Setting up a polymerase chain reaction assay for the detection of toxic cyanobacteria

*Corresp nding uthor Abstract: Cyanobacteria are aquatic and photosynthetic prokaryotes which form harmful algal blooms under certain conditions. Water blooms of cyanobacteria from the genus Microcystis are of increasing concern due to their production of microcystin, a cyclic heptapeptide which is formed nonribosomally by peptide and polyketide synthetases. A number of studies have targeted the PCR amplification of microcystin synthetase gene cluster for the identification of toxic cyanobacteria, both in cultivated strains and environmental samples. In this study, water samples collected from seven sites and the cultures originated from the cyanobacterial bloom of the Lake Beira were analyzed by polymerase chain reaction (PCR) using the cyanobacterial specific oligonucleotide primers for the 16S rRNA gene and genus specific oligonucleotide primers for the mcyE gene. All DNA samples submitted to PCR reactions yielded the unique fragments of about 450 bp for the 16S rRNA gene and 250bp for the mcyE gene. The results of the BLAST EF051239 showed 98% of the nucleotide homology to the Microcystis aeruginosa gene of 16S ribosomal RNA and EF051238, 95% homology to the mcy E gene of M. aeruginosa (PCC 7941).These results confirm the presence of microcystin producing M. aeruginosa in the Beira Lake and the need for rapid identification of toxin producing cyanobacteria from environmental samples.


IntroductIon
Cyanobacteria are aquatic and photosynthetic prokaryotes which form harmful algal blooms (HAB) under optimal conditions such as high light and calm weather.Mass presence of these organisms in freshwaters is of increasing concern worldwide due to their production of a range of hepatotoxins and neurotoxins.The microcystins, cyclic heptapeptide hepatotoxins, are by far the most prevalent of the cyanobacterial toxins and are produced by strains of distantly related cyanobacterial genera Microcystis, Anabaena, Plankthothrix, and more rarely Anabaenopsis, Hapalosiphon and Nostoc 1 .Hepatotoxins are synthesized and retained in cyanobacterial cells but during bloom senescence and cell lyses they are released into the surrounding water.The incidence of wild and domestic animal poisoning and human health problems attributed to exposure to cyanobacterial toxins have been well documented 2 .
Identification of a cyanobacterial genus by microscopic morphology and / or molecular analysis does not indicate the potential for toxin production.Different strains of one species can be morphologically identical but differ in toxigenicity 8 .The most common methods for monitoring microcystin concentrations have been high performance liquid chromatography (HPLC) combined with a UV-visible light diode array detector, protein phosphatase inhibition and enzyme linked immunosorbent assays (ELISA) 9 .But their analysis does not indicate which cyanobacteria produce the toxins, since several genera of cyanobacteria may produce similar microcystin variants 1 .

Many investigators have used PCR based methods
for the direct detection and identification of toxigenic strains in bloom samples 8 .Phycocyanin intergenic spacer region between the genes for the β and α subunits of the phycocyanin operon 10,11 , the 16S-23S rRNA internally transcribed spacer region 12 are some of the examples that used DNA amplifications to detect potential toxicity in cyanobacteria.A number of studies have targeted the PCR amplification of microcystin synthetase (mcy) gene cluster for the identification of toxic Microcystis strains 13 and this has enabled the development of specific oligonucleotide primers for genes common to production of all microcystins 14,15 .The mcyE gene, which encodes the glutamate activating adenylation domain can be used as a surrogate for microcystin -producing cyanobacteria 13 .
The Beira Lake is situated within the city of Colombo, and covers 653,000 m 2 with a catchment area of 448, 000 m 2 .It has a history of severe cyanobacterial blooms.Previous reports from Sri Lanka on water blooms have revealed the occurrence of toxic cyanobacteria, and a study by Jayatissa et al. 16 showed the presence of Microcystis spp. in fresh water bodies of Sri Lanka, including the Beira Lake.
In May 2006, Sri Lankan media reported the deaths of a large number of fish and the presence of a heavy growth of algae in the Beira Lake.Therefore, this study was undertaken to determine the value of using specific DNA amplification techniques for the direct detection of toxigenic strains of toxin producing cyanobacteria from environmental samples.

MEtHodS And MAtErIALS
Water samples from the Beira Lake, Colombo (6 0 56'N & 79 0 51'E) were collected in sterile brown glass containers (2.5L) on 24 th May 2006.The collections were carried out at seven sites, both on the surface and down the water column (0.1 & 1m) so that they represent the whole water body.
For culture, samples were concentrated by centrifugation (3500 rpm, 10 min) and the resulting pellet was serially diluted and inoculated onto BG11 medium and modified BG11 medium.Microscopic observations were made from the field samples.The culture plates and culture bottles were incubated at 28°C with 16-light dark cycling.The standard strains used in this study were obtained from the Pasteur Culture Collection (PCC), France.Lyngbya (PCC 8937), a filamentous non toxin producing strain and Microcystis aeruginosa (PCC 7941), a unicellular toxin producing strain were maintained in modified BG11 medium 17 .
Cyanobacterial cells from the culture as well as 500 µL pellets obtained from environmental samples were transferred to 500 μl of 1xTE buffer and three sequential heating (at 99 °C for 5 min) and freezing (at -5°C for 5 min) achieved lyses.Samples were centrifuged (12,000 rpm, 5 min) and to each resulting pellet, 40 μL of TES and 20 μL of lysozyme (10 mg /mL) was added, and incubated for 1 h at 37°C.Cells were then treated with 10 µL of proteinase K (20 mg/mL) and 40 µL of TE/ SDS and incubated at 55°C for 2 h to lyse the organisms further.Subsequently, proteinase K was inactivated by heating the sample at 95°C for 10 min.Finally, nucleic acids were purified by Boom's method 18 using silica particles and guanidium isothiocyanate.
The visualized PCR fragments were excised and purified using genElute TM Gel extraction Kit (SIGMA) according to the manufacturer's instructions.The PCR fragments for the 16S rRNA and mcyE genes were sequenced using the MegaBase 1000 (GE Healthcare Biosciences).The obtained sequences were surveyed in the GenBank using Blastn ® 20 .The nucleotide sequences were deposited in GenBank under accession numbers EF051238 and EF051239.

rESuLtS And dIScuSSIon
In this study, a method based on the PCR technique and genus specific mcyE primers 13 was used to detect the microcystin producer in the Beira Lake.When the samples from seven sites were subjected to microscopic examination, Microcystis sp. was recorded as the dominant cyanobacterial species in the water body.The second species recorded was Spirulina sp. which was comparatively very low in numbers.
The method employed for genomic DNA extraction resulted in high quality DNA in satisfactory amounts for amplification.All DNA samples submitted to PCR reactions from the water samples collected from the Lake Beira for the 16S rRNA gene yielded the unique fragment of about 450bp, using the cyanobacterial specific oligonucleotide primers of Cya 359F forward and Cya 781 Rb reverse (Figure 1a).According to the advice given by Boutte et al. 21we used the cyanobacterial specific reverse primers Cya 781 Ra and Cya 781 Rb 19 separately and also in eqimolar mixtures to determine the cyanobacterial community composition in the Beira Lake.The study by Boutte et al. 21showed that the primers Ra and Rb target filamentous and unicellular cyanobacteria, respectively in a cyanobacterial community.The use of the forward primer Cya 359 F and the reverse primer Cya 781Ra, as well as Cya 359F and the equimolar mixture of Cya 781 Ra + Cya 781Rb did not yield the fragment of 450bp.In our study, only the reverse primer Rb yielded the unique fragment for the environmental samples indicating the dominance of unicellular/colonial nature of the Microcystis species in the algal bloom.
A single amplification product was observed when genomic DNA from the standard microcystin -producing M. aeruginosa (PCC 7941) was used as a template in PCR with Microcystis genus specific primers.All DNA samples submitted to PCR reactions both from the water samples collected from the Beira Lake and from the cultured isolates originated from water samples for the mcyE gene, yielded the unique fragment of about 250bp, using the microcystin synthetase gene E forward primer (mcyE -F2) and genus specific reverse primer for Microcystis (MicmycE-R8) (Figure1b).The results of the BLAST of EF051239 showed 98% of the nucleotide homology to the M. aeruginosa gene for 16S ribosomal could be toxins in the cyanophyte, depletion of oxygen in the water, by the liberation of hydrogen sulphide and ammonia caused by cell decomposition or by clogging of the gills.
The results obtained from the study confirms the presence of microcystin producing M. aeruginosa in the Beira Lake and the usefulness of molecular biological techniques for the rapid identification of toxin producing cyanobacteria from environmental samples.RNA, partial sequence of AB 271211 while EF051238 showed 95% homology to the mcyE gene of Microcystis aeruginosa PCC 7941 ( AY382536.1).Therefore the presence of the gene mcyE in the analyzed water samples/ cultures originating from water samples indicates that the cyanobacterial strains have the genetic potential to produce microcystins.
Previous analysis using the technique of high performance liquid chromatography (HPLC), recorded 0.737 µgL -1 concentration of microcystins in water samples collected from the Beira Lake in 2000 16 .A study in 2002 8 has shown that the PCR assays, applied directly to environmental samples, were as sensitive as HPLC in providing a useful indicator of toxicity.The PCR based assays detect toxigenic cells rather than toxins and require little sample preparation and modest capital costs.Comparatively the DNA amplification technique described in this study does not require a large capital.This is the first study in Sri Lanka which used molecular markers and DNA sequencing to identify toxin generating strains of cyanobacteria in environmental water samples.Being a developing country, Sri Lanka is unable to make large investments for monitoring water quality.Therefore, molecular techniques combined with microscopy can be used to rapidly determine the presence of toxigenic cyanobacterial species in environmental samples.Cyanobacteria grow best in non turbulent, warm rivers, lakes and reservoirs.Microcystis occasionally forms a bloom, or dense aggregation of cells, that floats on the surface of the water forming a thick layer or 'mat'.Blooms usually occur during the warmest months of the year, especially when the water contains an over abundance of nitrogen (N) and phosphorus (P).Excessive P most often provides the stimulus for cyanobacterial blooms, especially if the total N to total P concentration ratio is less than 10 22 .According to the data of the Ports Authority of Sri Lanka, (CLEAN), on the day when an excessive fish death on the lake was observed, the total N to total P concentration ratio was around 1.9 [nitrate,1.5 mg L -1 (max -5 mg L -1 ) and phosphate, 0.8 mg L -1 (max -0.4mg L -1 )] which was less than 10.Therefore, it is possible that the excessive P in the lake has stimulated the cyanobacterial growth.
Cyanobacterial blooms potentially affect water quality as well as the health of human and animal life.Decomposition of large blooms can lower the concentration of dissolved oxygen in the water, resulting in hypoxia (low oxygen) or anoxia (no oxygen).Wild animal poisonings can occur after ingestion of cyanobacterial biomass and intake of toxins during drinking and feeding.In the case of fish deaths, the causes