Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.     

Characterization of a symbiotic,heterocystous, N2-fixing cyanobacterium fromCycas coralloid roots

A symbiotic, heterocystous, N₂-fixing blue-green alga, isolated from the coralloid roots of a xerophytic plant,Cycas revoluta, grew best in liquid medium supplemented with 4 mM NO ₃ ^– . Morphologically, the isolated alga was identical to that of the natural endophyte but the cell size had decreased markedly. The alga was heterotrophic. Intact coralloid roots had nearly 4 to 5 times more nitrogenase activity compared with natural- and laboratory-grown agla but nitrate reductase was inducible in both the forms. Plasmid(s) were found in both algal forms.     

Modeling culture profiles of the heterocystous N2-fixing cyanobacterium Anabaena flos-aquae

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

Phytochelatin plays a role in UV-B tolerance in N2-fixing cyanobacterium Anabaena doliolum

To study the role of Cd-induced phytochelatins in UV-B tolerance, lipid peroxidation, antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), glutathione arid phytochelatin contents were measured in buthionine sulphoximine treated and untreated cells of Anabaena doliolum. Cd-pretreatment of the cyanobacterium reduced the lipid peroxidation as well as the antioxidative enzymes in comparison to UV-B treatment alone, whereas the phytochelatin content demonstrated an increase. In contrast to this, buthionine sulphoximine-induced inhibition of phytochelatin synthase, dramatically decreased the Cd-induced co-tolerance against UV-B, hence demonstrating that phytochelatin not only protects the cyanobacterium from heavy metal but participates in UV-B tolerance as well.     

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 ? resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C(2)S(2)M(2) supercomplexes, to avoid overlap.     

The occurrence of fimbriae on a N 2 2 -fixing cyanobacterium which occurs in lichen symbiosis

The Nostoc cyanobiont of the lichen Peltigera canina when grown on N₂ possesses, in the motile stage, discrete unbranched non-flagellar appendages (fimbriae or pili). These arise from the host cell surface in a peritrichous manner, have an axial hole, are 7.0 ±0.3 nm in diameter and are up to 3 m long. They do not haemagglutinate guinea pig red blood corpuscles and differ from the major fimbrial types reported for Gram-negative heterotrophic bacteria and from sex pili. They may be involved in motility and specificity in symbiotic cyanobacteria.     

Assessment of Hg2+ toxicity to a N2-fixing cyanobacterium in long- and short-term experiments

Toxicological responses of the filamentous N₂-fixing cyanobacteriumNostoc calcicola Bréb. towards Hg²⁺ were studied to enumerate the decisive lethal events. In low-dose, long-term experiments (0.05–0.25 m Hg²⁺, 10 days), photoautotrophic growth was severely inhibited with concurrent loss of photosynthetic pigments (phycocyanin>chlorophyll >carotenoids) and nucleic acids. The termination of growth after a day 4 exposure to 0.25 m Hg²⁺ has been attributed to the complete inhibition ofin vivo photosynthetic activity in the cyanobacterium (O₂ evolution>¹⁴CO₂ incorporation). The elevated Hg²⁺ concentrations irreversibly damaged the cell membrance as observed under light microscopy, and as indicated by the leakage of intracellular electrolytes and phycocyanin. In high-dose, short-term experiments (0.5–20.0 m Hg²⁺, up to 6 h), thein vivo activities of selected enzymes (glutamine synthetase > nitrate reductase > nitrogenase) were less inhibited by Hg²⁺ than the uptake of nutrient ions (NH ₄ ⁺ >NO ₃ ^– >PO ₄ ^3– ).     

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.     

Physiological responses of the freshwater N2-fixing cyanobacterium Raphidiopsis raciborskii to Fe and N availabilities

The cyanobacterium Raphidiopsis raciborskii is of environmental and social concern in view of its toxicity, bloom-forming characteristics and increasingly widespread occurrence. However, while availability of macronutrients and micronutrients such as N and Fe are critically important for the growth and metabolism of this organism, the physiological response of toxic and non-toxic strains of R. raciborskii to varying Fe and N availabilities remains unclear. By determining physiological parameters as a function of Fe and N availability, we demonstrate that R. raciborskii growth and N₂-fixing activity are facilitated at higher Fe availability under N₂-limited conditions with faster growth of the CS-506 (cylindrospermopsin-producing) strain compared with that of CS-509 (the non-toxic) strain. Radiolabelled Fe uptake assays indicated that R. raciborskii acclimated under Fe-limited conditions acquires Fe at significantly higher rates than under Fe replete conditions, principally via unchelated Fe(II) generated as a result of photoreduction of complexed Fe(III). While N₂-fixation of both strains occurred during both day and night, the CS-506 strain overall exhibited higher N₂-fixing and Fe uptake rates than the CS-509 strain under N-deficient and Fe-limited conditions. The findings of this study highlight that Fe availability is of significance for the ecological advantage of CS-506 over CS-509 in N-deficient freshwaters.     

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO₂ on growth, CO₂-fixation and N₂-fixation rates by strains of the unicellular marine N₂-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO₂-acclimated cultures (190 ppm) of WH0401, growth, CO₂-fixation and N₂-fixation rates were significantly lower than those in cultures acclimated to higher (present-day ～385 ppm, or future ～750 ppm) pCO₂ treatments. Growth rates were not significantly different, however, in low-pCO₂-acclimated cultures of WH0402 in comparison with higher pCO₂ treatments. Unlike previous reports for C. watsonii (strain WH8501), N₂-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both light and pCO₂ had a significant negative effect on gross : net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO₂, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO₂-fixation rates of C. watsonii may be positively affected by elevated light and pCO₂ within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Three-dimensional ultrastructure of a unicellular cyanobacterium

The first complete three-dimensional ultrastructural reconstruction of a cyanobacterium was accomplished with high-voltage electron microscopy and computer-aided assembly of serial sections. The precise arrangement of subcellular features within the cell body was very consistent from one cell to another. Specialized inclusion bodies always occupied specific intracellular locations. The photosynthetic thylakoid membranes entirely surrounded the central portion of the cytoplasm, thereby compartmentalizing it from the rest of the cell. The thylakoid membranes formed an interconnecting network of concentric shells, merging only at the inner surface of the cytoplasmic membrane. The thylakoids were in contact with the cytoplasmic membrane at several locations, apparently to maintain the overall configuration of the thylakoid system. These results clarified several unresolved issues regarding structure-function relationships in cyanobacteria.     

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Gammaherpesviruses, including the human pathogens Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral–host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.     

Lipid content and fatty acid composition in N2-fixing cyanobacterium Anabaena doliolum as affected by molybdenum deficiency

Anabaena doliolum grown under molybdenum deficiency produced less biomass (on a dry wt basis) and the cells had lower protein content but higher carbohydrate content than Mo-grown material. Molybdenum deficiency led to a slight decrease in chlorophyll a, a 1.5-fold increase in carotenoids and a 1.4-fold increase in total lipid but there was no difference in the lipid profiles of Mo-enriched and Mo-deficient cells. Molybdenum deficiency caused increases in the cell contents of digalactosyl diacylglycerol and phosphatidylglycerol and decreases in monogalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol lipids. The concentration of unsaturated C₁₈ fatty acids was lower in the Mo-deficient cells.     

Active transport and accumulation of bicarbonate by a unicellular cyanobacterium

The rates of inorganic carbon accumulation and carbon fixation in light by the unicellular cyanobacterim Coccohloris peniocystis have been determined. Cells incubated in the light in medium containing H14CO3- were rapidly separated from the medium by centrifugation through silicone oil into a strongly basic terminating solution. Samples of these inactivated cells were assayed to determine total 14C accumulation, and acid-treated samples were assayed to determine 14C fixation. The rate of transport of inorganic into illuminated cells was faster than the rate of CO2 production in the medium from HCO3- dehydration. This evidence for HCO3- transport in these cells is in agreement with our previous results based upon measurements of photosynthetic O2 evolution. A substantial pool of inorganic carbon was bulit up within the cells presumably as HCO3- before the onset of the maximum rate of photosynthesis. Large accumulation ratios were observed, greater than 1,000 times the external HCO3- concentration. Accumulation did not occur in the dark and was greatly suppressed by the photosynthesis inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 3-chloro-carbonylcyanide phenylhydrazone. These results indicate that the accumulation of inorganic carbon in these cells involves a light-dependent active transport process.     

The effects of salt stress on photosynthetic electron transport and thylakoid membrane proteins in the cyanobacterium Spirulina platensis

The response of Spirulina (Arthrospira) platensis to high salt stress was investigated by incubating the cells in light of moderate intensity in the presence of 0.8 M NaCl. NaCl caused a decrease in photosystem II (PSII) mediated oxygen evolution activity and increase in photosystem I (PSI) activity and the amount of P700. Similarly maximal efficiency of PSII (Fv/Fm) and variable fluorescence (Fv/Fo) were also declined in salt-stressed cells. Western blot analysis reveal that the inhibition in PSII activity is due to a 40 % loss of a thylakoid membrane protein, known as D1, which is located in PSII reaction center. NaCl treatment of cells also resulted in the alterations of other thylakoid membrane proteins: most prominently, a dramatic diminishment of the 47-kDa chlorophyll protein (CP) and 94-kDa protein, and accumulation of a 17-kDa protein band were observed in SDS-PAGE. The changes in 47-kDa and 94-kDa proteins lead to the decreased energy transfer from light harvesting antenna to PSII, which was accompanied by alterations in the chlorophyll fluorescence emission spectra of whole cells and isolated thylakoids. Therefore we conclude that salt stress has various effects on photosynthetic electron transport activities due to the marked alterations in the composition of thylakoid membrane proteins.