Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp.
Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3,7-didesmethylmicrocystin-LR. Three open reading frames (mcyA, mcyB, mcyC) encoding microcystin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an N-methylation domain attached to the first and an epimerization domain attached to the second; McyB (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in mcyA, generated by homologous recombination, abolished the production of both microcystins in M. aeruginosa K-139. Primer extension analysis demonstrated light-dependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.
Nishizawa
T
T
Division of Biotechnology, School of Agriculture, Ibaraki University, Ami, Ibaraki, 300-0393, Japan.
Asayama
M
M
Fujii
K
K
Harada
K
K
Shirai
M
M
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D046931:Microcystis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010453:Peptide Synthases / Q000235:genetics*; D010456:Peptides, Cyclic / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D010456:Peptides, Cyclic; C070123:3,7-didesmethylmicrocystin LR; C070122:7-desmethylmicrocystin LR; D010453:Peptide Synthases