Journal of Chemical Ecology

, Volume 29, Issue 8, pp 1757–1770 | Cite as

Isolation, Characterization, and Quantitative Analysis of Microviridin J, a New Microcystis Metabolite Toxic to Daphnia

  • Thomas Rohrlack
  • Kirsten Christoffersen
  • Poul Erik Hansen
  • Wei Zhang
  • Olaf Czarnecki
  • Manfred Henning
  • Jutta Fastner
  • Marcel Erhard
  • Brett A. Neilan
  • Melanie Kaebernick


This paper describes the purification and characterization of microviridin J, a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Microcystis cyanobacteria Daphnia microviridin NMR microcystin environmental toxins 


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  1. Agrawal, M. K., Bagchi, D., and Bagchi, S. N. 2001. Acute inhibition of protease and suppression of growth in zooplankter, Moina macrocopa, by Microcystis blooms collected in Central India. Hydrobiologia 464:37–44.Google Scholar
  2. Allinger, N. 1997. MM2 (QCPE Quantum Exchange program).Google Scholar
  3. Bartels, C., Xia, T. H., Billeter, M., Güntert, P., and Wüthrich, K. 1995. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 6:1–10.Google Scholar
  4. Bieth, J., Spiess, B., and Wermuth, C. G. 1974. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem. Med. 11:350–357.Google Scholar
  5. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.Google Scholar
  6. Braunschweiler, L. and Ernst, R. R. 1983. Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy. J. Magn. Reson. 53:521–528.Google Scholar
  7. Carmichael, W. W. 1992. Cyanobacteria secondary metabolites—The cyanotoxins. J. Appl. Bacteriol. 72:445–459.Google Scholar
  8. Chorus, I. and Bartram, J. 1999. Toxic cyanobacteria in water. A guide to their public health consequences, monitoring and management. World Health Organization.Google Scholar
  9. Christoffersen, K. 1996. Ecological implications of cyanobacterial toxins in aquatic food webs. Phycologia 35:42–50.Google Scholar
  10. Codd, G. A. 1995. Cyanobacterial toxins: Occurrence, properties and biological significance. Water Sci. Technol. 32:149–156.Google Scholar
  11. DeMott, W. R. and Dhawale, S. 1995. Inhibition of in vitro protein phosphatase activity in three zooplankton species by microcystin-LR, a toxin from cyanobacteria. Arch. Hydrobiol. 134:417–424.Google Scholar
  12. Erhard, M., von Döhren, H., and Jungblut, P. R. 1999. Rapid identification of the new anabaenopeptin G from Planktothrix agardhii HUB 011 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spec. 13:337–343.Google Scholar
  13. Erlanger, B. F., Kokowsky, N., and Cohen, W. 1961. The preparation of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95:271–278.Google Scholar
  14. Fastner, J., Erhard, M., and von Döhren, 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.Google Scholar
  15. Fastner, J., Flieger, I., and Neumann, U. 1998. Optimised extraction of microcystins from field samples—A comparison of different solvents and procedures. Water Res. 32:3177–3181.Google Scholar
  16. Fujii, K., Sivonen, K., Naganawa, E., and Harada, K. 2000. Non-toxic peptides from toxic cyanobacteria, Oscillatoria agardhii. Tetrahedron 56:725–733.Google Scholar
  17. Gimenez, A. V. F., Garcia-Carreno, F. L., del Toro, M. A. N., and Fenucci, J. L. 2001. Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): Partial characterization and relationship with molting. Comp. Biochem. Phys. B 130:331–338.Google Scholar
  18. Haney, J. F., Forsyth, D. J., and James, M. R. 1994. Inhibition of zooplankton filtering rate by dissolved inhibitors produced by naturally-occurring cyanobacteria. Arch. Hydrobiol. 132: 1–13.Google Scholar
  19. Hasler, A. D. 1935. The physiology of digestion of plankton crustacea. Biol. Bull. Woods Hole 68:207–214.Google Scholar
  20. Ishitsuka, M. O., Kusumi, T., Kakisawa, H., Kaya, K., and Watanabe, M. M. 1990. Microviridin—A novel tricyclic depsipeptide from the toxic cyanobacterium Microcystis viridis. J. Am. Chem. Soc. 112:8180–8182.Google Scholar
  21. Jakobi, C., Rinehart, K. L., Neuber, R., Mez, K., and Weckessser, J. 1996. Cyanopeptolin SS, a disulfated depsipeptide from a water bloom in Leipzig (Germany): Structural elucidation and biological activities. Phycologia 35:111–116.Google Scholar
  22. Jochimsen, E. M., Carmichael, W. W., An, J. S., Cardo, D. M., Cooksen, S. T., Holmes, C. E. M., Antunes, M. B. D., de Melo, D. A., Lyra, T. M., Barreto, V. S. T., Azevedo, S. M. F. O., and Jarvis, W. R. 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338:873–878.Google Scholar
  23. John, B. K., Plant, D., and Hurd, R. E. 1992. Improved proton-detected heteronuclear correlation using gradient-enhanced z and zz filters. J. Magn. Reson. 101:113–117.Google Scholar
  24. Jongsma, M. A. and Bolter, C. 1997. The adaptation of insects to plant protease inhibitors. J. Insect Physiol. 43:885–895.Google Scholar
  25. Jungmann, D. 1992. Toxic compounds isolated from PCC7806 that are more active to Daphnia than two microcystins. Limnol. Oceanogr. 37:1777–1793.Google Scholar
  26. Kaebernick, M., Rohrlack, T., Christoffersen, K., and Neilan, B. A. 2001. A spontaneous mutant of microcystin biosynthesis: Genetic characterization and effect on Daphnia. Environ. Microbiol. 3:669–679.Google Scholar
  27. Kay, L., Keifer, P., and Saarinen, T. 1992. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114:10663–10665.Google Scholar
  28. Klüttgen, B., Dulmer, U., Engels, M., and Ratte, H. T. 1994. ADaM, an artificial fresh-water for the culture of zooplankton. Freshw. Biol. 28:743–746.Google Scholar
  29. Koppitz, H., Kühl, H., and Kohl, J.-G. 2000. Differences in morphology and C/N-balance between clones of Phragmites australis within a plantation at a degraded fen. Folia Geobot. 35:389–402.Google Scholar
  30. Kotai, J. 1972. Instructions for the Preparation of Modified Nutrient Solution Z8 for Algae. Publication B-11/69. Norsk institutt for vannforskning, Oslo, 5 pp.Google Scholar
  31. Laskowski, M. and Qasim, M. A. 2000. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? BBA-Protein Struct. M. 1477:324–337.Google Scholar
  32. Murakami, M., Sun, Q., Ishida, K., Matsuda, H., Okino, T., and Yamaguchi, K. 1997. Microviridins, elastase inhibitors from the cyanobacterium Nostoc minutum (NIES-26). Phytochemistry 45:1197–1202.Google Scholar
  33. Nagel, W., Willig, F., Peschke, W., and Schmidt, F. H. 1965. über die Bestimmung von Trypsin und Chymotrypsin mit Aminosäure-p-nitroaniliden. Hoppe-Seyler's Z. Physiol. Chem. 340:1–10.Google Scholar
  34. Okino, T., Matsuda, H., Murakami, M., and Yamaguchi, K. 1995. New microviridins, elastase inhibitors from the cyanobacterium Microcystis aeruginosa. Tetrahedron 51:10679–10686.Google Scholar
  35. Piantini, U., Sorensen, O. W., and Ernst, R. R. 1982. Multiple quantum filters for elucidating NMR coupling networks. J. Am. Chem. Soc. 104: 6800–6801.Google Scholar
  36. Pouria, S., de Andrade, A., Barbosa, J., Cavalcanti, R. L., Barreto, V. T. S., Ward, C. J., Preiser, W., Poon, G. K., Neild, G. H., and Codd, G. A. 1998. Fatal microcystin intoxication in a haemodialysis unit in Caruaru, Brazil. Lancet 352:21–26.Google Scholar
  37. Radau, G. 2000. Serine proteases inhibiting cyanopeptides. Pharmazie 55:555–560.Google Scholar
  38. Reinikainen, M. 1997. Acute and Sublethal Effects of Cyanobacteria with Different Toxic Properties on Cladocerean Zooplankton. PhD Thesis. åbo Akademi University, åbo, Finland.Google Scholar
  39. Riemann, B. and Christoffersen, K. 1993. Microbial trophodynamics in temperate lakes. Mar. Microb. Food Webs 7:69–100.Google Scholar
  40. Rohrlack, T., Dittmann, E., Börner, T., and Christoffersen, K. 2001. Effects of cell-bound microcystins on survival and feeding of Daphnia spp. Appl. Environ. Microbiol. 67:3523–3529.Google Scholar
  41. Rohrlack, T., Dittmann, E., Henning, M., Börner, T., and 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. 65:737–739.Google Scholar
  42. Shin, H. J., Murakami, M., Matsuda, H., and Yamaguchi, K. 1996. Microviridins D-F, serine protease inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-204). Tetrahedron 52:8159–8168.Google Scholar
  43. States, D. J., Haberkorn, R. A., and Reuben, D. J. 1982. A two-dimensional nuclear Overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson. 48:286–292.Google Scholar
  44. Weckesser, J., Martin, C., and Jakobi, C. 1996. Cyanopeptolins, depsipeptides from cyanobacteria. Syst. Appl. Microbiol. 19:133–138.Google Scholar
  45. Zor, T. and Seliger, Z. 1996. Linearization of the Bradford protein assay increases its sensitivity—Theoretical and experimental studies. Anal. Biochem. 236:302–308.Google Scholar

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Thomas Rohrlack
    • 1
  • Kirsten Christoffersen
    • 1
  • Poul Erik Hansen
    • 2
  • Wei Zhang
    • 2
  • Olaf Czarnecki
    • 3
  • Manfred Henning
    • 3
  • Jutta Fastner
    • 4
  • Marcel Erhard
    • 5
  • Brett A. Neilan
    • 6
  • Melanie Kaebernick
    • 7
  1. 1.Freshwater Biological LaboratoryUniversity of CopenhagenHillerødDenmark
  2. 2.Department of Life Sciences and ChemistryRoskilde UniversityRoskildeDenmark
  3. 3.Group Ecophysiology, Department of BiologyHumboldt-UniversityBerlinGermany
  4. 4.Biotechnology Center and Max Vollmer InstituteTechnical University BerlinBerlinGermany
  5. 5.AnagnosTec GmbH, Im Biotechnologiepark TGZ IILuckenwaldeGermany
  6. 6.School of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
  7. 7.Group Genetics, Department of BiologyHumboldt-UniversityBerlinGermany

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