Effects of extracellular pH on the metabolic pathways in sulfur-deprived,H2-producing Chlamydomonas reinhardtii cultures

Sustained photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, can be obtained by incubating cells in sulfur-deprived medium [Ghirardi et al. (2000b) Trends Biotechnol. 18: 506; Melis et al. (2000) Plant Physiol. 122: 127]. The current work focuses on (a) the effects of different initial extracellular pHs on the inactivation of photosystem II (PSII) and O(2)-sensitive H(2)-production activity in sulfur-deprived algal cells and (b) the relationships among H(2)-production, photosynthetic, aerobic and anaerobic metabolisms under different pH regimens. The maximum rate and yield of H(2) production occur when the pH at the start of the sulfur deprivation period is 7.7 and decrease when the initial pH is lowered to 6.5 or increased to 8.2. The pH profile of hydrogen photoproduction correlates with that of the residual PSII activity (optimum pH 7.3-7.9), but not with the pH profiles of photosynthetic electron transport through photosystem I or of starch and protein degradation. In vitro hydrogenase activity over this pH range is much higher than the actual in situ rates of H(2) production, indicating that hydrogenase activity per se is not limiting. Starch and protein catabolisms generate formate, acetate and ethanol; contribute some reductant for H(2) photoproduction, as indicated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone inhibition results; and are the primary sources of reductant for respiratory processes that remove photosynthetically generated O(2). Carbon balances demonstrate that alternative metabolic pathways predominate at different pHs, and these depend on whether residual photosynthetic activity is present or not.     

Modeling and optimization of photosynthetic hydrogen gas production by green alga Chlamydomonas reinhardtii in sulfur-deprived circumstance

Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH(4+) 8.00 mM, PO(4)(3-) 1.11 mM, and SO(4)(2-) 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h(-1). Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H(2) for a 10-mL culture of alga with density of 6 x 10(6) cells mL(-1) for 96 h under conditions of NH(4)(+) 9.20 mM, PO(4)(3-) 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency.     

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.     

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Proteomic analysis of high-CO(2)-inducible extracellular proteins in the unicellular green alga, Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.     

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.     

In vivo localization of centrin in the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii

In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.