Red Alga Cyanidium Caldarium Research Articles

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Biochemical and spectroscopic characterization of PSI-LHCI from the red alga Cyanidium caldarium.

Light-harvesting complexes (LHCs) have been diversified in oxygenic photosynthetic organisms, and play an essential role in capturing light energy which is transferred to two types of photosystem cores to promote charge-separation reactions. Red algae are one of the groups of photosynthetic eukaryotes, and their chlorophyll(Chl) a-binding LHCs are specifically associated with photosystem I (PSI). In this study, we purified three types of preparations, PSI-LHCI supercomplexes, PSI cores, and isolated LHCIs, from the red alga Cyanidium caldarium, and examined their properties. The polypeptide bands of PSI-LHCI showed characteristic PSI and LHCI components without contamination by other proteins. The carotenoid composition of LHCI displayed zeaxanthins, β-cryptoxanthins, and β-carotenes. Among the carotenoids, zeaxanthins were enriched in LHCI. On the contrary, both zeaxanthins and β-cryptoxanthins couldnot be detected from PSI, suggesting that zeaxanthins and β-cryptoxanthins are bound to LHCI but not PSI. A Qy peak of Chl a in the absorption spectrum of LHCI was shifted to a shorter wavelength than those in PSI and PSI-LHCI. This tendency is in line with the result of fluorescence-emission spectra, in which the emission maxima of PSI-LHCI, PSI, and LHCI appeared at 727, 719, and 677nm, respectively. Time-resolved fluorescence spectra of LHCI represented no 719 and 727-nm fluorescence bands from picoseconds to nanoseconds. These results indicate that energy levels of Chls around/within LHCIs and within PSI are changed by binding LHCIs to PSI. Based on these findings, we discuss the expression, function, and structure of red algal PSI-LHCI supercomplexes.

Assessment of Phycocyanin Extraction from Cyanidium caldarium by Spark Discharges, Compared to Freeze-Thaw Cycles, Sonication, and Pulsed Electric Fields.

Phycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.

Microbial Analysis and Efficiency of Biofiltration Packing Systems for Hydrogen Sulfide Removal from Wastewater Off Gas

Hydrogen sulfide (H2S) is commonly detected among waste gases from wastewater treatment plants causing atmospheric pollution, which affects human and environmental health. The aim of this study is to characterize the microbial diversity present within two different H2S biofiltration systems packed with either pozzolan or marble and to assess their efficiency for H2S removal. Few examples were reported about the relationship between the characteristics of the packing material and biofilter performance. Physicochemical parameters such as sulfate concentrations and pH are measured along this study. Sulfate concentrations produced by H2S oxidation were higher using marble biofilter and a higher efficiency of H2S removal was observed. Microbial diversity was characterized using a culture-independent 16S rRNA gene approach. Our results show that the biofilter packed with marble is very acidic (pH <3) and exhibits a higher bacterial diversity with three dominant bacterial families: Xanthomonadaceae, Hydrogenophilaceae, and Spirillaceae. However the biofilter packed with pozzolan is less acidic (pH 5.7–6.8) and shows completely different phylotypes where the plastid 16S rRNA gene of the red algae Cyanidium caldarium and Acidithiobacillus bacterium were retrieved. Such results show that the selection of an appropriate packing material is essential to the odor removal performance of a biofilter system and should allow for the design of better reactor with optimal operating conditions.

Phycobilisome Mobility and Its Role in the Regulation of Light Harvesting in Red Algae.

Red algae represent an evolutionarily important group that gave rise to the whole red clade of photosynthetic organisms. They contain a unique combination of light-harvesting systems represented by a membrane-bound antenna and by phycobilisomes situated on thylakoid membrane surfaces. So far, very little has been revealed about the mobility of their phycobilisomes and the regulation of their light-harvesting system in general. Therefore, we carried out a detailed analysis of phycobilisome dynamics in several red alga strains and compared these results with the presence (or absence) of photoprotective mechanisms. Our data conclusively prove phycobilisome mobility in two model mesophilic red alga strains, Porphyridium cruentum and Rhodella violacea. In contrast, there was almost no phycobilisome mobility in the thermophilic red alga Cyanidium caldarium that was not caused by a decrease in lipid desaturation in this extremophile. Experimental data attributed this immobility to the strong phycobilisome-photosystem interaction that highly restricted phycobilisome movement. Variations in phycobilisome mobility reflect the different ways in which light-harvesting antennae can be regulated in mesophilic and thermophilic red algae. Fluorescence changes attributed in cyanobacteria to state transitions were observed only in mesophilic P. cruentum with mobile phycobilisomes, and they were absent in the extremophilic C. caldarium with immobile phycobilisomes. We suggest that state transitions have an important regulatory function in mesophilic red algae; however, in thermophilic red algae, this process is replaced by nonphotochemical quenching.

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222 1 with unit cell constants of a = 146.8 Å, b = 176.9 Å, and c = 353.7 Å, and the other one had a space group of P2 12 12 1 with unit cell constants of a = 209.2 Å, b = 237.5 Å, and c = 299.8 Å. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 Å resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 Å resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.

External Iron Regulates Polyphosphate Content in the Acidophilic, Thermophilic Alga Cyanidium caldarium

Transmission electron microscopy revealed the presence of electron-dense bodies (EDB) in the cytosol of the acidophilic, thermophilic red alga Cyanidium caldarium. These bodies contain almost exclusively Fe, P, and O and can play a role in Fe storage. (31)P-nuclear magnetic resonance analysis identified a sharp signal at -23.3 ppm, which was attributed to the phosphate groups of the inner portions of polyphosphate chains. From this evidence, as well as that of a previous ESR study (Nagasaka et al., BioMetals 16:465-470, 2003), it can be concluded that polyphosphates are the major anionic constituents of the EDB. Omission of Fe from the culture medium resulted in substantially decreased polyphosphate levels, demonstrating the control of cellular polyphosphate content by the Fe status of the culture medium.

Aromatic structure of Tyrosine-92 in the extrinsic PsbU protein of red algal Photosystem II is important for its functioning

PsbU is one of the extrinsic proteins in red algal Photosystem II (PSII) and functions to optimize the availability of Ca 2+ and Cl − cofactors for water oxidation. To determine the functional residue of PsbU, we constructed various PsbU mutants from a red alga Cyanidium caldarium and reconstituted these mutants with the red algal PSII. The results revealed that Tyr-92 of PsbU, especially its aromatic ring, was essential for maintaining its function. From the crystal structure of PSII, Tyr-92 is located close to Pro-340 of D1, suggesting that the aromatic ring of Tyr-92 interacts with the CH group of Pro-340 of D1, and this CH/π interaction is important for the optimal function of the Mn 4Ca-cluster.

Subcellular localization of ferredoxin-NADP+ oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms

Ferredoxin-NADP(+) oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP(+)-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP(+)-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ′) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.

Metal metabolism in the red alga Cyanidium caldarium and its relationship to metal tolerance.

The unicellular red alga Cyanidium caldarium is tolerant to high levels of various metal ions. Cells of this alga cultured with divalent metal ions at 5 mM contained an elevated concentration of each metal, with the highest level for Zn followed by Mn > Ni > Cu. This order is in fair agreement with the toxicity levels reported previously, with the exception of Mn, which shows a toxicity level comparable to that of Ni. Transmission electron microscopy indicated the presence of electron-dense bodies in the algal cells, and elemental analysis by energy dispersive X-ray spectrometry showed high levels of Fe and P in these bodies. Accumulation of Zn was found in these particles in Zn-treated algal cells, whereas no such deposition was found for Cu, Ni, or Mn in cells treated with the respective metals. Although trapping of Zn in the intracellular bodies may contribute to reduction of metal activity in the cells, this effect can be overcome by high intracellular levels of Zn that result in a high degree of toxicity. The correlation between intracellular concentration and toxic levels of metal ions implies that the reduced incorporation of the metals is a major detoxification mechanism in this alga.

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).

Cloning and expression of a cDNA encoding the extrinsic 20 kDa protein of photosystem II from a red alga Cyanidium caldarium

Oxygen-evolving PS II complex purified from a red alga, Cyanidium caldarium, contains four extrinsic proteins; they are 12 kDa, 20 kDa, 33 kDa proteins and cytochrome (cyt) c-550 [Enami et al. (1995) B.B.A. 1232, 208 - 216]. In this study, the 20 kDa protein was cloned and expressed in E. coli. The 20 kDa protein was purified from CaCl2-treated extracts of purified PS II complex and its N-terminal amino acid sequence was determined. Oligonucleotides synthesized on the basis of its N-amino acid sequence were used as primers for degenerated PCR, and complete sequence of the gene was determined using modified 5'- and 3'- RACE methods. The resulted open reading frame encodes a protein of 218 amino acid residues of which, the first 62 residues were assigned as leader sequences according to the N-terminal sequence of the mature protein. The resulted mature protein contains 146 amino acid residues with a molecular mass of 16386 Da. A search with the Swiss-Prot database revealed that the Cyanidium caldarium 20 kDa protein is 36.3% and 30.3% identical to the extrinsic 17 kDa protein from Volvox and Chlamydomonas, respectively, suggesting that the red algal 20 kDa protein is possibly an ancestor of the 17 kDa protein in green algae. An expression vector containing the 20 kDa gene was constructed in pCAL-n-EK plasmid, and the successful expression of the protein has been confirmed. We are currently performing reconstitution experiments with the recombinant 20 kDa protein to CaCl2-washed red algal PS II to study the binding and functional properties of the protein.

Reconstitution of the extrinsic 23 and 17 kDa proteins with spinach PSII which had been exchanged for the 33 kDa protein from different plant species

The 33 kDa protein of photosystem II (PSII) plays an important role in maintaining the function of the Mn cluster, and is common to all of the organisms and totally exchangeable in binding to PSII from various different organisms (Enami et al., 2000, Plant Cell Physiol. 41, 1354-1364). The amino acid sequence of the 33 kDa protein showed a relatively high homology above 40% from cyanobacteria to higher plants. Thus, the structure of the 33 kDa protein has been considered to be conserved during evolution from cyanobacteria to higher plants. In this study, reconstitution experiments were performed for the extrinsic 23 and 17 kDa proteins from spinach with spinach PSII which had been exchanged for the 33 kDa protein from different sources. Spinach PSII was treated with 2.6 M urea/0.2 M NaCl to remove all of the extrinsic proteins, and then reconstituted with the 33 kDa protein from a cyanobacterium (Synechococcus vulcanus), a red alga (Cyanidium caldarium) or a higher plant (Spinach). The results showed that, while spinach PSII bearing the higher plant 33kDa protein retained the full ability of binidng the 23 kDa and 17 kDa proteins, PSII of which the 33 kDa protein had been replaced with the red algal or cyanobacterial 33kDa protein partially rebound the 23kDa protein but totally failed to bind the 17kDa protein. This suggests that the structure of the 33kDa protein interacting with other extrinsic proteins is different between higher plants and red algae or cyanobacteria.

Screening of algal strains for metal removal capabilities

Eight algal species were tested for their ability to remove five toxic metalsduring 30-min exposures to single-metal (1 mg L-1) solutions at pH7. Efficacy of metal bioremoval varied according to algal species and metal. Al+3 was best removed by the thermophilic blue-green alga(cyanobacterium) Mastigocladus laminosus, Hg+2 and Zn+2 by the thermophilic and acidophilic red alga Cyanidiumcaldarium, and Cd+2 by C. caldarium and the green alga Scenedesmus quadricauda. All of these alga/metal combinations resultedin >90% metal removal. However, none of the eight algal speciesremoved more than 10% of Cr+6. Results indicate that some toxicmetals are more readily removed than others are by algae and that selectionof appropriate strains could potentially enhance bioremoval of specificmetals from wastewater at neutral pH.

Isolation, characterization, and chromosomal mapping of an ftsZ gene from the unicellular primitive red alga Cyanidium caldarium RK-1.

The FtsZ protein is involved in eukaryote plastid division, but there is little information on its involvement in the plastid-dividing apparatus. To investigate the relationship between FtsZ and the plastid-dividing ring, the ftsZ gene was isolated from the unicellular primitive red alga Cyanidium caldarium RK-1. Comparison of several prokaryotic and eukaryotic FtsZ proteins shows that there are six highly conserved domains in the core region of FtsZ. To determine the chromosomal location of ftsZ, we first determined the electrophoretic karyotype of C. caldarium RK-1. Southern-hybridization analysis combined with CHEF revealed the chromosomes on which the ftsZ gene exist. Northern-hybridization analysis indicated that the C. caldarium RK-1 ftsZ gene is transcribed as a 1.9-kb molecule, and that the transcripts specifically accumulate just before plastid division. Phylogenetic analysis indicated that C. caldarium RK-1 and other eukaryotic ftsZ genes are the descendants of cyanobacterial ftsZ genes, supporting the current agreement that FtsZ is involved in plastid division.

Cloning, Expression of the psbU Gene, and Functional Studies of the Recombinant 12-kDa Protein of Photosystem II from a Red Alga Cyanidium caldarium

The encoding extrinsic 12-kDa protein of oxygen-evolving PS II complex from a red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure. The gene encodes a putative polypeptide of 154 amino acids with a calculated molecular mass of 16,714 Da. The full sequence of the protein includes two characteristic transit peptides, one for transfer across the chloroplast envelope and another for targeting into the thylakoid lumen. This indicates that the protein is encoded in the nuclear genome. The mature protein consists of 93 amino acids with a calculated molecular mass of 10,513 Da. The cloned gene was successfully expressed in Escherichia coli and the resulting protein was purified, reconstituted to CaCl2-washed PS II complex together with the other extrinsic proteins of 33 and 20 kDa and cyt c-550. The recombinant 12-kDa protein bound completely with the PSII complex, which resulted in a restoration of oxygen evolution equal to the level achieved by binding of the native 12-kDa protein.

The primitive red algae Cyanidium caldarium and Cyanidioschyzon merolae as model system for investigating the dividing apparatus of mitochondria and plastids

The Cyanidiophyceae species Cyanidium caldarium and Cyanidioschyzon merolae have played important roles in showing the division mechanisms of mitochondria and plastids. The apparatus regulating mitochondrial and plastid divisions was formerly unknown. We first identified the division apparatus of plastids, called the plastid-dividing ring (PD ring), in C. caldarium and the division apparatus of mitochondria, called the mitochondrion-dividing ring (MD ring), in C. merolae. Eukaryotic cell division is therefore controlled by at least three dividing apparati (rings)—a contractile ring, an MD ring, and a PD ring—while bacterial division is controlled by a single bacterial contractile FtsZ ring. BioEssays 20:344-354, 1998.© 1998 John Wiley & Sons, Inc.

Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714.

Isopentenyl diphosphate, the universal isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1-13C]glucose and analysis of the resulting labelling patterns found in the isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for isoprenoid biosynthesis. All isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus, which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast isoprenoids (phytol in Cy. caldarium and O. danica and beta-carotene in O. danica) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium Synechocystis PCC 6714 using [1-13C]- and [6-13C]-glucose. The two isoprenoids examined, phytol and beta-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route.