Detection of Microcystin-Producing Cyanobacteria in Finnish Lakes with Genus-Specific Microcystin Synthetase Gene E (mcyE) PCR and Associations with Environmental Factors

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.     

Quantitative real-time PCR for determination of microcystin synthetase e copy numbers for microcystis and anabaena in lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanj?rvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanj?rvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanj?rvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Identification of hepatotoxin-producing cyanobacteria by DNA-chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena , Microcystis , Planktothrix , Nostoc and Nodularia . Genus-specific probe pairs were designed for the detection of the microcystin ( mcyE ) and nodularin synthetase genes ( ndaF ) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE / ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE / ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1–5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.     

Molecular characterization of potential microcystin-producing cyanobacteria in Lake Ontario embayments and nearshore waters

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 microg MC-LReq liter(-1). MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.     

Use of a generalized additive model to investigate key abiotic factors affecting microcystin cellular quotas in heavy bloom areas of Lake Taihu

Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation.     

Nitrogen Forms Influence Microcystin Concentration and Composition via Changes in Cyanobacterial Community Structure

The eutrophication of freshwaters is a global health concern as lakes with excess nutrients are often subject to toxic cyanobacterial blooms. Although phosphorus is considered the main element regulating cyanobacterial biomass, nitrogen (N) concentration and more specifically the availability of different N forms may influence the overall toxicity of blooms. In this study of three eutrophic lakes prone to cyanobacterial blooms, we examined the effects of nitrogen species and concentrations and other environmental factors in influencing cyanobacterial community structure, microcystin (MC) concentrations and MC congener composition. The identification of specific MC congeners was of particular interest as they vary widely in toxicity. Different nitrogen forms appeared to influence cyanobacterial community structure leading to corresponding effects on MC concentrations and composition. Total MC concentrations across the lakes were largely explained by a combination of abiotic factors: dissolved organic nitrogen, water temperature and ammonium, but Microcystis spp. biomass was overall the best predictor of MC concentrations. Environmental factors did not appear to affect MC congener composition directly but there were significant associations between specific MC congeners and particular species. Based on redundancy analyses (RDA), the relative biomass of Microcystis aeruginosa was associated with MC-RR, M. wesenbergii with MC-LA and Aphanizomenon flos-aquae with MC-YR. The latter two species are not generally considered capable of MC production. Total nitrogen, water temperature, ammonium and dissolved organic nitrogen influenced the cyanobacterial community structure, which in turn resulted in differences in the dominant MC congener and the overall toxicity.     

Diversity and dynamics of microcystin—Producing cyanobacteria in China's third largest lake, Lake Taihu

Microcystins (MC), the most prevalent group of harmful cyanobacterial hepatotoxins, are primarily produced by strains of cyanobacteria in Microcystis, Anabaena and Planktothrix. Lake Taihu, which is the third largest freshwater lake in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, PCR-DGGE was used to evaluate the diversity of potential MC-producing cyanobacteria and real-time PCR was used to analyze the dynamics of this population based on the presence of the mcy gene in samples collected during a year long study. The results revealed that all MC-producing genotypes detected belonged to the genus Microcystis. In addition, the MC-producing genotype communities were more diverse during the bloom season than the non-bloom season, and the diversity in the late bloom period was lower than the diversity in the early bloom period. Furthermore, the abundance of MC-producing genotypes increased dramatically during the bloom development period, reaching its peak in late summer (September). The results also suggested that the highest mcy gene concentration lagged behind the highest MC concentration, and the potential MC-producing cyanobacterial community shift lagged behind the development of blooms.     

Widespread distribution and identification of eight novel microcystins in antarctic cyanobacterial mats

The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [D-Asp(3)] MC-RR and [D-Asp(3)] MC-LR containing glycine [Gly(1)] rather than alanine and combinations of homoarginine [hAr(2)] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda(5)] substitutions. Nostoc sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing Nostoc sp. from multiple samples.     

Use of electrospray tandem mass spectrometry for identification of microcystins during a cyanobacterial bloom event

Drastic environmental conditions such as elevated temperature, abrupt pH variation, low turbulence, and high nutrient inputs can enhance the development of toxic cyanobacterial blooms in lakes and reservoirs. This study describes the occurrence of four microcystin variants (MC) in a bloom in the eutrophic reservoir Billings, in S?o Paulo City. The bloom sample was collected in October 2003, and Microcystis were the main genus found. The MC were separated and purified by reverse phase high performance liquid chromatography (RP-HPLC). Their structures were elucidated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and four MC variants were determined: MC-RR, MC-LR, MC-YR, and MC-hRhR. MC-hRhR is described for the first time as a new variant of MC with two homoarginines at positions 2 and 4 in its structure. ESI-MS/MS analysis thus provides a powerful and convenient tool for the determination of variants of MC. These results represent an important contribution to the knowledge of the biochemistry of toxic cyanobacteria and their toxins, specifically in S?o Paulo State.     

Survey of microcystins in water between 1995 and 1996 in Paraná, Brazil using ELISA

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml(-1)) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming-fishing sites at Itaipu dam, 6380 and 10,000 pg ml(-1)) and human supplies (6627 pg ml(-1)) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl- reduced MCs levels, although 267 pg ml(-1) remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region.     

Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90

The cluster of microcystin synthetase genes from Anabaena strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (mcyA-mcyB-mcyC) is transcribed in the opposite direction from the second operon (mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI) and the third operon (mcyH). The genes mcyA, mcyB, and mcyC encode nonribosomal peptide synthetases (NRPS), while mcyD codes for a polyketide synthase (PKS), and mcyG and mcyE are mixed NRPS-PKS genes. The genes mcyJ, mcyF, and mcyI are similar to genes coding for a methyltransferase, an aspartate racemase, and a D-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of mcyB coding for the adenylation domain was found to be 96% identical with the corresponding part of mcyC, suggesting a recent duplication of this fragment and a replacement in mcyB. In Anabaena strain 90, the order of the domains encoded by the genes in the two sets (from mcyG to mcyI and from mcyA to mcyC) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in Anabaena strain 90 differs from the arrangement found in two other cyanobacterial species, Microcystis aeruginosa and Planktothrix agardhii. The average sequence match between the microcystin synthetase genes of Anabaena strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.     

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.     

Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin

The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Water-blooming and toxin-producing cyanobacteria in Swedish fresh and bracish waters, 1981–1995

During the period 1981–1995 331 phytoplankton samples from 153stations in different parts of Sweden were analysed regardingspecies composition and possible toxicity. Toxins were indicated in156 samples which gives a percentage of 47. The toxicity wasconfirmed by mouse bioassay. All samples were collected fromwater-blooming lakes, mainly containing taxa belonging to thecyanobacterial genera Anabaena, Aphanizomenon, Microcystis and Planktothrix. In addition to informationabout occurrence of neuro- or hepatotoxins, knowledge about thedistribution of water-blooming lakes in the country was obtained aswell as about their predominating taxa. Microcystisaeruginosa, Anabaena flos-aquae and Aphanizomenonflos-aquae were the most frequently occurring taxa in the lakes.Among the taxa observed, nine different cyanobacterial species withcertainty produce toxins. Microcystis spp. most frequentlyproduced toxic blooms (only hepatotoxins). Anabaena spp.,however, caused the majority of waterblooms. The Anabaenaflos-aquae-group produced neurotoxins [incl. anatoxin-a(s)],neurotoxins + hepatotoxins or only hepatotoxins. Aphanizomenon flos-aquae was only recorded as toxin-producing inthree Swedish lakes, which may be compared with the conditions inFinland, where it was toxic in 36 bloom samples. Aspects are givenon toxicity versus lake acidification and trophic conditions, aswell as on health effects on animals and humanbeings.     

An extracellular glycoprotein is implicated in cell-cell contacts in the toxic cyanobacterium Microcystis aeruginosa PCC 7806

Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis aeruginosa; however, the role of the peptide for the producing organism is poorly understood. Differences in the cellular aggregation of M. aeruginosa PCC 7806 and a microcystin-deficient Delta mcyB mutant guided the discovery of a surface-exposed protein that shows increased abundance in PCC 7806 mutants deficient in microcystin production compared to the abundance of this protein in the wild type. Mass spectrometric and immunoblot analyses revealed that the protein, designated microcystin-related protein C (MrpC), is posttranslationally glycosylated, suggesting that it may be a potential target of a putative O-glycosyltransferase of the SPINDLY family encoded downstream of the mrpC gene. Immunofluorescence microscopy detected MrpC at the cell surface, suggesting an involvement of the protein in cellular interactions in strain PCC 7806. Further analyses of field samples of Microcystis demonstrated a strain-specific occurrence of MrpC possibly associated with distinct Microcystis colony types. Our results support the implication of microcystin in the colony specificity of and colony formation by Microcystis.     

Immunogold localisation of microcystins in cryosectioned cells of Microcystis

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC(+)) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC(-)) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC(+)); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC(+)); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.     

Competition between microcystin- and non-microcystin-producing Planktothrix agardhii (cyanobacteria) strains under different environmental conditions

The factors that control the production of microcystins (hepatotoxins) during cyanobacterial blooms, and the function of these metabolites remain largely unknown. In an attempt to provide answers to these questions, we compared the fitness of microcystin (MC)-producing and non-MC-producing Planktothrix agardhii strains under various experimental conditions. More specifically, we investigated the effects of temperature, light intensity and nitrate concentrations on several MC-producing and non-MC-producing strains in monoculture and competition experiments. In the monoculture experiments, no significant difference in cell growth rates was found for any of the environmental conditions tested. On the other hand, at the end of the competition experiments, we found that when the environmental conditions limited cell growth, MC-producing strains were clearly winning out over the non-MC-producing ones. This suggested that, under growth-limiting conditions, the benefits of producing MC outweigh the cost. Moreover, the reverse was found under non-growth-limiting conditions, suggesting that under environmental conditions that favour cyanobacterial growth, the cost of MC production must outweigh its benefits. These findings suggest that environmental factors may have an indirect effect on the MC production rate, and on the selection of MC-producing and non-MC-producing strains, via their direct impact on both the cell growth rate and the cell densities in the cultures. Several hypotheses have been advanced concerning the possible function of MCs, but none of them seems to be supported by our data.     

Nontoxic strains of cyanobacteria are the result of major gene deletion events induced by a transposable element

Blooms that are formed by cyanobacteria consist of toxic and nontoxic strains. The mechanisms that result in the occurrence of nontoxic strains are enigmatic. All the nontoxic strains of the filamentous cyanobacterium Planktothrix that were isolated from 9 European countries were found to have lost 90% of a large microcystin synthetase (mcy) gene cluster that encoded the synthesis of the toxic peptide microcystin (MC). Those strains still contain the flanking regions of the mcy gene cluster along with remnants of the transposable elements that are found in between. The majority of the strains still contain a gene coding for a distinct thioesterase type II (mcyT), which is putatively involved in MC synthesis. The insertional inactivation of mcyT in an MC-producing strain resulted in the reduction of MC synthesis by 94 +/- 2% (1 standard deviation). Nontoxic strains that occur in shallow lakes throughout Europe form a monophyletic lineage. A second lineage consists of strains that contain the mcy gene cluster but differ in their photosynthetic pigment composition, which is due to the occurrence of strains that contain phycocyanin or large amounts of phycoerythrin in addition to phycocyanin. Strains containing phycoerythrin typically occur in deep-stratified lakes. The rare occurrence of gene cluster deletion, paired with the evolutionary diversification of the lineages of strains that lost or still contain the mcy gene cluster, needs to be invoked in order to explain the absence or dominance of toxic cyanobacteria in various habitats.     

Molecular detection of uncultured cyanobacteria and aminotransferase domains for cyanotoxin production in sediments of different Kenyan lakes

PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments was used to identify the cyanobacterial phylotypes in sediments and plankton of saline–alkaline and freshwater lakes of Kenya. The detection of the aminotransferase domain located on modules mcyE and ndaF using specific molecular markers confirmed the presence of potential toxin-producing cyanobacteria. The eight nucleotide sequences obtained from DGGE bands were placed in three divergent cyanobacterial clusters. Five nucleotide sequences were close to members of the genera Anabaenopsis and Umezakia ( Nostocales ), two sequences fell in the cluster with Arthrospira sp. ( Oscillatoriales ) and one sequence was related to Chroococcidiopsis sp. ( Pleurocapsales ). The presence of the latter taxon was demonstrated de novo in the investigated lakes. All nine attained nucleotide sequences of the aminotransferase region belonged to the mcyE module. Five sequences of the aminotransferase domain were included in the cluster having the nucleotide sequence of Anabaena sp. but showed a separate lineage. Other four aminotransferases were placed in the cluster represented by nucleotide sequence of Microcystis aeruginosa . To our knowledge, this is the first report on molecular detection of cyanobacterial phylotypes in sediments of African lakes and aminotransferase domains for cyanotoxin production from sediment samples in general.