Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml⁻¹ of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.     

Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of Finland

The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena . Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena . Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland.     

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.     

Diversity of toxic and nontoxic nodularia isolates (cyanobacteria) and filaments from the Baltic Sea

Cyanobacteria of the genus Nodularia form toxic blooms in brackish waters worldwide. In addition, Nodularia spp. are found in benthic, periphytic, and soil habitats. The majority of the planktic isolates produce a pentapeptide hepatotoxin nodularin. We examined the morphologic, toxicologic, and molecular characters of 18 nodularin-producing and nontoxic Nodularia strains to find appropriate markers for distinguishing the toxic strains from the nontoxic ones in field samples. After classical taxonomy, the examined strains were identified as Nodularia sp., Nodularia spumigena, N. baltica, N. harveyana, and N. sphaerocarpa. Morphologic characters were ambiguous in terms of distinguishing between the toxic and the nontoxic strains. DNA sequences from the short 16S-23S rRNA internally transcribed spacer (ITS1-S) and from the phycocyanin operon intergenic spacer and its flanking regions (PC-IGS) were different for the toxic and the nontoxic strains. Phylogenetic analysis of the ITS1-S and PC-IGS sequences from strains identified as N. spumigena, and N. baltica, and N. litorea indicated that the division of the planktic Nodularia into the three species is not supported by the ITS1-S and PC-IGS sequences. However, the ITS1-S and PC-IGS sequences supported the separation of strains designated N. harveyana and N. sphaerocarpa from one another and the planktic strains. HaeIII digestion of PCR amplified PC-IGS regions of all examined 186 Nodularia filaments collected from the Baltic Sea produced a digestion pattern similar to that found in toxic isolates. Our results suggest that only one planktic Nodularia species is present in the Baltic Sea plankton and that it is nodularin producing.     

A MOLECULAR AND PHENOTYPIC ANALYSIS OF NODULARIA (CYANOBACTERIA) FROM THE BALTIC SEA

The filamentous diazotrophic cyanobacterium Nodularia forms water blooms each year in the Baltic Sea. Filaments isolated from such water blooms vary in their trichome width, degree of coiling, and properties of their gas vesicles; previously, these characters have been used to classify individuals to species level. To test the validity of such a phenotypic classification, we determined the nucleotide sequences for a region of the phycocyanin locus that includes a noncoding intergenic spacer (PC-IGS), the IGS between two adjacent copies of the gvpA gene (which encodes the main structural gas vesicle protein) and the rDNA internal transcribed spacer (rDNA-ITS), for 13 clonal Nodularia isolates from the Baltic Sea during August 1994. The complete 16S-rDNA sequence was determined for three isolates and was found to be identical in each of them. Molecular sequences for noncoding regions of the genome were used to assign isolates to three groups on the basis of PC-IGS, two groups on the basis of gvpA -IGS, and three groups on the basis of rDNA-ITS. No consistent correlation was found between genotype and any of the phenotypic features examined, and no link was found between any of these features themselves, indicating that these characters are not useful for placing Nodularia isolates into meaningful taxonomic groups. The PC-IGS, gvpA -IGS, and rDNA-ITS genotypic groupings were not congruent. This might indicate that gene flow occurs between individuals in Nodularia populations.     

Deposit-feeders accumulate the cyanobacterial toxin nodularin

Blooms of toxic cyanobacteria may potentially affect food web productivity and even be a human health hazard. In the Baltic Sea, regularly occurring summer blooms of nitrogen-fixing cyanobacteria are often dominated by Nodularia spumigena, which produces the potent hepatotoxin nodularin. Evidence of sedimentation of these blooms indicates that benthic fauna can be exposed to nodularin. In a one month experiment, we simulated the settling of a summer bloom dominated by N. spumigena in sediment microcosms with three species of sediment-dwelling, deposit-feeding macrofauna, the amphipods Monoporeia affinis and Pontoporeia femorata and the bivalve Macoma balthica, and analyzed nodularin in the animals by HPLC–ESI–MS (high-performance liquid chromatography–electrospray ionization–mass spectrometry). We found nodularin in quantities of 50–120 ng g⁻¹ DW. The results show that deposit-feeding macrofauna in the Baltic Sea may contribute to trophic transfer of nodularin.     

Unattended monitoring of potentially toxic phytoplankton species in the Baltic Sea in 1993

Variabilities in chlorophyll a fluorescence, temperature andsalinity in the surface were recorded unattended on board twomerchant ships in the Baltic Sea. When these recordings werecomplemented by automated water sampling, it was possible toanalyze the phytoplankton species composition in 426 samples.In total, 22 potentially toxic phytoplankton species or generawere detected. Nodularia spumigena was the only species thatformed extensive blooms The system has proved to be an effectiveearly warning method for exceptional and eventually harmfulalgal blooms. The possibilities for using this method as analternative, or a complement, to conventional methods in marinephytoplankton monitoring are discussed.     

Sedimentation of Nodularia spumigena and distribution of nodularin in the food web during transport of a cyanobacterial bloom from the Baltic Sea to the Kattegat

Nodularia spumigena is a toxic cyanobacteria that blooms in the Baltic Sea every year. In the brackish water of the Baltic Sea, its toxin, nodularin, mainly affects the biota in the surface water due to the natural buoyancy of this species. However, the fate of the toxin is unknown, once the cyanobacteria bloom enters the more saline waters of the Kattegat. In order to investigate this knowledge gap, a bloom of N. spumigena was followed during its passage, carried by surface currents, from the Baltic Sea into the Kattegat area, through the Öresund strait. N. spumigena cells showed an increased cell concentration through the water column during the passage of the bloom (up to 130 10³ cells ml⁻¹), and cells (4.2 10³ cells ml⁻¹) could be found down to 20 m depth, below a pycnocline. Sedimentation trap samples from below the pycnocline (10–12 m depth) also showed an increased sedimentation of N. spumigena filaments during the passage of the bloom. The toxin nodularin was detected both in water samples (0.3–6.0 μg l⁻¹), samples of sedimenting material (a toxin accumulation rate of 20 μg m^-2 day⁻¹), zooplankton (up to 0.1 ng ind.⁻¹ in copepods), blue mussels (70–230 μg kg⁻¹ DW), pelagic and benthic fish (herring (1.0–3.4 μg kg⁻¹ DW in herring muscle or liver) and flounder (1.3-6.2 μg kg⁻¹ DW in muscle, and 11.7-26.3 μg kg⁻¹ DW in liver). A laboratory experiment showed that N. spumigena filaments developed a decreased buoyancy at increased salinities and that they were even sinking with a rate of up to 1,7 m day⁻¹ at the highest salinity (32 PSU). This has implications for the fate of brackish water cyanobacterial blooms, when these reach more saline waters. It can be speculated that a significant part of the blooms content of nodularin will reach benthic organisms in this situation, compared to blooms decaying in brackish water, where most of the bloom is considered to be decomposed in the surface waters.     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin.

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth, Nitrogen Fixation, and Nodularin Production by Two Baltic Sea Cyanobacteria

In late summer, nitrogen-fixing cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae form blooms in the open Baltic Sea. N. spumigena has caused several animal poisonings, but Baltic A. flos-aquae is not known to be toxic. In this laboratory study, performed with batch cultures, the influences of environmental conditions on the biomass and nitrogen fixation rate of N. spumigena and A. flos-aquae were compared and the toxin (nodularin) concentration produced by N. spumigena was measured. Several differences in the biomasses and nitrogen fixation rates of N. spumigena and A. flos-aquae were observed. A. flos-aquae preferred lower irradiances, salinities, and temperatures than N. spumigena. The biomass of both species increased with high phosphate concentrations and with accompanying bacteria and decreased with unnaturally high inorganic nitrogen concentrations. Nodularin concentrations in cells and growth media, as well as nitrogen fixation rates, were generally highest under the conditions that promoted growth. Intracellular nodularin concentrations increased with high temperature, high irradiance, and high phosphate concentration and decreased with low and high salinities and high inorganic nitrogen concentrations. Nodularin concentrations in growth media increased with incubation time, indicating that intracellular nodularin was released when cells lysed. The different responses of A. flos-aquae and N. spumigena to changes in salinity, irradiance, and temperature may explain the different spatial and temporal distribution of these species in the Baltic Sea. According to the results, toxic N. spumigena blooms may be expected in late summer in areas of the Baltic Sea with high phosphorus concentrations and moderate salinity.     

Correlation of the content of hepatotoxin nodularin and glycosidic carotenoids, 4-ketomyxol-2′-fucoside and novel 1′-O-methyl-4-ketomyxol-2′-fucoside,in 20 strains of the cyanobacterium Nodularia spumigena

The carotenoids of 19 different strains of Nodularia spumigena and one Nodularia sphaerocarpa from different global locations were investigated. The molecular structure of the diagnostic pigment in N. spumigena of the Baltic Sea, tentatively named ‘4-keto-myxoxanthophyll-like pigment’ in Schlüter, L., Garde, K., Kaas, H., [2004. A 4-keto-myxoxanthophyll-like pigment is a diagnostic pigment for the toxic cyanobacteria Nodularia spumigena in the Baltic Sea. Mar. Ecol. Prog. Ser. 275, 69–78.] was determined to be a 4-ketomyxol-2′-fucoside. In most of the strains an additional carotenoid was found, identified as the novel 1′-O-methyl-4-ketomyxol-2′-fucoside by 2D NMR. This glycosidic carotenoid methyl ether was found to be a more important diagnostic pigment than the 4-ketomyxol-2′-fucoside for the toxic N. spumigena in the Baltic Sea. Out of the 20 strains 15 were found to produce the hepatotoxin nodularin. The content of carotenoids and nodularin was found to increase relative to chlorophyll a at increasing light intensity and at stationary growth, and nodularin was significantly correlated to both 4-ketomyxol-2′-fucoside and 1′-O-methyl-4-ketomyxol-2′-fucoside, and particular to the sum of these two pigments.     

Estimating nodularin content of cyanobacterial blooms from abundance of Nodularia spumigena and its characteristic pigments—a case study from the Baltic entrance area

The presence of toxin-producing cyanobacteria is of concern for the management of recreational waters. The abundance of toxic cyanobacteria and environmental concentrations of their toxins may fluctuate substantially within a short time emphasising the need for rapid methods to estimate toxin concentrations in the water. During late June-early September 2002 the abundance, biomass and characteristic pigments of Nodularia spumigena Mertens and the hepatotoxin nodularin produced by N. spumigena were analysed in water samples collected from the Baltic entrance area. Significant relationships were found between cell-bound concentrations of nodularin and the abundance and biomass of N. spumigena with a relationship of approximately 1 pg nodularin per Nodularia-cell. It is suggested that simple counts of Nodularia under the microscope may be used as a rapid on-site technique to estimate potential nodularin concentrations in recreational waters. Comparison with data from Australia shows that cell-bound concentration of nodularin per Nodularia-cell differs between geographically distant areas and therefore such relationships should be established for individual areas. The carotenoids echinenone, canthaxanthin and a cis-canthaxanthin-like caroteniod, identified from a laboratory culture of N. spumigena isolated from the Baltic Sea, were also significantly correlated with concentrations of cell-bound nodularin. However, Aphanizomenon and Anabaena, two genera commonly co-occurring with Nodularia, also contain these pigments and thus the significant correlations obtained presumably originate from Nodularia being the dominant cyanobacterium in all samples collected in 2002.     

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena . We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.     

Allelopathy of Baltic Sea cyanobacteria: no evidence for the role of nodularin

Extracts of Aphanizomenon flos-aquae and Nodularia spumigena,the two most common cyanobacteria forming recurrent blooms inthe Baltic Sea, decrease the abundance of some phytoplanktonspecies via the release of allelopathic substances. We investigatedhow cell-free filtrates of the two cyanobacteria, as well aspurified hepatotoxin nodularin, produced by N. spumigena affectedcell numbers, chlorophyll a content and ¹⁴CO₂ uptake of thecryptophyte Rhodomonas sp. Both cyanobacterial filtrates significantlyretarded the growth of Rhodomonas sp., A. flos-aquae filtrateup to 46%, whereas purified nodularin showed no significanteffect on any of the growth parameters of the cryptophyte. Theseresults suggest that the allelopathic effect of N. spumigenais most probably due to metabolite(s) other than nodularin,possibly acting via the damage of the target cells.     

GENETIC VARIATION IN APHANIZOMENON (CYANOBACTERIA) COLONIES FROM THE BALTIC SEA AND NORTH AMERICA

Aphanizomenon Morren is an important member of the cyanobacterial community in the Baltic Sea, but studies of this genus have been hampered by the difficulty of growing it in laboratory culture. PCR amplification of DNA from colonies picked directly from water samples has circumvented this problem and made it possible to carry out an analysis of genetic diversity within the Baltic Sea and in two small North American lakes separated by just a few kilometers. The nucleotide sequence of the phycocyanin intergenic spacer and partial flanking coding regions of cpcB and cpcA was determined for 32 colonies of Aphanizomenon , 26 from the Baltic Sea, and 6 from the North American lakes. No variation was detected among the 26 Baltic Sea colonies, but two alleles, differing at 19 nucleotide positions (5.4%), were found in the North American lake colonies. Surprisingly, the two North American types were less closely related to each other than to the Baltic Sea genotype. The Baltic Sea Aphanizomenon is clearly distinct from A. flos-aquae at both the cpcB–cpcA and 16S rDNA loci, which lends phylogenetic support to their tentative separation based on ultrastructural analysis. We conclude that although there is significant genetic diversity in the genus Aphanizomenon , the population in the Baltic Sea is, in contrast to the Nodularia population, genetically homogeneous.     

Effects of Cyanobacteria on Survival and Reproduction of the Littoral Crustacean Gammarus zaddachi (Amphipoda)

Toxic cyanobacteria can have harmful or fatal impacts on aquatic organisms. In the archipelagos of the northern Baltic Sea, the open sea blooms often drift into littoral areas, where they decompose and release toxins and other chemical compounds in the water. However, the effects of cyanobacteria on the littoral organisms have not previously been investigated. We studied the effects of three cyanobacteria species (toxic Nodularia spumigena, non-toxic N. sphaerocarpa and non-toxic Aphanizomenon flos-aquae) and purified dissolved nodularin (produced by N. spumigena) on a common littoral amphipod Gammarus zaddachi. Nodularin was transferred to eggs, juveniles and adults of G. zaddachi, but no significant negative effects of dissolved nodularin were detected on adults, eggs or juveniles. However, survival of adults decreased by the exposure to toxic N. spumigena cells. The egg hatching rate and juvenile survival were not affected when exposed to the three cyanobacteria species. In contrast, a weak decrease in the egg production and an increased abortion of embryos from the brood pouch of females was observed, the later indicating a failure in parental care. Further, a decrease in grazing rate on the filamentous green alga Enteromorpha intestinalis was observed. The results suggest that toxic cyanobacteria blooms are not extremely fatal, but may have, in high concentrations, negative effects on the adult survival, fecundity, and feeding behaviour of gammarids inhabiting the littoral zone.     

Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status

In lakes, benthic micro-algae and cyanobacteria (periphyton) can contribute significantly to total primary productivity and provide important food sources for benthic invertebrates. Despite recognition of their importance, few studies have explored the diversity of the algal and cyanobacterial composition of periphyton mats in temperate lakes. In this study, we sampled periphyton from three New Zealand lakes: Tikitapu (oligotrophic), ōkāreka (mesotrophic) and Rotoiti (eutrophic). Statistical analysis of morphological data showed a clear delineation in community structure among lakes and highlighted the importance of cyanobacteria. Automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity. Despite the close geographic proximity of the lakes, cyanobacterial species differed markedly. The 16S rRNA gene sequence analysis identified eight cyanobacterial OTUs. A comparison with other known cyanobacterial sequences in GenBank showed relatively low similarities (91-97%). Cyanotoxin analysis identified nodularin in all mats from Lake Tikitapu. ndaF gene sequences from these samples had very low (≤ 89%) homology to sequences in other known nodularin producers. To our knowledge, this is the first detection of nodularin in a freshwater environment in the absence of Nodularia. Six cyanobacteria species were isolated from Lake Tikitapu mats. None were found to produce nodularin. Five of the species shared low (< 97%) 16S rRNA gene sequence similarities with other cultured cyanobacteria.