The mosaic structure of the mcyABC operon in Microcystis.

An extensive study of the mcyABC genes and regions flanking the mcy gene cluster was performed in naturally occurring Microcystis strains. Lack of methylation in strains producing only desmethyl(7)-microcystin was found to be associated with point mutations in substrate-binding sequence motifs of the N-methyltransferase (NMT) domain in McyA. Multiple recombination events giving rise to 'phylogenetic mosaics' were detected within the NMT-domain-encoding mcyA sequences and the adenylation (A) domain sequences of mcyB and mcyC. Recombination leading to exchanges between the mcyB and mcyC regions encoding A domains in modules McyB1 and McyC was also detected. A previously reported replacement of the A domain in McyB1 was found to involve the region between the conserved motifs A3 and A8/A9. In all microcystin-producing strains the mcy gene cluster was flanked by the genes uma1 and dnaN. Clear indications of recombination, an insertion element and footprints of IS elements were found in the dnaN-mcyJ intergenic region. Among the non-microcystin producers, uma1 and dnaN were linked in some, but not all strains. Most non-producing strains lacked all mcy genes, while one strain possessed a partially deleted mcy operon. Our results show that frequent horizontal gene transfer events in addition to point mutations and insertions/deletions contribute to variation in the mcy gene cluster.

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains.

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.