Genera Anabaena Research Articles

First report of an Anabaena Bory strain containing microcystin-LR in a freshwater body in Africa

In South Africa, little is known about the production of microcystin by the genus Anabaena Bory. In April 2012, during a cyanobacterial bloom event in Theewaterskloof Dam, Western Cape province, the plankton was sampled on 10 occasions. The dominant algae belonged to the genus Anabaena, a family of filamentous cyanobacteria known to produce cyanotoxins such as anatoxin-a, harmful to humans and the aquatic foodweb. The specimens isolated lacked the characteristic akinetes and/or heterocysts associated with this genus. Therefore the 16S rRNA gene was Sanger sequenced and a maximum parsimony tree was constructed, confirming its identity as Anabaena ucrainica (Schkorbatow) M. Watanabe. Enzyme-linked immunosorbent assay (ELISA) confirmed the presence of microcystin-LR in the isolated A. ucrainica field sample, while PCR analysis and sequencing further confirmed the presence of mcy genes in this species. It was speculated from the data that prevailing low water-column temperatures and strong gusty winds may have resulted in the lack of akinete or heterocyst production. The Anabaena strain isolated from Theewaterskloof Dam is the first report of a strain containing microcystin-LR belonging to this genus in a freshwater body in Africa.

First evidences of PAMAM dendrimer internalization in microorganisms of environmental relevance: A linkage with toxicity and oxidative stress

This article reports novel results on the toxic mechanisms of action of amine- and hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers toward microorganisms of environmental relevance, namely a cyanobacterium of the genus Anabaena and the green alga Chlamydomonas reinhardtii. We used PAMAM ethylenediamine core dendrimers from generations G2 to G4, which displayed a positive charge, measured as ζ-potential, in culture media. All amine-terminated and most remarkably the G4 hydroxyl-terminated dendrimer inhibited the growth of both microorganisms. The effect on the growth of the green alga was significantly higher than that on the cyanobacterium. With concentrations expressed in terms of molarity, there was a clear relationship between dendrimer generation and toxicity, with higher toxicity for higher generation. Hormesis was observed for hydroxyl-terminated dendrimers at low concentrations. The cationic dendrimers and G4-OH significantly increased the formation of reactive oxygen species (ROS) in both organisms. ROS formation was not related with the chloroplast or photosynthetic membranes and photosystem II photochemistry was unaffected. Cell damage resulted in cytoplasm disorganization and cell deformities and was associated to an increase in ROS formation and lipid peroxidation in mitochondria in the green alga; cell wall and membrane disruption with apparent loss of cytoplasmic contents was found in the cyanobacterium. It was determined for the first time that cationic PAMAM dendrimers were quickly and largely internalized by both organisms. These results warn against the generalization of the use of dendrimers, which may pose significant risk for the environment and particularly for primary producers which are determinant for the health of natural ecosystems.

Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway.

Cyanobacteria produce a wide variety of cyclic peptides, including the widespread hepatotoxins microcystins and nodularins. Another class of peptides, cyclic glycosylated lipopeptides called hassallidins, show antifungal activity. Previously, two hassallidins (A and B) were reported from an epilithic cyanobacterium Hassallia sp. and found to be active against opportunistic human pathogenic fungi. Bioinformatic analysis of the Anabaena sp. 90 genome identified a 59-kb cryptic inactive nonribosomal peptide synthetase gene cluster proposed to be responsible for hassallidin biosynthesis. Here we describe the hassallidin biosynthetic pathway from Anabaena sp. SYKE748A, as well as the large chemical variation and common occurrence of hassallidins in filamentous cyanobacteria. Analysis demonstrated that 20 strains of the genus Anabaena carry hassallidin synthetase genes and produce a multitude of hassallidin variants that exhibit activity against Candida albicans. The compounds discovered here were distinct from previously reported hassallidins A and B. The IC50 of hassallidin D was 0.29-1.0 µM against Candida strains. A large variation in amino acids, sugars, their degree of acetylation, and fatty acid side chain length was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon, Cylindrospermopsis raciborskii, Nostoc, and Tolypothrix. These compounds may protect some of the most important bloom-forming and globally distributed cyanobacteria against attacks by parasitic fungi.

남조류 Anabaena 휴면포자의 연구 동향 및 방향

남조류 Anabaena 휴면포자의 연구 동향 및 방향

Characterization of freshwater benthic biofilm-forming Hydrocoryne (Cyanobacteria) isolates from Antarctica.

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886-1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.

Microbial-Activated Sediment Traps Associated with Oncolite Formation Along a Peritidal Beach, Northern Arabian (Persian) Gulf, Kuwait

Identification of microbial communities within shoreline sediments and sediment precipitates from the Tigris-Euphrates delta (northern Kuwait) were determined by microscopic/nanoscopic studies, and by molecular analysis. Oncolites are syn-diagenetic carbonate precipitates that are surviving in a shallow subtidal to intertidal siliciclastic environment with periodically excessive hydraulic energy, extreme salinity (up to 47 per mil), and high concentrations of organic matter. X-ray diffraction techniques reveal that oncolite cortices are predominantly composed of calcite, quartz, halite and dolomite, associated with minor fractions of clay minerals. Quantitative analysis of the Corey Shape Factor reveals distinct morphological populations but with local overlap. A plot of the Equivalent Diameter vs. Corey Shape Factor provided the best indicator of the morphological relationships within the total oncolite population, indicating a hydrodynamically controlled morphological distribution defining intertidal and subtidal oncolite classes. Direct microscopic examination of the samples indicates that diatoms are the most abundant eukaryotic algae in subtidal sediments and within actively precipitating carbonate cements, especially the genus Navicula. In contrast, filamentous cyanobacteria from the genus Anabaena are most abundant in the intertidal zone sediments. The PCR-DGGE of the 16SrRNA gene of the cyanobacteria shows a higher diversity for this genus of bacteria in all sediment samples and that the cyanobacterial population in the diagenetically precipitating oncolites are closely related to the population found in the subtidal sediments. Dunaliella viridis dominates the culturable algae obtained from the four tidal zones. Our results indicate that a range of microbial populations are actively contributing to the formation of microbially-induced sedimentary structures in the extreme conditions of the southern Tigris-Euphrates delta.

Anabaenolysins, Novel Cytolytic Lipopeptides from Benthic Anabaena Cyanobacteria

Two novel cyclic lipopeptides, anabaenolysin A and anabaenolysin B, were isolated from two benthic cyanobacterial strains of the genus Anabaena. This novel class of cyanobacterial lipopeptides has a general structure of a small peptide ring consisting of four amino acids from which two are proteinogenic and two unusual; glycine1, glycine2, 2-(3-amino-5-oxytetrahydrofuran-2-yl)-2-hydroxyacetic acid3 and a long unsaturated C18 β-amino acid4 with a conjugated triene structure. They are distinguished by the presence of a conjugated dienic structure in the C18 β-amino acid present in anabaenolysin A but not in anabaenolysin B. Conjugated triene structure generates a typical UV spectrum for anabaenolysins for easy recognition. Anabaenolysin A constituted up to 400 ppm of the cyanobacterial dry weight. We found evidence of thirteen variants of anabaenolysins in one cyanobacterial strain. This suggests that the anabaenolysins are an important class of secondary metabolites in benthic Anabaena cyanobacteria. Both anabaenolysin A and B had cytolytic activity on a number of mammalian cell lines.

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk (Southern Baltic Sea). Application of DGGE in taxonomic studies on environmental samples

Abstract The aim of this paper was a taxonomic verification of cyanobacteria of the genus Anabaena that occur in the Gulf of Gdańsk. Classical taxonomic methods were combined with modern molecular taxonomic methods (polyphasic approach). Analysis of the species diversity of cyanobacteria from the genus Anabaena was based on the microscopic analysis and on the analysis of 16S rRNA and ITS region. Comparison of the obtained results with sequences in GenBank showed 97.8–100% similarity for 16S rRNA and 98.8-100% similarity in the case of the ITS fragment. Similarity of the 16S rRNA and ITS sequence of 98.5% seems to be sufficient to determine the species.

Influence of phosphorus and pH on the fungicidal potential of Anabaena strains

The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains - A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.

Modulation of fungicidal potential of Anabaena strains by light and temperature

The regulation of fungicidal and hydrolytic enzyme activity was investigated in a set of cyanobacterial strains belonging to the genus Anabaena (Anabaena laxa RPAN8, Anabaena iyengarii RPAN9, Anabaena variabilis RPAN59 and Anabaena oscillarioides RPAN69), with A. variabilis RPAN16 serving as negative control. Time course studies undertaken with cultures incubated under different light and temperature conditions revealed enhancement in growth and fungicidal activity under continuous light (CL) and light dark (LD, 16:8) conditions and temperature of 30 °C and 40 °C. A significant increase of 3-18 % in chitosanase activity was recorded in all the 4-week-old cultures under CL condition and at 40 °C. Endoglucanase activity of RPAN8 and 9 was twofolds higher than the other strains under all light/dark conditions and temperature in the 4-week-old cultures, while continuous dark (CD) enhanced CMCase activity in RPAN69. This study provided useful information regarding the most suitable conditions of light and temperature for maximizing hydrolytic enzyme activity and fungicidal activity, as a prelude to their effective use as biocontrol agents.

Two newly recorded species of <i>Anabaena</i>(Nostocales, Cyanobacteria) in China

迄今为止,全球范围共报道出100多种鱼腥藻,中国记录报道的仅有50余种,但有些种类的描述仍较模糊.最近在福建、浙江等地进行野外调查时,发现了多种丝状蓝藻.其中就有2种鱼腥藻在中国尚未报道:威格鱼腥藻(Anabaena viguieri Denis et Frémy 1923)和史密斯鱼腥藻(Anabaena smithii(Komárek) Watanabe 1992).;So far, more than 100 species of genus Anabaena have been recorded in the world, and about 50 taxa were described in China. During our recent field investigations for water-bloom forming cyanobacteria in China, several filamentous cyanobacterial species were examined in the samples. In this study, we demonstrated that two Anabaena species as Anabaena viguieri Denis et Frémy 1923 and Anabaena smithii (Komárek) Watanabe 1992, had never been reported in China, and they are newly described and discussed in the present study.

Taxonomic studies of some cultured strains of Cyanobacteria (Nostocales) isolated from the Yenisei River basin

Twenty-one strains of cyanobacteria representing the genera Anabaena, Cylindrospermum, Mojavia, Nostoc, Trichormus, and Wollea (Nostocales, Cyanobacteria) isolated from algocenoses of the Yenisei River basin (eastern Siberia, Russia) were taxonomically studied. New taxa characteristic of this region were discovered. The properties of pure cultures of Anabaena sedovii, A. zinserlingii,Cylindrospermum stagnale f. tortuosum, Nostoc kihlmani, Trichormus variabilis f. tenuis, and Wollea saccata have never been described before. The primary taxonomic features (the position of akinetes and heterocysts, the width of vegetative cells, akinetes and heterocysts, and the shape of terminal cells) of these cyanobacteria when cultured were shown to fit the diagnosis of the identified taxa.

Anabaena fuscovaginata (Nostocales), a new cyanobacterial species from periphyton of the freshwater alkaline marsh of Everglades, South Florida, USA.

A new species of the nostocalean genus Anabaena was discovered in periphytic cyanobacterial mats of the Everglades marsh, South Florida, USA. In some of the 10 samples where it occurred, the species dominated the cyanobacterial community, however, it was always part of a rich assemblage of microbiota, including many types characteristic for alkaline herbaceous wetlands of Caribbean America or freshwater tropical marshes. On the basis of typical morphological traits and occurrence in a periphytic habitat, I suggest that the species is a member of the genus Anabaena in its original sense, valid after recent separation of planktic taxa. As the most distinctive morphological feature, it has firm, coloured sheaths that have never been reported for this genus before.

Non‐autonomous transposable elements associated with inactivation of microcystin gene clusters in strains of the genus Anabaena isolated from the Baltic Sea

Microcystins are potent peptide toxins produced by a range of distantly related cyanobacteria. They are assembled on a large enzyme complex encoded by the 55 kb microcystin synthetase (mcy) gene cluster. Here we report two strains of the genus Anabaena isolated from the Baltic Sea, which contain the entire mcy gene cluster but do not produce microcystins. Transcription analysis demonstrated that mcy genes were not expressed in these strains. We identified short insertion elements interrupting the mcyA, mcyD and mcyE genes in the two strains isolated in different years from different parts of the Baltic Sea. The 126-207 bp insertion elements encoded both terminal inverted repeats and direct repeats but lacked transposases. However, we found evidence for transposition of these elements despite the absence of genes encoding transposases, suggesting that they are non-autonomous mobile miniature inverted-repeat transposable elements (MITEs) recently described from cyanobacteria. The MITE insertion elements were present in mcyD genes amplified directly from the cyanobacterial community present in the Baltic Sea blooms from 2005. The results demonstrate that mcy gene cluster mutants can make up a stable proportion of the mcy gene pool in the Baltic Sea population.

The apoptosis-inducing activity towards leukemia and lymphoma cells in a cyanobacterial culture collection is not associated with mouse bioassay toxicity

Cyanobacteria (83 strains and seven natural populations) were screened for content of apoptosis (cell death)-inducing activity towards neoplastic cells of the immune (jurkat acute T-cell lymphoma) and hematopoetic (acute myelogenic leukemia) lineage. Apoptogenic activity was frequent, even in strains cultured for decades, and was unrelated to whether the cyanobacteria had been collected from polar, temperate, or tropic environments. The activity was more abundant in the genera Anabaena and Microcystis compared to Nostoc, Phormidium, Planktothrix, and Pseudanabaena. Whereas the T-cell lymphoma apoptogens were frequent in organic extracts, the cell death-inducing activity towards leukemia cells resided mainly in aqueous extracts. The cyanobacteria were from a culture collection established for public health purposes to detect toxic cyanobacterial blooms, and 54 of them were tested for toxicity by the mouse bioassay. We found no correlation between the apoptogenic activity in the cyanobacterial isolates with their content of microcystin, nor with their ability to elicit a positive standard mouse bioassay. Several strains produced more than one apoptogen, differing in biophysical or biological activity. In fact, two strains contained microcystin in addition to one apoptogen specific for the AML cells, and one apoptogen specific for the T-cell lymphoma. This study shows the potential of cyanobacterial culture collections as libraries for bioactive compounds, since strains kept in cultures for decades produced apoptogens unrelated to the mouse bioassay detectable bloom-associated toxins.

Fingerprinting of repetitive DNA sequences in the genus Anabaena using PCR- based techniques

In this study, ten species of Anabaena were used to test the congruence between the traditional morphological classification system and the present molecular classification system. The electrophoretic patterns for 10 cyanobacterial strains belonging to the genera (Anabaena) were used for molecular analysis using the repeated PCR technique. Genetic diversity was assessed using the banding patterns of repetitive DNA sequences including the HIP1 sequences, enterobacterial repetitive intergenic consensus [ERIC] sequences and REP sequences that are present in the cyanobacterial genome. PCR with different sets of repetitive oligonucleotides as primers were used to evaluate the various repetitive DNA sequences. The dendrograms based on the HIP1, ERIC and REP sequences did not show a clear correlation with morphological dendrogram based on traditional classification. Key words: Anabaena, cyanobacteria, highly iterated palindromic sequences, enterobacterial repetitive intergenic consensus sequences, repetitive extragenic palindromic sequences, polymerase chain reaction.

Modulation of IAA Production in Cyanobacteria by Tryptophan and Light

Cyanobacteria represent less a investigated group of prokaryote, in terms of their plant growth promoting potential, especially in relation to the production of phytohormones. The present investigation was aimed towards analyzing growth kinetics, indole acetic acid (IAA) production and acetylene reduction activity (ARA) as an index of nitrogen fixation in two selected cyanobacterial strains belonging to the genus Anabaena, as influenced by tryptophan supplementation and light:dark conditions. Interesting observations were recorded in terms of enhancement of IAA production accompanied by protein and chlorophyll accumulation in the two cyanobacterial strains grown in media without tryptophan and incubated under light:dark or continuous light conditions. Colorimetric and chromatographic analyses supported the observations that tryptophan is not essential as a precursor for IAA biosynthesis in these cyanobacteria. Further study is in progress to identify genes involved in the tryptophan independent pathway for IAA biosynthesis.

Occurrence of anatoxin-a(s) during a bloom of Anabaena crassa in a water-supply reservoir in southern Brazil

Cyanobacterial blooms and the accompanying production of cyanotoxins are a serious global problem. Toxic blooms of Anabaena species are common in lagoons and reservoirs of southern Brazil. Worldwide, species of the genus Anabaena produce the majority of the known hepatotoxins (microcystins) and neurotoxins [anatoxin-a, anatoxin-a(s), and saxitoxins]. This report links a bloom of Anabaena crassa in the Faxinal Reservoir, the main water supply for the city of Caxias do Sul (400,000 inhabitants) in southern Brazil, to the occurrence of anatoxin-a(s) in the water. During the bloom period, the reservoir was strongly stratified, with higher temperatures and a deep anoxic hypolimnion. Two methods for sample concentration (direct and complete extraction) were tested, and direct extraction of samples proved to be more efficient. Water samples collected during the bloom showed 9% acetylcholinesterase inhibition at 50 mg mL−1, corresponding to 0.61 μg of anatoxin-a(s) per gram of lyophilized powder. At these concentrations, symptoms of neurotoxicity and mortality were not observed in tests with Swiss albino mice. Although the concentrations of anatoxin-a(s) in the Faxinal Reservoir were low, these results are important because this is the first record of the toxin for A. crassa. Furthermore, this cyanotoxin is not yet included in Brazilian legislation for drinking-water monitoring, because of the lack of information about toxicity levels and risk calculation for oral doses. The data presented here contribute to the basis for the future inclusion of this toxin in Brazilian legislation for drinking-water quality control, and for the development of analytical methods for this toxin.

Genotypic and phenotypic diversity of Anabaena isolates from diverse rice agro‐ecologies of India

The genus Anabaena is one of the commonly observed genera in the rice fields in South-east Asia. Diversity analyses of a set of 70 Anabaena strains (including 67 strains isolated from diverse rice agro ecologies of India, and three International Reference/Type strains), was carried out using morphological and molecular datasets. The pattern of growth in liquid and solid medium and microscopic observations revealed tremendous diversity in the Anabaena germplasm analysed. The species wise distribution in different soil types and soil pH revealed that Anabaena iyengarii was present at pH ranging from 5.5-8.5 and all the species of Anabaena except A. oscillarioides were present in alluvium soils. Molecular profiling using primers based on HipTG, STRR(mod) and STRR1A sequences generated specific fingerprints for individual isolates. STRR1A was observed to be the most informative and useful for differentiating the isolates. Analyses of a combined dataset, including both morphological and molecular data, proved highly effective in discerning the genetic relationships among the 70 Anabaena strains. The present study provided useful information for the development of a comprehensive database based on the distribution of Anabaena strains in diverse agro ecologies of India and identified useful primers for PCR based differentiation of isolates.

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.