Nonribosomal peptide synthesis and toxigenicity of cyanobacteria

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryo tes by the thiotemplate function of large, modular enzyme complexes known c ollectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use i n unnatural-product or combinatorial biosynthesis (R. McDaniel, S, Ebert-Kh osla, D, A. Hopwood, and C. Khosla, Science 262: 1546-1557, 1993; T, Stache lhaus, A. Schneider, and M, A. Marahiel, Science 269:69-72, 1995), Most non ribosomal peptides from microorganisms are classified as secondary metaboli tes; that is, they rarely have a role in primary metabolism, growth, or rep roduction but have evolved to somehow benefit the producing organisms, Cyan obacteria produce a myriad array of secondary metabolites, including alkalo ids, polyketides, and nonribosomal peptides, some of which are potent toxin s. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide sy nthetase genes was achieved by use of degenerate primers directed to conser ved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacteria l toxin microcystin was shown for both cultured and uncultured samples. Blo t hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cy anobacteria. The results demonstrate a molecular approach to assessing pree xpression microbial functional diversity in uncultured cyanobacteria, The n onribosomal peptide biosynthetic pathways detected may lead to the discover y and engineering of novel antibiotics, immunosuppressants, or antiviral ag ents.