Lipid composition of the plasma-membrane of the halotolerant alga,Dunaliella salina

The major lipids of the isolated plasma-membrane of the halotolerant alga Dunaliella salina are diacylglyceroltrimethylhomoserine (DGTS, 23.5%), sterol peroxides (7-dehydroporiferasterol peroxide and ergosterol peroxide, 22%), phosphatidylcholine (13%) and phosphatidylethanolamine (11%). Free sterols comprised 5% of the lipids and contained predominantly 7-dehydroporiferasterol and ergosterol. The major fatty acids of the plasma-membrane were palmitic (31%), oleic (13%), linoleic (20%) and γ-linolenic (17%) acids. In constrast to the whole cells, the plasma-membrane contained less (11%) α-linolenic acid and no 16-carbon unsaturated fatty acids. Sterol peroxides were identified by ¹H-NMR and ¹³C-NMR spectroscopy, mass spectrometry, and by comparison on thin-layer chromatography to the product of ergosterol photooxygenation. We believe that this is the first report on the occurrence of sterol peroxides as major constituents of a biological membrane. It is suggested that they may play a role in the unusual membrane-permeability properties of the plasma-membrane of Dunaliella.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

Temperature-regulated expression of a glycine-rich RNA-binding protein in the halotolerant alga Dunaliella salina

Acclimation of the halotolerant alga Dunaliella salina to low temperature induced the accumulation of a 12.4 kDa protein (DsGRP-1) and reduction of a 13.1 kDa protein (DsGRP-2). DsGRP-1 and DsGRP-2 are boiling-stable proteins that are localised in the cytoplasm, as revealed by sub-cellular fractionation and by immuno-localisation. The proteins were partially purified and their corresponding genes were cloned. The predicted sequences are homologous to Glycine-Rich RNA-binding Proteins (GRPs) from plants and cyanobacteria. The nucleotide sequences of grp1 and grp2 differ in a short insert encoding 9 amino acids in the glycine-rich domain of DsGRP-2. grp2 contains a single intron at position 179 indicating that DsGRP-1 and DsGRP-2 are not derived from alternative splicing of a common gene. The level of grp mRNA increased at 7 degrees C and was rapidly depressed at 24 degrees C. Analysis of binding to ribonucleotide homopolymers revealed that DsGRP-1 and DsGRP-2 bind preferentially to poly-G and to poly-U indicating that they are RNA-binding proteins. It is proposed that DsGRP-1 and DsGRP-2 are encoded by distinct genes which are differentially regulated by temperature.     

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.     

盐藻PDS基因的同源克隆及在大肠杆菌中的表达

【目的】八氢番茄红素脱氢酶PDS为真核膜结合蛋白,我们通过更换不同的表达策略,探索在大肠杆菌中表达真核膜结合蛋白的方式。【方法】利用RACE的方法克隆盐藻PDS的全长c DNA序列。利用原核表达载体p ET-28a构建p ET-28a-PDS表达载体;使用PLtac启动子替换T7启动子构建p ET-PLtacPDS表达载体;合成Mistic序列融合入p ET-28 a中构建了p ET-Mistic-PDS融合表达载体。分别转化入大肠杆菌BL21(DE3)中进行原核表达。【结果】获得了盐藻PDS基因的全长c DNA序列2237 bp,开放阅读框为1749 bp,共编码582个氨基酸(NCBI登录号为GQ923693.1)。利用p ET-28a-PDS和p ET-PLtacPDS表达的PDS蛋白表达量低,并以包涵体形式存在;利用p ET-Mistic-PDS载体表达的PDS蛋白表达量明显提高,且大部分以可溶蛋白形式存在,具有脱氢酶活性。【结论】实验结果表明Mistic作为促溶标签能促进膜蛋白的正确折叠,提高蛋白的可溶性。蛋白酶活测定结果证明了Mistic的融合可以保持蛋白的天然活性。     

Lipid profile of the halophilic alga,Dunaliella salina

The lipid composition comprised more than 50% of the cellular organic material. Greater than 30% of the total lipid composition consisted of hydrocarbons. The aliphatic hydrocarbons consisted of C-17 and C-19 saturated and unsaturated ones, some of them with an internal methyl branch. The remaining lipids consisted of a large quantity of pigmented hydrocarbons together with at least six sterol derivatives, six phospholipids, two glycolipids, one sulfolipid and numerous other components that were not fully identified. This wall-less alga is an immensely rich source of a wide variety of lipids.     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.     

Contrasting fates of the paraquat monocation radical in Escherichia coli B and in Dunaliella salina

Anaerobic cultures of Escherichia coli exposed to paraquat (PQ2+) accumulated the corresponding monocation radical PQ+., both within the cells and in the suspending medium. The green alga, Dunaliella salina, which is susceptible to a light- and O2-dependent toxicity of PQ2+, was nevertheless unable to cause accumulation of PQ+. when illuminated anaerobically and could, moreover, discharge the ESR signal and the blue color of PQ+. accumulated by E. coli. Spin trapping allowed demonstration of the photoproduction of O2- within D. salina and of the augmentation of that O-2 production by PQ2+. D. salina appears to contain an electron sink and a heat-labile mechanism for transferring electrons from PQ+. to that sink. This mechanism was demonstrable anaerobically but did not prevent PQ+.-mediated O2- production under aerobic conditions.     

The role of intracellular orthophosphate in triggering osmoregulation in the alga Dunaliella salina

A new hypothesis is presented for the mechanism of metabolic response during osmoregulation in the alga Dunaliella salina. We propose that the osmotic response is initiated by differential volume changes of the cytoplasm and the chloroplast (observed using the electron microscope) which alter the cytoplasmic orthophosphate concentration. This triggers a flow through the Pi/triose-phosphate shuttle, activating chloroplast enzymes in the direction of either starch or glycerol synthesis. The Pi-dependent response was investigated in vivo using NMR. The rates of glycerol synthesis or elimination following osmotic shocks were modulated by the intracellular Pi level as predicted by the hypothesis.