Microbial Ecology

, Volume 58, Issue 2, pp 323–333 | Cite as

Distribution and Abundance of Nontoxic Mutants of Cyanobacteria in Lakes of the Alps

  • Veronika Ostermaier
  • Rainer Kurmayer
Microbiology of Aquatic Systems


The filamentous cyanobacterium Planktothrix rubescens frequently occurs in deep and stratified lakes in the temperate region of the northern hemisphere and is a known producer of the hepatotoxic secondary metabolite microcystin. These cyclic heptapeptides are synthesized nonribosomally via large enzyme complexes encoded by the microcystin (mcy) synthetase gene cluster. The occurrence of cyanobacterial strains lacking microcystin, but containing the mcy gene cluster has been reported repeatedly; it was shown that this inactivation is due to mutations such as gene deletion events and the insertion of transposable elements. In the present study, 12 lakes in Austria, Germany, and Switzerland were sampled from July 2005 to October 2007, and the proportion of inactive mcy genotypes was quantified in relation to the total population of the red-pigmented filamentous cyanobacterium Planktothrix by means of quantitative polymerase chain reaction. In total, four different mutations were quantified, namely two insertions affecting mcyD, one insertion affecting mcyA, and a deletion within mcyH and mcyA. The mutations occurred over a wide range of population densities (40–570,000 filaments L−1), and their abundance was found to be positively correlated with population density. However, on average, all nontoxic mutants were found in a low proportion only (min 0%, mean 6.5% ± 1.1 (SE), max 52% of the total population). The genotype containing the mcyHA deletion had a significantly higher proportion (min 0%, mean 3.7% ± 1, max 52%) when compared with all the genotypes containing insertions within the mcy gene cluster (min 0%, mean 2.8% ± 0.7, max 24%). The results demonstrate that the occurrence of inactive mcy genotypes is linearly related to the population density, and selective sweeps of nontoxic mutants did not occur during the transition from prebloom to bloom conditions.


Insertion Sequence Filamentous Cyanobacterium Adenylation Domain Peptide Composition Microcystis Strain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank Guntram Christiansen for his help in molecular biological techniques. We are most grateful to Arno Stöckli (Department Bau, Verkehr und Umwelt, Kanton Aarau, Switzerland), Ferdinand Schanz (University of Zürich), Günther Bruschek and Karl Mayrhofer (BAW Scharfling, Austria) for the provision of water samples. Marcel Erhard did the automated MALDI-TOF MS measurements on peptide composition in single filaments. We appreciated the comments of two anonymous reviewers. This study was financed by the Austrian Science Fund project P18185 “Microevolution of toxin synthesis in cyanobacteria”.

Supplementary material

248_2009_9484_MOESM1_ESM.doc (242 kb)
Fig. S1 Relationship between Planktothrix biovolume as determined by the TNA and estimated from the Lugol fixed samples using the sedimentation method. For details on the regression curve, see text (DOC 242 kb)
248_2009_9484_MOESM2_ESM.doc (380 kb)
Fig. S2 Relationship between the Planktothrix biovolume as determined by 16S rDNA-TNA and the biovolume of genotypes containing the IS element as determined by TNA (TIB). For details on the regression curve, see text (DOC 379 kb)


  1. 1.
    Anagnostidis K, Komárek J (1988) Modern approach to the classification system of cyanophytes, 3-Oscillatoriales. Arch Hydrobiol Suppl Algol Stud 80(50–53):327–472Google Scholar
  2. 2.
    Barker GLA, Handley BA, Vacharapiyasophon P, Stevens JR, Hayes PK (2000) Allele-specific PCR shows that genetic exchange occurs among genetically diverse Nodularia (Cyanobacteria) filaments in the Baltic Sea. Microbiology 146:2865–2875PubMedGoogle Scholar
  3. 3.
    Berg OG, Kurland CG (2002) Evolution of microbial genomes: sequence acquisition and loss. Mol Biol Evol 19:2265–2276PubMedGoogle Scholar
  4. 4.
    Chorus I, Bartram J (1999) Toxic cyanobacteria in water. A guide to their public health consequences, monitoring and management. WHO, E & FN Spon, London, p 416Google Scholar
  5. 5.
    Christiansen G, Fastner J, Erhard M, Börner T, Dittmann E (2003) Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J Bacteriol 185:564–572PubMedCrossRefGoogle Scholar
  6. 6.
    Christiansen G, Kurmayer R, Liu Q, Börner T (2006) Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp. Appl Environ Microbiol 72:117–123PubMedCrossRefGoogle Scholar
  7. 7.
    Christiansen G, Molitor C, Philmus B, Kurmayer R (2008) Nontoxic strains of cyanobacteria are the result of major gene deletion events induced by a transposable element. Mol Biol Evol 25:1695–1704PubMedCrossRefGoogle Scholar
  8. 8.
    Edmondson WT, Litt AH (1982) Daphnia in Lake Washington. Limnol Oceanogr 27:272–293CrossRefGoogle Scholar
  9. 9.
    Fastner J, Erhard M, von Döhren H (2001) Determination of oligopeptide diversity within a natural population of Microcystis (Cyanobacteria) by typing single colonies by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 67:5069–5076PubMedCrossRefGoogle Scholar
  10. 10.
    Hesse K, Dittmann E, Börner T (2001) Consequences of impaired microcystin production for light-dependent growth and pigmentation of Microcystis aeruginosa PCC 7806. FEMS Microbiol Ecol 37:39–43CrossRefGoogle Scholar
  11. 11.
    ISO (1992) Water quality - measurement of biochemical parameters - spectrometric determination of the chlorophyll-a concentration. International Organisation for Standardization, Geneve, p 12Google Scholar
  12. 12.
    Jacquet S, Briand J-F, Leboulanger C, Avois-Jacquet C, Oberhaus L, Tassin B, Vincon-Leite B, Paolini G, Druart J-C, Anneville O, Humbert J-F (2005) The proliferation of the toxic cyanobacterium Planktothrix rubescens following restoration of the largest natural French lake (Lac du Bourget). Harmful Algae 4:651–672CrossRefGoogle Scholar
  13. 13.
    Kaebernick M, Rohrlack T, Christoffersen K, Neilan BA (2001) A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia. Environ Microbiol 3:669–679PubMedCrossRefGoogle Scholar
  14. 14.
    Kardinaal W, Tonk L, Janse I, Hol S, Slot P, Huisman J, Visser P (2007) Competition for light between toxic and nontoxic strains of the harmful cyanobacterium Microcystis. Appl Environ Microbiol 73:2939–2946PubMedCrossRefGoogle Scholar
  15. 15.
    Kurmayer R, Christiansen G, Chorus I (2003) The abundance of microcystin-producing genotypes correlates positively with colony size in Microcystis and determines its microcystin net production in Lake Wannsee. Appl Environ Microbiol 69:787–795PubMedCrossRefGoogle Scholar
  16. 16.
    Kurmayer R, Christiansen G, Fastner J, Börner T (2004) Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. Environ Microbiol 6:831–841PubMedCrossRefGoogle Scholar
  17. 17.
    Kurmayer R, Gumpenberger M (2006) Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii. Mol Ecol 15:3849–3861PubMedCrossRefGoogle Scholar
  18. 18.
    Kurmayer R, Jüttner F (1999) Strategies for the co-existence of zooplankton with the toxic cyanobacterium Planktothrix rubescens in Lake Zürich. J Plankt Res 21:659–683CrossRefGoogle Scholar
  19. 19.
    Kurmayer R, Kutzenberger T (2003) Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp. Appl Environ Microbiol 69:6723–6730PubMedCrossRefGoogle Scholar
  20. 20.
    Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371PubMedCrossRefGoogle Scholar
  21. 21.
    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 185:2774–2785PubMedCrossRefGoogle Scholar
  22. 22.
    Mira A, Ochman H, Moran NA (2001) Deletional bias and the evolution of bacterial genomes. Trends Genet 17:589–596PubMedCrossRefGoogle Scholar
  23. 23.
    Naselli-Flores L, Barone R, Chorus I, Kurmayer R (2007) Toxic cyanobacterial blooms under a semiarid mediterranean climate: The magnification of a problem. Environ Toxicol 22:399–404PubMedCrossRefGoogle Scholar
  24. 24.
    Nishizawa T, Asayama M, Fujii K, Harada K, Shirai M (1999) Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J Biochem 126:520–529PubMedGoogle Scholar
  25. 25.
    Nürnberg GK, LaZerte BD (2003) An artificially induced Planktothrix rubescens surface bloom in a small kettle lake in Southern Ontario compared to blooms world-wide. Lake Res Manag 19:307–322CrossRefGoogle Scholar
  26. 26.
    Padisák J, Scheffler W, Kasprzak P, Koschel R, Krienitz L (2003) Interannual variability in the phytoplankton composition of Lake Stechlin (1994-2000). Arch Hydrobiol/Advanc Limnol 58:101–133Google Scholar
  27. 27.
    Pridmore R, Etheredge M (1987) Planktonic cyanobacteria in New Zealand inland waters: distribution and population dynamics. N Z J Mar Freshw Res 21:491–502CrossRefGoogle Scholar
  28. 28.
    Rantala A, Fewer DP, Hisbergues M, Rouhiainen L, Vaitomaa J, Börner T, Sivonen K (2004) Phylogenetic evidence for the early evolution of microcystin synthesis. Proc Natl Acad Sci USA 101:568–573PubMedCrossRefGoogle Scholar
  29. 29.
    Rippka R (1988) Isolation and purification of cyanobacteria. Meth Enzymol 167:3–27PubMedCrossRefGoogle Scholar
  30. 30.
    Rohrlack T, Dittmann E, Henning M, Börner T, Kohl J-G (1999) Role of microcystins in poisoning and food ingestion inhibition of Daphnia galeata caused by the cyanobacterium Microcystis aeruginosa. Appl Environ Microbiol 65:737–739PubMedGoogle Scholar
  31. 31.
    Rohrlack T, Edvardsen B, Skulberg R, Halstvedt CB, Utkilen HC, Ptacnik R, Skulberg OM (2008) Oligopeptide chemotypes of the toxic freshwater cyanobacterium Planktothrix can form subpopulations with dissimilar ecological traits. Limnol Oceanogr 53:1279–1293Google Scholar
  32. 32.
    Rouhiainen L, Vakkilainen T, Siemer BL, Buikema W, Haselkorn R, Sivonen K (2004) Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90. Appl Environ Microbiol 70:686–692PubMedCrossRefGoogle Scholar
  33. 33.
    Schober E, Kurmayer R (2006) Evaluation of different DNA sampling techniques for the application of the real-time PCR method for the quantification of cyanobacteria in water. Lett Appl Microbiol 42:412–417PubMedCrossRefGoogle Scholar
  34. 34.
    Schober E, Werndl M, Laakso K, Korschinek I, Sivonen K, Kurmayer R (2007) Interlaboratory comparison of Taq Nuclease Assays for the quantification of the toxic cyanobacteria Microcystis sp. J Microbiol Meth 69:122–128CrossRefGoogle Scholar
  35. 35.
    Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M (2006) ISfinder: the reference centre for bacterial insertion sequences. Nucl Acid Res 34:D32–D36CrossRefGoogle Scholar
  36. 36.
    Sokal R, Rohlf F (1995) Biometry. The principles and practice of statistics in biological research, 3rd edn. W.H. Freeman and Company, New York, p 886Google Scholar
  37. 37.
    Suda S, Watanabe MM, Otsuka S, Mahakahant A, Yongmanitchai W, Nopartnaraporn N, Liu Y, Day JG (2002) Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria. Int J Syst Evol Microbiol 52:1577–1595PubMedCrossRefGoogle Scholar
  38. 38.
    Tillett D, Dittmann E, Erhard M, vonDöhren H, Börner T, Neilan BA (2000) Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem Biol 7:753–764PubMedCrossRefGoogle Scholar
  39. 39.
    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 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl Environ Microbiol 67:2810–2818PubMedCrossRefGoogle Scholar
  40. 40.
    Travisano M, Velicer GJ (2004) Strategies of microbial cheater control. Trends Microbiol 12:72–78PubMedCrossRefGoogle Scholar
  41. 41.
    Utermöhl H (1958) Zur Vervollkommnung der quantitativen Phytoplanktonmethodik. Mitt Internat Verein Limnol 2:1–38Google Scholar
  42. 42.
    Vollenweider RA, Kerekes J (1982) Eutrophication of waters. Monitoring, assessment and control. OECD Cooperative programme on monitoring of inland waters (Eutrophication control). Environmental Directorate, OECD, Paris, p 154Google Scholar
  43. 43.
    Walsby AE, Avery A (1996) Measurement of filamentous cyanobacteria by image analysis. J Microbiol Meth 26:11–20CrossRefGoogle Scholar
  44. 44.
    Walsby AE, Ng G, Dunn C, Davis PA (2004) Comparison of the depth where Planktothrix rubescens stratfies and the depth where the daily insolation supports its neutral buoyancy. New Phytol 162:133–145CrossRefGoogle Scholar
  45. 45.
    Welker M, Christiansen G, von Döhren H (2004) Diversity of coexisting Planktothrix (cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides. Arch Microbiol 182:288–298PubMedCrossRefGoogle Scholar
  46. 46.
    Welker M, Erhard M (2007) Consistency between chemotyping of single filaments of Planktothrix rubescens (Cyanobacteria) by MALDI-TOF and the peptide patterns of strains determined by HPLC-MS. J Mass Spectrom 42:1062–1068PubMedCrossRefGoogle Scholar
  47. 47.
    Wetzel RG, Likens GE (2000) Limnological analyses, 3rd edn. Springer-Verlag, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Austrian Academy of SciencesInstitute for LimnologyMondseeAustria

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