Reconstitution of carotenoids into the light-harvesting pigment-protein complex from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26

Two carotenoids, neurosporene and spheroidene, have been successfully added to chromatophores from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26. Carotenoids reconstituted in this way into the B-850 light-harvesting pigment-protein complex both sensitise bacteriochlorophyll fluorescence and protect the complex from the photodynamic reaction.     

The isolation and partial characterisation of the light-harvesting pigment-protein complement of Rhodopseudomonas acidophila

The photosynthetic membranes of two strains of Rhodopseudomonas acidophila (7750 and 7050) have been resolved into their constituent light-harvesting pigment-protein complexes. Four different types of antenna complexes (B880, B800–830 and two types of B800–850) have been isolated and partially purified. In each case the light-harvesting pigments (bacteriochlorophyll a and carotenoids) are bound to rather low molecular weight polypeptides (in the 5000–9000 region).     

On the location of the carotenoids in the light-harvesting pigment-protein complexes of the photosynthetic bacterium Rhodopseudomonas capsulata

Measurements of pronase-induced shifts of the absorption spectrum and of the isobestic point of the light-induced difference spectrum of the carotenoids show that the pool responsible for the light-induced absorption changes in Rhodopseudomonas capsulata wild type is more sensitive to pronase treatment than the bulk carotenoids. The most likely explanation for this, in the context of the work of Kakitani et al. (Kakitani, T., Honig, B. and Crofts, A.R. (1982) Biophys. J. 39, 57–63), is that the field indicating carotenoids, or at least that part of the molecules which determines their spectral characteristics, are imbedded in the LHC II pigment-protein complexes, close to the membrane surface. The importance of the location of the carotenoids for the measurement of the electrical potential differences is briefly discussed.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

Energy transfer within the isolated light-harvesting B800–850 pigment-protein complex of Rhodobacter sphaeroides

We have used picosecond absorption spectroscopy with low intensity (5 · 10¹¹–5 · 10¹² photons · pulse⁻¹ · cm⁻²) continuously tunable infrared (800–900 nm) pulses to study the energy transfer dynamics in the isolated B800–850 pigment-protein complex of Rhodobacter sphaeroides. Our results suggest the following picture of the energy transfer dynamics: (i) a fast transfer, within approx. 1 ps, from BChl 800 to BChl 850; (ii) transfer among different BChl 800's with a rate which is at the most of the same order of magnitude as that of BChl 800 → BChl 850 transfer; (iii) very fast transfer (k > 1 · 10¹² s⁻¹) between BChl 850 molecules. Assuming Förster type of energy transfer maximum distances of about 22 and 15 Å are obtained for the BChl 800–BChl 850 and BChl 850–BChl 850 separations, respectively.     

The cooperativity phenomena in a pigment-protein complex of light-harvesting antenna revealed by picosecond absorbance difference spectroscopy

A model of the cooperative changes in optical properties of light-harvesting bacteriochlorophyll molecules of complex B890 in response to the absorption of light quanta is proposed. According to the model, each antenna chromophore may persist in either of two optically non-excited states, R and T. The occurrence of at least one excitation per complex causes all optically non-excited chromophores of the complex to be converted from state R to state T. The theory is shown to be in good agreement with experimental ‘light curves’ (ΔAvs intensity of picosecond excitation pulse) for the ‘minor’ and ‘major’ signals of light-harvesting bacteriochlorophylls of complex B890 from Chromatium minutissimum.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dialysis, the dialysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c₂ photooxidation and cytochrome b photoreduction were demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane.

The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides.

Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c₂ photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greater membrane cytochrome c₂ photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.     

The secondary structure content of pigment-protein complexes from the thylakoids of two Chromophyte algae

The light-harvesting complex and Photosystem I have been isolated from thylakoids of two chromophyte algae by digitonin solubilisation and sucrose-gradient centrifugation. The -helic and β-structure content was determined by ultraviolet circular dichroism. The values obtained were approx. 40% -helix and approx. 14% β-structure for the light-harvesting complex of both Pavlova lutherii and Phaeodactylum tricornutum. For Photosystem I the values were approx. 55% -helix and 7% β-structure for both algae. It is concluded that for all photosynthetic antennae containing chlorophyll the dominant secondary structure is -helix.     

Correlation of membrane-potential-sensing carotenoid to pigment-protein complex II in Rhodopseudomonas sphaeroides

The changes in carotenoid absorbance induced by illumination or by a diffusion potential were larger in chromatophores from cells cultured under low light intensity than those in chromatophores from high-light culture in a photosynthetic bacterium, Rhodopseudomonas sphaeroides. The carotenoid molecules which are associated with the pigment-protein complex (with the infrared bacteriochlorophyll peaks at 800 and 850 nm) (complex II) probably respond to the electrical field changes in the chromatophore membrane.     

Effect of the in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer in light-harvesting complex from Rhodobacter sphaeroides 601

The oxidation of bacteriochlorophylls (BChls) in peripheral light-harvesting complexes (LH2) from Rhodobacter sphaeroides was investigated by spectroelectrochemistry of absorption, fluorescence emission, and femtosecond (fs) pump-probe, with the aim obtaining information about the effect of in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer within LH2. The experimental results revealed that: (a) the generation of the BChl radical cation in both B800 and B850 rings dramatically induced bleaching of the characteristic absorption in the NIR region and quenching of the fluorescence emission from the B850 ring for the electrochemical oxidized LH2; (b) the BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process.     

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.     

Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CA_ext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO₂-grown cells. CA_ext was detected only in UTEX 640 grown under CO₂-limited conditions and present in trace amounts in cells grown on high CO₂. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO₂-grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CA_ext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO₂-inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO₂-grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CA_ext was located in the periplasmic space and on the cell membrane, whereas in high CO₂-grown cells only internal CA was detected.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Changes in the content of pigment-protein complexes in Rhodobacter sphaeroides forma sp. denitrificans grown under photosynthetic and photo-denitrifying conditions

Changes in the relative content of pigment-protein complexes, RC-B880 and B800-850, were studied in membranes of Rhodobacter sphaeroides forma sp. denitrificans cultured under various anaerobic conditions. The content of each pigment-protein complex was determined by the decomposition of the absorption spectra of membranes in the near-infrared region into the spectra of RC-B880 and B800-850. The standard spectrum of each complex in the membranes was obtained using two absorption spectra of membranes with different ratios of the complexes by eliminating the spectrum of first one than the other complex. Spectra composed from the two standard spectra were in good agreement with original membrane spectra after subtraction of the contribution of scattering in various membrane samples. Bacteriochlorophyll (BChl) content in the membrane was dependent on the light intensity during growth. The relation between the total BChl content in the membrane and BChl content in the RC-B880 and B800-850 complex was linear above 15 nmol BChl per mg membrane protein, regardless of the culturel conditions, photosynthetic or photo-denitrifying. The linear relationship reached a point where all BChl molecules were contained in RC-B880 at 13 nmol BChl per mg membrane protein. This means that only RC-B880 would be synthesized below the threshold, and above the threshold additional BChl was distributed between RC-B880 and B800-850 in a constant ratio (1:5.7). The results suggest that the syntheses of B800-850 and RC-B880 are not regulated independently.     

Difference picosecond spectroscopy of pigment-protein complexes of photosystem I from higher plants

Using a difference picosecond spectrophotometer with a time resolution of 10 ps, we investigated excitation energy transfer and charge separation in pigment-protein complexes of Photosystem I from bean leaves (chlorophyll/P-700 = 60). Under 20 ps excitation at 650 or 667 nm, the difference absorption spectra in the spectral region 600–720 nm were measured. They are associated with transition of antenna chlorophylls into singlet excited states and P-700 photooxidation. It was shown that the excited states in the whole inhomogeneous antenna were generated within 10 ps and deactivated with three-component kinetics, the t_1/e values being 20–45, 100–300 and over 500 ps. Formation of P-700⁺ has a rise time of 15–30 ps. The fast component of the depletion of the antenna excited states is suggested to be due to transfer of excitation energy from antenna pigments to reaction centers and its trapping. The kinetics of the fast component is independent of excitation energy and a redox state of P-700.     

三角褐指藻甘油酯对氮胁迫的响应

【背景】三角褐指藻作为生物燃料潜在的生产者,在胁迫条件下能通过改变其甘油酯组成来适应外部环境的变化,同时伴随着生物燃料原料甘油三酯(TAG)的积累,研究三角褐指藻甘油酯对氮胁迫的响应机制有利于深入认识TAG的积累过程。【目的】通过分析三角褐指藻在正常和氮胁迫条件下各类脂质含量及其脂肪酸成分的变化,揭示氮胁迫诱导积累的TAG酰基主要来源,以及在胁迫前生成的各极性甘油酯脂肪酸的去向,从而为进一步认识三角褐指藻对氮胁迫的响应机制提供新信息。【方法】利用高效薄层色谱结合气相色谱法分析三角褐指藻在正常和氮胁迫条件下的脂肪酸及甘油酯组分的变化。【结果】三角褐指藻在氮胁迫条件下TAG含量增加至57.8 mg/g时,总甘油酯含量几乎不变,但各甘油酯含量变化差异很大,表现为各极性脂含量显著降低。在此期间,各类甘油酯脂肪酸组成含量的变化表明,三角褐指藻TAG主要积累饱和及单不饱和脂肪酸,即16:0和16:1n7,分别以从头合成及原有极性脂转化为主,极性脂的部分二十碳五烯酸(EPA)作为酰基供体也向TAG发生了转化;此外组成极性脂的多不饱和脂肪酸16:2n4、16:3n4及EPA分解导致其含量显著下降。【结论】当氮胁迫诱导的三角褐指藻TAG含量为57.8 mg/g时,积累的TAG酰基中有48%来自从头合成,52%来自极性脂转化;而氮胁迫诱导所减少的极性脂酰基中有54%转化成TAG,46%发生了分解。     

Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.

In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Production of 13C polyunsaturated fatty acids from the microalga Phaeodactylum tricornutum

An integrated process for the indoor production of ¹³C labelled PUFA from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor from which the exhaust gas from the reactor is returned to the culture by a low pressure compressor. To avoid accumulation of dissolved oxygen in the culture medium, the exhaust gas is bubbled through a sodium sulphite solution before returning it to the reactor. Carbon is removed from the medium before inoculating the alga, then labelled ¹³CO₂ is injected for pH control and carbon supply. The reactor has been operated in semicontinuous mode at a dilution rate of 0.01 h^–1, a biomass productivity of 0.1 g L^–1 d^–1 being obtained. Under this conditions both pH and dissolved oxygen were correctly controlled and the adequacy of the system for autotrophic production of labelled biomass was demonstrated. Analysis by GC-MS revealed that the fatty acids content of the biomass obtained was 10% d.wt., the content of eicosapentaenoic acid was 2.5% d.wt. All the fatty acids were labelled, more that 90% of the carbon present in these fatty acids was ¹³C. Element analysis of biomass and supernatant showed that 59.5% of injected carbon was assimilated into the biomass whereas 33% remained in the supernatant, and 7.5% remained undetected. Due to the high cost of ¹³CO₂ different strategies for the optimisation of labelled carbon use are proposed.