Combined remediation and lipid production using Chlorella sorokiniana grown on wastewater and exhaust gases.

Substitution of conventional feedstock with waste based alternatives is one route towards both remediation and reducing costs associated with production of algal biomass. This work explores whether exhaust gases and wastewater can replace conventional feedstock in the production of biomass from Chlorella sorokiniana. Exhaust gases were used to augment production in final effluent, anaerobic digester centrate or in standard medium. Cultures were grown in 1L bottles under illumination of 80 µmol m(-2) s(-1). The results showed an average µmax ranging between 0.04 and 0.07 h(-1), whilst the final biomass yield in different media ranged between 220 and 330 mg L(-1). Lipid yield was increased over time to 31 mg L(-1). CO2 addition resulted in complete nitrogen removal between 48 and 96 h in both final effluent and centrate. The results also indicated that levels of carbon monoxide, carbon dioxide and nitrogen oxides in the exhaust gases can be reduced by between 20% and 95%.

Evidence from time resolved studies of the P700(.+)/A1(.-) radical pair for photosynthetic electron transfer on both the PsaA and PsaB branches of the photosystem I reaction centre.

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild-type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron-sulphur centres, Fe-S(X/A/B), can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.

Chlamydomonas nuclear mutants that fail to assemble respiratory or photosynthetic electron transfer complexes.

We are using a molecular-genetic approach to investigate the role of nuclear genes in the biogenesis of the electron transfer complexes of mitochondria and chloroplasts. Our analysis of nuclear mutants of the green alga Chlamydomonas that are defective in respiration or photosynthesis has led to the identification of genes encoding factors required for the expression of specific organellar genes, and genes encoding structural components of the complexes.

The sites of interaction of triphenyltetrazolium chloride with mitochondrial respiratory chains.

The inability of cells and microorganisms to reduce the colourless electron acceptor triphenyltetrazolium chloride (TTC) to a red formazan precipitate is commonly used as a means of screening for cells that have a dysfunctional respiratory chain. The site of reduction of TTC is often stated to be at the level of cytochrome c oxidase where it is assumed to compete with oxygen for reducing equivalents. However, we show here that TTC is reduced not by cytochrome c oxidase but instead by dehydrogenases, particularly complex I, probably by accepting electrons directly from low potential cofactors. The reduction rate is fastest in coupled membranes because of accumulation in the matrix of the positively charged TTC+ cation. However, the initial product of TTC reduction is rapidly reoxidised by molecular oxygen, so that generation of the stable red formazan product from this intermediate occurs only under strictly anaerobic conditions. Colonies of mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions to allow the intermediate to form the stable red formazan. This revision of the mode of interaction of TTC with respiratory chains has implications for the types of respiratory-defective mutants that might be detected by TTC screening.

Site-directed mutagenesis of PsaA residue W693 affects phylloquinone binding and function in the photosystem I reaction center of Chlamydomonas reinhardtii.

To investigate the environment of the phylloquinone secondary electron acceptor A(1) within the photosystem I reaction center, we have carried out site-directed mutagenesis of two tryptophan residues (W693 and W702) in the PsaA subunit of Chlamydomonas reinhardtii. One of these conserved tryptophans (W693) is predicted to be close to the phylloquinone and has been implicated in the interaction of A(1) with an aromatic residue through pi--pi stacking. We find that replacement of W702 with either histidine or leucine has no effect on the electronic structure of A(1)(*-) or on forward electron transfer from A(1)(*-) to the iron--sulfur center F(x). In contrast, the same mutations of W693 alter the electronic structure of the photoaccumulated A(1)(*-) and slow forward electron transfer as measured by the decay of the electron spin-polarized signal arising from the P700(*+)/A(1)(*-) radical pair. These results provide support for the hypothesis that W693 has a role in poising the redox potential of A(1)/A(1)(*-) so it can reduce F(x), and they indirectly provide evidence for electron transfer along the PsaA-side branch of cofactors in PSI.

Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii.

Although most eukaryotic cells are sensitive to the 80S ribosome inhibitor cycloheximide (CYH), naturally occurring CYH resistance is widespread amongst yeast species. The primary determinant of resistance appears to be a single residue within ribosomal protein L41; resistance is acquired by the substitution of a conserved proline (P56) by a glutamate residue. We have isolated the L41 gene (RPL41) from the green alga Chlamydomonas reinhardtii, and investigated the molecular basis of CYH resistance in various mutant strains. In both the wild-type strain and the mutant act-1, a proline is found at the key position in L41. However, analysis of six independently isolated act-2 mutants reveals that all have point mutations that replace the proline with either leucine or serine. Of the two changes, the leucine mutation confers significantly higher levels of CYH resistance. This work identifies the ACT-2 locus as RPL41 and provides a possible dominant marker for nuclear transformation of C. reinhardtii.

Isolation and characterisation of chemotactic mutants of Chlamydomonas reinhardtii obtained by insertional mutagenesis.

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker.

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.

The 9-kDa phosphoprotein of photosystem II. Generation and characterisation of Chlamydomonas mutants lacking PSII-H and a site-directed mutant lacking the phosphorylation site.

The chloroplast gene psbH encodes a 9-10 kDa thylakoid membrane protein (PSII-H) that is associated with photosystem II and is subject to light-dependent phosphorylation at a threonine residue located on the stromal side of the membrane. The function of PSII-H is not known, neither is it clear what regulatory role phosphorylation may play in the control of PSII activity. Using particle gun-mediated transformation, we have created chloroplast transformants of Chlamydomonas reinhardtii in which the synthesis of PSII-H is prevented by the disruption of psbH, or in which the phosphorylatable threonine is replaced by alanine through site-directed mutagenesis of the gene. The mutants lacking PSII-H have a photosystem II-deficient phenotype, with no detectable functioning PSII complex present in whole cells or isolated thylakoid membranes. In contrast, the alanine mutant (T3A) grows photoautotrophically, and PSII activity is comparable to wild-type cells as determined by various biochemical and biophysical assays.

The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.

A chimeric gene composed of the coding sequence of the ble gene from Streptoalloteichus hindustanus fused to the 5' and 3' untranslated regions of the Chlamydomonas reinhardtii nuclear gene RBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome of C. reinhardtii by co-transformation with the ARG7 marker yields Arg+ transformants of which approximately 80% possess the ble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against the ble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of the ble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome of C. reinhardtii and provides a useful dominant marker for nuclear transformation.

Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome b6f complex.

The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes. One mutant fails to accumulate transcript for cytochrome f, whilst a second displays a severely reduced accumulation of the cytochrome b6 transcript. Two other mutants appear to be affected in the insertion of the haem co-factor into cytochrome b6. The fifth mutant displays no detectable defect in the synthesis of any of the known subunits of the complex. Genetic analysis of the mutants demonstrates that in three cases, the mutant phenotype co-segregates with the introduced DNA. For the mutant affected in the accumulation of the cytochrome f transcript, we have used the introduced DNA as a tag to isolate the wild-type version of the affected gene.

Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii.

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A0, A1 and Fe-SX. Fe-SX is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-SX is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-SA/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii.

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library.

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3 x 10(9) treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus.

The argininosuccinate lyase (ASL) gene of Chlamydomonas reinhardtii has been cloned using four oligonucleotide probes corresponding to highly conserved regions of the ASL polypeptide sequence. The identity of the gene was confirmed by partial sequencing. It is unique, contains several introns and spans a region less than 7.8 kb that includes highly repetitive sequences. Using a particle gun, a reliable nuclear transformation system has been established by complementing three mutants deficient in ASL activity with the wild-type ASL gene. Analysis of the transformants reveals variable patterns of integration of the transforming DNA into the nuclear genome. Previous work has mapped the mutations in the mutants arg2 and arg7 to either end of the ARG7 locus 1.0 to 1.6 recombination map units apart. Our transformation results show that these two mutations are located within a region of 7.8 kb. This allows for the first correlation of the recombination map and the molecular map at the ARG7 locus and indicates a high recombination frequency in this region of the nuclear genome.

The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts.

The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.