Molecular (PCR-DGGE) versus morphological approach: analysis of taxonomic composition of potentially toxic cyanobacteria in freshwater lakes

Aquatic Biosystems

Aleksandra Bukowska¹  Aleksandra Bielczyńska¹  Anna Karnkowska²  Ryszard J Chróst¹  Iwona Jasser¹ 

[1] Microbial Ecology Department, Faculty of Biology, Institute of Botany, University of Warsaw, ul. Miecznikowa 1, Warsaw, Poland

[2] Department of Systematics and Plant Geography, Faculty of Biology, Institute of Botany, University of Warsaw, Aleje Ujazdowskie 4, Warsaw, Poland

关键词: Microscopic analysis;    Microcystins;    mcy genes;    ITS;    DGGE;    Cyanobacteria;   

Others  :  793919
DOI  :  10.1186/2046-9063-10-2

 received in 2013-07-25, accepted in 2014-01-29,  发布年份 2014

【 摘 要 】

Background

The microscopic Utermöhl method is commonly used for the recognition of the presence and taxonomic composition of potentially toxic cyanobacteria and is especially useful for monitoring reservoirs used as drinking water, recreation and fishery resources. However, this method is time-consuming and does not allow potentially toxic and nontoxic cyanobacterial strains to be distinguished. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) of the marker gene ITS and the mcy-gene cluster, and DNA sequencing. We have attempted to calibrate the DGGE-method with a microscopic procedure, using water samples taken in 2011 from four lakes of the Great Mazurian Lakes system.

Results

Results showed that the classic microscopic method was much more precise and allowed the classification of the majority of cyanobacterial taxa to the species or genus. Using the molecular approach, most of the sequences could only be assigned to a genus or family. The results of DGGE and microscopic analyses overlapped in the detection of the filamentous cyanobacteria. For coccoid cyanobacteria, we only found two taxa using the molecular method, which represented 17% of the total taxa identified using microscopic observations. The DGGE method allowed the identification of two genera of cyanobacteria (Planktothrix and Microcystis) in the studied samples, which have the potential ability to produce toxins from the microcystins group.

Conclusions

The results confirmed that the molecular approach is useful for the rapid detection and taxonomic distinction of potentially toxic cyanobacteria in lake-water samples, also in very diverse cyanobacterial communities. Such rapid detection is unattainable by other methods. However, with still limited nucleotide sequences deposited in the public databases, this method is currently not sufficient to evaluate the entire taxonomic composition of cyanobacteria in lakes.

【 授权许可】

   
2014 Bukowska et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Reynolds CS: The plant life of the pelagic. Verh int Ver theor angew Limnol 1996, 26:97-113.
• [2]Sivonen K, Jones G: Cyanobacterial toxins. In Toxic Cyanobacteria In Water. Edited by Chorus I, Bartram J. London: E & FN Spon; 1999:55-124.
• [3]Pearson LA, Neilan BA: The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk. Curr Opin Biotech 2008, 19:281-288.
• [4]Christiansen G, Molitor C, Philmus B, Kurmayer R: Nontoxic strains of Cyanobacteria are the result of major gene deletion events induced by a transposable element. Mol Biol Evol 2008, 25:1695-1704.
• [5]Utermöhl H: Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Int Verein Theoretische Angew Limnol 1958, 9:1-38.
• [6]Muyzer G, de Waal EC, Uitterlinden AG: Profiling of complex microbial populations by denaturing gradient gel electrophoresis analyses of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 1993, 59:695-700.
• [7]Janse I, Meima M, Kardinaal WE, Zwart G: High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis. Appl Environ Microbiol 2003, 69:6634-6643.
• [8]Hisbergues M, Christiansen G, Rouhiainen L, Sivonen K, Borner T: PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera. Arch Microbiol 2003, 180:402-410.
• [9]Kwok S, Kellog DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ: Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus 1 model studies. Nucleic Acids Res 1990, 18:999-1005.
• [10]Rychlik W: Selection of primers for polymerase chain reaction. Mol Biotechnol 1995, 3:129-134.
• [11]Spiegelman D, Whissell G, Greer CW: A survey of the methods for the characterization of microbial consortia and communities. Can J Microbiol 2005, 51:355-386.
• [12]Chróst RJ, Siuda W: Microbial production, utilization, and enzymatic degradation of organic matter in the upper trophogenic water layer in the pelagial zone of lakes along the eutrophication gradient. Limnol Oceanogr 2006, 51:749-762.
• [13]Starmach K: Plankton roślinny wód słodkich: metody badania i klucze do oznaczania gatunków występujących w wodach Europy Środkowej. Warszawa–Kraków: PWN; 1989.
• [14]Starmach K: Flora słodkowodna Polski 2. Warszawa: PWN; 1966.
• [15]Hindák F: Sladkovodné riasy. Bratislava: Slovenské Pedagogické Nakladatelstvo; 1978.
• [16]Pliński M, Komárek J: Flora Zatoki Gdańskiej i wód przyległych (Bałtyk Południowy). Sinice - Cyanobakterie (Cyanoprokaryota). Gdańsk: Wydawnictwo Uniwersytetu Gdańskiego; 2007.
• [17]Komárek J, Anagnostidis K: Cyanoprokaryota 1. Teil: chroococcales. In Süsswasserflora von Mitteleuropa. Volume 19. Edited by Ettl H, Gerloff J, Heying H, Mollenhauer D. Heidelberg: Spektrum Akademischer Verlag; 2008. vol. 1
• [18]Komárek J, Anagnostidis K: Cyanoprokaryota 2. Teil oscillatoriales. In Süsswasserflora von Mitteleuropa. 19th edition. Edited by Büdel B, Gärtner G, Krienitz L, Schagerl M, Büdel B, Gärtner G, Krienitz L, Schagerl M. Heidelberg: Spektrum Akademischer Verlag; 2008. 9 vol. 2
• [19]Hindák F: Colour Atlas of Cyanophytes. Bratislava: VEDA; 2008.
• [20]Komárek J: Review of the cyanoprokaryotic genus Romeria. Czech Phycol 2001, 1:5-19.
• [21]Komárek J, Komárkova J: Review of the European Microcystis morphospecies (Cyanoprokaryotes) from nature. Czech Phycol 2002, 2:1-24.
• [22]Komárek J, Komárkova J: Taxonomic review of the cyanoprokaryotic genera Planktothrix and Planktothricoides. Czech Phycol 2004, 4:1-18.
• [23]Komárek J, Komárkova J: Diversity of Aphanizomenon-like cyanobacteria. Czech Phycol 2006, 6:1-32.
• [24]Komárek J, Zapomělová E: Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum – 1. part: coiled types. Fottea 2007, 7:1-31.
• [25]Komárek J, Zapomělová E: Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum – 2. part: straight types. Fottea 2008, 8:1-14.
• [26]Jasser I, Karnkowska-Ishikawa A, Kozłowska E, Królicka A, Łukomska-Kowalczyk M: Composition of picocyanobacteria community in the Great Mazurian Lakes: isolation of phycoerythrin-rich and phycocyanin-rich ecotypes from the system - comparison of two methods. Pol J Microbiol 2010, 59:21-31.
• [27]Wilmotte A, Van der Aauwera G, De Wachter R: Structure of the 16 S ribosomal RNA of the thermophilic cyanobacterium chlorogloeopsis HTF (‘mastigocladus laminosus HTF’) strain PCC7518, and phylogenetic analyses. FEBS Lett 1993, 317:96-100.
• [28]Ye W, Liu X, Tan J, Li D, Yang H: Diversity and dynamics of microcystin—producing cyanobacteria in China’s third largest lake, Lake Taihu. Harmful Algae 2009, 8:637-644.