Determination of cyanobacterial diversity during algal blooms in Daechung Reservoir, Korea, on the basis of cpcBA intergenic spacer region analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.     

Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers

Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Méjean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.     

Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.     

Stimulation of aquatic bacterial activity by cyanobacteria

The time-course response of natural bacterial populations and isolates from lake water to various densities of the filamentous cyanobacteriaAphanizomenon flos-aquae andLyngbya birgei collected from the same lake is reported. The cyanobacteria were separated from the bacteria by dialysis membranes that allowed only dissolved cyanobacterial products to pass. Bacterial³H-thymidine incorporation and cell number were significantly (p<0.05) correlated with cyanobacterial density for both species. Estimated dissolved organic carbon (DOC) utilization, based on bacterial biomass changes over time, were usually significantly (p<0.01) correlated with cyanobacterial density and the decrease in bulk pool DOC for both species. Bacterial volume per cell increased significantly (p<0.05) in response to cyanobacterial density on day 5 of the experiments; cell volume remained unchanged on day 1. Bacterial cell numbers on outer surfaces of the tubular membrane containing the cyanobacteria (on the side exposed to the test bacteria) were significantly (p<0.01) correlated with cyanobacterial density. Statistical analysis inferred that bacteria closely associated with cyanobacteria (i.e. attached) responded more strongly to cyanobacterial products than free-living bacteria. Overall, our results indicate that cyanobacterial products have a potentially important role in regulating bacterioplankton productivity in aquatic systems.     

Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria

The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis , Lyngbya , Pseudanabaena , Aphanizomenon , Nodularia , Anabaena , and Nostoc , based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gram-negative bacteria and the Archea.     

Effects of experimentally induced cyanobacterial blooms on crustacean zooplankton communities

SUMMARY 1. Large in situ enclosures were used to study the effects of experimentally induced cyanobacterial blooms on zooplankton communities. A combination of N and P was added to shallow (2 m) and deep enclosures (5 m) with the goal of reducing the TN : TP ratio to a low level (∼5 : 1) to promote cyanobacterial growth. After nutrient additions, high biomass of cyanobacteria developed rapidly in shallow enclosures reaching levels only observed during bloom events in eutrophic lakes.
2. In the shallow enclosures, particulate phosphorus (PP) was on average 35% higher in comparison with deep enclosures, suggesting that depth plays a key role in P uptake by algae. Phytoplankton communities in both deep and shallow enclosures were dominated by three cyanobacteria species – Aphanizomenon flos-aquae , Anabaena flos-aquae and Microcystis aeruginosa – which accounted for up to 70% of total phytoplankton biomass. However, the absolute biomass of the three species was much higher in shallow enclosures, especially Aphanizomenon flos-aquae . The three cyanobacteria species responded in contrasting ways to nutrient manipulation because of their different physiology.
3. Standardised concentrations of the hepatotoxic microcystin-LR increased as a result of nutrient manipulations by a factor of four in the treated enclosures. Increased biomass of inedible and toxin producing cyanobacteria was associated with a decline in Daphnia pulicaria biomass caused by a reduction in the number of individuals with a body length of >1 mm. Zooplankton biomass did not decline at moderate cyanobacteria biomass, but when cyanobacteria reached high biomass large cladocerans were reduced.
4. Our results demonstrate that zooplankton communities can be negatively affected by cyanobacterial blooms and therefore the potential to use herbivory to reduce algal blooms in such eutrophic lakes appears limited.     

THE CONTRIBUTION OF SUB‐SAHARAN AFRICAN STRAINS TO THE PHYLOGENY OF CYANOBACTERIA: FOCUSING ON THE NOSTOCACEAE (NOSTOCALES,CYANOBACTERIA)1

To date, phylogenies have been based on known gene sequences accessible at GenBank, and the absence of many cyanobacterial lineages from collections and sequence databases has hampered their classification. Investigating new biotopes to isolate more genera and species is one way to enrich strain collections and subsequently enhance gene sequence databases. A polyphasic approach is another way of improving our understanding of the details of cyanobacterial classification. In this work, we have studied phylogenetic relationships in strains isolated from freshwater bodies in Senegal and Burkina Faso to complement existing morphological and genetic databases. By comparing 16S rDNA sequences of African strains to those of other cyanobacteria lineages, we placed them in the cyanobacterial phylogeny and confirmed their genus membership. We then focused on the Nostocaceae family by concatenated analysis of four genes (16S rDNA, hetR, nifH, and rpoC1 genes) to characterize relationships among Anabaena morphospecies, in particular, Anabaena sphaerica var. tenuis G. S. West. Using a polyphasic approach to the Nostocaceae family, we demonstrate that A. sphaerica var. tenuis is more closely related to Cylindrospermospsis/Raphidiopsis than to other planktonic Anabaena/Aphanizomenon. On the basis of phylogeny and morphological data, we propose that these three significantly different clusters should be assigned to three genera.     

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.     

Distribution of nitrogen-fixing cyanobacteria (Nostocaceae) during rice cultivation in fertilized and unfertilized paddy fields

This study describes the development of nitrogen-fixing cyanobacteria (Nostocaceae) during different stages of rice seedlings plantation in fertilized and unfertilized paddy fields in north Bihar, India. A heterogeneous population of cyanobacteria was randomly harvested around day 20, 40 and 60 of rice seedlings plantation, and the diversity was analyzed. Thirtytwo species (7 genera) were identified from unfertilized fields, of which Anabaena was represented by 12 species, Anabaenopsis and Aulosira by 3 each, Cylindrospermum by 4, Nostoc by 8 and Aphanizomenon and Nodularia by 1 species each. However, fertilized fields contained only 25 species (7 genera), of which 8 each belonged to Nostoc and Anabaena , 3 to Cylindrospermum , 2 to each Anabaenopsis and Aulosira and 1 to each Aphanizomenon and Nodularia . Although Nostoc and Anabaena were dominant in both fertilized and unfertilized paddy fields, a marked decrease in nitrogen-fixing cyanobacteria was recorded from fertilized fields. In both treatments, the diversity of nitrogen-fixing cyanobacteria was at a maximum around day 60 after seedling plantation. The current study concludes that there is a negative effect of nitrogenous fertilizers on the development of heterocystous cyanobacteria in rice fields. It is proposed that early appearing efficient nitrogen-fixers should be used as nitrogen fertilizers in the management for better establishment and exploitation of heterocystous cyanobacteria for sustainable agricultural practices.     

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.     

Consumption of cyanobacteria by roach (Rutilus rutilus): useful or harmful to the fish?

1. The ability of roach to use cyanobacterial food is generally believed to be one reason for the dominance of roach over perch in eutrophic European lakes. The aim of this study was to test whether cyanobacteria really are a suitable food for juvenile roach. Special attention was paid to differences between the two cyanobacteria species Aphanizomenon and Microcystis which are common in eutrophic lakes and are ingested by roach there.
2. We performed growth and behaviour experiments with juvenile roach fed with zooplankton and the different cyanobacteria. Growth rate with Aphanizomenon was lower than with Daphnia but significantly higher than without food, whereas growth rate with Microcystis was as low as without food.
3. In cultivation experiments of roach faeces, Microcystis was found not to have been digested and grew exponentially after passing through the gut whereas Aphanizomenon stayed at low biomass. Differences in growth were not related to the toxin content of cyanobacteria. Investigations of roach motility showed no differences whether fed with Aphanizomenon or Microcystis .
4. In contrast to Microcystis , Aphanizomenon can be regarded as a suitable food source for juvenile roach probably because of its better digestability. We conclude that the ability to feed on cyanobacteria is not a general competitive advantage for roach, but the outcome depends on the species composition of the cyanobacteria.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

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.     

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.     

Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum.

We have studied the genetic polymorphism at 10 Plasmodium falciparum loci that are considered potential targets for specific antimalarial vaccines. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsynonymous substitutions indicates that natural selection may account for the polymorphism observed at seven of the 10 loci studied. This inference depends on the assumption that synonymous substitutions are neutral, which we test by analyzing codon bias and G+C content in a set of 92 gene loci. We find evidence for an overall trend towards increasing A+T richness, but no evidence for mutation bias. Although the neutrality of synonymous substitutions is not definitely established, this trend towards an A+T rich genome cannot explain the accumulation of substitutions at least in the case of four genes (AMA-1, CSP, LSA-1, and PF48/45) because the Gleft and right arrow C transversions are more frequent than expected. Moreover, the Tajima test manifests positive natural selection for the MSP-1 and, less strongly, MSP-3 polymorphisms; the McDonald-Kreitman test manifests natural selection at LSA-1 and PF48/45. We conclude that there is definite evidence for positive natural selection in the genes encoding AMA-1, CSP, LSA-1, MSP-1, and Pfs48/45. For four other loci, EBA-175, MSP-2, MSP-3, and RAP-1, the evidence is limited. No evidence for natural selection is found for Pfs25.     

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea)

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.     

Evolutionary Processes Acting on Candidate cis-Regulatory Regions in Humans Inferred from Patterns of Polymorphism and Divergence

Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

Distribution of alkaline phosphatase genes in cyanobacteria and the role of alkaline phosphatase on the acquisition of phosphorus from dissolved organic phosphorus for cyanobacterial growth

Phosphorus is a vital nutrient for cyanobacterial growth. Aside from dissolved inorganic phosphorus, dissolved organic phosphorus (DOP) is used by cyanobacterial species via the activity of alkaline phosphatase (APase), which likely plays an important role in acquiring phosphorus for algal growth in the same manner as it does in other bacteria. In this work, APase genes phoA, phoD, and phoX were found distributed in the cyanobacterial strains included in the algal genome collection of the NCBI database. PhoX has a wider distribution than the classical phoA and phoD. Furthermore, multiple types of APase genes were simultaneously identified in a single strain or genome. Anabaena flos-aquae FACHB-245 was selected as a typical strain to study the performance of cyanobacteria growing on DOP. In algal growth involving AMP or lecithin, APase regulates the release of phosphorus from DOP as confirmed by the relative quantification of phoD and phoX expression levels. Our results confirmed that the distribution of APase is prevalent in cyanobacteria and thus provides a new insight into the potential role of cyanobacterial APase on phosphorus acquisition in natural environment.     

Direct evidence for production of microcystins by Anabaena strains from the Baltic Sea

Anabaena is a filamentous, N(2)-fixing, and morphologically diverse genus of cyanobacteria found in freshwater and brackish water environments worldwide. It contributes to the formation of toxic blooms in freshwater bodies through the production of a range of hepatotoxins or neurotoxins. In the Baltic Sea, Anabaena spp. form late summer blooms, together with Nodularia spumigena and Aphanizomenon flos-aquae. It has been long suspected that Baltic Sea Anabaena may produce microcystins. The presence of microcystins has been reported for the coastal regions of the Baltic proper, and a recent report also indicated the presence of the toxin in the open Gulf of Finland. However, at present there is no direct evidence linking Baltic Sea Anabaena spp. to microcystin production. Here we report on the isolation of microcystin-producing strains of the genus Anabaena in the open Gulf of Finland. The dominant microcystin variants produced by these strains included the highly toxic MCYST-LR as well as [d-Asp(3)]MCYST-LR, [d-Asp(3)]MCYST-HtyR, MCYST-HtyR, [d-Asp(3),Dha(7)]MCYST-HtyR, and [Dha(7)]MCYST-HtyR variants. Toxic strains were isolated from the coastal Gulf of Finland as well as from the easternmost open-sea sampling station, where there were lower salinities than at other stations. This result suggests that lower salinity may favor microcystin-producing Anabaena strains. Furthermore, we sequenced 16S rRNA genes and found evidence for pronounced genetic heterogeneity of the microcystin-producing Anabaena strains. Future studies should take into account the potential presence of microcystin-producing Anabaena sp. in the Gulf of Finland.