Eukaryotic Green Alga Chlamydomonas Reinhardtii Research Articles

High-throughput identification of novel heat tolerance genes via genome-wide pooled mutant screens in the model green alga Chlamydomonasreinhardtii.

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome-wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat-sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high-confidence Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products.Key points• A protein expression vector using double-antibiotic resistance genes was designed• Double antibiotic selection causes fewer colonies with more positive for phenotype• Coupling the new vector with FACS improves microalgal screening efficiency > 60%

Chlamydomonas reinhardtii LFO1 Is an IsdG Family Heme Oxygenase.

Heme is essential for respiration across all domains of life. However, heme accumulation can lead to toxicity if cells are unable to either degrade or export heme or its toxic by-products. Under aerobic conditions, heme degradation is performed by heme oxygenases, enzymes which utilize oxygen to cleave the tetrapyrrole ring of heme. The HO-1 family of heme oxygenases has been identified in both bacterial and eukaryotic cells, whereas the IsdG family has thus far been described only in bacteria. We identified a hypothetical protein in the eukaryotic green alga Chlamydomonas reinhardtii, which encodes a protein containing an antibiotic biosynthesis monooxygenase (ABM) domain consistent with those associated with IsdG family members. This protein, which we have named LFO1, degrades heme, contains similarities in predicted secondary structures to IsdG family members, and retains the functionally conserved catalytic residues found in all IsdG family heme oxygenases. These data establish LFO1 as an IsdG family member and extend our knowledge of the distribution of IsdG family members beyond bacteria. To gain further insight into the distribution of the IsdG family, we used the LFO1 sequence to identify 866 IsdG family members, including representatives from all domains of life. These results indicate that the distribution of IsdG family heme oxygenases is more expansive than previously appreciated, underscoring the broad relevance of this enzyme family. IMPORTANCE This work establishes a protein in the freshwater alga Chlamydomonas reinhardtii as an IsdG family heme oxygenase. This protein, LFO1, exhibits predicted secondary structure and catalytic residues conserved in IsdG family members, in addition to a chloroplast localization sequence. Additionally, the catabolite that results from the degradation of heme by LFO1 is distinct from that of other heme degradation products. Using LFO1 as a seed, we performed phylogenetic analysis, revealing that the IsdG family is conserved in all domains of life. Additionally, C.reinhardtii contains two previously identified HO-1 family heme oxygenases, making C.reinhardtii the first organism shown to contain two families of heme oxygenases. These data indicate that C.reinhardtii may have unique mechanisms for regulating iron homeostasis within the chloroplast.

Purification and photobiochemical profile of photosystem 1 from a high-salt tolerant, oleaginous Chlorella (Trebouxiophycaea, Chlorophyta).

The eukaryotic green alga Chlamydomonas reinhardtii has been studied extensively within the biofuel industry as a model organism, as researchers look towards algae to provide chemical feedstocks (i.e., lipids) for the production of liquid transportation fuels. C. reinhardtii, however, is unsuitable for high-level production of such precursors due to its relatively poor lipid accumulation and fresh-water demand. In this study we offer insight into the primary light harvesting and electron transfer reactions that occur during phototropic growth in a high-salt tolerant strain of Chlorella (a novel strain introduced here as NE1401), a single-celled eukaryotic algae also in the phylum Chlorophyta. Under nutrient starvation many eukaryotic algae increase dramatically the amount of lipids stored in lipid bodies within their cell interiors. Microscopy and lipid analyses indicate that Chlorella sp. NE1401 may become a superior candidate for algal biofuels production. We have purified highly active Photosystem 1 (PS1) complexes to study in vitro, so that we may understand further the photobiochemisty of this promising biofuel producer and how its characteristics compare and contrast with that of the better understood C. reinhardtii. Our findings suggest that the PS1 complex from Chlorella sp. NE1401 demonstrates similar characteristics to that of C. reinhardtii with respect to light-harvesting and electron transfer reactions. We also illustrate that the relative extent of the light state transition performed by Chlorella sp. NE1401 is smaller compared to C. reinhardtii, although they are triggered by the same dynamic light stresses.

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.

Crystallization and preliminary X-ray crystallographic studies of CrArsM, an arsenic(III)S-adenosylmethionine methyltransferase fromChlamydomonas reinhardtii

Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å.

Transcriptome sequencing (RNA-seq) analysis of the effects of metal nanoparticle exposure on the transcriptome of Chlamydomonas reinhardtii.

The widespread use of nanoparticles (NPs) raises concern over their potential toxicological effects in humans and ecosystems. Here we used transcriptome sequencing (RNA-seq) to evaluate the effects of exposure to four different metal-based NPs, nano-Ag (nAg), nano-TiO2 (nTiO2), nano-ZnO (nZnO), and CdTe/CdS quantum dots (QDs), in the eukaryotic green alga Chlamydomonas reinhardtii. The transcriptome was characterized before and after exposure to each NP type. Specific toxicological effects were inferred from the functions of genes whose transcripts either increased or decreased. Data analysis resulted in important differences and also similarities among the NPs. Elevated levels of transcripts of several marker genes for stress were observed, suggesting that only nZnO caused nonspecific global stress to the cells under environmentally relevant conditions. Genes with photosynthesis-related functions were decreased drastically during exposure to nTiO2 and slightly during exposures to the other NP types. This pattern suggests either toxicological effects in the chloroplast or effects that mimic a transition from low to high light. nAg exposure dramatically elevated the levels of transcripts encoding known or predicted components of the cell wall and the flagella, suggesting that it damages structures exposed to the external milieu. Exposures to nTiO2, nZnO, and QDs elevated the levels of transcripts encoding subunits of the proteasome, suggesting proteasome inhibition, a phenomenon believed to underlie the development and progression of several major diseases, including Alzheimer's disease, and used in chemotherapy against multiple myeloma.

Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways inChlamydomonas reinhardtii

A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.

Composition and Structure of the 80 S Ribosome from the Green Alga Chlamydomonas reinhardtii: 80 S Ribosomes are Conserved in Plants and Animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.

NADP-Malate Dehydrogenase from Unicellular Green Alga Chlamydomonas reinhardtii. A First Step toward Redox Regulation?

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii.

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38–40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.

Characteristics and Sequence of Phosphoglycolate Phosphatase from a Eukaryotic Green Alga Chlamydomonas reinhardtii

Phosphoglycolate phosphatase (PGPase), a key enzyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The enzyme was an approximately 65-kDa homodimer with a pI value of 5.1 composed of approximately 32-kDa subunits not connected by any S-S bridges. It was also highly specific for phosphoglycolate with a K(m) value of 140 microm and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl(2), and it recovered competitively following the addition of MgCl(2) or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl(2), indicating that the enzyme binds to Ca(2+). The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophenylphosphatase-like proteins, although the purified PGPase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase complemented the PGPase-deficient mutant pgp1. Possible regulatory mechanisms during adaptation to limiting CO(2) were discussed based on the characteristics of the purified PGPase and the deduced amino acid sequence.

Tryptophan-heme pi-electrostatic interactions in cytochrome f of oxygenic photosynthesis.

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.

Crystal structure of the W35A mutant thioredoxin h from Chlamydomonas reinhardtii: the substitution of the conserved active site Trp leads to modifications in the environment of the two catalytic cysteines.

The conformational analysis of W35A thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii in the solid state has been carried out by x-ray diffraction, with the aim to clarify the role of Trp in the catalysis. Comparative analysis of W35A mutant with wild-type (WT) thioredoxin shows that, even if the structural motif of thioredoxin is not perturbed, the substitution of Trp35 by an Ala leads to significant changes in protein conformation near the active site. This rearrangement increases its solvent exposure and explains the change of the pKa values of the catalytic cysteines. The substitution of the Trp residue also influences the crystal packing as well as the recognition ability of thioredoxin. The solid state analysis suggests that the Trp residue has a structural function both to force the active site in the bioactive conformation, and to mediate the protein-protein recognition.

Light induces accumulation of isocitrate lyase mRNA in a carotenoid-deficient mutant of Chlamydomonas reinhardtii.

A cDNA with sequence similarity to isocitrate lyase (ICL) genes was isolated from the unicellular eukaryotic green alga Chlamydomonas reinhardtii as a light-induced mRNA in the carotenoid biosynthetic mutant strain FN68. The 416 amino acid open reading frame shows significant sequence similarity to isocitrate lyases of bacteria (70%), molds (48%), yeasts (45%), and plants (47%). Expression of the Chlamydomonas ICL gene was tested in the mutant strain FN68, which when grown in the dark fails to accumulate carotenoids and is deficient in chlorophyll, and in CC400G, a strain that accumulates wild-type levels of carotenoids and chlorophyll. In vegetative CC400G cells, ICL mRNA accumulated to a high level in the dark and declined to a barely detectable level within 30 min of exposure to light. This response was more sensitive to white (tungsten filament) or red light than green or blue light, excluding cryptochrome and rhodopsin as the photoreceptor. These results are consistent with excitation by chlorophyll and/or a phytochrome-related photoreceptor. In vegetative FN68 cells, ICL mRNA abundance was very low in the dark, but increased dramatically in response to light. At intensities above threshold, excitation by far-red or red light-induced ICL mRNA accumulation to the highest levels. The threshold of the response was lowest for far-red and blue light. These results are consistent with excitation of a photochromic far-red-responsive pigment.

Thiosulfate reductase from Chlamydomonas

Summary Thiosulfate reductase (thiosulfate-thiol sulfurtransferase, (EC 2.8.1.3) TSR) and rhodanese (thiosulfate-cyanide sulfurtransferase, (EC 2.8.1.1) RDN) activities have been identified in crude extracts from the eukaryotic green alga Chlamydomonas reinhardtii . These activities co-purified after several purification steps and no isoforms have been separated. Kinetic studies indicate that dithiols (DTE) exert a competitive inhibition on rhodanese activity, the affinity for DTE being 28-fold higher than that for cyanide. These results support the idea that both activities are mediated by the same protein. The activities are not sensitive to thiol reagents, whereas they are inhibited by specific arginine-modifying reagents. The physiological role of this enzyme could be basically the assimilation of S-sulfane by the alga.

1H,13C,15N-NMR Resonance Assignments of Oxidized Thioredoxin h from the Eukaryotic Green Alga Chlamydomonas reinhardtii Using New Methods based on Two-Dimensional Triple-Resonance NMR Spectroscopy and Computer-Assisted Backbone Assignment

The cytosolic thioredoxin h (112 amino acids, 11.8 kDa) cDNA from the eukaryotic green alga Chlamydomonas reinhardtii has been over-expressed in Escherichia coli and the protein was uniformly labelled with 13C and 15N. For the backbone resonance assignments (HN, N, C alpha, CO and H alpha) we used a new set of two-dimensional triple-resonance correlation experiments [Simorre, J.-P., Brutscher, B., Caffrey, M. S. & Marion, D. (1994) J. Biomol. NMR 4, 325-333; Brutscher, B., Simorre, J.-P., Caffrey, M. S. & Marion, D. (1994) J. Magn. Reson. B 105, 77-82] and the new computer assignment protocol ALPS (Assignment for Labelled Protein Spectra) developed in our laboratory [Morelle, N., Brutscher, B., Simorre, J.-P. & Marion, D. (1995) J. Biomol. NMR 5, 154-160]. The assignment was extended to the side chains using three-dimensional HC(C)H-TOCSY correlation experiments together with a set of regular two-dimensional proton spectra. The secondary and super-secondary structures were deduced from the C alpha chemical-shift differences to the random-coil values, the measure of the exchange rates of slow-exchanging amide protons using 15N-HSQC spectroscopy, and the assignment of medium and long-range NOEs. The oxidized C. reinhardtii thioredoxin h is based on an open alpha/beta motif consisting of a five-stranded beta-sheet surrounded by four helices in a manner similar to the bacterial E. coli thioredoxin [Katti, S. K., LeMaster, D. M. & Eklund, H. (1990) J. Mol. Biol. 212, 167-184; Jeng, M.-F., Campbell, A. P., Begley, T., Holmgren, A., Case, D. A., Wright, P. E. & Dyson, H. J. (1994) Structure 2, 853-868] and the human thioredoxin [Qin, J., Clore, G. M. & Gronenborn, A. M. (1994) Structure 2, 503-522] for which C. reinhardtii thioredoxin h shares 32 and 44 sequence identities, respectively. From the analysis of the secondary structure characteristics, the C. reinhardtii thioredoxin h is closer to the human thioredoxin structure than to the bacterial thioredoxin structure. Conversely, the latter would share several structural features with the previously reported [Lancelin, J.-M., Stein, M. & Jacquot, J.-P. (1993) J. Biochem. (Tokyo) 114, 421-431] highly thermostable chloroplast C. reinhardtii thioredoxin m that is involved in the thiol-redox enzymic control of photosynthetic intermediate carbon metabolism.

Assembly of the Photosystem II oxygen-evolving complex is inhibited in psbA site-directed mutants of Chlamydomonas reinhardtii. Aspartate 170 of the D1 polypeptide.

Photosystem II catalyzes the photooxidation of water to molecular oxygen, providing electrons to the photosynthetic electron transfer chain. The D1 and D2 chloroplast-encoded reaction center polypeptides bind cofactors essential for Photosystem II function. Transformation of the chloroplast genome of the eukaryotic green alga Chlamydomonas reinhardtii has allowed us to engineer site-directed mutants in which aspartate residue 170 of D1 is replaced by histidine (D170H), asparagine (D170N), threonine (D170T), or proline (D170P). Mutants D170T and D170P are completely deficient in oxygen evolution, but retain normal (D170T) or 50% (D170P) levels of Photosystem II reaction centers. D170H and D170N accumulate wild-type levels of PSII centers, yet evolve oxygen at rates approximately 45% and 15% those of control cells, respectively. Kinetic analysis of chlorophyll fluorescence in the mutants reveals a specific defect in electron donation to the reaction center. Measurements of oxygen flash yields in D170H show, however, that those reaction centers capable of evolving oxygen function normally. We conclude that aspartate residue 170 of the D1 polypeptide plays a critical role in the initial binding of manganese as the functional chloroplast oxygen-evolving complex is assembled.

Growth and adaptation of the green alga Chlamydomonas reinhardtii on diesel exhaust particle extracts

SUMMARY: We have examined the growth and photosynthesis of liquid cultures of the eukaryotic green alga Chlamydomonas reinhardtii incubated with iso-octane extracts of diesel particulate exhaust. At concentrations up to 0.125% (v/v), the extracts led to a dose-dependent growth retardation. Higher concentrations caused cell death. After a prolonged lag phase, air-grown illuminated algae, stressed with sublethal concentrations of diesel exhaust extracts, adapted to the pollutants. Further addition of toxic extract up to a concentration of 2.5 (v/v) affected neither growth nor photosynthesis. Media of adapted and non-adapted cells were analysed by HPLC and GC-MS and the concentrations of the remaining toxic compounds were determined. The results showed that the amounts of certain components decreased during incubation with adapted cells.

Sequence analysis and regulation of expression of a gene coding for carbonic anhydrase in Chlamydomonas reinhardtii

Using immunological and molecular biological techniques, we studied the localization, gene organization, and regulation of expression of the extracellular carbonic anhydrase in the eukaryotic green alga Chlamydomonas reinhardtii. Electron microscopy data using immunogold labelling suggest an association of the protein with the inner face of the cell wall. The same polyclonal antibody was previously used to select an immunoreactive 2.5-kb genomic DNA fragment coding for a portion of the carbonic anhydrase monomer. Using the known carbonic anhydrase cDNA sequence and sequence analysis of the genomic DNA fragment, we have deduced the exon–intron organization of the genomic clone. The similarities between Chlamydomonas and higher plant carbonic anhydrase amino acid sequences and the effect of photoheterotrophic growth on the expression of the algal carbonic anhydrase are also examined. Key words: carbonic anhydrase, Chlamydomonas, immunogold labelling, sequence, expression.