Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806

Abstract

SummarySeveral bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloomforming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and nontoxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

An integrated method for removal of harmful cyanobacterial blooms in eutrophic lakes

“…This algal control method focuses on inactivating the photosynthetic apparatus but not destroying the whole cell, which is not consistent with some previous reports that only electron transport is inhibited by H 2 O 2 (Samuilov et al, 2001). As a consequence, the intracellular cyanotoxins should not be released to the environment where they can threaten humans and wildlife (Jones and Orr, 1994;Dittmann et al, 1997). In this case, the photosynthetic activities of all kinds of algae expressed with Fv/Fm decreased to 0 in few hours.…”

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

“…Transformation with exogenous DNA, either plasmid, intergeneric or interspecifi c has been successful in a few unicellular species, including Synechococcus sp. PCC 7002 (Stevens and Porter 1980), Synechocystis (Grigorieva and Shestakov 1982), Microcystis aeruginosa PCC 7806 (Dittmann et al, 1997) and Thermosynechococcus elongatus BP-1 (Onai et al, 2004), as well as the heterocystous cyanobacteria Nostoc muscorum and Anabaena tortulosa (Trehan and Sinha 1982) where reciprocal transformation has been exemplifi ed. One of the main diffi culties in establishing a genetic transfer system in cyanobacteria, particularly the fi lamentous strains, is the presence of various restriction-modifi cation systems in these species.…”

Effects of nonylphenol on the growth and microcystin production of Microcystis strains

“…Some toxic strains isolated from field experiments can spontaneously lose their toxicities under laboratory conditions (Schatz et al, 2005). It is recognized that toxicity occurrence of a M. aeruginosa strain is determined by its microcystin production genes (mcy genes) (Dittmann et al, 1997;Kaebernick and Neilan, 2001). On the other hand, environmental factors can adjust and control the expression of microcystin genes.…”