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.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.     

Molecular genetic and chemotaxonomic characterization of the terrestrial cyanobacterium Nostoc commune and its neighboring species

The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP? oxidoreductase and by random amplified polymorphic DNA analysis. The genotypes of N. commune were distributed in Japan without regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease was widely distributed in the genus Nostoc, suggesting that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein was only found in N. commune and Nostoc verrucosum, suggesting that these Nostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.     

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.     

The UV-B stimulon of the terrestrial cyanobacterium Nostoc commune comprises early shock proteins and late acclimation proteins

The UV-B and desiccation-tolerant terrestrial cyanobacterium Nostoc commune was grown under defined UV irradiation. Proteome changes were monitored in the membrane and the cytosolic and the extracellular fractions. Tools were developed to separate stress-triggered from growth stage-dependent changes. UV-B changed the relative cellular concentration of 493 out of 1,350 protein spots at least by a factor of three, rendering the UV-B stimulon of N. commune the most complex one described so far. It comprises two different parts: an early shock response influencing 214 proteins and a late acclimation response involving 279 proteins. The shock response comprised many membrane or membrane-associated proteins, whereas the acclimation response mainly changed cytosolic proteins. Most of the shock-induced changes were transient and did not overlap with the acclimation response. In the extracellular fraction, UV irradiation induced superoxide dismutase and the water stress protein. In total, 27 intracellular, UV-B-induced proteins were partially sequenced by electrospray ionization tandem mass spectrometry. Three functional classes were identified: proteins involved in lipid metabolism, in carbohydrate metabolism and in regulatory pathways. About 50% of the sequenced proteins were homologous to cyanobacterial database entries with un-known function. Interestingly, all of these proteins belong to the UV-B acclimation response. We conclude that the UV-B shock response and the UV-B acclimation response represent two completely different and remarkably complex strategies of N. commune to protect itself against UV-B radiation in its natural environment.     

Recovery of photosynthetic systems during rewetting is quite rapid in a terrestrial cyanobacterium, Nostoc commune

Recovery processes of photosynthetic systems during rewetting were studied in detail in a terrestrial, highly drought-tolerant cyanobacterium, Nostoc commune. With absorption of water, the weight of N. commune colony increased in three phases with half-increase times of about 1 min, 2 h and 9 h. Fluorescence intensities of phycobiliproteins and photosystem (PS) I complexes were recovered almost completely within 1 min, suggesting that their functional forms were restored very quickly. Energy transfer from allophycocyanin to the core-membrane linker peptide (L(CM)) was recovered within 1 min, but not that from L(CM) to PSII. PSI activity and cyclic electron flow around PSI recovered within 2 min, while the PSII activity recovered in two phases after a time lag of about 5 min, with half times of about 20 min and 2 h. Photosynthetic CO(2) fixation was restored almost in parallel with the first recovery phase of the PSII reaction center activity. Although the amount of absorbed water became more than 20 times the initial dry weight of the N. commune colony in the presence of sufficient water, about twice the initial dry weight was enough for recovery and maintenance of the PSII activity.     

Four chemotypes of the terrestrial cyanobacterium Nostoc commune characterized by differences in the mycosporine‐like amino acids

The cyanobacterium Nostoc commune is adapted to terrestrial environments and has a cosmopolitan distribution. Four genotypes of N. commune can be identified based on differences in their 16S rRNA genes, and these genotypes are distributed throughout Japan without regional specificity. Mycosporine‐like amino acids (MAAs) are UV‐absorbing pigments, and novel glycosylated MAA derivatives with radical scavenging activities have been identified in N. commune. In this study, we investigated the consistency of the relationship between MAA compositions and N. commune genotypes. The MAA compositions were different in a genotype‐specific manner, suggesting that the types of MAA derivatives can feasibly be used as chemotaxonomic markers to characterize N. commune. The novel 756‐Da MAA, which was identified as an aglycone of the 1050‐Da MAA and named nostoc‐756, occurred in genotype C of N. commune. Nostoc‐756 functioned as a radical scavenger in vitro. In conclusion, N. commune is classified into four groups representing genetically different chemotypes, namely, the arabinose‐bound porphyra‐334 producer (chemotype A), the glycosylated nostoc‐756 producer (chemotype B), the nostoc‐756 producer (chemotype C) and the glycosylated palythine‐threonine producer (chemotype D). Either the molecular taxonomical method or chemical analysis of a characteristic secondary metabolite is sufficient to identify the types of N. commune; however, there are no obvious ecophysiological differences that allow us to distinguish them.     

Protein synthesis and proteolysis in immobilized cells of the cyanobacterium Nostoc commune UTEX 584 exposed to matric water stress.

Cells of the cyanobacterium Nostoc commune UTEX 584 in exponential growth were subjected to acute water stress by immobilizing them on solid supports and drying them at a matric water potential (psi m) of -99.5 MPa. Cells which had been grown in the presence of Na235SO4 before immobilization and rapid drying continued to incorporate 35S into protein for 90 min. This incorporation was inhibited by chloramphenicol. No unique proteins appeared to be synthesized during this time. Upon further drying, the level of incorporation of 35S in protein began to decrease. In contrast, there was an apparent increase in the level of certain phycobiliprotein subunits in solubilized protein extracts of these cells. Extensive proteolysis was detected after prolonged desiccation (17 days) of the cells in the light, although they still remained intact. Phycobilisomes became dissociated in both light- and dark-stored desiccated material.     

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, showsunique responses to dehydration. These responses are: (i) lossof PSII activity in parallel with the loss of photosynthesis;(ii) loss of PSI activity; and (iii) dissipation of light energyabsorbed by pigment–protein complexes. In this study,the deactivation of PSII is shown to be important in avoidingphotoinhibition when the Calvin–Benson cycle is repressedby dehydration. Furthermore, our evidence suggests that dissipationof light energy absorbed by PSII blocks photoinhibition understrong light in dehydrated states.     

Effect of UV-B radiation on the synthesis of UV-absorbing compounds in a terrestrial cyanobacterium, Nostoc flagelliforme

Effects of two intensities (1 and 5 W?m^?2) of UV-B radiation on the synthesis of UV-absorbing compounds in a terrestrial cyanobacterium Nostoc flagelliforme were investigated. UV-B radiation resulted in lower biomass. Short period (less than 12 h) of UV-B radiation caused an increase of chlorophyll a content, but subsequent duration of treatment (more than 24 h) resulted in a rapid decrease. N. flagelliforme synthesized UV-absorbing compounds such as scytonemin and mycosporine-like amino acids (MAAs) in response to UV-B radiation. Upon 48 h of exposure to UV-B radiation, scytonemin content in cells increased by 103.8 and 164.0 % at 1 and 5 W?m^?2, respectively. Oligosaccharide-linked mycosporine-like amino acids increased by 145.5 % after 12 h at 5 W?m^?2 and 114.5 % after 48 h at 1 W?m^?2 UV-B radiation. HPLC analysis showed that nine MAAs existed in N. flagelliforme cells both from liquid suspension culture and field colony. But the concentration and kinds of them were different. At the two distinct levels of UV-B radiation, the content of particular MAAs increased, declined, or remained unchanged. Moreover, the appearance of two new MAAs was observed.     

The ultrastructure of immobilised desiccated cells of the cyanobacterium Nostoc commune UTEX 584

Cells of the cyanobacterium Nostoc commune UTEX 584 were immobilised, subjected to acute matric water stress (ψ_m = −128 MPa) and then desiccated. Their ultrastructure was investigated by the use of an anhydrous fixation procedure. Although shrunken and bleached, the integrity of the vegatative cells at the ultrastructural level was apparently preserved. The ease with which certain cyanobacterial cells can recover from desiccation may be consequent upon the maintenance of cellular organisation at the ultrastructural level.     

Antioxidative activity and chemical constituents of edible terrestrial alga Nostoc commune Vauch

The extract of terrestrial alga Nostoc commune Vauch. has high antioxidative activity. Our study on N. commune Vauch. resulted in the isolation of two β-ionone derivatives, nostocionone and 3-oxo-β-ionone, together with four indole alkaloids, scytonemin, reduced scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine. The structures of the isolated compounds were determined on the basis of 1D and 2D NMR and MS analyses. Among these isolates, nostocionone and reduced scytonemin demonstrated strong antioxidative activities which were assessed by using a β-carotene oxidation assay.     

Purification and properties of an enzyme capable of degrading the polysaccharide of the cyanobacterium,Nostoc commune

A novel Nostoc commune-polysaccharide (NPS)-degrading enzyme with a molecular mass of 128.5 kDa was purified from Paenibacillus glycanilyticus DS-1. The optimum pH and temperature of the enzyme activity were 5.5 and 35 degrees C, respectively. The enzyme completely degraded NPS to oligosaccharides, ranging from tetra to hexasaccharides and could degrade the xylan weakly whereas xanthan, gellan, cellulose, curdlan and p-nitrophenyl-beta-D-xylopyranoside were not degraded. Homology analysis of the N-terminal amino acid sequence of the NPS-degrading enzyme against the PIR and SWISS-PROT databases indicated that the sequence was not homologous to any other polysaccharide-degrading enzyme.     

Phycoerythrin synthesis is induced by solar UV-B in the cyanobacterium Nostoc

Solar UV-B (280–315 nm) induces the synthesis of phycoerythrin (PE) in a Nostoc species isolated from the Andean high altitude lake Yanaqocha. The outdoor experiments were carried out in a small lake in Erlangen, Germany, using natural conditions. After 2- and 4-h exposure to solar radiation, the immunodetection signal using monoclonal antibodies anti-PE was lower in control cells (exposed to PAR + UV-A) than in cells exposed to total solar radiation (PAR + UV-A + UV-B). Cells exposed at depths in which no UV-B penetrated showed no differences from control cells regarding PE content. When exposed to monochromatic radiation of 280, 300 or 360 nm, purified PE was photodegraded in a wavelength dependent manner resulting in different polypeptide fragments carrying chromophore groups. Immunodetection revealed active synthesis of PE in parallel to photodamage by solar UV-B indicating that PE is important for photoadaptation to shorter wavelengths in the cyanobacterium Nostoc sp.     

The odd behaviour of carbonic anhydrase in the terrestrial cyanobacterium Nostoc flagelliforme during hydration-dehydration cycles

The terrestrial cyanobacterium Nostoc flagelliforme , inhabiting arid areas, withstands prolonged periods of dehydration. How dehydration and occasional wetting affect inorganic C acquisition in this organism is not well known. As inorganic C acquisition in cyanobacteria often involves carbonic anhydrases (CA), we studied the effect of cycles of hydration and dehydration on the extracellular and intracellular CA activities, at the pH values presumably associated with dew or rain wetting. The external CA of N. flagelliforme (or of the microorganismal consortium of which N. flagelliforme is the main component) is activated by hydration, especially at low pH, and it may facilitate inorganic C acquisition when N. flagelliforme colonies are wetted by dew. Internal CA is present in dry colonies and is rapidly inactivated upon rehydration, therefore an anaplerotic role for this enzyme is proposed.     

Effects of different UV radiation on photoprotective pigments and antioxidant activity of the hot‐spring cyanobacterium Leptolyngbya cf. fragilis

UV‐induced synthesis/accumulation of photoprotective pigments and antioxidant activity were investigated in the hot‐spring cyanobacterium Leptolyngbya cf. fragilis. The results indicated that UV radiation may induce biosynthesis of carotenoids, allophycocyanin, phycoerythrin, and scytonemin while phycocyanin degrades in response to longtime UV radiation. Moreover, pigment composition of L. cf. fragilis was significantly altered with increasing UV radiation times, probably due to destruction and resynthesis of accessory pigments as an adaptation strategy to UV stress. The in vitro antioxidant analysis of different extracts of UV treated cyanobacteria exhibited concentration‐dependent antioxidant activity. Ethyl acetate extract of 72 h UV treatment showed maximum total antioxidant activity (IC50 = 71.73 ± 5.3 μg mL^?1) followed by ethyl acetate control (non‐UV irradiated) extract (IC50 = 109.43 ± 2.76 μg mL^?1). This is the first report for the UV‐induced synthesis of photoprotective pigments and their antioxidant activity in L. cf. fragilis.