Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions.
Reliable cyanotoxin monitoring in water reservoirs is difficult because of, among other reasons, unpredictable changes in cyanobacteria biomass, toxin production, and inadequate sampling frequency. Therefore, it would be useful to identify potentially microcystin-producing strains of cyanobacterial populations in field samples. With this aim, we developed a methodology to distinguish microcystin-producing from non-producing Microcystis strains by amplifying six characteristic segments of the microcystin synthetase mcy cluster, three corresponding to the nonribosomal peptide synthetase, genes mcyA, mcyB, and mcyC, and three to the polyketide synthase, genes mcyD, mcyE, and mcyG. For this purpose five new primer sets were designed and tested using purified DNA, cultured cells, and field colonies as DNA sources. Simultaneous amplification of several genes in multipex PCR reactions was performed in this study. The results obtained showed that: (i) the expected specific amplicons were obtained with all microcystin-producing strains but not with nonproducing strains; (ii) cells could be directly used as DNA templates, 2000 cells being a sufficient number in most cases; (iii) simultaneous amplification of several gene regions is feasible both with cultured cells and with field colonies. Our data support the idea that the presence of various mcy genes in Microcystis could be used as a criterion for ascribing potential toxigenicity to field strains, and the possibility of applying whole-cell assays for the simultaneous amplification of various genes may contribute significantly to simplifying toxigenicity testing.
Ouahid
Youness
Y
Plant Physiology Laboratory, Department of Biology, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Pérez-Silva
Gonzalo
G
del Campo
Francisca F
FF
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D004784:Environmental Monitoring; D004791:Enzyme Inhibitors / Q000032:analysis*; D052998:Microcystins; D046931:Microcystis / Q000472:pathogenicity*; D021141:Nucleic Acid Amplification Techniques; D010453:Peptide Synthases / Q000032:analysis / Q000235:genetics*; D010456:Peptides, Cyclic / Q000032:analysis* / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D014881:Water Supply
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D052998:Microcystins; D010456:Peptides, Cyclic; C078588:microcystin; D010453:Peptide Synthases; C432283:microcystin synthetase A