Morphological, biochemical and molecular characterization of Anabaena, Aphanizomenon and Nostoc strains (Cyanobacteria, Nostocales) isolated from Portuguese freshwater habitats

Abstract

Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.

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References

  1. Andersen, R. A. & M. Kawachi, 2005. Traditional microalgae isolation techniques. In Andersen, R. A. (ed.), Algal Culturing Techniques. Elsevier Academic Press, Tokyo: 83–100.

    Google Scholar 

  2. Ariosa, Y., D. Carrasco, A. Quesada & E. Fernandez-Valiente, 2006. Incorporation of different N sources and light response curves of nitrogenase and photosynthesis by cyanobacterial blooms from rice fields. Microbial Ecology 51: 394–403.

    CAS  Article  PubMed  Google Scholar 

  3. Castenholz, R. W., 1989. Subsection IV. Order Nostocales. In Staley, J. T., M. P. Bryant, N. Pfennig & J. G. Holt (eds), Bergey’s Manual of Systematic Bacteriology, Vol. 3. Williams & Wilkins, Baltimore: 1780–1793.

    Google Scholar 

  4. Castenholz, R. W. & J. B. Waterbury, 1989. Group I. Cyanobacteria. In Staley, J. T., M. P. Bryant, N. Pfennig & J. G. Holt (eds), Bergey’s Manual of Systematic Bacteriology, Vol. 3. Williams & Wilkins, Baltimore: 1710–1727.

    Google Scholar 

  5. Caudales, R. & J. M. Wells, 1992. Differentiation of the free-living Anabaena and Nostoc cyanobacteria on the basis of fatty acid composition. International Journal of Systematic and Evolutionary Microbiology 42: 246–251.

    CAS  Google Scholar 

  6. de Figueiredo, D. R., A. S. S. P. Reboleira, S. C. Antunes, N. Abrantes, U. Azeiteiro, F. Gonçalves & M. J. Pereira, 2006. The effect of environmental parameters and cyanobacterial blooms on phytoplankton dynamics of a Portuguese temperate lake. Hydrobiologia 568: 145–157.

    CAS  Article  Google Scholar 

  7. de Figueiredo, D. R., A. Alves, M. J. Pereira & A. Correia, 2010. Molecular characterization of bloom-forming Aphanizomenon strains isolated from Vela Lake (Western Central Portugal). Journal of Plankton Research 32: 239–252.

    Article  Google Scholar 

  8. Desikachary, T. V., 1959. Cyanophyta. Indian Council of Agricultural Research, New Delhi, India.

    Google Scholar 

  9. Galhano, V., F. Peixoto, J. Gomes-Laranjo & E. Fernández-Valiente, 2009. Differential effects of bentazon and molinate on Anabaena cylindrica, an autochthonous cyanobacterium of Portuguese rice field agro-ecosystems. Water, Air and Soil Pollution 197: 211–222.

    CAS  Article  Google Scholar 

  10. Geitler, L., 1932. Cyanophyceae. In Kolkwitz, R. (ed.), Rabenhorst’s Kriptogamenflora von Deutschland, Österreich und der Schweiz. Akademische Verlagsgesellschaft, Leipzig, Federal Republic of Germany: 1–1196.

    Google Scholar 

  11. Gillis, M., P. Vandamme, P. De Vos, J. Swings & K. Kersters, 2005. Polyphasic taxonomy. In Brenner, D. J., N. R. Krieg, J. T. Staley & G. M. Garrity (eds), Bergey’s Manual of Systematic Bacteriology. Springer, New York: 43–48.

    Google Scholar 

  12. Gugger, M., C. Lyra, P. Henriksen, A. Couté, J.-F. Humbert & K. Sivonen, 2002a. Phylogenetic comparison of the cyanobacterial genera Anabaena and Aphanizomenon. International Journal of Systematic and Evolutionary Microbiology 52: 1867–1880.

    CAS  Article  PubMed  Google Scholar 

  13. Gugger, M., C. Lyra, I. Suominen, I. Tsitko, J.-F. Humbert, M. S. Salkinoja-Salonen & K. Sivonen, 2002b. Cellular fatty acids as chemotaxonomic markers of the genera Anabaena, Aphanizomenon, Microcystis, Nostoc and Planktothrix (cyanobacteria). International Journal of Systematic and Evolutionary Microbiology 52: 1007–1015.

    CAS  Article  PubMed  Google Scholar 

  14. Guillard, R. R. L., 2005. Purification methods for microalgae. In Andersen, R. A. (ed.), Algal Culturing Techniques. Elsevier Academic Press, Tokyo: 117–132.

    Google Scholar 

  15. Han, D., Y. Fan & Z. Hu, 2009. An evaluation of four phylogenetic markers in Nostoc: Implications for cyanobacterial phylogenetic studies at the intrageneric level. Current Microbiology 58: 170–176.

    CAS  Article  PubMed  Google Scholar 

  16. Hayes, P. K., N. A. El Semary & P. Sánchez-Baracaldo, 2007. The taxonomy of cyanobacteria: molecular insights into a difficult problem. In Brodie, J. & J. Lewis (eds), Unravelling The Algae: The Past, Present, and Future of Algal Systematics. CRC Press/Taylor & Francis Group, Boca Raton: 93–101.

    Google Scholar 

  17. Henson, B. J., L. E. Watson & S. R. Barnum, 2002. Molecular differentiation of the heterocystous cyanobacteria, Nostoc and Anabaena, based on complete NifD sequences. Current Microbiology 45: 161–164.

    CAS  Article  PubMed  Google Scholar 

  18. Hindák, F., 2000. Morphological variation of four planktic nostocalean cyanophytes – members of the genus Aphanizomenon or Anabaena? Hydrobiologia 438: 107–116.

    Article  Google Scholar 

  19. Janson, S., A. Matveyev & B. Bergman, 1998. The presence and expression of hetR in the non-heterocystous cyanobacterium Symploca PCC 8002. FEMS Microbiology Letters 168: 173–179.

    CAS  Article  PubMed  Google Scholar 

  20. Kenyon, C. N., R. Rippka & R. Y. Stanier, 1972. Fatty acid composition and physiological properties of some filamentous blue-green algae. Archives of Microbiology 83: 216–236.

    CAS  Google Scholar 

  21. Komárek, J., 2006. Cyanobacterial taxonomy: current problems and prospects for the integration of traditional and molecular approaches. Algae 21: 349–375.

    Article  Google Scholar 

  22. Komárek, J., 2010. Modern taxonomic revision of planktic nostocacean cyanobacteria: a short review of genera. Hydrobiologia 639: 231–243.

    Article  Google Scholar 

  23. Komárek, J. & K. Anagnostidis, 1989. Modern approach to the classification system of Cyanophytes. 4: Nostocales. Archive für Hydrobiologie Supplement/Algological Studies 82(3/56): 247–345.

    Google Scholar 

  24. Komárek, J. & J. Komárková, 2006. Diversity of Aphanizomenon-like cyanobacteria. Czech Phycology, Olomouc 6: 1–32.

    Google Scholar 

  25. Lachance, M., 1981. Genetic relatedness of heterocystous cyanobacteria by deoxyribonucleic acid-deoxyribonucleic acid reassociation. International Journal of Systematic and Evolutionary Microbiology 31: 139–147.

    Google Scholar 

  26. Lane, D. J., 1991. 16S/23S rRNA sequencing. In Stackebrandt, E. & M. Goodfellow (eds), Nucleic Acid Techniques in Bacterial Systematics. Wiley, Chichester, UK: 115–175.

    Google Scholar 

  27. Li, R. & M. M. Watanabe, 2001. Fatty acid profiles and their chemotaxonomy in planktonic species of Anabaena (Cyanobacteria) with straight trichomes. Phytochemistry 57: 727–731.

    CAS  Article  PubMed  Google Scholar 

  28. Li, R. & M. M. Watanabe, 2004. Fatty acid composition of planktonic species of Anabaena (Cyanobacteria) with coiled trichomes exhibited a significant taxonomic value. Current Microbiology 49: 376–380.

    CAS  Article  PubMed  Google Scholar 

  29. Li, R., S. W. Wilhelm, W. W. Carmichael & M. M. Watanabe, 2008. Polyphasic characterization of water bloom forming Raphidiopsis species (cyanobacteria) from central China. Harmful Algae 7: 146–153.

    CAS  Article  Google Scholar 

  30. Liu, X.-J., F. Chen & Y. Jiang, 2003. Differentiation of Nostoc flagelliforme and its neighboring species using fatty-acid profiling as a chemotaxonomic tool. Current Microbiology 47: 467–474.

    CAS  Article  PubMed  Google Scholar 

  31. Lyra, C., S. Suomalainen, M. Gugger, C. Vezie, P. Sundman, L. Paulin & K. Sivonen, 2001. Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. International Journal of Systematic and Evolutionary Microbiology 51: 513–526.

    CAS  PubMed  Google Scholar 

  32. Marques, C. R., N. Abrantes, D. R. de Figueiredo, M. J. Pereira & F. Gonçalves, 2008. Are Pseudokirchneriella subcapitata and Chlorella vulgaris affected by environmental samples from a rice field? Water, Air and Soil Pollution 189: 49–59.

    CAS  Article  Google Scholar 

  33. Murata, N., H. Wada & Z. Gombos, 1992. Modes of fatty-acid desaturation in cyanobacteria. Plant Cell Physiology 33: 933–941.

    CAS  Google Scholar 

  34. Nayak, S., R. Prasanna, B. M. Prasanna & D. B. Sahoo, 2007. Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters. World Journal of Microbiology and Biotechnology 23: 1575–1584.

    CAS  Article  Google Scholar 

  35. Nübel, U., F. Garcia-Pichel & G. Muyzer, 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology 63: 3327–3332.

    PubMed  Google Scholar 

  36. Oudra, B., M. D. E. Andaloussi & V. M. Vasconcelos, 2009. Identification and quantification of microcystins from a Nostoc muscorum bloom occurring in Ouka meden River (High-Atlas mountains of Marrakech, Morocco). Environmental Monitoring and Assessment 149: 437–444.

    CAS  Article  PubMed  Google Scholar 

  37. Page, R. D. M., 1996. TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357–358.

    CAS  PubMed  Google Scholar 

  38. Pan, X. J., F. Y. Chang, L. J. Kang, G. B. Li, D. H. Li, Y. D. Liu, Y. W. Shen & Z. H. Wei, 2008. Morphological characteristics and phylogenetic relationship of Anabaena species from Lakes Dianchi and Erhai, China. Hydrobiologia 614: 353–362.

    CAS  Article  Google Scholar 

  39. Pereira, P., R. H. Li, W. W. Carmichael, E. Dias & S. Franca, 2004. Taxonomy and production of paralytic shellfish toxins by the freshwater cyanobacterium Aphanizomenon gracile LMECYA40. European Journal of Phycology 39: 361–368.

    CAS  Article  Google Scholar 

  40. Petkov, G. & G. Garcia, 2007. Which are fatty acids of the green alga Chlorella? Biochemical Systematics and Ecology 35: 281–285.

    CAS  Article  Google Scholar 

  41. Quesada, A., E. Moreno, D. Carrasco, T. Paniagua, L. Wormer, C. De Hoyos & A. Sukenik, 2006. Toxicity of Aphanizomenon ovalisporum (Cyanobacteria) in a Spanish water reservoir. European Journal of Phycology 41: 39–45.

    CAS  Article  Google Scholar 

  42. Rajaniemi, P., P. Hrouzek, K. Kaštovská, R. Willame, A. Rantala, L. Hoffmann, J. Komárek & K. Sivonen, 2005. Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (Nostocales, Cyanobacteria). International Journal of Systematic and Evolutionary Microbiology 55: 11–26.

    CAS  Article  PubMed  Google Scholar 

  43. Rannala, B. & Z. H. Yang, 1996. Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. Journal of Molecular Evolution 43: 304–311.

    CAS  Article  PubMed  Google Scholar 

  44. Rasmussen, B., I. R. Fletcher, J. J. Brocks & M. R. Kilburn, 2008. Reassessing the first appearance of eukaryotes and cyanobacteria. Nature 455: 1101–1104.

    CAS  Article  PubMed  Google Scholar 

  45. Rastogi, R. P. & R. P. Sinha, 2009. Biotechnological and industrial significance of cyanobacterial secondary metabolites. Biotechnology Advances 27: 521–539.

    CAS  Article  PubMed  Google Scholar 

  46. Rippka, R., J. Deruelles, J. B. Waterbury, M. Herdman & R. Y. Stanier, 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111: 1–61.

    Google Scholar 

  47. Rodríguez, F., J. L. Oliver, A. Marín & J. R. Medina, 1990. The general stochastic model of nucleotide substitutions. Journal of Theoretical Biology 142: 485–501.

    Article  PubMed  Google Scholar 

  48. Ronquist, F. & J. P. Huelsenbeck, 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.

    CAS  Article  PubMed  Google Scholar 

  49. Rosa, D. & A. Catalá, 1998. Fatty acid profiles and non enzymatic lipid peroxidation of microsomes and mitochondria from bovine liver, kidney, lung and heart. Archives of Physiology and Biochemistry 106: 33–37.

    CAS  Article  PubMed  Google Scholar 

  50. Rücker, J., A. Stüken, B. Nixdorf, J. Fastner, I. Chorus & C. Wiedner, 2007. Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes. Toxicon 50: 800–809.

    Article  PubMed  Google Scholar 

  51. Saker, M., C. Moreira, J. Martins, B. Neilan & V. M. Vasconcelos, 2009. DNA profiling of complex bacterial populations: toxic cyanobacterial blooms. Applied Microbiology and Biotechnology 85: 237–252.

    CAS  Article  PubMed  Google Scholar 

  52. Sato, N. & N. Murata, 1988. Membrane lipids. Methods in Enzymology 167: 251–259.

    CAS  Article  Google Scholar 

  53. Schaeffer, D. J. & V. S. Krylov, 2000. Anti-HIV activity of extracts and compounds from algae and cyanobacteria. Ecotoxicology and Environmental Safety 45: 208–227.

    CAS  Article  PubMed  Google Scholar 

  54. Schleifer, K. H., 2009. Classification of Bacteria and Archaea: past, present and future. Systematic and Applied Microbiology 32: 533–542.

    Article  PubMed  Google Scholar 

  55. Stüken, A., R. J. Campbell, A. Quesada, A. Sukenik, P. K. Dadheech & C. Wiedner, 2009. Genetic and morphologic characterization of four putative cylindrospermopsin producing species of the cyanobacterial genera Anabaena and Aphanizomenon. Journal of Plankton Research 31: 465–480.

    Article  Google Scholar 

  56. Swofford, D. L., 2003. PAUP*. Phylogenetic Analysis Using Parsimony (and other Methods), Version 4.0b10 [Computer Program CD-ROM]. Sinauer Associates, Sunderland, MA.

    Google Scholar 

  57. Temina, M., H. Rezankova, T. Rezanka & V. M. Dembitsky, 2007. Diversity of the fatty acids of the Nostoc species and their statistical analysis. Microbiological Research 162: 308–321.

    CAS  Article  PubMed  Google Scholar 

  58. Thacker, R. W. & V. J. Paul, 2004. Morphological, chemical, and genetic diversity of tropical marine cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales). Applied and Environmental Microbiology 70: 3305–3312.

    CAS  Article  PubMed  Google Scholar 

  59. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin & D. G. Higgins, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882.

    CAS  Article  PubMed  Google Scholar 

  60. Valério, E., P. Pereira, M. L. Saker, S. Franca & R. Tenreiro, 2005. Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters. Harmful Algae 4: 1044–1052.

    Article  Google Scholar 

  61. Wacklin, P., L. Hoffmann & J. Komárek, 2009. Nomenclatural validation of the genetically revised cyanobacterial genus Dolichospermum (RALFS ex BORNET et FLAHAULT) comb. nova. Fottea 9: 59–64.

    Google Scholar 

  62. Wada, H. & N. Murata, 1998. Membrane lipids in cyanobacteria. In Siegenthaler, P.-A. & N. Murata (eds), Lipids in Photosynthesis: Structure, Function and Genetics. Kluwer Academic Publishers, Dordrecht: 65–81.

    Google Scholar 

  63. Wartiainen, I., T. Eriksson, W. Zheng & U. Rasmussen, 2008. Variation in the active diazotrophic community in rice paddy: nifH PCR-DGGE analysis of rhizosphere and bulk soil. Applied Soil Ecology 39: 65–75.

    Article  Google Scholar 

  64. Welker, M. & H. von Döhren, 2006. Cyanobacterial peptides – nature’s own combinatorial biosynthesis. FEMS Microbiology Reviews 30: 530–563.

    CAS  Article  PubMed  Google Scholar 

  65. Whitton, B. A., 2000. Soils and rice-fields. In Whitton, B. A. & M. Potts (eds), The Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht: 233–255.

    Google Scholar 

  66. Willame, R., C. Boutte, S. Grubisic, A. Wilmotte, J. Komárek & L. Hoffmann, 2006. Morphological and molecular characterization of planktonic cyanobacteria from Belgium and Luxembourg. Journal of Phycology 42: 1312–1332.

    CAS  Article  Google Scholar 

  67. Wilmotte, A., 1994. Molecular evolution and taxonomy of the cyanobacteria. In Bryant, D. A. (ed.), The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers, Dordrecht: 1–25.

    Google Scholar 

  68. Zancan, S., R. Trevisan & M. G. Paoletti, 2006. Soil algae composition under different agro-ecosystems in North-Eastern Italy. Agriculture Ecosystems and Environment 112: 1–12.

    Article  Google Scholar 

  69. Zapomělová, E., K. Řehaková, J. Jezberová & J. Komárková, 2010. Polyphasic characterization of eight planktonic Anabaena strains (Cyanobacteria) with reference to the variability of 61 Anabaena populations observed in the field. Hydrobiologia 639: 99–113.

    Article  Google Scholar 

  70. Zehr, J. P. & L. A. McReynolds, 1989. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Applied and Environmental Microbiology 55: 2522–2526.

    CAS  PubMed  Google Scholar 

  71. Zwart, G., M. P. Kamst-van Agterveld, I. van der Werff-Staverman, F. Hagen, H. L. Hoogveld & H. J. Gons, 2005. Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake. Environmental Microbiology 7: 365–377.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

This study was supported by PhD grants to Victor Galhano (SFRH/BD/17582/2004) and Daniela de Figueiredo (SFRH/BD/23864/2005) of Portuguese Science and Technology Foundation (FCT), Ministry of Science, Technology and Higher Education, Portuguese Government. The authors are indebted to Prof. Fernando Nunes (Chemistry Department, UTAD, Vila Real) for instrumental laboratory facilities on lipid analysis and to Dr. Fátima Santos (Botany Department, FCTUC, Coimbra) for her kind help on morphological identification and loan of precious historical publications. Further, we are thankful to Prof. Luigi Naselli-Flores (Department of Botanical Sciences, University of Palermo, Palermo, Italy) and to the anonymous reviewer for their constructive and helpful comments on the manuscript.

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Galhano, V., de Figueiredo, D.R., Alves, A. et al. Morphological, biochemical and molecular characterization of Anabaena, Aphanizomenon and Nostoc strains (Cyanobacteria, Nostocales) isolated from Portuguese freshwater habitats. Hydrobiologia 663, 187–203 (2011). https://doi.org/10.1007/s10750-010-0572-5

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Keywords

  • Polyphasic taxonomy
  • Cyanobacteria
  • 16S rRNA
  • hetR
  • nifH
  • Fatty acid methyl esters