. 4). The observation that the PKS and PS genes are either both present or absent in strains examined suggests that they are physically linked.

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