Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (T_m) of desiccated cells of N. commune CHEN was low (T_{m dry} = 8 °C) and was comparable to the T_m of rehydrated cells((T_m)_H20 = 6 °C). The EPS was not responsible for the depression of T_m. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low T_{m dry} in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

Spectroscopical and functional characterization of the hemoglobin of Nostoc commune UTEX 584 (Cyanobacteria)

Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al. (1992) Science 256, 1690–1692), is presented. Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity. There was no evidence for decreased stability of the pigment at 37°C. Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin. The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins. The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin. Met-cyanoglobin possesses spectral characteristics at pH 7.0–9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum. The spin-state character of met-cyanoglobin is pH-dependent. Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0. The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH. Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure. Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another.     

Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O2 evolution was not detected in desiccated colonies, the ability of the cells to evolve O2 rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O2 evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O2, but O2 evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O2 evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.     

Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modified during long-term (decades) desiccation but is protected from oxidative damage and degradation

Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5′-GWCWATCGCC-3′) based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.     

Variation in Phospholipid Ester-Linked Fatty Acids and Carotenoids of Desiccated Nostoc commune (Cyanobacteria) from Different Geographic Locations

Profiles of phospholipid fatty acids and carotenoids in desiccated Nostoc commune (cyanobacteria) collected from China, Federal Republic of Germany, and Antarctica and in axenic cultures of the desiccation-tolerant strains N. commune UTEX 584 and Hydrocoleum strain GOEI were analyzed. The phospholipid fatty acid contents of the three samples of desiccated Nostoc species were all similar, and the dominant compounds were 16:1ω7c, 16:0, 18:2ω6, 18:3ω3, and 18:1ω7c. In comparison with the field materials, N. commune UTEX 584 had a much higher ratio of 18:2ω6 to 18:3ω3 (5.36) and a significantly lower ratio of 18:1ω7c to 18:1ω9c (1.86). Compound 18:3 was present in large amounts in the samples of desiccated Nostoc species which had been subject, in situ, to repeated cycles of drying and rewetting, but represented only a small fraction of the total fatty acids of the strains grown in liquid culture. This finding is in contrast to the data obtained from studies on the effects of drought and water stress on higher plants. Field materials of Nostoc species contained, in contrast to the axenic strains, significant amounts of apocarotenoids and a P384 pigment which, upon reduction with NaBH₄, yielded a mixture of a chlorophyll derivative and a compound with an absorption maximum of 451 nm. A clear distinction can be made between the carotenoid contents of the axenic cultures and the desiccated field materials. In the former, β-carotene and echinenone predominate; in the latter, canthaxanthin and the β-γ series of carotenoids are found.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Biochemistry and structure of the glycan secreted by desiccation-tolerantNostoc commune (Cyanobacteria)

Summary Filaments of the desiccation-tolerant cyanobacteriumNostoc commune are embedded within, and distributed throughout, a dense glycan sheath. Analysis of the glycan of field materials and of pure cultures ofN. commune DRH 1 through light and electron microscopy, immunogold labelling and staining with dyes, revealed changes in the pattern of differentiation in glycan micro-structure, as well as localized shifts in pH, upon rehydration of desiccated field material. A Ca/Si rich external (pellicular) layer of the glycan acts as a physical barrier to epiphytic bacteria on the surface ofN. commune colonies. A purified fraction (>12 kDa) of an aqueous extract of the glycan from desiccated field material contained glucose, N-acetylglucosamine, glucosamine, mannose, and galactosamine with ratios of 3.11.410.10.06, respectively. Lipid soluble extracts ofN. commune contained trehalose and sucrose and the levels of both became undetectable following cell rehydration. Intracellular cyanobacterial trehalase was identified using immunoblotting and its synthesis was detected upon rehydration of desiccated field cultures. Elemental analysis of glycan extracts showed a flux in the concentrations of salts in the glycan matrix following rehydration of desiccated colonies. Water-stress proteins (Wsp; most abundant proteins in glycan), a water soluble UV-A/B-absorbing pigment, the lipid-soluble UV-protective pigment scytonemin (in both its oxidized and reduced forms), as well as two unidentified cyanobacterial glycoproteins (75 kDa and 110 kDa), were found within the glycan matrix. An unidentified 68 kDa protein, the second most abundant protein in aqueous extracts of the glycan, was isolated and its N-terminal sequence was determined as AFIFGTISPNNLSGTSGNSGIVGSA. Gene bank searches with this sequence identified significant homologies (35–45%) with various carbohydrate-modifying enzymes. The role of the glycan in the desiccation tolerance ofN. commune is discussed with respect to structure/function relationships.Abbreviations EPS extracellular polysaccharides - Wsp water-stress protein - SEM scanning electron microscopy - TEM transmission electron microscopy - EDX energy dispersive X-ray analysis - FPLC fast performance liquid chromatography - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography - UV ultra-violet radiation - UTEX University of Texas Culture Collection     

UV-B-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium Nostoc commune.

Liquid cultures of the terrestrial cyanobacterium Nostoc commune derived from field material were treated with artificial UV-B and UV-A irradiation. We studied the induction of various pigments which are though to provide protection against damaging UV-B irradiation. First, UV-B irradiation induced an increase in carotenoids, especially echinenone and myxoxanthophyll, but did not influence production of chlorophyll a. Second, an increase of an extracellular, water-soluble UV-A/B-absorbing mycosporine occurred, which was associated with extracellular glycan synthesis. Finally, synthesis of scytonemin, a lipid-soluble, extracellular pigment known to function as a UV-A sunscreen, was observed. After long-time exposure, the UV-B effect on carotenoid and scytonemin synthesis ceased whereas the mycosporine content remained constantly high. The UV-B sunscreen mycosporine is exclusively induced by UV-B (< 315 nm). The UV-A sunscreen scytonemin is induced only slightly by UV-B (< 315 nm), very strongly by near UV-A (350 to 400 nm), and not at all by far UV-A (320 to 350 nm). These results may indicate that the syntheses of these UV sunscreens are triggered by different UV photoreceptors.     

Annual carbon fixation in terrestrial populations of Nostoc commune (Cyanobacteria) from an Antarctic dry valley is driven by temperature regime

Nostoc commune Vaucher (a cyanobacterium) is a very conspicuous terrestrial primary producer in Victoria Land, continental Antarctica. Because polar ecosystems are considered to be especially sensitive to environmental changes, understanding the environmental constraints on net carbon (C) fixation by N. commune is necessary to determine the effects of environmental changes on the ecological functioning of ice‐free areas of the continent. A model describing net C fixation in terrestrial populations of N. commune in an Antarctic dry valley was constructed using field and laboratory measurements in which N. commune colonies were exposed to different combinations of incident irradiance (400–700 nm), temperature, and degree of desiccation. For desiccated N. commune mats with water content ≤ 30% saturation, net C fixation was highly variable between replicates and could not be modelled. However, for colonies at > 30% saturation, rates of net C fixation and dark respiration depended strongly on irradiance and temperature. Net C fixation reached a maximum rate of 21.6 μg C m^{− 2} s^{− 1} at irradiance of approximately 250 μmol m^{− 2} s^{− 1} and the optimum temperature of 20.5 °C. Agreement between predicted short‐term net C fixation and field and laboratory measurements allowed estimation of total seasonal fixation, using previously published environmental data. Annual net C fixation was estimated in the range 14.5–21.0 g C fixed m^{− 2}Nostoc mat, depending on year/season. Estimates for different seasons correlated with thermal time (accumulated hours above 0 °C during the year) rather than irradiance, in contrast to communities in local lacustrine environments, where irradiance is the main driver of primary productivity. In the terrestrial habitat, N. commune appears to compromise between an ability to capitalize on short periods of higher temperature and efficient utilization of lower irradiance at low temperature. The relationship between thermal time and net annual C fixation by N. commune is strongly linear.     

Exopolysaccharide of Nostoc muscorum (Cyanobacteria) in the aggregation of soil particles

The effects on a saline-sodic soil of exopolysaccaride isolated from Nostoc Muscorum or the addition of a cyanobacterial mass proliferation were evaluated in a greenhouse experiment. By day 180 exopolysaccharide increased soluble C by 100%, microbial activity by 366% and the amount of water-stable aggregates larger than 250μm by 12 times. Inoculation with living cyanobacterial mass increased at the end of 365 oxidizable C by 11%, soluble C by 66%, microbial activity by 73% and aggregates larger than 250 μm by66%. A slimy film 3–5 mm thick, with N. Muscorum predominating, covered all the surface of inoculated soils. The higher soil aggregate stability produced by both treatments is a consequence of increased microbial activity and concentrating the soil polysaccharide. The high percentage of clays favours the creation of firm and long-lasting slime-mineral joints. Addition of isolated exopolysaccharide produces a faster and higher increase in soil aggregate stability than cyanobacterial mass inoculation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Desiccation‐induced changes in photochemical processes of photosynthesis and spectral reflectance in Nostoc commune (Cyanobacteria,Nostocales) colonies from polar regions

Cyanobacterium Nostoc commune is a species highly resistant against desiccation. In this study, we investigated changes in photochemical processes of photosynthesis and spectral reflectance indices during controlled desiccation of the colonies from Antarctica. In a dehydration process, water potential (WP) reached ?3 MPa and values of potential (F _v/F _m) and effective quantum yields (ΦPSII) of photosystem II were kept to high value until 90% of water was lost from the colony, and these values decreased rapidly by further loss of water. This indicates that the colony loses water mostly from the exopolysaccharidic envelope, not from cells during the initial part of dehydration (relative water content, RWC = 100–10%). Other suggestions of inhibition of photosynthetic processes after 90% loss of water were the increase of the chlorophyll fluorescence parameter F _p/F _s. The F _m′ was higher than F _m in hydrated colonies because of state transition which change energy distribution between PS I and PS II, but decreased to same level as F _m in dehydrated colonies. The Normalized Difference Vegetation Index (NDVI) and Photochemical Reflectance Index (PRI) showed concave‐ and convex‐curvilinear relationship with RWC, respectively. The changes of NDVI values were, however, statistically insignificant. PRI values were predominantly below 0 because of phycobiliprotein involvement. These results were compared with the same species in the Arctic region. This is, according to our best knowledge, the first measurement of changes in spectral reflectance indices during desiccation of cyanobacteria.     

A Structural and Utrastructural Study of Nostoc commune

Nostoc commune which belongs to blue-green algae has been observed with microscope, scanning and transmission electron microscopes. The following special structures are found: 1. The structure in thallus is spongiform with an uneven surface. 2. The filaments made up of torulose cells are tortuous with different length, some are piled up, some are branchy, and the others arrange in a circle. 3. The cell wall of torulose cell is five-layered. The outermost layer is ripply and inlays with neighbour cells. 4. Torulose cells are typical prokaryote. The thylakoids disperse in the peripheral plasma. The nucleoplasm exists in the center of the cell. There are structured granules, polyhedral and polyphosphate bodies interspersing in cytoplasm. Ribosomes and phycobilins are found on the surface of thylakoid. 5. The heterocyst is surrounded by a thicking sheath. This is an additional envelope outside the cell wall and can be called “cell envelope”. In the cell envelope exist a transparent layer and a dense electron layer with hemispherical nodule in which there is an channel interlinking up with neighbour cell. 6. The reproductive manner of N. commune is of horizontal split, and the direction of split is different.     

The exo-proteome and exo-metabolome of Nostoc punctiforme (Cyanobacteria) in the presence and absence of nitrate

The ability of nitrogen-fixing filamentous Cyanobacteria to adapt to multiple environments comes in part from assessing and responding to external stimuli, an event that is initiated in the extracellular milieu. While it is known that these organisms produce numerous extracellular substances, little work has been done to characterize both the metabolites and proteins present under standard laboratory growth conditions. We have assessed the extracellular milieu of Nostoc punctiforme when grown in liquid culture in the presence and absence of a nitrogen source (nitrate). The extracellular proteins identified were enriched in integrin β-propellor domains and calcium-binding sites with sequences unique to N. punctiforme, supporting a role for extracellular proteins in modulating species-specific recognition and behavior processes. Extracellular proteases are present and active under both conditions, with the cells grown with nitrate having a higher activity when normalized to chlorophyll levels. The released metabolites are enriched in peptidoglycan-derived tetrasaccharides, with higher levels in nitrate-free media.