Effects of Modification of Light Parameters on the Production of Cryptophycin, Cyanotoxin with Potent Anticancer Activity, in Nostoc sp.

Cryptophycin-1 is a cyanotoxin produced by filamentous cyanobacteria. It has been evaluated as an anticancer agent with great potential. However, its synthesis provides insufficient yield for industrial use. An alternative solution for metabolite efficient production is to stress cyanobacteria by modifying the environmental conditions of the culture (Nostoc sp. ATCC 53789). Here, we examined the effects of light photoperiod, wavelength, and intensity. In light photoperiod, photoperiods 24:0 and 16:8 (light:dark) were tested while in wavelength, orange-red light was compared with blue. Medium, high, and very high light intensity experiments were performed to test the effect of light stress. For a 10-day period, growth was measured, metabolite concentration was calculated through HPLC, and the related curves were drawn. The differentiation of light wavelength had a major effect on the culture, as orange-red filter contributed to noticeable increase in both growth and doubled the cyanotoxin concentration in comparison to blue light. Remarkably, constant light provides higher cryptophycin yield, but slightly lower growth rate. Lastly, the microorganism prefers medium light intensities for both growth and metabolite expression. The combination of these optimal conditions would contribute to the further exploitation of cryptophycin.

Phylogenetic evaluation of the genus Nostoc and description of Nostoc neudorfense sp. nov., from the Czech Republic.

Cyanobacterial strain ARC8 was isolated from seepage coming into the river Dracice, Frantiskov, Czech Republic, and was characterized using a polyphasic approach. Strain ARC8 showed a typical Nostoc-like morphology and in-depth morphological characterization indicated that it is a member of the genus Nostoc. Furthermore, in the 16S rRNA gene phylogeny inferred using Bayesian inference, maximum likelihood and neighbour joining methods, strain ARC8 clustered within the Nostoc sensu stricto clade. The phylogenetic distance and the positioning of strain ARC8 also indicated that it is a member of the genus Nostoc. Furthermore, the rbcL gene phylogeny along with the 16S-23S ITS secondary structure analysis also supported the findings from the 16S rRNA gene tree. In accordance with the International Code of Nomenclature for Algae, Fungi and Plants we describe a novel species of Nostoc with the name Nostoc neudorfense sp. nov.

Interactions of superabsorbent polymers based on acrylamide substances with microorganisms occurring in human dwellings.

Superabsorbent polymers (SAPs) are most often used in hygienic goods or in the agricultural sector but the range of their application is much broader, including the utilization in advanced building materials. Although SAPs were studied widely during the last decades, the data related to the interactions between the natural environment and various organisms occurring on their surface are still lacking. In addition, SAPs can create a variable gel-forming matter in the presence of water but standard ecotoxicological bioassays are mostly not suitable for testing such type of materials. In this study, the SAPs potential for reducing/supporting unwanted indoor microorganism settlement was analyzed by biological methods under controlled laboratory conditions. Three commonly used SAPs (Cabloc CT, Creasorb SIS, Hydropam) were exposed to selected organisms representing green algae (Hematococcus pluvialis), cyanobacteria (Nostoc sp.), yeasts (Saccharomyces cerevisiae), wood-destroying fungi (Gleophyllium trabeum), and aerial molds. The obtained results indicated that Hydropam provided favorable conditions for Hematococcus pluvialis, Nostoc sp., and Saccharomyces cerevisiae. All three tested SAPs inhibited, both with and without nutrient addition, the growth of Gleophyllium trabeum and aerial molds.

Description of two new species of Aliinostoc and one new species of Desmonostoc from India based on the Polyphasic Approach and reclassification of Nostoc punensis to Desmonostoc punense comb. nov.

Three heterocytous cyanobacterial strains were isolated from different habitats of Central India, and initial morphological studies indicated them to be members of the genus Nostoc or other closely related genera. Subsequent studies using morphological, ecological, molecular and phylogenetic methods indicated the three strains to be new members of the genera Aliinostoc and Desmonostoc. Folding of the D1-D1' helix of the ITS region clearly differentiated the three strains from the other closely related strains, thus providing final indications of the strains being different and new additions to the genera Aliinostoc and Desmonostoc. In accordance with the International Code of Nomenclature for algae, fungi and plants, we establish three new species: Aliinostoc tiwarii sp. nov, Aliinostoc soli sp. nov. and Desmonostoc magnisporum sp. nov. along with reclassifying Nostoc punensis as Desmonostoc punense comb. nov.

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.

Nostoc thermotolerans sp. nov., a soil-dwelling species of Nostoc (Cyanobacteria).

A filamentous, soil-dwelling cyanobacterial strain (9C-PST) was isolated from Mandsaur, Madhya Pradesh, India, and is described as a new species of the genus Nostoc. Extensive morphological and molecular characterization along with a thorough assessment of ecology was performed. The style of filament orientation, type and nature of the sheath (e.g. distribution and visibility across the trichome), and vegetative and heterocyte cell dimensions and shape were assessed for over one year using both the laboratory grown culture and the naturally occurring samples. Sequencing of the 16S rRNA gene showed 94 % similarity with Nostocpiscinale CENA21 while analyses of the secondary structures of the 16S-23S ITS region showed unique folding patterns that differentiated this strain from other species of Nostoc. The level of rbcl and rpoC1 gene sequence similarity was 91 and 94 % to Nostocsp. PCC 7524 and Nostocpiscinale CENA21, respectively, while the nifD gene sequence similarity was found to be 99 % with Nostocpiscinale CENA21. The phenotypic, ecological, genetic and phylogenetic observations indicate that the strain 9C-PST represents a novel species of the genus Nostoc with the name proposed being Nostoc thermotolerans sp. nov. according to the International Code of Nomenclature for Algae, Fungi, and Plants.

Nitrogen fixation activity in biological soil crusts dominated by cyanobacteria in the Subpolar Urals (European North-East Russia).

The nitrogen fixation by biological soil crusts with a dominance of cyanobacteria was studied using the acetylene reduction assay in the territory of the Subpolar Urals (65°11' N, 60°18' E), Russia. The field measurements of nitrogen fixation activity were conducted in situ for two different types of soil crusts dominated by Stigonema (V1 type) and Nostoc with Scytonema (V2 type). The nitrogen fixation process had similar dynamics in both crusts but nitrogen fixation rates were different. The crusts of the V2 type showed a significantly higher acetylene reduction activity, with ethylene production rate of 1.76 ± 0.49 g C2H4 m(-2) h(-1) at 15°C, compared with V1-type soil crusts, with a rate of 0.53 ± 0.21 mg C2H4 m(-2) h(-1) at 15°C. The daily value of acetylene reduction activity in V2-type soil crusts was 32.7 ± 6.2 mg C2H4 m(-2) d(-1) and in V1-type crusts, 12.3 ± 1.8 mg C2H4 m(-2) d(-1) After recalculation for N, the daily values of nitrogen fixation were in the range 3.3-22.3 mg N m(-2) d(-1), which is a few times higher than the values of N input from the precipitation to the soil in the studied regions. The dependence of nitrogen-fixation activity on temperature and light intensity of biological soil crusts was investigated. On the basis of temperature models obtained from the dependence, the nitrogen balance was calculated for the growing season (approximately 120 days). The crusts dominated by Stigonema species were fixing 0.3 g N m(-2) (ethylene production rate, 1.10 g C2H4 m(-2)) and crusts dominated by Nostoc and Scytonema were fixing 1.3 g N m(-2) (4.10 g C2H4 m(-2)).

Complete genome sequence of cyanobacterium Nostoc sp. NIES-3756, a potentially useful strain for phytochrome-based bioengineering.

To explore the diverse photoreceptors of cyanobacteria, we isolated Nostoc sp. strain NIES-3756 from soil at Mimomi-Park, Chiba, Japan, and determined its complete genome sequence. The Genome consists of one chromosome and two plasmids (total 6,987,571 bp containing no gaps). The NIES-3756 strain carries 7 phytochrome and 12 cyanobacteriochrome genes, which will facilitate the studies of phytochrome-based bioengineering.

Effect of Light Intensity and Photoperiod on Growth and Biochemical Composition of a Local Isolate of Nostoc calcicola.

A study was conducted to investigate the effect of light intensity (21, 42, and 63 µmol photons m(-2) s(-1)) and photoperiod (8:16, 12:12, and 16:8 h light/dark) on the biomass production and its biochemical composition (total carotenoids, chlorophyll a, phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC), total protein, and carbohydrates) of a local isolate of Nostoc calcicola. The results revealed that N. calcicola prefers dim light; however, the most of the levels of light intensity and photoperiod investigated did not have a significant impact on biomass production. Increasing light intensity biomass content of chlorophyll a, PE, PC, APC, and total protein decreased, while total carotenoids and carbohydrate increased. The same behavior was observed also when light duration (photoperiod) increased. The interaction effect of increasing light intensity and photoperiod resulted in an increase of carbohydrate and total carotenoids, and to the decrease of chlorophyll a, PE, PC, APC, and total protein content. The results indicate that varying the light regime, it is capable to manipulate the biochemical composition of the local isolate of N. calcicola, producing either valuable phycobiliproteins or proteins under low light intensity and shorter photoperiods, or producing carbohydrates and carotenoids under higher light intensities and longer photoperiods.

Antifungal compounds from cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133.

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H2O2 could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.

Environmental context shapes the bacterial community structure associated to Peltigera cyanolichens growing in Tierra del Fuego, Chile.

The structure of the associated bacterial community of bipartite cyanolichens of the genus Peltigera from three different environmental contexts in the Karukinka Natural Park, Tierra del Fuego, Chile, was assessed. The sampling sites represent different habitat contexts: mature native forest, young native forest and grassland. Recently it has been determined that the bacterial community associated to lichens could be highly structured according to the mycobiont or photobiont identities, to the environmental context and/or to the geographic scale. However, there are some inconsistencies in defining which of these factors would be the most significant on determining the structure of the microbial communities associated with lichens, mainly because most studies compare the bacterial communities between different lichen species and/or with different photobiont types (algae vs. cyanobacteria). In this work bipartite lichens belonging to the same genus (Peltigera) symbiotically associated with cyanobacteria (Nostoc) were analyzed by TRFLP to determine the structure of the bacterial community intimately associated with the lichen thalli and the one present in the substrate where they grow. The results indicate that the bacterial community intimately associated differs from the one of the substrate, being the former more influenced by the environmental context where the lichen grows.

Genetic diversity of terricolous Peltigera cyanolichen communities in different conservation states of native forest from southern Chile

Decreasing quality of forest habitats is among the major factors leading to a loss of epiphytic lichen diversity. However, there is little information about how this factor influences the diversity of terricolous lichens, which do not grow over living trees and could be less susceptible to such disturbances. In this work we describe the genetic diversity of Peltigeraterricolous cyanolichens and their cyanobiont (Nostoc) from three habitats at the Karukinka Natural Park (Tierra del Fuego, southern Chile), which represent different conservation states: native mature-forest (low disturbance intensity), native young forest (medium disturbance intensity) and grassland (high disturbance intensity). In both forest contexts, a higher diversity and a higher number of unique OTUs (operational taxonomic units) were found. In contrast, in the grassland, the diversity was lower and the Peltigera species were mostly cosmopolitan. The presence of unique OTUs and the higher diversity of lichens in native forest areas highlight the importance of their preservation, indicating that decreasing forest quality also has a negative impact on terricolous lichens diversity (AU)

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Molecular identification and ultrastructural and phylogenetic studies of cyanobacteria from association with the white sea hydroid Dynamena pumila (L., 1758).

Three new cyanobacterial strains, that have been previously purified from the hydroid Dynamena pumila (L., 1758), isolated from the White Sea, were studied using scanning and transmission electron microscopy methods and were characterized by using almost complete sequence of the 16S rRNA gene, internal transcribed spacer 16S-23S rRNA, and part of the gene for 23S rRNA. The full nucleotide sequences of the rRNA gene clusters were deposited to GenBank (HM064496.1, GU265558.1, JQ259187.1). Comparison of rRNA gene cluster sequences of Synechococcus cyanobacterium 1Dp66E-1, Oscillatoriales cyanobacterium 2Dp86E, and Nostoc sp. 10Dp66E with all sequences present at the GenBank shows that these cyanobacterial strains do not have 100% identity with any organisms investigated previously. Furthermore, for the first time heterotrophic bacterium, associated with Nostoc sp. 10Dp66E, was identified as a member of the new phylum Gemmatimonadetes, genus of Gemmatimonas (GenBank accession number is JX437625.1). Phylogenetic analysis showed that cyanobacterium Synechococcus sp. 1Dp66E-1 forms the unique branch and belongs to a cluster of Synechococcus, including freshwater and sea strains. Oscillatoriales cyanobacterium 2Dp86E belongs to a cluster of Leptolyngbya strains. Isolate Nostoc sp. 10Dp66E forms unique branch and belongs to a cluster of the genus Nostoc, with the closest relative of Nostoc commune isolates.

Diversity of endosymbiotic Nostoc in Gunnera magellanica from Tierra del Fuego, Chile [corrected].

Global warming is causing ice retreat in glaciers worldwide, most visibly over the last few decades in some areas of the planet. One of the most affected areas is the region of Tierra del Fuego (southern South America). Vascular plant recolonisation of recently deglaciated areas in this region is initiated by Gunnera magellanica, which forms symbiotic associations with the cyanobacterial genus Nostoc, a trait that likely confers advantages in this colonisation process. This symbiotic association in the genus Gunnera is notable as it represents the only known symbiotic relationship between angiosperms and cyanobacteria. The aim of this work was to study the genetic diversity of the Nostoc symbionts in Gunnera at three different, nested scale levels: specimen, population and region. Three different genomic regions were examined in the study: a fragment of the small subunit ribosomal RNA gene (16S), the RuBisCO large subunit gene coupled with its promoter sequence and a chaperon-like protein (rbcLX) and the ribosomal internal transcribed spacer (ITS) region. The identity of Nostoc as the symbiont was confirmed in all the infected rhizome tissue analysed. Strains isolated in the present study were closely related to strains known to form symbioses with other organisms, such as lichen-forming fungi or bryophytes. We found 12 unique haplotypes in the 16S rRNA (small subunit) region analysis, 19 unique haplotypes in the ITS region analysis and 57 in the RuBisCO proteins region (rbcLX). No genetic variability was found among Nostoc symbionts within a single host plant while Nostoc populations among different host plants within a given sampling site revealed major differences. Noteworthy, interpopulation variation was also shown between recently deglaciated soils and more ancient ones, between eastern and western sites and between northern and southern slopes of Cordillera Darwin. The cell structure of the symbiotic relationship was observed with low-temperature scanning electron microscopy, showing changes in morphology of both cyanobiont cells (differentiate more heterocysts) and plant cells (increased size). Developmental stages of the symbiosis, including cell walls and membranes and EPS matrix states, were also observed.

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae).

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400 bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.

Fungal lectin of Peltigera canina induces chemotropism of compatible Nostoc cells by constriction-relaxation pulses of cyanobiont cytoskeleton.

A glycosylated arginase acting as a fungal lectin from Peltigera canina is able to produce recruitment of cyanobiont Nostoc cells and their adhesion to the hyphal surface. This implies that the cyanobiont would develop organelles to motility towards the chemoattractant. However when visualized by transmission electron microscopy, Nostoc cells recently isolated from P. canina thallus do not reveal any motile, superficial organelles, although their surface was covered by small spindles and serrated layer related to gliding. The use of S-(3,4-dichlorobenzyl)isothiourea, blebbistatin, phalloidin and latrunculin A provide circumstantial evidence that actin microfilaments rather than MreB, the actin-like protein from prokaryota, and, probably, an ATPase which develops contractile function similar to that of myosin II, are involved in cell motility. These experimental facts, the absence of superficial elements (fimbriae, pili or flagellum) related to cell movement, and the appearance of sunken cells during of after movement verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin act as a chemoattractant.

Microcystin producing cyanobacterium Nostoc sp. BHU001 from a pond in India.

The cyanobacterium Nostoc sp. BHU001 is a new isolate from a pond in India. The cyanobacterium produces more than ten peptides including five microcystin (MC) variants, MC-LR, -WR, -AR, -LA and methylated MC-LR, and a new peptide similar to cyanopeptolin. Total MC content determined by ELISA was 25.2 microg g(-1) dry wt of the cyanobacterium, dominated by MC-LR (54%). This is the first report of MC producing Nostoc strain from India.

Isolation and characterization of the gene associated with geosmin production in cyanobacteria.

Geosmin is a secondary metabolite responsible for earthy tastes and odors in potable water supplies. Geosmin continues to be a challenge to water utility management regimes and remains one of the most common causes of consumer complaints, as the taste of "dirty" water may suggest a failed disinfection regime and that the water may be unsafe to drink. Although cyanobacteria have been reported to be largely responsible for these taste and odor events, the answer as to how or why geosmin is produced has eluded researchers. We describe here for the first time the mechanism by which geosmin is produced in a model cyanobacterium, Nostoc punctiforme PCC 73102 (ATCC 29133), which we demonstrate utilizes a single enzyme to catalyze the cyclization of farnesyl diphosphate to geosmin. Using this information, we have developed a PCR-based assay that allows the rapid detection of geosmin-producing cyanobacteria. This test may be utilized to confirm and track the emergence of taste and odor-producing cyanobacteria in any given water body and thus can be used as an early warning system by managers of water bodies that may suffer from adverse taste and odor episodes.