Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.     

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Aims: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. Method and Results: A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml⁻¹ for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml⁻¹ and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. Conclusions: The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. Significance and Impact of the Study: This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.     

Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii,and taxonomically diverse freshwater cyanobacteria and green algae

Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.     

Application of real-time PCR in the assessment of the toxic cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis> abundance and toxicological potential

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.     

Identification and Phylogenetic Analysis of Toxigenic Cyanobacteria by Multiplex Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA PCR was used to generate unique and identifying DNA profiles for members of the cyanobacterial genera Anabaena and Microcystis, which are responsible for much of the production of nuisance blooms in various freshwater systems, including recreational and drinking water supplies. A method based on the combination of two 10-mer oligonucleotides in a single PCR was developed to provide specific and repeatable DNA fingerprints for cyanobacterial isolates. The strain-specific randomly amplified polymorphic DNA profiles made it possible to discriminate among all toxigenic cyanobacteria studied to the three taxonomic levels of genus, species, and strain. Analysis of DNA typing results obtained by the described method clearly distinguishes between the genera Anabaena and Microcystis. The markers produced for each strain were also applied to a phylogenetic analysis to infer genetic relatedness in this group of prokaryotes.     

Recent invader or indicator of environmental change? A phylogenetic and ecological study of Cylindrospermopsis raciborskii in New Zealand

Cylindrospermopsis raciborskii is a diazotrophic and potentially toxic cyanobacterium that was initially thought to be confined to tropical freshwaters. Recently it appears to have expanded its range to more temperate regions of the globe. There are contrasting hypotheses to explain this spread including; dispersal of highly adapted strains or localised spread from warm refuges as climatic or environmental conditions change. C. raciborskii was first detected in the isolated island nation of New Zealand in 2003, providing a unique opportunity to explore whether this recent identification is due to a new incursion or resultant from climatic or environmental change. Phylogenetic analysis (nifH, ITS1-L, ITS1-S, and rpoC1) of six strains isolated from two New Zealand lakes showed they were most closely related to those from South America, and suggest that the recent detection of this species was not due to a new incursion. Ten years of environmental data from three lakes (Waaki, Waikare and Whangape) experiencing blooms were analysed to identify potential reasons for recent C. raciborskii blooms. This analysis showed that the relatively recent (within the last 20–30 years) collapses of extensive macrophyte stands in lakes Waaki, Waikare and Whangape have resulted in increased turbidity’, low water column dissolved reactive phosphorus and seasonal shifts in the dissolved inorganic nitrogen availability, all conditions known to facilitate C. raciborskii dominance. Collectively these data indicate that C. raciborskii has always been present in New Zealand, and that recent changes in environmental conditions in these lakes are now facilitating bloom events.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.     

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.     

Genetic characterisation of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) isolates from Africa and Europe

The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading in temperate freshwater habitats worldwide and is of major concern due to its ability to produce potent toxins. It is, therefore, important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In the present study, new isolates of C. raciborskii from Europe and Africa were genetically characterised by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate a variation among the populations of C. raciborskii from different geographical regions. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian–African groups, whereas the PC-IGS and rpoC1 separated into American and European/Australian/African groups. An analysis of concatenated data supported the division into American, European and African/Australian groups, and even indicated a subdivision into an African and an Australian group. Our findings do not strongly support any of the existing hypotheses on the phylogeography of C. raciborskii, and most likely a combination of these hypotheses is the best approach to understand the evolution and dispersal of this species.     

Phylogeny of culturable cyanobacteria from Brazilian mangroves

The cyanobacterial community from Brazilian mangrove ecosystems was examined using a culture-dependent method. Fifty cyanobacterial strains were isolated from soil, water and periphytic samples collected from Cardoso Island and Bertioga mangroves using specific cyanobacterial culture media. Unicellular, homocytous and heterocytous morphotypes were recovered, representing five orders, seven families and eight genera (Synechococcus, Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya, Phormidium, Nostoc and Microchaete). All of these novel mangrove strains had their 16S rRNA gene sequenced and BLAST analysis revealed sequence identities ranging from 92.5 to 99.7% when they were compared with other strains available in GenBank. The results showed a high variability of the 16S rRNA gene sequences among the genotypes that was not associated with the morphologies observed. Phylogenetic analyses showed several branches formed exclusively by some of these novel 16S rRNA gene sequences. BLAST and phylogeny analyses allowed for the identification of Nodosilinea and Oxynema strains, genera already known to exhibit poor morphological diacritic traits. In addition, several Nostoc and Leptolyngbya morphotypes of the mangrove strains may represent new generic entities, as they were distantly affiliated with true genera clades. The presence of non-ribosomal peptide synthetase, polyketide synthase, microcystin and saxitoxin genes were detected in 20.5%, 100%, 37.5% and 33.3%, respectively, of the 44 tested isolates. A total of 134 organic extracts obtained from 44 strains were tested against microorganisms, and 26% of the extracts showed some antimicrobial activity. This is the first polyphasic study of cultured cyanobacteria from Brazilian mangrove ecosystems using morphological, genetic and biological approaches.     

Fingerprinting of Cyanobacteria Based on PCR with Primers Derived from Short and Long Tandemly Repeated Repetitive Sequences

The presence of repeated DNA (short tandemly repeated repetitive [STRR] and long tandemly repeated repetitive [LTRR]) sequences in the genome of cyanobacteria was used to generate a fingerprint method for symbiotic and free-living isolates. Primers corresponding to the STRR and LTRR sequences were used in the PCR, resulting in a method which generate specific fingerprints for individual isolates. The method was useful both with purified DNA and with intact cyanobacterial filaments or cells as templates for the PCR. Twenty-three Nostoc isolates from a total of 35 were symbiotic isolates from the angiosperm Gunnera species, including isolates from the same Gunnera species as well as from different species. The results show a genetic similarity among isolates from different Gunnera species as well as a genetic heterogeneity among isolates from the same Gunnera species. Isolates which have been postulated to be closely related or identical revealed similar results by the PCR method, indicating that the technique is useful for clustering of even closely related strains. The method was applied to nonheterocystus cyanobacteria from which a fingerprint pattern was obtained.     

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.     

Identification of cyanobacteria by polymorphisms of PCR-amplified ribosomal DNA spacer region

The 16S-23S ribosomal DNA spacer region of selected cyanobacterial strains was amplified by the polymerase chain reaction using primers to conserved flanking sequences. Single or multiple rDNA amplification products were generated depending on the strain and primer pair. Species could generally be distinguished on the basis of size heterogeneity of the products. Analysis of restriction digests of the amplified rDNAs indicated polymorphisms useful in identification. Four enzymes (HinfI, DdeI, AluI, TaqI) generated restriction fragment length patterns that could discriminate between the cyanobacteria to the taxonomic levels of genus and species. This approach should prove useful in the rapid identification of cyanobacteria.     

Identification of common,unique and polymorphic microsatellites among 73 cyanobacterial genomes

Microsatellites also known as Simple Sequence Repeats are short tandem repeats of 1–6 nucleotides. These repeats are found in coding as well as non-coding regions of both prokaryotic and eukaryotic genomes and play a significant role in the study of gene regulation, genetic mapping, DNA fingerprinting and evolutionary studies. The availability of 73 complete genome sequences of cyanobacteria enabled us to mine and statistically analyze microsatellites in these genomes. The cyanobacterial microsatellites identified through bioinformatics analysis were stored in a user-friendly database named CyanoSat, which is an efficient data representation and query system designed using ASP.net. The information in CyanoSat comprises of perfect, imperfect and compound microsatellites found in coding, non-coding and coding-non-coding regions. Moreover, it contains PCR primers with 200 nucleotides long flanking region. The mined cyanobacterial microsatellites can be freely accessed at www.compubio.in/CyanoSat/home.aspx. In addition to this 82 polymorphic, 13,866 unique and 2390 common microsatellites were also detected. These microsatellites will be useful in strain identification and genetic diversity studies of cyanobacteria.     

Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake

We have studied the diversity of pelagic cyanobacteria in Lake Loosdrecht, The Netherlands, through recovery and analysis of small subunit ribosomal RNA gene sequences from lake samples and cyanobacterial isolates. We used an adapted protocol for specific amplification of cyanobacterial rDNA for denaturing gradient gel electrophoresis (DGGE) analysis. This protocol enabled direct comparison of cyanobacterial community profiles with overall bacterial profiles. The theoretical amplification specificity of the primers was supported by sequence analysis of DNA from excised DGGE bands. Sequences recovered from these bands, in addition to sequences obtained by polymerase chain reaction (PCR) and cloning from lake DNA as well as from cyanobacterial isolates from the lake, revealed a diverse consortium of cyanobacteria, among which are representatives of the genera Aphanizomenon, Planktothrix, Microcystis and Synechococcus. One numerically important and persistent cyanobacterium in the lake, Prochlorothrix hollandica, appeared to co-occur with an unknown but related species. However, the lake is dominated by filamentous species that originally have been termed 'Oscillatoria limnetica-like'. We show that this is a group of several related cyanobacteria, co-occurring in the lake, which belong to the Limnothrix/Pseudanabaena group. The available variation among the coexisting strains of this group can explain the persistent dominance of the group under severe viral pressure.     

Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus.

Cyanobacteria are a highly diverse group in relation to form, function, and habitat. Current cyanobacterial systematics relies on the observation of minor and plastic morphological characters. Accurate and reliable delineation of toxic and bloom-forming strains of cyanobacteria has not been possible by traditional methods. We have designed general primers to the phycocyanin operon (cpc gene) and developed a PCR which allows the amplification of a region of this gene, including a variable intergenic spacer sequence. Because of the specificity of this PCR for cyanobacterial isolates, the assay is appropriate for the rapid and reliable identification of strains in freshwater samples. Successive restriction endonuclease digestion of this amplification product, with a total of nine enzymes, yielded many identifying DNA profiles specific to the various taxonomic levels of cyanobacteria. The restriction enzyme profiles for MspI, RsaI, and TaqI were conserved for strains within each of the eight genera (40 strains) studied and clearly discriminated among these genera. Intrageneric delineation of strains was revealed by the enzymes AluI, CfoI, and HaeIII for members of the genus Microcystis, while strains of genus Anabaena were differentiated by the digestion patterns provided by AluI, CfoI, and ScrFI. Phenetic and cladistic analyses of the data were used to infer the genetic relatedness and evolution of toxic and bloom-forming cyanobacteria.