Construction of cyanobacterial-bacterial consortium on the basis of axenic cyanobacterial cultures and heterotrophic bacteria cultures for bioremediation of oil-contaminated soils and water ponds

The ways of the creation of cyanobacterial-bacterial communities with a high remediation potential for cleaning of oil-contaminated soils and water reservoirs are considered. A special methodology was elaborated for the obtaining of bacteriologically pure (axenic) cultures of cyanobacteria: Phormidium sp. K-1, Oscillatoria sp. A-2, and Oscillatoria sp. C-3. It was shown that 14 strains of bacteria associated with these cyanobacteria manifest a hydrocarbon-oxidizing activity. They belong to the genera Pseudomonas, Alcaligenes, Arthrobacter, and Bacillus. The usage of oil-degrading bacteria, which we isolated from water reservoirs or took from collections, allowed us to construct artificial cyanobacterial-bacterial communities with a high hydrocarbon-oxidizing activity. In field experiments on the landfill site Khimpromservis Aktobe, the high remediatory effect of cyanobacterial-bacterial associations between Phormidium sp. K-1 sp. and bacteria Pseudomonas stutzeri A1, Pseudomonas sp. N2, and P. alcaligenes A5 was demonstrated. The results obtained are the basis for the elaboration of microbiological technologies of environment protection using cyanobacteria.     

The rise of toxic benthic Phormidium proliferations: A review of their taxonomy,distribution, toxin content and factors regulating prevalence and increased severity

There has been a marked increase in the distribution, intensity and frequency of proliferations of some species of the benthic mat-forming, toxin-producing genus Phormidium in rivers globally over the last decade. This review summarises current knowledge on their taxonomy, distribution, toxin content, environmental drivers of proliferations, and monitoring and management strategies in New Zealand. Although toxic Phormidium proliferation occurs in rivers worldwide little is known about these factors in most countries. Proliferations, defined as >20% cover of a riverbed, have been identified in 103 rivers across New Zealand. Morphological and molecular data indicate the main species responsible is Phormidium autumnale. In New Zealand Phormidium produces anatoxins (anatoxin-a, homoanatoxin-a, dihydroanatoxin-a, and dihydrohomoanatoxin-a) and these were detected in 67% of 771 samples from 40 rivers. The highest concentration measured was 712 mg kg⁻¹ dried weight (Oreti River, Southland), with considerable spatial and temporal variability in anatoxin concentrations between and within rivers. A synthesis of field based studies suggests that Phormidium proliferations are most likely when there is some enrichment of dissolved inorganic nitrogen but when water-column dissolved reactive phosphorus is less than 0.01 mg L⁻¹. Once established Phormidium-dominated mats trap sediment and internal mat biogeochemistry can mobilise sediment-bound phosphorus, which is then available for growth. Removal of Phormidium-dominated mats is primarily due to shear stress and substrate disturbance, although there is also evidence for autogenic detachment. A combination of factors including; changes to riparian margins, increased nitrate and fine sediment loads, and alterations in flow regimes are likely to have contributed to the rise in Phormidium proliferations.     

The direct role of aerobic heterotrophic bacteria associated with cyanobacteria in the degradation of oil compounds

This study aimed at evaluating the role of cyanobacteria and their associated aerobic heterotrophic bacteria in biodegradation of petroleum compounds. We investigated the potential of ten non-axenic typical mat-forming cyanobacterial strains to degrade phenanthrene, pristane, n-octadecane, and dibenzothiophene. Five strains (Aphanothece halophyletica, Dactyolococcopsis salina, Halothece strain EPUS, Oscillatoria strain OSC, and Synechocystis strain UNIGA) were able to degrade n-alkanes. In case of the other five strains (Microcoleus chthonoplastes, Oscillatoria sp. MPI 95 OS 01, Halothece strain EPUG, Halomicronema exentricum, and Phormidium strain UNITF) alkanes were not significantly affected. Moderate changes in the concentration of the aromatic compounds were observed for three isolates only. In follow-up experiments with Oscillatoria strain OSC, we demonstrated that the cyanobacteria-associated aerobic heterotrophic bacteria were responsible for the observed biodegradation. The cyanobacteria themselves apparently do not degrade petroleum compounds, but more likely play a significant, indirect role in biodegradation by supporting the growth and activity of the actual degraders.     

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus <Emphasis Type="Italic">Phormidium</Emphasis> (Oscillatoriales) from different habitats and geographical sites

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.     

Low-molecular mass carbohydrate accumulation in cyanobacteria from a marine microbial mat in response to salt

Forty seven strains of cyanobacteria, all isolated from microbial mats of intertidal sediments of the island of Mellum (North Sea), were analyzed for the presence of organic osmotica. The cyanobacteria examined belonged to taxonomically different groups and were classified according to their salt optimum and salt tolerance as either freshwater, brackish or marine. Except betaine, all organic osmotica known to occur in cyanobacteria, were found. The results showed no clear correlation between the chemical nature of the organic solute and the salt optimum or salt tolerance of the cyanobacteria examined, indicating that these solutes are not specific to this marine habitat. All strains belonging to the Nostoc/Anabea-group accumulated sucrose as the sole organic osmoticum. The marine, heterocystous Calothrix sp. accumulated trehalose. All strains of the LPP-group (Lyngbya, Plectonema, Phormidium) accumulated glucosylglycerol as sole or primary organic solute. Some LPP-strains accumulated a disaccharide as a secundary solute, e.g. sucrose or trehalose. Gloeocapsa, Synechocystis and Spirulina accumulated glucosylglycerol. Two marine Oscillatoria accumulated trehalose, whereas a freshwater Oscillatoria with a broad salinity tolerance, accumulated sucrose.Analysis of field samples of the microbial mats demonstrated the presence of glycerol, glucosylglycerol, sucrose and trehalose. The relative abundance of the different compounds was related to the species composition as could be predicted from laboratory observations. These data suggest that these carbohydrates have a function in maintaining osmotic balance in the organisms within the microbial mat.     

Characterization of Phormidium lacuna strains from the North Sea and the Mediterranean Sea for biotechnological applications

In biotechnological applications, cyanobacteria are employed for conversion of CO₂ into bioproducts with sunlight as sole energy source. We describe the isolation of motile filamentous cyanobacteria from rockpools of the North Sea or the Mediterranean Sea and their characterization by physiological assays and genome sequencing. The five isolated lines are genetically highly similar, we regard them as strains of the same species. Phylogenetic studies placed the strains in the genus Phormidium; the species is termed Phormidium lacuna. Under liquid media growth conditions or in photobioreactors, Phormidium growth rates were comparable with the single celled model cyanobacterium Synechocystis PCC6803. However, Phormidium strains tolerate different media that can contain up to 3.7× the salt concentration of seawater and grows at temperatures up to 50 °C. Growth in medium free of NH₃ or NO₃⁻ suggests that Phormidium can fix atmospheric dinitrogen by nitrogenase even in the presence of light. Genome data confirmed the presence of nitrogenase and revealed its evolutionary position close to anoxygenic δ-proteobacteria. Genes for photosynthesis, photoreceptors, nitrogen metabolism, hydrogenases, tryptophan synthesis, glucose uptake, and fermentative pathways are discussed in the context of biotechnological applications.     

Emergence of toxic cyanobacterial species in the Ganga River,India, due to excessive nutrient loading

Despite of several efforts by the Government of India, pollution in National River Ganga is rising. The aim of the present study is to investigate the pollution in Ganga River in relation to appearance of toxic cyanobacterial strains. Jajmau area of Kanpur city is the industrial hub of Uttar Pradesh and is the main source of adding unwanted discharge into Ganga River. Water samples were randomly collected from the most polluted stretch of Ganga River (Kanpur, Uttar Pradesh, India). Samples were also collected from other major water of Uttar Pradesh to compare their water chemistry with Ganga River. Physico-chemical parameters of water bodies were estimated periodically for three years 2013–2015. Pearson product-mean correlation showed strong correlation between water parameters of sampling sites. Regression analysis showed seasonal variation in water parameters of Ganga River. Cyanobacteria prevalence in Ganga River was highest in May while lowest in August month. Fourteen cultivable cyanobacteria were isolated from Ganga River. Two new isolates, Oscillatoria sp. RBD01 and Leptolyngbya sp. RBD05 were found to be toxic and showed the presence of algal toxin (microcystin). Phylogenetic relatedness of toxic cyanobacterial isolates with their close homologues was established using 16S rRNA sequence analysis. Microcystin content in water samples (extracellular release) and in cyanobacterial isolates (intracellular content) was estimated by enzyme linked immunosorbent assay. Ganga River was found to be positive for microcystin with concentration ≥2 ppb which is above the permissible limit of WHO. Toxic cyanobacteria Oscillatoria sp. RBD01 and Leptolyngbya sp. RBD05 showed the presence of 23 and 17 ppb of microcystin in cells. Growth of the toxic cyanobacteria Oscillatoria sp. RBD01 showed very strong correlation with phosphate (0.834) and nitrate (0.761) content of water. Toxic Oscillatoria sp. RBD01 growing in moderate combination of nitrate (16x) and phosphate (4x) showed optimum growth and protein content. Periodic assessment of water quality and monitoring of toxic cyanobacteria would be helpful in identification and regulation of toxins which are responsible for destroying its sanctity and making it unsafe for human consumption.     

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.     

The Genome Sequence of the Cyanobacterium Oscillatoria sp. PCC 6506 Reveals Several Gene Clusters Responsible for the Biosynthesis of Toxins and Secondary Metabolites

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites.The presence in the environment of cyanobacteria producing toxins represents a risk for human and animal health (5). It is thus important to develop methodologies to detect these toxic microorganisms in water reservoirs, and so far, PCR-based amplification of genes responsible for the biosynthesis of these toxins is the best method (1). We have been interested in the study of cyanobacteria producing the neurotoxins anatoxin-a and homoanatoxin-a (2, 17, 18). Our goals were first to identify the genes responsible for the biosynthesis of these toxins and to deepen our knowledge of this interesting group of bacteria. For that, we chose Oscillatoria sp. PCC 6506 (4, 6, 14), a filamentous benthic cyanobacterium, as our model microorganism and initiated the sequencing of its genome.Whole-genome shotgun sequencing of Oscillatoria sp. PCC 6506 was performed using the 454 technology (GATC, Konstanz, Germany), giving 677,304 reads, with a total number of 164 megabases, representing an estimated coverage of 24. Assembly yielded 419 contigs consisting of 6,729,249 bases with a G+C content of 43.40%. Automatic annotation of this genome using the MaGe pipeline system (16) predicted 6,007 coding sequences (CDS), 61 fragments of CDS, and 84 genes coding for RNA (4 rRNA genes, 70 tRNA genes, and 10 small noncoding RNAs [ncRNAs]).Oscillatoria sp. PCC 6506 is an aerobic photosynthetic and diazotrophic cyanobacterium that shows transient and dispersed gas vesicles (4, 6). We found 37 genes coding for proteins involved in photosynthesis and the nif gene cluster, responsible for nitrogen fixation. However, we could not identify any gene coding for a hydrogenase (15), as for Synechococcus sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511 (8). Four genes were annotated as gas vesicle protein (gvp) genes (13).Oscillatoria sp. PCC 6506 produces anatoxin-a and homoanatoxin-a, and we recently identified the ana cluster of genes, responsible for the biosynthesis of these alkaloids (3, 10, 11). We also identified the cluster of genes (cyr) responsible for the biosynthesis of cylindrospermopsin (9, 12). Cyanobacteria are known to produce a wide range of secondary metabolites (7), and 3.7% of the total annotated CDS in this genome were dedicated to secondary metabolism. We identified, beside the ana and cyr clusters, four other clusters of genes containing polyketide synthase and nonribosomal peptide synthase coding genes, likely involved in the biosynthesis of secondary metabolites.We found 51 transposase genes in the Oscillatoria sp. PCC 6506 genome, some close to the biosynthetic gene clusters identified, suggesting horizontal gene transfer events.In conclusion, we present here the sequence of the first genome of an Oscillatoria cyanobacterium strain, a very common genus found in the environment, and the first genome of a neurotoxin-producing cyanobacterium.     

Phylogenetic relationships between geographically separate Phormidium cyanobacteria: is there a link between north and south polar regions?

We present a phytogeographical comparison between polar (Arctic and Antarctic) and non-polar strains of the cyanobacterial genus Phormidium, which plays a key role in Arctic and Antarctic ecosystems as primary producer. A total of 26 Phormidium strains were studied using a polyphasic approach, 18 from Arctic (Svalbard, Ellesmere Island and Scandinavian Arctic—Abisko) and Antarctic (Antarctic Peninsula—King George and James Ross Island) regions, and 8 from temperate sites (mostly situated in Central Europe). A phylogenetic tree was constructed and compared with similar 16S rRNA sequences retrieved from Genbank. Within the Phormidium autumnale cluster, genetic similarity of 16S rDNA was more related to geographical proximity of strain origin than to morphological similarity. No genetic identity of Phormidium strains from north and south polar regions was found. The cluster Phormidium autumnale apparently belongs to generic entities in which geographical limitation plays a prominent role. However, the cyanobacterial strains found in Europe suggest that the distribution areas of some Phormidium cyanobacteria overlap. The Phormidium autumnale cluster is evidently a very characteristic type and represents an isolated clade within the traditional genus Phormidium. According to morphological features and the structure of trichomes, it is most similar and thus probably belongs to the genus Microcoleus.     

Molecular Detection and Ecological Significance of the Cyanobacterial Genera Geitlerinema and Leptolyngbya in Black Band Disease of Corals

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.     

Fresh Water Cyanobacteria Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as an Anticancer Drug Resource

An increasing number of cancer patients worldwide, especially in third world countries, have raised concern to explore natural drug resources, such as the less explored fresh water filamentous cyanobacteria. Six strains of cyanobacteria (Phormidium sp. CCC727, Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Phormidium sp. CCC731, Phormidium sp. CCC730, and Leptolyngbya sp. CCC732) were isolated (paddy fields and ponds in the Banaras Hindu University, campus) and five strains screened for anticancer potential using human colon adenocarcinoma (HT29) and human kidney adenocarcinoma (A498) cancer cell lines. Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 were the most potent as determined by examination of morphological features and by inhibition of growth by graded concentrations of crude extracts and thin-layer chromatography (TLC) eluates. Cell cycle analysis and multiplex assays using cancer biomarkers also confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as cancer drug resources. Apoptotic studies in the cells of A498 (cancer) and MCF-10A (normal human epithelial) exposed to crude extracts and TLC fractions revealed no significant impact on MCF-10A cells emphasizing its importance in the development of anticancer drug. Identification of biomolecules from these extracts are in progress.     

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments. Received: February 25, 2001 / Accepted: July 27, 2001     

Entrapped Sediments as a Source of Phosphorus in Epilithic Cyanobacterial Proliferations in Low Nutrient Rivers

Proliferations of the benthic mat-forming cyanobacteria Phormidium have been reported in rivers worldwide. Phormidium commonly produces natural toxins which pose a health risk to animal and humans. Recent field studies in New Zealand identified that sites with Phormidium proliferations consistently have low concentrations of water column dissolved reactive phosphorus (DRP). Unlike other river periphyton, Phormidium mats are thick and cohesive, with water and fine sediment trapped in a mucilaginous matrix. We hypothesized that daytime photosynthetic activity would elevate pH inside the mats, and/or night time respiration would reduce dissolved oxygen. Either condition could be sufficient to facilitate desorption of phosphates from sediment incorporated within mats, thus allowing Phormidium to utilize it for growth. Using microelectrodes, optodes and pulse amplitude modulation fluorometry we demonstrated that photosynthetic activity results in elevated pH (>9) during daytime, and that night-time respiration causes oxygen depletion (<4 mg L^-1) within mats. Water trapped within the mucilaginous Phormidium mat matrix had on average 320-fold higher DRP concentrations than bulk river water and this, together with elevated concentrations of elements, including iron, suggest phosphorus release from entrapped sediment. Sequential extraction of phosphorus from trapped sediment was used to investigate the role of sediment at sites on the Mangatainoka River (New Zealand) with and without Phormidium proliferations. Deposition of fine sediment (<63 μm) was significantly higher at the site with the most extensive proliferations and concentrations of biological available phosphorus were two- to four- fold higher. Collectively these results provide evidence that fine sediment can provide a source of phosphorus to support Phormidium growth and proliferation.     

Metal biosorption by two cyanobacterial mats in relation to pH, biomass concentration, pretreatment and reuse

The pH-dependent metal sorption by Oscillatoria- and Phormidium-dominated mats was effectively expressed by the Hill function. The estimated Hill functions can fruitfully predict the amount of metal sorbed at a particular initial pH. Pretreatment of biomass with 0.1 mmol L⁻¹ HCl was more effective than pretreatment with CaCl₂, HNO₃, NaOH, and SDS in enhancing metal sorption ability of the biomass. Desorption of metal ions in the presence of 100 mmol L⁻¹ HCl from metal-loaded mat biomass was completed within 1 h. After six cycles of metal sorption/desorption, sorption decreased by 6-15%. Only 6% and 11% of the biomass derived from the Oscillatoria sp.- and Phormidium sp.-dominated mats was lost during the cycling. The cyanobacterial mats seem to have better potential than several biomass types for use in metal sorption from wastewaters as they are ubiquitous, self-immobilized, and have good reusability.     

A Study of the Effect of Red Mud Amendments on the Growth of Cyanobacterial Species

Red mud is a bauxite residue, a by-product of the Bayer process for extraction of alumina from bauxite ore. It is highly alkaline in nature and contains heavy metal oxides and a variety of minor trace elements. Its disposal is the paramount environmental issue in alumina refining. Establishment of vegetation on red mud dumping sites is difficult because of alkaline pH and high concentrations of soluble ions and toxic compounds. The bioremediation of red mud seems to be a realistically convenient way for environmentally safe disposal of this industrial residue. The present study specifically deals with the utilization of cyanobacteria to bioremediate red mud. Being predominantly a nitrogen-fixing photosynthetic microorganism, it has considerable potential for bioremediation of bauxite residue. As a prelude step, four cyanobacterial species, viz. Oscillatoria sp., Lyngbya sp., Phormidium sp., and Microcystis sp. were selected to study their capability to grow on red mud–amended medium. The results indicate that Oscillatoria and Phormidium performed well on red mud–amended medium, followed by Lyngbya. Growth of Microcystis was inhibited significantly even at lower concentrations of red mud amendments. These findings point out the potential of these microorganisms, to be utilized as active biological agents for bioremediation of red mud.     

席藻ITS序列拷贝类型及进化关系分析

由于协同进化未完成,蓝藻的16S-23S rDNA基因间隔序列(ITS)存在多种拷贝类型,目前蓝藻ITS序列的拷贝类型分布以及演化规律尚未研究清楚。该文采用本地采集的席藻属样品,测定其ITS序列,并结合基因库中已有的席藻ITS序列,对席藻属蓝藻ITS序列拷贝类型及其之间的进化关系进行探讨。结果显示,席藻属ITS序列的PCR产物有两种情况,即单一条带(仅出现ITS-IA或ITS-I型)和两条条带(同时出现ITS-IA和ITS-N型),其中以单一条带的ITS-IA型最为普遍;在基于ITS序列的系统发生树中,ITS-IA型和ITS-I型均各自聚为一个组群,且ITS-IA型的组群节点出现较早,表明席藻属ITS-I型极有可能是由其原来的ITS-IA型缺失tRNAAla编码区进化而来,ITS-IA型应为席藻ITS序列的基本结构。     

Rapid Determination of Cytokinins and Auxin in Cyanobacteria

Five cyanobacterial strains, Anabaena sp. Ck1, Oscillatoria sp. Ck2, Phormidium sp. Ck3, Chroococcidiopsis sp. Ck4, and Synechosystis sp. Ck5 were selected for their positive cytokinins-like activity using cucumber cotyledon bioassay and GUS assay in Arabidopsis ARR5::GUS. Classical cucumber cotyledon bioassay was modified for direct screening of cyanobacteria avoiding need for extraction and purification. Cytokinins from cyanobacteria were absorbed onto filter paper which was then assayed for cytokinins-like activity. A rapid chromatographic method was developed for the simultaneous determination of cytokinins and indole-3-acetic acid (IAA). Cyanobacterial biomass (50–100 mg) and cell-free culture filtrate were extracted in Bieleski buffer and purified by solid-phase extraction. The extract was used to determine phytohormones by ultra performance liquid chromatography and electrospray ionization-tandem mass spectrometry in positive and negative modes, respectively, with multiple reactions monitoring. Stable isotope-labeled cytokinins and IAA standards were added in the samples to follow recovery of the compounds and method validation. Five cytokinins determined in the selected strains were Zeatin (cis and trans isomers), Zeatin riboside, Dihydrozeatin riboside, and zeatin-o-glucoside. The strains were shown to accumulate as well as release the phytohormones.     

Neurotoxic effects evoked by cyanobacterial extracts suggest multiple receptors involved in electrophysiological responses of molluscan (CNS, heart) models

The responses of the snail central neurons (Helix pomatia, Lymnaea stagnalis) and the isolated Helix heart were characterized evoked by cyanobacterial extracts (Cylindrospermopsis raciborskii ACT strains) isolated from Lake Balaton (Hungary). The nicotinergic acetylcholine (ACh) receptors in the CNS (both excitatory and inhibitory) were blocked by the extracts of ACT 9502 and ACT 9505 strains and the anatoxin- a (homoanatoxin-a) producing reference strain of Oscillatoria sp. (PCC 6506), similar to the inhibitory effects of the pure anatoxin-a. The enhancement of the ACh responses by the ACT 9504 extract suggests additional, probably acetylcholine esterase inhibitory mechanisms. On the isolated Helix heart the crude ACT 9505 and PCC 6506 extracts evoked frequency increase and transient twitch contraction, opposite to the ACh evoked heart relaxation. Anatoxin-a similarly contracted the heart but did not increase its contration frequency. These data suggest the involvement of some non-cholinergic mechanisms, acting very likely by direct modulation of the electrical or contractile system of the isolated heart. Diversity of the effects evoked by the cyanobacterial extracts in the CNS and heart suggest pharmacologically different neuroactive components among the secondary metabolites of the cyanobacteria acting on both (anatoxin-a like) cholinergic and (unidentified) non-cholinergic receptors.     

Associative Cyanobacteria Isolated from the Roots of Epiphytic Orchids

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of A. papillosa and D. moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrixwere isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria,and representatives of the LPP group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP group, and Scytonema in D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root rhizoplane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi in this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.