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Cyanobacteria are some of the primary producers in extremely cold biospheres such as the Arctic, Antarctic, and vast ice sheets. Many genera of cyanobacteria are identified from these harsh environments, but their specific mechanisms for cold adaptation are not fully understood. Nostoc sp. strain SO-36 is a cyanobacterium isolated in Antarctica more than 30 years ago and regarded as a psychrotolelant species. To determine whether the strain is psychrotolelant or psychrophilic, it was first grown at 30 °C and 10 °C. The cells grew exponentially at 30 °C, but their growth stopped at 10 °C, indicating that the strain is only psychrotolerant. Microscopic analysis revealed that the morphology of the cells grown at 30 °C was filamentous and differentiated heterocysts, which are specialized cells for gaseous nitrogen fixation under nitrogen-deprived conditions, indicating that the strain can grow diazotrophically. The cells grown at 10 °C have a smaller size, shortened filament length and decreased chlorophyll content per cell. At 10 °C, the cells are aggregated with extracellular polymeric substrates (EPSs), which is a common mechanism to protect cells from ultraviolet light. These results imply that segmentation into short filaments was induced by photodamage at low temperatures. To fully understand the adaptation mechanisms of Nostoc sp. strain SO-36 for low-temperature conditions, next-generation sequencing analyses were conducted. Complete genome sequence of the strain revealed that it has one main chromosome of approximately 6.8 Mbp with 4 plasmids, including 6855 coding sequences, 48 tRNA genes, 4 copies of rRNA operons, and 5 CRISPR regions. Putative genes for EPS biosynthesis were found to be conserved in Nostocaceae regardless of their habitat. These results provide basic information to understand the adaptation mechanisms at low temperatures, and the strain can be a model organism to analyze adaptation to extreme environments.

Mibyou has been defined in traditional oriental medicine as a certain physiological condition whereby an individual is not ill but not healthy; it is also often referred to as a sub-healthy condition. In a society focused on longevity, “Mibyou-care” becomes of primary importance for healthy lifespan expenditure. Functional foods can play crucial roles in Mibyou-care; thus, the search for novel resources of functional food is an important and attractive research field. Mushrooms are the target of such studies because of their wide variety of biological functions, such as immune modulation and anti-obesity and anticancer activities, in addition to their nutritional importance. Basidiomycetes-X (BDM-X; Shirayukidake in Japanese) is a mushroom which has several attractive beneficial health functions. A metabolome analysis revealed more than 470 components of both nutritional and functional interest in BDM-X. Further isolation and purification studies on its components using radical scavenging activity and UV absorbance identified ergosterol, (10E,12Z)-octadeca-10,12-dienoic acid (CLA), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), formyl pyrrole analogues (FPA), including 4-[2-foemyl-5-(hydroxymethyl)-1H-pyrrole-1-yl] butanamide (FPAII), adenosine and uridine as major components. Biological activities attributed to these components were related to the observed biological functions of BDM-X, which suggest that this novel mushroom is a useful resource for Mibyou-care functional foods and medicines.

UV radiation, desiccation, and starvation induce some cyanobacteria to produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although the accumulation of scytonemin is recognizable as dark sheaths through optical microscopes, it has been nontrivial to identify its redox status and obtain its subcellular distribution in response to physiological conditions. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of non-UV-induced scytonemins with distinct redox statuses in a filamentous cyanobacterium with a single nitrogen-fixing cell at the basal end. Cellular differentiations and scytonemin redox conditions were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The molecular imaging results give insights into not only secretion mechanisms of the sunscreen pigment but also interdependence between photosynthesis, nitrogen fixation, and redox homeostasis in one of the simplest forms of multicellular organisms.

The terrestrial cyanobacterium Nostoc commune has a cosmopolitan distribution. It is edible, and dry thalli are sold as a food in China under the name of Di Pi Cai. The pigment composition and the genotypes were characterized to identify the cyanobacterium Di Pi Cai from China as N. commune. Myxol glycosides and ketocarotenoids were detected, as expected in Nostoc sp., but β-carotene and hydroxylated carotenoids were not detected. Nostoc-756, mycosporine-2-(4-deoxygadusoyl-ornitine), was found to be a main mycosporine-like amino acid, which indicates that Di Pi Cai belongs to the N. commune chemotype C. However, the 16S rRNA gene and the petH gene encoding ferredoxin-NADP+ oxidoreductase of Di Pi Cai did not exactly match those of genotype C found in Japan. These results suggest the unique molecular genetic features of Di Pi Cai and the global diversity of N. commune.

Some cyanobacteria produce a UVA-absorbing pigment, scytonemin, at extracellular sheaths. Although scytonemin-containing dark sheaths are recognizable through optical microscopes and its redox changes have been known for decades, there has been no report to obtain images of both oxidized and reduced scytonemins at subcellular resolution. Here, we show that a spontaneous Raman scattering spectral microscopy based on an excitation-laser-line-scanning method unveil 3D subcellular distributions of both the oxidized and reduced scytonemins in a filamentous cyanobacterium. The redox changes of scytonemin were supported by comparison in the Raman spectra between the cyanobacterial cells, solid-state scytonemin and quantum chemical normal mode analysis. Distributions of carotenoids, phycobilins, and the two redox forms of scytonemin were simultaneously visualized with an excitation wavelength at 1064 nm that is virtually free from the optical screening by the dark sheaths. The redox differentiation of scytonemin will advance our understanding of the redox homeostasis and secretion mechanisms of individual cyanobacteria as well as microscopic chemical environments in various microbial communities. The line-scanning Raman microscopy based on the 1064 nm excitation is thus a promising tool for exploring hitherto unreported Raman spectral features and distribution of nonfluorescent molecules embedded below nontransparent layers for visible light, while avoiding interference by autofluorescence.

The terrestrial cyanobacterium Nostoc commune has a cosmopolitan distribution. It is edible, and dry thalli are sold as a food in China under the name of Di Pi Cai. The pigment composition and the genotypes were characterized to identify the cyanobacterium Di Pi Cai from China as N. commune. Myxol glycosides and ketocarotenoids were detected, as expected in Nostoc sp., but β-carotene and hydroxylated carotenoids were not detected. Nostoc-756, mycosporine-2-(4-deoxygadusoyl-ornitine), was found to be a main mycosporine-like amino acid, which indicates that Di Pi Cai belongs to the N. commune chemotype C. However, the 16S rRNA gene and the petH gene encoding ferredoxin-NADP

The terrestrial cyanobacterium Nostoc commune forms macroscopic colonies in its natural habitats, and these colonies consist of both cellular filaments and massive extracellular matrixes. In this study, the biochemical features of the extracellular matrix components were investigated. Naturally growing N. commune was tolerant to desiccation, and produced massive extracellular polysaccharides that contained both neutral sugars and glucuronic acid as constituent monosaccharides. The extracellular polysaccharide contents and desiccation tolerance were compared in laboratory culture strains of Nostoc species. The laboratory culture of N. commune strain KU002 was sensitive to desiccation and produced smaller amounts of extracellular polysaccharides, unlike the field-isolated naturally growing colonies. Nostoc punctiforme strain M-15, which is genetically closed to N. commune, was able to tolerate desiccation, although the other Nostoc strains were desiccation-sensitive. A laboratory culture strain of the aquatic cyanobacterium Nostoc sphaericum produced massive extracellular polysaccharides but was sensitive to desiccation, suggesting that extracellular matrix production is not enough to make this strain tolerant to desiccation. WspA (water stress protein) and SodF (superoxide dismutase) were found to be characteristic protein components of the extracellular matrix of N. commune. Because the WspA proteins were heterogeneous, the wspA genes were highly diverse among the different genotypes of N. commune, although the sodF gene was rather conservative. The heterogeneity of the WspA proteins suggests their complex roles in the environmental adaptation mechanism in N. commune.

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for >90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.

We present the fundamental architecture and functions of quantum key distribution (QKD) network that support multiple applications. As applications on the QKD network, we introduce a quantum key distribution-advanced encryption standard (QKD-AES) hybrid system and an encrypted smartphone system.

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.

The terrestrial cyanobacterium Nostoc commune forms macroscopic colonies in its natural habitats, and these colonies consist of both cellular filaments and massive extracellular matrixes. In this study, the biochemical features of the extracellular matrix components were investigated. Naturally growing N. commune was tolerant to desiccation, and produced massive extracellular polysaccharides that contained both neutral sugars and glucuronic acid as constituent monosaccharides. The extracellular polysaccharide contents and desiccation tolerance were compared in laboratory culture strains of Nostoc species. The laboratory culture of N. commune strain KU002 was sensitive to desiccation and produced smaller amounts of extracellular polysaccharides, unlike the field-isolated naturally growing colonies. Nostoc punctiforme strain M-15, which is genetically closed to N. commune, was able to tolerate desiccation, although the other Nostoc strains were desiccation-sensitive. A laboratory culture strain of the aquatic cyanobacterium Nostoc sphaericum produced massive extracellular polysaccharides but was sensitive to desiccation, suggesting that extracellular matrix production is not enough to make this strain tolerant to desiccation. WspA (water stress protein) and SodF (superoxide dismutase) were found to be characteristic protein components of the extracellular matrix of N. commune. Because the WspA proteins were heterogeneous, the wspA genes were highly diverse among the different genotypes of N. commune, although the sodF gene was rather conservative. The heterogeneity of the WspA proteins suggests their complex roles in the environmental adaptation mechanism in N. commune.

A UV-absorbing compound was purified and identified as a novel glycosylated mycosporine-like amino acid (MAA), 13-O-β-galactosyl-porphyra-334 (β-Gal-P334) from the edible cyanobacterium Nostoc sphaericum, known as "ge xian mi" in China and "cushuro" in Peru. Occurrence of the hexosylated derivative of shinorine (hexosyl-shinorine) was also supported by LC-MS/MS analysis. β-Gal-P334 accounted for about 86.5% of total MAA in N. sphaericum, followed by hexosyl-shinorine (13.2%) and porphyra-334 (0.2%). β-Gal-P334 had an absorption maximum at 334nm and molecular absorption coefficient was 46,700 at 334nm. Protection activity of β-Gal-P334 from UVB and UVA+8-methoxypsoralen induced cell damage on human keratinocytes (HaCaT) was assayed in comparison with other MAA (porphyra-334, shinorine, palythine and mycosporine-glycine). The UVB protection activity was highest in mycosporine-glycine, followed by palythine, β-Gal-P334, porphyra-334 and shinorine in order. β-Gal-P334 had highest protection activity from UVA+8-methoxypsoralen induced cell damage followed by porphyra-334, shinorine, mycosporine-glycine and palythine. We also found an antioxidant (radical-scavenging) activity of β-Gal-P334 by colorimetric and ESR methods. From these findings, β-Gal-P334 was suggested to play important roles in stress tolerant mechanisms such as UV and oxidative stress in N. sphaericum as a major MAA. We also consider that the newly identified MAA, β-Gal-P334 has a potential for use as an ingredient of cosmetics and toiletries.

Mycosporine-like amino acids (MAAs) are UV absorbing pigments, and structurally distinct MAAs have been identified in taxonomically diverse organisms. Two novel MAAs were purified from the cyanobacterium Nostoc commune, and their chemical structures were characterized. An MAA with an absorption maximum at 335 nm was identified as a pentose-bound porphyra-334 derivative with a molecular mass of 478 Da. Another identified MAA had double absorption maxima at 312 and 340 nm and a molecular mass of 1050 Da. Its unique structure consisted of two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen and two pentose and hexose sugars. These MAAs had radical scavenging activity in vitro; the 1050-Da MAA contributed approximately 27% of the total radical scavenging activities in a water extract of N. commune. These results suggest that these glycosylated MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. © 2011 Elsevier B.V. All rights reserved.

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies, which consist of both cellular filaments and massive extracellular matrix material. In this study, the physiological features of N. verrucosum were investigated and compared with those of the anhydrobiotic cyanobacterium Nostoc commune. Nostoc verrucosum cells were sensitive to desiccation, but tolerant of freeze-thawing treatment in terms of both cell viability and photosynthetic O(2) evolution. Natural colonies of these cyanobacteria contained similar levels of chlorophyll a, carotenoids, the UV-absorbing pigments scytonemin and mycosporine-like amino acids, and uronic acid [a component of extracellular polysaccharides (EPS)]. EPS from both N. verrucosum and N. commune indicated low acidity and a high affinity for divalent cations, although their sugar compositions differed. The WspA protein, known to be a major component of the extracellular matrix of N. commune, was detected in N. verrucosum. Desiccation caused similarly high levels of trehalose accumulation in both cyanobacteria. Although previously considered relevant to anhydrobiosis in the terrestrial cyanobacterium N. commune, the data presented here suggest that extracellular matrix production and trehalose accumulation are not enough for standing extreme desiccation in N. verrucosum.

SUMMARY 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.

SUMMARY The ntcA and ntcB genes, encoding cAMP-receptor-protein (CRP) type and LysR type transcriptional regulators, respectively, were isolated from the transformable marine cyanobacterium Synechococcus sp. strain PCC 7002 to study the inactivation of these genes by interposon mutagenesis. The ntcA gene could not be disrupted in this cyanobacterial strain, indicating that the ntcA gene is essential for cell growth on either urea or nitrate as the sole nitrogen source. The ntcB mutant cells grew very slowly on nitrate or nitrite as the sole nitrogen source but could grow on urea at the same rate as wild-type cells. The consumption of nitrate and nitrite was barely detectable in the ntcB mutant cells, indicating that nitrate and nitrite use was impaired in the ntcB mutant. In wild-type cells, mRNA levels for the three genes required for nitrate assimilation, nrtP (nitrate permease), narB (nitrate reductase) and nirA (nitrite reductase), increased when cells were shifted to nitrate growth conditions, but no increase in transcription of these genes occurred in the ntcB mutant. The mRNA level of ntcA was nearly identical in the wild-type and ntcB mutant cells. These results demonstrate that NtcB specifically controls the transcription of the three genes for nitrate assimilation in this marine cyanobacterium.

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and forms a visible colony in which the cells are embedded in extracellular polysaccharides (EPSs), which play a crucial role in the extreme desiccation tolerance of this organism. When natural colonies were immersed in water, degradation of the colonies occurred within 2 days and N. commune cells were released into the water. The activities that hydrolyze glycoside bonds in various N. commune fractions were examined using artificial nitrophenyl-linked sugars as substrates. A beta-D-glucosidase purified from the water-soluble fraction was resistant to 20 min of boiling. The beta-D-glucosidase, with a molecular mass of 20 kDa, was identified as a cyanobacterial fasciclin protein based on its N-terminal amino-acid sequence. The 36-kDa major protein in the water-soluble fraction was purified, and the N-terminal amino-acid sequence of the protein was found to be identical to that of the water-stress protein (WspA) of N. commune. This WspA protein also showed heat-resistant beta-D-galactosidase activity. The fasciclin protein and WspA in the extracellular matrix may play a role in the hydrolysis of the EPSs surrounding the cells, possibly as an aid in the dispersal of cells, thus expanding the colonies of this cyanobacterium.

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and omega 3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the omega 3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22 degrees C. However, the mRNA levels of the desA (delta 12 desaturase), desB (omega 3 desaturase) and desC (delta 9 desaturase) genes increased within 15 min after a temperature shift-down to 22 degrees C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38 degrees C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22 degrees C and 38 degrees C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.

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 O 2 evolution was not detected in desiccated colonies, the ability of the cells to evolve O 2 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 O 2 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 O 2 , but O 2 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, O 2 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 effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m−2 s−1, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m−2 s−1, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m−2 s−1. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria.

金沢大学理工研究域生命理工学系, Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two genotypes of N. commune colonies with different water extract UV-absorption spectra. We found structurally distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum with an absorption maximum at 335 nm. The extract contained the following compounds: 7-O-(β-arabinopyranosyl)-porphyra-334 (478 Da), pentose-bound shinorine (464 Da), hexose-bound porphyra-334 (508 Da) and porphyra-334 (346 Da). The water extract from genotype B showed a characteristic UV-absorbing spectrum with double absorption maxima at 312 and 340 nm. The extract contained hybrid MAAs (1050 Da and 880 Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen linked to 2-O-(β-xylopyranosyl)-β-galactopyranoside. A novel 273-Da MAA with an absorption maximum at 310 nm was also identified in genotype B. The MAA consisted of a 3-aminocyclohexen-1-one linked to a γ-aminobutyric acid chain. These MAAs had potent radical scavenging activities in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively produced their own characteristic glycosylated MAAs, which supports that MAA composition could be a chemotaxonomic marker for the classification of N. commune.

Synechococcus sp. PCC 7002 cells appeared to be nitrogen limited when grown on urea without nickel in the growth medium; the amounts of chlorophyll a and phycobiliproteins decreased and glycogen accumulated to high levels. When nickel was added to the urea medium, the diagnostics of nitrogen limitation was not observed. The activity of urease was dependent on the presence of nickel no matter what nitrogen source was used for cell growth. The ureC gene encoding the α-subunit of urease was constitutively transcribed in cells grown on all nitrogen sources tested. The addition of nickel to the growth medium rapidly led to a 15-fold increase in urease activity in 2 hours. These results indicate that cells of Synechococcus sp. PCC 7002 require a nickel supplement (5 μM NiSO4 in the growth medium) to utilize urea efficiently, and that Synechococcus sp. PCC 7002 cells are nickel-starved under the laboratory conditions regularly used for this strain. In two fresh water strains of cyanobacteria, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, high levels of urease activity were detected without addition of nickel to the growth medium, suggesting that these fresh water strains have a high-affinity uptake system for nickel or that their nickel requirements are lower than Synechococcus sp. PCC 7002.

The nrtP and narB genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium Synechococcus sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An nrtP mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The nrtP mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A narB mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the narB mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the nrtP and narB genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the nrtP and narB genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in Synechococcus sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth.Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m−2 s−1) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m−2 s−1) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.

Strong light leads to damage to photosynthetic machinery, particularly at low temperatures, and the main site of the damage is the D1 protein of the photosystem II (PSII) complex. Here we describe that transformation of Synechococcus sp. PCC 7942 with the desA gene for a [delta]12 desaturase increased unsaturation of membrane lipids and enhanced tolerance to strong light. To our knowledge, this is the first report of the successful genetic enhancement of tolerance to strong light. Analysis of the light-induced inactivation and of the subsequent recovery of the activity of the PSII complex revealed that the recovery process was markedly accelerated by the genetic transformation. Labeling experiments with [35S]L-methionine also revealed that the synthesis of the D1 protein de novo at low temperature, which was a prerequisite for the restoration of the PSII complex, was much faster in the transformed cells than in the wild-type cells. These findings demonstrate that the ability of membrane lipids to desaturate fatty acids is important for the photosynthetic organisms to tolerate strong light, by accelerating the synthesis of the D1 protein de novo.

Cyanobacteria acclimate to low-temperature conditions by desaturating their membrane lipids. The desB (omega 3 desaturase) and desC (delta 9 desaturase) genes of Synechococcus sp. strain PCC 7002 were cloned and characterized, and the expression of the desA (delta 12 desaturase), desB and desC genes was studied as a function of temperature. The steady-state mRNA abundance for the desA gene was threefold higher in cells grown at 22 degrees C than in cells grown at 38 degrees C. desB transcripts were not detected at 38 degrees C, but were abundant in cells grown at 22 degrees C. Levels of desC mRNA were similar at both growth temperatures. The mRNA levels of each desaturase gene increased within 15 min of a temperature shift-down to 22 degrees C, and mRNA levels recovered within 15 min after a shift-up to 38 degrees C. The cold-induced accumulation of transcripts from the desA and desB genes was suppressed by the addition of chloramphenicol, but the transient elevation of the desC transcript levels at 22 degrees C was not affected by chloramphenicol. The half-lives of the desA and desB mRNAs were significantly longer in cells grown at 22 degrees C than in cells grown at 38 degrees C, but the desC mRNA had a similar half-life at both temperatures. These studies reveal three patterns of temperature regulation for the desaturase genes, whose expression is tightly controlled by a combination of mRNA synthesis and stabilization. These studies demonstrate that elevation of desaturase mRNA levels is not the rate-limiting event during the low-temperature acclimation of cyanobacteria.

A cyaC gene encoding an adenylate cyclase of the filamentous cyanobacterium Spirulina platensis was sequenced. The predicted amino acid sequence of the C-terminal region of cyaC is similar to the catalytic domains of adenylate cyclases in other cyanobacteria and eukaryotes. The sequences of other regions are similar to those of proteins consisting of the bacterial two-component signal transduction system: the sensory kinase and the response regulator. The predicted gene product of cyaC contains, from the N-terminal end, a receiver domain of the response regulator protein (R1), a domain similar to the ETR1 of Arabidopsis thaliana, a transmitter domain of the sensory kinase protein, a receiver domain of the response regulator protein (R2), and a catalytic domain of adenylate cyclase. The cyaC gene was expressed as an affinity-tagged protein in Escherichia coli, and the recombinant protein was purified. The purified protein had adenylate cyclase activity which was activated by Mu2+. The results of Western blotting using an anti-CyaC antiserum and the S.platensis cell extract confirmed that cyaC gene is expressed in S. platensis.

A cyaA gene, encoding an adenylate cyclase, was isolated from a filamentous cyanobacterium, Spirulina platensis, by functional complementation of a cya mutant of Escherichia coli, defective in adenylate cyclase activity. The predicted gene product of cyaA contains a signal peptide-like domain, a putative sensor domain similar to the gene product of vsrA of Pseudomonas solanacearum, a putative membrane-spanning domain and an adenylate cyclase-like catalytic domain. Two other positive clones that complemented the E. coli mutant were isolated from the same cyanobacterium, suggesting that several cya genes are functioning in S. platensis.

Scytonemin is a 544-Da hydrophobic pigment that can absorb UV-A radiation. It is present in cyanobacterial sheaths and is thought to function as a UV protectant. In this study, scytonemin was purified from the terrestrial cyanobacterium Nostoc commune, and its radical-scavenging activity was characterized. The purified scytonemin quenched an organic radical in vitro and accounted for up to 10% of the total activity of an ethanol extract of N. commune. These results suggest that the extracellular UV-absorbing pigment scytonemin has multiple roles, functioning as a UV sunscreen and an antioxidant relevant to anhydrobiosis in N. commune.

Cyanobacteria respond to a decrease in temperature by desaturating fatty acids of membrane lipids to compensate for the decrease in membrane fluidity. Among various desaturation reactions in cyanobacteria, the desaturation of the omega 3 position of fatty acids is the most sensitive to the change in temperature. In the present study, we isolated a gene, designated desB, for the omega 3 desaturase from the cyanobacterium, Synechocystis sp. PCC 6803. The desB gene encodes a protein a 359 amino-acid residues with molecular mass of 41.9 kDa. The desB gene is transcribed as a monocistronic operon that produced a single transcript of 1.4 kb. The level of the desB transcript in cells grown at 22 degrees C was 10 times higher than that in cells grown at 34 degrees C. In order to manipulate the fatty-acid unsaturation of membrane lipids, the desB gene in Synechocystis sp. PCC 6803 was mutated by insertion of a kanamycin-resistance gene cartridge. The resultant mutant was unable to desaturate fatty acids at the omega 3 position. The desA gene, which encodes the delta 12 desaturase of Synechocystis sp. PCC 6803, and the desB gene were introduced into Synechococcus sp. PCC 7942. Whilst the parent cyanobacterium can only desaturate membrane lipids at the delta 9 position of fatty acids, the resultant transformant was able to desaturate fatty acids of membrane lipids at the delta 9, delta 12 and omega 3 positions. These results confirm the function of the desB gene and demonstrate that it is possible to genetically manipulate the fatty-acid unsaturation of membrane lipids in cyanobacteria.

The phylogeny of the terrestrial cyanobacterium N ostoc commune and its neighboring N ostoc 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 N ostoc , 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 N ostoc verrucosum , suggesting that these N ostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of N ostoc and A nabaena 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. N ostoc commune and N ostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.