Cyanobacterial protease inhibitor microviridin J causes a lethal molting disruption in Daphnia pulicaria

Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.     

Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins

The role of zooplankton in the control of cyanobacterial bloomsand the transfer of cyanotoxins to higher trophic levels areof great importance to the management of water resources. Manystudies have focused on the cyanobacterium Microcystis, butfew have examined the interactions between zooplankton and filamentouscyanobacteria. In this study, we provide experimental evidencefor the potential grazing of two toxic strains of filamentouscyanobacteria, Planktothrix rubescens and P. agardhii, by Daphniapulicaria, and for transfer of toxins in the planktonic foodchain. We determined clearance rates (CRs) by adult and juvenileD. pulicaria of the two Planktothrix strains, Scenedesmus acutusand a mixture of S. acutus cells with P. rubescens culture filtrate.Filament lengths were analyzed, and microcystin (MCY) presencein Daphnia was assessed using the Protein Phosphatase-2A (PP-2A)Inhibition Assay. The two Planktothrix strains were equallygrazed by D. pulicaria, but at lower CRs than S. acutus. Potentialanti-grazer toxins in P. rubescens filtrate did not inhibitDaphnia grazing. Small P. rubescens (<100 µm) filamentswere preferentially grazed by adult D. pulicaria, suggestingtheir limited ability to control a Planktothrix population duringa bloom. Large quantities of MCYs were found in unstarved Daphniapreviously exposed to Planktothrix, whereas quantities weresignificantly smaller in individuals starved for 24 h beforepreservation. This indicated a potential for transfer of toxinsin the food chain by Daphnia, especially immediately after ingestionof toxic cyanobacteria.     

Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient‐rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin‐inhibitor‐containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC₅₀ values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour‐joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.     

Cyanobacterial Protease Inhibitor Microviridin J Causes a Lethal Molting Disruption in Daphnia pulicaria

Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.     

Identification of peptide metabolites of Microcystis (Cyanobacteria) that inhibit trypsin-like activity in planktonic herbivorous Daphnia (Cladocera)

Cyanobacteria are recognized as producers of a broad variety of bioactive metabolites. Among these, the peptides synthesized by the non-ribosomal peptide synthetase pathway occur in high structural variability. One class of cyanobacterial peptides, the cyanopeptolins or micropeptins, have been shown to be strong inhibitors of vertebrate serine proteases, like trypsin. In the present study we screened extracts of ten strains of the unicellular cyanobacterium Microcystis sp. for their potential to inhibit trypsin-like activity in the planktonic crustacea Daphnia, the main herbivores in freshwater ecosystem. Respective standardized IC(50)'s varied for nearly two orders of magnitude. In HPLC fractions we could identify mainly cyanopeptolins as active compounds by MALDI-TOF mass spectrometry. Cyanopeptolins were found in 22 structural variants with 13 variants produced by one strain alone. Peptides of the microviridin class were moderately active while no activity was evident for microginins and microcystins. Among the cyanopeptolins only those were active that had an arginine or lysine residue N-terminal to the modified amino acid 3-amino-6-hydroxy-piperidone. Structural variants that had a tyrosine residue at this particular position did not inhibit trypsin-like activity. The highly variable composition of the side chain of cyanopeptolins had no marked effect on the activity. Among the six cyanobacterial strains we tested intensively two did not produce any cyanopeptolins and were accordingly less active as crude extracts. The present study underlines the potential importance of the biochemistry of cyanobacteria for the feeding ecology of a planktonic herbivore.     

Possible implications of chytrid parasitism for population subdivision in freshwater cyanobacteria of the genus Planktothrix

Populations of the cyanobacterium Planktothrix comprise multiple coexisting oligopeptide chemotypes that can behave differently in nature. We tested whether this population subdivision can, in principle, be driven by parasitic chytrid fungi, which are almost neglected agents of Planktothrix mortality. Two chytrid strains, Chy-Lys2009 and Chy-Kol2008, were isolated from Planktothrix-dominated lakes in Norway. The two strains shared 98.2% and 86.2% of their 28S and internal transcribe spacer rRNA gene sequences, respectively. A phylogenetic analysis placed them in the order Rhizophydiales family Angulomycetaceae. Chy-Lys2009 and Chy-Kol2008 could completely lyse Planktothrix cultures within days, while they failed to infect other filamentous cyanobacteria. The effect on Planktothrix was chemotype dependent, and both chytrid strains showed distinct chemotype preferences. These findings identify chytrid fungi infecting Planktothrix as highly potent and specialized parasites which may exert strong selective pressure on their hosts. According to established hypotheses on host-parasite coevolution, parasitism with the above properties may result in subdivision of Planktothrix populations into coexisting chemotypes and periodic shifts in the relative Planktothrix chemotype composition. These predictions are in agreement with field observations. Moreover, a genetic analysis verified the co-occurrence of Chy-Lys2009 and Chy-Kol2008 or related chytrid strains along with distinct Planktothrix chemotypes in at least one water body. Our findings are consistent with a scenario where chytrid parasitism is one driving force of Planktothrix population subdivision, which in turn leads to polymorphism in parasitic chytrid fungi. Future studies should test the validity of this scenario under field conditions.     

16S rDNA analyses of the cyanobacterial microbiota through the water-column in a boreal lake with a metalimnic Planktothrix population

The Planktothrix population in Lake Steinsfjord has attracted particular attention, due to the potential development of toxic blooms. This population is special in the sense that mass developments of Planktothrix occur in the metalimnion. We investigated the distribution of Planktothrix, as well as other cyanobacteria, through the water-column during a Planktothrix mass development at 10-16 m depth. The analyses were done by chlorophyll measurements, microscopy, and by a recently developed 16S rDNA array-based method.These analyses showed that Planktothrix dominated the cyanobacterial community at 11 m, while cyanobacteria belonging to the order Nostocales were predominant at 4 m. The combination of analytical methods presented in this work provides a powerful tool to analyze cyanobacterial communities. We have developed a concept that enables both relative (16S rDNA array analyses) and absolute quantification (chlorophyll a measurements) of cyanobacteria through water-columns. Such approaches will be important in better understanding cyanobacterial microbiota and bloom dynamics.     

Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

Effects of nutrient and light availability on production of bioactive anabaenopeptins and microviridin by the cyanobacterium <Emphasis Type="Italic">Planktothrix agardhii</Emphasis>

Cyanobacteria produce a large number of bioactive oligopeptides with yet unknown functions. Here the effect of environmental factors on the production of two anabaenopeptins and a microviridin by Planktothrix agardhii was investigated. The results were compared with previous findings on the production of a third family of oligopeptides, the highly toxic microcystins, to test the hypothesis that environmental factors affect the production of different types of cyanobacterial bioactive oligopeptides in a similar manner. Despite marked changes in culture conditions, variations in the amount of cell-bound anabaenopeptins and microviridin I per biomass unit did not exceed a factor of 5. High amounts of cell-bound anabaenopeptins and microviridin I per Planktothrix biovolume were associated with a high availability of nitrogen and phosphorus. The production of anabaenopeptins and microviridin continued as long as the Planktothrix cultures grew. In all cases the oligopeptide net production rate was linearly correlated to the growth rate of Planktothrix, identifying the growth activity as a major regulator of anabaenopeptin and microviridin production. The concentration of anabaenopeptins and microviridins in nature may therefore be estimated from the biomass concentration of their producers. These findings are similar to those previously reported for microcystins and thus support the idea that different types of bioactive cyanobacterial oligopeptides may share common regulation patterns. This may be seen as a hint to a mutual function of cyanobacterial oligopeptides, although further studies are needed to draw final conclusions on this issue. Handling editor: J. Padisak     

微囊藻毒素在滇池鱼体内的积累水平及分布特征

为了解富营养化水体中鱼体内微囊藻毒素(MC)的积累水平及其分布特征,2003年4月和9月份两次在滇池试验区采集了鲢、鳙和草鱼等鱼种,用ELISA方法对鱼体中肝、肾、空肠、胆、肌肉等不同组织中MC的含量进行了检测。结果表明,MC在所有样品中均能检测到,且主要分布在鱼体的肝肾脏和消化道等器官,而肌肉和非消化道器官中毒素含量相对较低。不同鱼种不同组织对MC的富集程度也明显不同,鲢鳙中肝脏和肾脏这两个主要的靶器官对MC的蓄积能力就远高于草鱼。同时,不同季节MC在鱼体内的积累水平也明显不同,4月份鱼样中MC的含量普遍低于9月份鱼样中MC的含量。最后按照WHO生活饮用水安全标准的建议进行推算,所有鱼肉中的MC均没有超过其推荐的人体每日可允许摄入量(≤0.04μg/kg人体重),初步推断鱼肉中MC暂时还未危及到人体健康,但仍具有潜在的风险性。     

Quantitative PCR enumeration of total/toxic Planktothrix rubescens and total cyanobacteria in preserved DNA isolated from lake sediments

The variability of spatial distribution and the determinism of cyanobacterial blooms, as well as their impact at the lake scale, are still not understood, partly due to the lack of long-term climatic and environmental monitoring data. The paucity of these data can be alleviated by the use of proxy data from high-resolution sampling of sediments. Coupling paleolimnological and molecular tools and using biomarkers such as preserved DNA are promising approaches, although they have not been performed often enough so far. In our study, a quantitative PCR (qPCR) technique was applied to enumerate total cyanobacterial and total and toxic Planktothrix communities in preserved DNA derived from sediments of three lakes located in the French Alps (Lake Geneva, Lake Bourget, and Lake Annecy), containing a wide range of cyanobacterial species. Preserved DNA from lake sediments was analyzed to assess its quality, quantity, and integrity, with further application for qPCR. We applied the qPCR assay to enumerate the total cyanobacterial community, and multiplex qPCR assays were applied to quantify total and microcystin-producing Planktothrix populations in a single reaction tube. These methods were optimized, calibrated, and applied to sediment samples, and the specificity and reproducibility of qPCR enumeration were tested. Accurate estimation of potential inhibition within sediment samples was performed to assess the sensitivity of such enumeration by qPCR. Some precautions needed for interpreting qPCR results in the context of paleolimnological approaches are discussed. We concluded that the qPCR assay can be used successfully for the analysis of lake sediments when DNA is well preserved in order to assess the presence and dominance of cyanobacterial and Planktothrix communities.     

The relationship between temperature,competition and the potential for colonization of a subtropical pond by Daphnia magna

The role that temperature plays in excluding large daphnid species from subtropical and tropical ponds, and competitive relationships between Daphnia laevis and Daphnia magna, are explored. D. magna, a large temperate species is probably not excluded from subtropical or tropical systems by elevated temperatures. However, D. magna was a poorer competitor, under a restricted set of laboratory conditions, than D. laevis, the only pond dwelling daphnid in subtropical Florida. Competition is proposed as one mechanism that may limit the number of daphnid species in subtropical and tropical ponds and lakes. Reduced environmental fluctuations in subtropical and tropical systems (compared with temperate systems) may allow zooplankton populations to reach an environmental carrying capacity where competition limits the number of similar, coexisting species in a habitat.     

Applications of cyanobacteria in biotechnology

Cyanobacteria have gained a lot of attention in recent years because of their potential applications in biotechnology. We present an overview of the literature describing the uses of cyanobacteria in industry and services sectors and provide an outlook on the challenges and future prospects of the field of cyanobacterial biotechnology. Cyanobacteria have been identified as a rich source of biologically active compounds with antiviral, antibacterial, antifungal and anticancer activities. Several strains of cyanobacteria were found to accumulate polyhydroxyalkanoates, which can be used as a substitute for nonbiodegradable petrochemical-based plastics. Recent studies showed that oil-polluted sites are rich in cyanobacterial consortia capable of degrading oil components. Cyanobacteria within these consortia facilitated the degradation processes by providing the associated oil-degrading bacteria with the necessary oxygen, organics and fixed nitrogen. Cyanobacterial hydrogen has been considered as a very promising source of alternative energy, and has now been made commercially available. In addition to these applications, cyanobacteria are also used in aquaculture, wastewater treatment, food, fertilizers, production of secondary metabolites including exopolysaccharides, vitamins, toxins, enzymes and pharmaceuticals. Future research should focus on isolating new cyanobacterial strains producing high value products and genetically modifying existing strains to ensure maximum production of the desired products. Metagenomic libraries should be constructed to discover new functional genes that are involved in the biosynthesis of biotechnological relevant compounds. Large-scale industrial production of the cyanobacterial products requires optimization of incubation conditions and fermenter designs in order to increase productivity.     

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.     

Effects of Daphnia exudates and sodium octyl sulphates on filament morphology and cell wall thickness of Aphanizomenon gracile (Nostocales), Cylindrospermopsis raciborskii (Nostocales) and Planktothrix agardhii (Oscillatoriales)

Grazing is recognized as one of the selective factors shaping the morphology and physiology of cyanobacteria. A recent study has shown that the filamentous cyanobacterium Aphanizomenon gracile strain SAG 31.79 thickened in the presence of Daphnia (Cladocera) and its exudates. The aims of our study were: (1) to determine whether this type of response to Daphnia cues is common for other strains of A. gracile, and other species of filamentous cyanobacteria, (2) to test whether the response is due to nutrients recycled by Daphnia, or kairomone induced, and (3) whether it is related to toxin production. Prior to the experiment, cyanobacterial strains were inspected using chromatographic methods for the presence of two toxins, cylindrospermopsin (CYN) and three homologues of microcystin (MC-RR, MC-YR, MC-LR). HPLC analyses showed that all strains were free of cylindrospermopsin, whereas microcystins were detected only in one strain (Planktothrix agardhii). We then tested whether Daphnia exudates can cause thickening of cyanobacterial filaments, which would suggest the morphological changes in cyanobacterial filaments are caused by recycled nutrients. Cyanobacteria were also exposed to sodium octyl sulphate (a commercially available Daphnia kairomone). Transmission electron microscopy (TEM) was used to check whether Daphnia exudates and sodium octyl sulphate trigger thickening of cyanobacterial cell walls, which would be a defence mechanism against grazing. The TEM analysis revealed no significant effect of either Daphnia exudates or kairomone (sodium octyl sulphate) on the cell wall thickness of cyanobacteria. However, our study showed that Daphnia exudates triggered filament thickening in nostocalean cyanobacteria, while filaments of the oscillatorialean strain P. agardhii did not show this response. It was also demonstrated that sodium octyl sulphate alone can also cause filament thickening, which suggests that this might be a specific defence response to the presence of grazers.     

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.     

Macrophyte and fish chemicals suppress Daphnia growth and alter life-history traits

Daphnids undergoing diel horizontal migration (DHM) to seek daytime refuge in the littoral zones of shallow lakes are likely to confront chemical cues from littoral-associated predators and macrophytes. In field experiments, we investigated how the natural suite of chemicals occurring in a wholly vegetated lake as well as within plant-free mesocosms with artificial macrophytes and epiphytes (either fishless or containing small fish) influenced individual daphnid growth. In laboratory experiments, we further examined how water containing chemicals from either a submerged macrophyte (waterweed, Elodea canadensis ), a planktivorous fish (roach, Rutilus rutilus ) or both impacted daphnid growth and life-history traits. In the field, we found the greatest suppression of daphnid growth in vials containing water from the wholly vegetated lake relative to growth of daphnids housed in vials containing spring water. Water from the mesocosm with fish also suppressed daphnid growth. Daphnid growth in water from the fishless mesocosm, which contained plastic plants colonized by epiphytes, did not differ from that of daphnids grown in spring water. In the lab experiment, daphnids exposed to Elodea chemicals took longer to mature and possessed fewer eggs than daphnids in media without Elodea chemicals. Daphnids exposed to chemicals from both Elodea and roach reproduced the earliest and at a smaller size. Daphnids exposed to only roach chemical cues did not significantly differ from daphnids in control media for age or size at first reproduction although they did possess fewer eggs. Daphnia responses to chemicals from either roach or Elodea alone did not predict how Daphnia responded to the combined influence of multiple chemical cues. Our results suggest that prolonged exposure to macrophyte chemicals incurs costs for Daphnia .     

Low fish predation pressure in London reservoirs: II. Consequences to zooplankton community structure

The 1992 survey of zooplankton structure in fourteen London supply reservoirs showed the overall dominance of large-bodied zooplankton, mainly species of Daphnia. These reservoirs can be considered as ‘anti-fish’ by virtue of their steeply sloping concrete or brick sides. The average biomass of large Daphnia spp (retained on a 710 μm sieve) in the total zooplankton biomass was higher than 20% for twelve out of fourteen reservoirs. The cladoceran-copepod ratio was inversely correlated with both dominance of large-bodied Daphnia magna and cladoceran body-size structure. Parallelly, there were tendency of more efficient utilization of lowered algal crops in reservoirs dominated by large-bodied Daphnia spp. A graphical model is presented which relates daphnid species composition and zooplankton size structure to a presumed gradient of fish biomass in these reservoirs.     

Preparation of l-proline based aeruginosin 298-A analogs: Optimization of the P1-moiety

Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug development. In order to understand the structure–activity relationship (SAR) and to find selective thrombin inhibitors, we synthesized a series of aeruginosin 298-A analogs, in which the P₂ bicyclic amino acid was replaced by a l-proline residue. The structure optimization was focused on modification of the P₁ position. In choosing the P₁ group, an effort was made to avoid using the highly basic guanidine groups present in nearly all naturally occurring aeruginosins. The synthesis and enzyme assays of these aeruginosin analogs against thrombin and trypsin are reported. We found that several compounds with neutral P₁ groups exhibit excellent selectivity over trypsin and good potency against thrombin. The SAR data of the P₁ groups obtained here can be used in preparing other thrombin inhibitors with better selectivity against trypsin.     

Site-specific restriction endonucleases in cyanobacteria

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.