Isolation of Porphyran-Degrading Marine Microorganisms from the Surface of Red Alga,Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

Cadmium Accumulation by the Marine Red Alga Porphyra umbilicalis

The characteristics of cadmium accumulation by the marine red alga Porphyra umbilicalis L. in culture are reported. The time course of uptake under various light conditions shows that cadmium is concentrated as the result of an on-going anabolic process and not as a consequence of a pH gradient as provided by photo-synthesis. The effect of cycloheximide is in agreement with de novo protein-synthesis being a prerequisite For cadmium accumulation. Autoradiography suggests a specific intracellular location for bound cadmium—apparently the nucleus.     

Identification of Calmodulin in the Red Alga Susabi Laver (Porphyra yezoensis)

To test whether calmodulin is present in the red alga Susabi laver (Porphyra yezoensis), materials was concentrated from this alga, using hydrophobic chromatography. In this case, 0.25 m (NH₄)₂SO₄ should be added to bind the protein to the column in the presence of 5 mm CaCl₂. Calmodulin is present in the marine red alga, although at a very low concentration.     

Purification and Properties of NADH:Nitrate Reductase from the Red Alga Porphyra yezoensis

Assimilatory nitrate reductase (NADH) (EC 1.6.6.1 [EC] ) from thered alga Porphyra yezoensis was purified 5,700-fold by a combinationof polyethylene glycol (PEG) treatment, ammonium sulfate fractionation,chromatography on columns of butyl-Toyopearl 650-M, Blue SepharoseCL-6B, DEAE-cellulose (DE 52), and hydroxyapatite, gel filtrationon Sephacryl S-400. The purest preparation of the enzyme hada specific activity of 12.5 units mg^–1 protein. A singleband of protein was detected after polyacrylamide gel electrophoresisunder nondenaturing conditions. This band corresponded to aband that stained positive for reduced methyl viologen-nitratereductase activity. The molecular weight of the native enzymewas estimated to be 220,000. A single band of a protein witha molecular weight of 100,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis. These results indicatethat the native nitrate reductase is composed of two identicalsubunits. The homogeneous preparation of nitrate reductase hada UV/visible spectrum typical of a b-type cytochrome. The K_mvalues for NADH and KNO₃ were 23 µM and 64 µM, respectively.The pH optimum for the reaction catalyzed by the nitrate reductasewas 8.3, while pH values that supported maximum partial activitiesranged from 7.0 to 8.5. Sulfhydryl reagents, such as p-HMB andNEM, inhibited full and NADH-utilizing partial activities, whilecyanide and azide were effective inhibitors of full and nitrate-reducingpartial activities. (Received March 3, 1993; Accepted September 6, 1993)     

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Twenty-two monoclonal antibodies were raised against the nativeform of nitrate reductase (NR) from Porphyra yezoensis, a marinered alga. All the antibodies were able to bind to NR from P.yezoensis, with resultant inhibition of full (NADH:NR) and/orpartial (NADH:FR, NADH:CR, FMNH₂:NR, and MV:NR) enzymatic activity.Fifteen of the antibodies recognized the denatured form of theenzyme. Size-exclusion gel nitration and immunoblotting of theproducts of the limited proteolysis of NR from P. yezoensisby trypsin or Staphylococcus aureus V8 protease revealed that2 out of the 15 subunit-recognizing antibodies bound to theFAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterindomain. The products of limited proteolysis of NR from P. yezoensisalso revealed that the sites of proteolytic cleavage that encompassedthe heme domain were inverted as compared to the analogous sitesin NRs of higher plants. Some of the monoclonal antibodies cross-reactedwith NRs from plants belonged to different phyla. From threeto five of the antibodies bound subunits of NR from multicellularred algae, but failed to bind NRs from unicellular red algae.Three or four of the antibodies crossreacted with NRs from higherplants, such as tobacco and spinach. One of the antibodies boundNRs from several types of plant, namely, members of Cryptophyta,Chromophyta, and Chlorophyta. All of the monoclonal antibodiesthat cross-reacted with NRs from plants other than the red algaewere specific for the Mo-pterin domain of NR from P. yezoensis. (Received May 10, 1994; Accepted September 7, 1994)     

Light-Harvesting for Photosynthesis in Four Strains of the Red Alga Porphyra yezoensis Having Different Phycobilin Contents

The light-harvesting system of photosynthesis was studied infour strains of Porphyra yezoensis differing in their phycoerythrin(PE) content; the red strain, richer in PE than the wild strain,and the green and the yellow strains, poorer in PE. Specialattention was given to possible alteration of pigment systemin response to PE content, especially in the green and the yellowstrains. The relative quantum yields of Chi a fluorescence at–196°C and O² evolution were compared. Four strains commonly showed a low yield in pigment system II(PS II) fluorescence on Chl a excitation. The yield was as lowas in those algae in which PS II has only a small portion ofChl a as the light harvester. Measurement of O₂ evolution gavethe same results. Results indicate that the functional compositionof Chl a system remains unaltered in four strains with differentPE content. PS II in the green and the yellow strains reflectsa reduction in the size of light-harvesting components, suggestingthat pigmentation in these strains is fixed genetically as asun-type. ⁴ On leave from University of Washington, Seatle, Washington,U.S.A. (Received September 18, 1982; Accepted December 25, 1982)     

红藻条斑紫菜R-藻红蛋白晶体的时间分辨偏振荧光研究

本文在皮秒时域内研究红藻条斑紫菜Ｒ－藻红蛋白单晶在不同晶轴取向下的偏振荧光动力学过程。荧光衰减表现为二指数过程,即与色素之间的能量传递和激发平衡有关的快过程（τ１≤６０ｐｓ）和色素荧光跃迁过程（τ２～３００ｐｓ）;由于色素间的能量传递,荧光明显退偏振;由于晶体中色素的光学跃迁矩取向倾向于沿主晶轴方向分布,在不同的晶轴的取向下各向异性荧光衰减过程明显不同。     

The First Symbiont-Free Genome Sequence of Marine Red Alga,Susabi-nori (Pyropia yezoensis)

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.     

Phycobilisome Heterogeneity in the Red Alga Porphyra umbilicalis

Phycobilisomes were isolated from Rhodophyceae brought from the field (Porphyra umbilicalis) or grown in culture under laboratory conditions (Antithamnion glanduliferum). In P. umbilicalis two kinds of well-coupled (ellipsoidal and hemidiscoidal) phycobilisomes were detected, in contrast to A. glanduliferum cultured algae in which only one kind of well-coupled, ellipsoidaltype phycobilisome appeared. The new phycobilisome-type particle detected in P. umbilicalis is characterized by an impoverishment in R-phycoerythrin and by sedimentation at lower density. The comparison between both phycobilisomes of P. umbilicalis allows determination of the presence of one colorless linker polypeptide (30 kilodaltons) associated with R-phycocyanin and allophycocyanin and two (40 and 38 kilodaltons) associated to R-phycoerythrin. The percentage of linker polypeptides associated with this pigment is low in the new phycobilisome-like particle detected. This suggests that part of the R-phycoerythrin is less strongly bound to the phycobilisome than the other pigments. This feature could probably explain the existence of two kinds of phycobilisomes as intermediary steps of phycobilisome organization in algae exposed to rapid changes in environmental factors. In contrast, algae growing in culture and adapted to specific conditions do not present intermediary organization steps. Polypeptide composition and identification are given for this phycobilisome-like particle.     

红藻条斑紫菜凝集素的纯化及其色氨酸化学修饰对其活性的抑制作用

为了探索条斑紫菜凝集素(Porphyra yezoensis Ueda lectin, PYL)的作用机理,对其进行了分离和纯化．条斑紫菜经磷酸盐缓冲液浸泡、20%～75%硫酸铵分级、DEAE 纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到PYL纯品. Sephadex G-200分子筛层析测得其分子量为63.2 kD,在非还原SDS-PAGE上显示1条蛋白染色带,分子量为63.1 kD,还原SDS-PAGE显示1条蛋白染色带,亚基分子量为15.8 kD．PYL在对兔、大鼠、鸡、羊、狗血细胞的凝集作用中,对大鼠红细胞的凝集活性最高.PYL在pH 6.50～10.53范围内均有活性,在pH 8.40～8.91活性最高．经42 ℃热处理10 min后,仍然对大鼠红细胞血凝活性保留12.5%,其活性最大温度范围为4 ℃～20 ℃, 48 ℃加热10 min后,其活性完全丧失．EDTA对PYL的凝集活性有抑制作用,最小抑制浓度为156 mmol/L,而 Ca2+和Mg2+未发生凝集抑制现象．PYL凝集大鼠红细胞的作用不被D 果糖、葡萄糖、D-半乳糖、D-甘露糖、菊粉、γ球蛋白、牛甲状腺球蛋白等所抑制,但可被蔗糖和麦芽糖抑制,最小抑制浓度蔗糖为20 mmol/L,麦芽糖为40 mmol/L．用N 溴代丁二酰亚胺(NBS) 对PYL分子中的Trp残基进行化学修饰,有2.1个Trp残基被修饰,修饰后PYL活性丧失, 表明Trp残基是PYL凝集活性所必需的基团．     

Isolation of Hyphomonas Strains that Induce Normal Morphogenesis in Protoplasts of the Marine Red Alga Pyropia yezoensis

Marine macroalgae cannot develop normal morphology under axenic conditions although normal morphogenesis can be sustained when certain bacteria are present. In this study, bacteria that induced normal morphogenesis in the red alga Pyropia yezoensis (Nori) were identified. The bacteria were isolated from algal media, thalli, tissue debris, and purified protoplasts during protoplast isolation from P. yezoensis laboratory cultures. 16S rRNA gene sequence analysis showed these bacterial isolates belonged to α-Proteobacteria (12 groups), γ-Proteobacteria (3 groups), and Flavobacteria (2 groups). Axenic protoplasts of P. yezoensis generated by removing epiphytic bacteria were co-cultured along with the bacterial isolates. Most axenic protoplasts showed irregular morphogenetic and anaplastic cells; cells with normal morphology were scarce. However, inoculation with 11 strains of Hyphomonas (α-Proteobacteria) led to significantly higher normal morphogenetic rates (4.5–7.3 %, P?Hyphomonas strains were recovered from all experiments; thus, certain Hyphomonas strains can induce normal morphogenesis in P. yezoensis protoplasts. Direct inoculation of the Hyphomonas strain exhibited higher morphogenetic activity than inoculation of its extracellular and intracellular products. This is the first study demonstrating the influence of specific bacteria on protoplast morphology in marine macroalgae.     

Visualization of Phosphoinositides via the Development of the Transient Expression System of a Cyan Fluorescent Protein in the Red Alga Porphyra yezoensis

Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal, and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins; however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cδ1 and Akt1, but not Bruton’s tyrosine kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular localization of other proteins in P. yezoensis and other red algal cells.     

Photosystem I-Mediated Regulation of Water Splitting in the Red Alga, Porphyra sanjuanensis

The marine red alga, Porphyra sanjuanensis is found mainly in the high intertidal zone and at low tide subject to frequent and extreme water stress, often accompanied by high temperatures and light intensities. Such exposures can lead to severe desiccation which is accompanied by the progressive loss of photosynthetic activity. Even following the loss of more than 90% of the thallus water content the alga recovers rapidly when returned to seawater. This stress-induced, reversible inactivation of photosynthesis is believed to be a protective adaptation which prevents photodamage to the exposed alga. Effects of light, inhibitors of water splitting, and electron donors to PSI on variable fluorescence and water splitting suggest that activity of the oxygen evolving complex is regulated by the PSI-driven reduction of a component of intersystem electron transport.     

The Manipulation of the Fatty Acid Composition of Glycerolipids in Cyanobacteria Using Exogenous Fatty Acids

The fatty acid composition of the major diacylglycerolipidsof Anacystis nidulans R2 cells was modified by the careful additionof exogenous fatty acids to the growth medium of the cyanobacteria.In this way it is possible to incorporate significant levelsof different fatty acids into the membranes of the cells andchange their physical properties without changing other environmentalconditions. The exogenous fatty acid reduces or inhibits thebiosynthesis of native fatty acids and substitutes for themin all the major diacylglycerolipids. Although modifying thefatty acid composition in this way is lethal above certain concentrations,the data demonstrate a remarkable ability of the cells to growand develop over a wide range of fatty acid compositions. Similardata show that the fatty acid composition of Synechocystis sp.6803 cells also undergo significant changes in fatty acid compositionon the addition of exogenous fatty acid to the growth mediumand can tolerate similar changes in the degree of unsaturationof the fatty acids of the diacylglycerolipids of the cell membranes. (Received September 4, 1989; Accepted March 13, 1990)     

Algicidal Activity of Glycerolipids from Brown Alga Ishige sinicola toward Red Tide Microalgae

Bioassay-guided fractionation of a methanol extract of the brown alga, Ishige sinicola, led to the isolation of five algicidal compounds. Their structures were determined to be α-monoglycerides of eicosa-5Z,8Z,11Z,14Z-tetraenoic (arachidonic) acid, octadeca-6Z,9Z,12Z,15Z-tetraenoic acid, linoleic acid and oleic acid, and 1-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol on the basis of spectroscopic data and a comparison with the data in the literature. These glycerolipids showed moderate-to-high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo, Karenia mikimotoi and Alexandrium catenella, at a concentration of 20 μg/mL.