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Typing of toxinogenic Microcystis from environmental samples by multiplex PCR

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Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.

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  1. Ansari MZ, Yadav G, Gokhale RS, Mohanty D (2004) NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res 32:W405–W413

  2. Baker JA, Neilan BA, Entsch B, McKay DB (2001) Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysis. Environ Toxicol 16:472–482

  3. Bittencourt-Oliveira MC (2003) Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker. Harmful Algae 2:51–60

  4. Corbett LL, Parker DL (1976) Viability of lyophilized cyanobacteria (blue-green algae). Appl Environ Microbiol 32:777–780

  5. Christiansen G, Fastner J, Erhard M, Borner T, Dittmann E (2003) Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J Bacteriol 185:564–572

  6. Dyble J, Fahnenstiel GL, Litaker RW, Millie DF, Tester PA (2008) Microcystin concentrations and genetic diversity of Microcystis in the lower Great Lakes. Environ Toxicol 23:507–516

  7. El Herry S, Fathalli A, Rejeb AJ, Bouaicha N (2008) Seasonal occurrence and toxicity of Microcystis spp. and Oscillatoria tenuis in the Lebna Dam, Tunisia. Water Res 42:1263–1273

  8. Fastner J, Erhard M, von Dohren H (2001) Determination of oligopeptide diversity within a natural population of Microcystis spp. (cyanobacteria) by typing single colonies by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 67:5069–5076

  9. Hisbergues M, Christiansen G, Rouhiainen L, Sivonen K, Borner T (2003) PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera. Arch Microbiol 180:402–410

  10. Hotto A, Satchwell M, Boyer G (2005) Seasonal production and molecular characterization of microcystins in Oneida Lake, New York, USA. Environ Toxicol 20:243–248

  11. Hotto AM, Satchwell MF, Boyer GL (2007) Molecular characterization of potential microcystin-producing cyanobacteria in Lake Ontario embayments and nearshore waters. Appl Environ Microbiol 73:4570–4578

  12. Komárek J, Anagnostidis K (1999) Cyanoprokaryota. 1. Teil: Chroococcales, 19/1. In: Ettl H, Gärtner G, Heynig H, Mollenhauer D (eds) Süβwasserflora von Mitteleuropa. Gustav Fischer, Stuttgart, pp 224–236

  13. Kurmayer R, Dittmann E, Fastner J, Chorus I (2002) Diversity of microcystin genes within a population of the toxic cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany). Microb Ecol 43:107–118

  14. Kurmayer R, Christiansen G, Fastner J, Borner T (2004) Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. Environ Microbiol 6:831–841

  15. Mankiewicz-Boczek J, Izydorczyk K, Romanowska-Duda Z, Jurczak T, Stefaniak K, Kokocinski M (2006) Detection and monitoring toxigenicity of cyanobacteria by application of molecular methods. Environ Toxicol 21:380–387

  16. Mikalsen B, Boison G, Skulberg OM, Fastner J, Davies W, Gabrielsen TM, Rudi K, Jakobsen KS (2003) Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J Bacteriol 185:2774–2785

  17. Neilan BA, Dittmann E, Rouhiainen L, Bass RA, Schaub V, Sivonen K, Borner T (1999) Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J Bacteriol 181:4089–4097

  18. Nishizawa T, Ueda A, Asayama M, Fujii K, Harada K, Ochi K, Shirai M (2000) Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin. J Biochem 127:779–789

  19. Oberholster PJ, Botha AM (2007) Use of PCR based technologies for risk assessment of a winter cyanobacterial bloom in Lake Midmar, South Africa. Afr J Biotechnol 6:1794–1805

  20. Ouahid Y, Perez-Silva G, del Campo FF (2005) Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions. Environ Toxicol 20:235–242

  21. Ouahid Y, Zaccaro MC, Zulpa G, Storni M, Stella AM, Bossio JC, Tanuz M, del Campo FF. A toxic Microcystis bloom from Río de La Plata, Argentina with an unsual major microcystin. Int J Environ Anal Chem (in press)

  22. Ouellette AJ, Handy SM, Wilhelm SW (2006) Toxic Microcystis is widespread in Lake Erie: PCR detection of toxin genes and molecular characterization of associated cyanobacterial communities. Microb Ecol 51:154–165

  23. Pearson LA, Hisbergues M, Borner T, Dittmann E, Neilan BA (2004) Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Appl Environ Microbiol 70:6370–6378

  24. Pearson LA, Barrow KD, Neilan BA (2007) Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806. J Biol Chem 282:4681–4692

  25. Rinta-Kanto JM, Ouellette AJ, Boyer GL, Twiss MR, Bridgeman TB, Wilhelm SW (2005) Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR. Environ Sci Technol 39:4198–4205

  26. Saker ML, Welker M, Vasconcelos VM (2007) Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption. Appl Microbiol Biotechnol 73:1136–1142

  27. Sielaff H, Dittmann E, Tandeau De Marsac N, Bouchier C, Von Dohren H, Borner T, Schwecke T (2003) The mcyF gene of the microcystin biosynthetic gene cluster from Microcystis aeruginosa encodes an aspartate racemase. Biochem J 373:909–916

  28. Tillett D, Dittmann E, Erhard M, von Dohren H, Borner T, Neilan BA (2000) Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem Biol 7:753–764

  29. Tillett D, Parker DL, Neilan BA (2001) Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16 S rRNA and phycocyanin operon (Phycocyanin Intergenic Spacer) phylogenies. Appl Environ Microbiol 67:2810–2818

  30. Via-Ordorika L, Fastner J, Kurmayer R, Hisbergues M, Dittmann E, Komarek J, Erhard M, Chorus I (2004) Distribution of microcystin-producing and non-microcystin-producing Microcystis sp. in European freshwater bodies: detection of microcystins and microcystin genes in individual colonies. Syst Appl Microbiol 27:592–602

  31. Xu Y, Wu Z, Yu B, Peng X, Yu G, Wei Z, Wang G, Li R (2008) Non-microcystin producing Microcystis wesenbergii (Komarek) Komarek (Cyanobacteria) representing a main waterbloom-forming species in Chinese waters. Environ Pollut 156:162–167

  32. Yoshida M, Yoshida T, Takashima Y, Kondo R, Hiroishi S (2005) Genetic diversity of the toxic cyanobacterium Microcystis in Lake Mikata. Environ Toxicol 20:229–234

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We wish to thank the CEDEX for providing the environmental samples from Cazalegas, Vega de Jabalón and Vicario, Dr. Susana Romo for the Albufera lake sample, and Lars Wörmer and Samuel Cires for the Cogotas sample. Thanks also to Dr. María Verdugo for the cyanobacteria identification from Vega de Jabalón and Vicario. This research was funded by the Spanish AECID (project A/017389/08) and the Community of Madrid (CCG07-UAM/AMB-1883/07).

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Correspondence to Francisca Fernández del Campo.

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Ouahid, Y., del Campo, F.F. Typing of toxinogenic Microcystis from environmental samples by multiplex PCR. Appl Microbiol Biotechnol 85, 405–412 (2009). https://doi.org/10.1007/s00253-009-2249-4

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  • mcy genes
  • NRPS
  • PKS
  • Microcystis
  • Multiplex PCR
  • Bloom biomass