Detection of Toxigenicity by a Probe for the Microcystin Synthetase A Gene ( mcyA) of the Cyanobacterial Genus Microcystis : Comparison of Toxicities with 16S rRNA and Phycocyanin Operon (Phycocyanin Intergenic Spacer) Phylogenies

Abstract

ABSTRACT The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for the N -methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37 Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMT-specific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function ( uma1 ) at a conserved distance from mcyC . All nontoxic strains also contained uma1 , which is not cotranscribed with mcyABC . The consistent linkage of mcyC to uma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the various Microcystis clades.