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

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 NMTspecific 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.Microcystis spp., cyanobacteria that frequently occur as noxious blooms in eutrophic freshwaters, are of major concern because many strains produce cyclic heptapeptide toxins called microcystins (5). The microcystins are members of a family of more than 65 heptapeptides and share the common structure, where L-X and L-Z are variable L-amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid, DMeAsp is 3-methyl-aspartic acid, and Mdha is N-methyldehydroalanine (38). Toxicity is mediated through the active transport of microcystin into hepatocytes by the bile acid organic anion transport system, followed by inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A (12, 17). Acute poisoning, leading to death from massive hepatic hemorrhage, has been reported to occur in both animals and humans (3,19,37). Chronic ingestion of sublethal doses has been demonstrated to induce primary hepatocellular carcinoma in rodents (33) and has been epidemiologically linked to primary liver cancer in humans (56,57).Water resource management has been complicated by the inability to differentiate between toxic and nontoxic Microcystis blooms without isolation and testing for toxin production. Several morphological studies (25, 54) and molecular studies have attempted to resolve the ambiguous relationship...

Benthic cyanobacteria of the genus Nodularia are non-toxic, without gas vacuoles, able to glide and genetically more diverse than planktonic Nodularia

“…These results show that the presence of ndaF is a good indicator of nodularin production. In the future, ndaF from Nodularia, similarly to mcyA and mcyE from Anabaena and Microcystis strains (Vaitomaa et al, 2003;Tillet et al, 2001), may prove to be a valuable tool for detection and early warning tests of nodularin-producers in water environments. The sequence divergence of ndaF among N. spumigena and Nodularia sp.…”

Characterization of the Biocidal Spectrum of Extracellular Filtrates of Microcystis aeruginosa

“…Also, the genetic aspect of toxin production i.e. the enzyme responsible for toxin production, enzyme structure and genes involved in toxin production by this cyanobacterial genus has also been investigated by several workers [19][20][21][22]. Studies have revealed that there is significant influence of culture age on microcystin production.…”

Isolation and preliminary characterization of cyanobacteria strains from freshwaters of Greece

“…For monitoring and mcyE gene in all the MC-producing strains [31]. The mcyE gene codes for the glutamate-1-semialdehyde aminotransferase (GSA-AMT) domain, whose role in MC biosynthesis is to supply the glutamate group to Adda [32]. In addition, the HEPF/HEPR primer pair targets the AMT domain of either mcyE or ndaF, involved in the production of MC or nodularin, respectively [33].…”