Unicellular Red Alga Cyanidium Caldarium Research Articles

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.

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.

Phycobilin biosynthetic reactions in extracts of cyanobacteria

Phycobilins are the chromophores of phycobiliproteins, the light-harvesting pigments of cyanobacteria, red algae and cryptophytes. Phycobilins are biosynthesized from heme by the action of heme oxygenase, which converts heme to biliverdin, followed by the action of other enzymes that convert biliverdin to the phycobilins. We previously reported on the enzymes and biosynthetic intermediates of phycobilin formation in extracts of the unicellular red alga Cyanidium caldarium. Heme oxygenase activity has now been obtained from extracts of the cyanobacterium Synechocystis sp. PCC 6701. The reaction requirements are similar to those for the C. caldarium enzyme: heme substrate, reduced ferredoxin, and a second reductant such as ascorbate or Trolox. The enzymatic nature of the reaction was verified by two criteria in addition to the requirement for cell extract: production of only the IXα isomer of the bilin product and inhibition by the substrate analog Sn-protoporphyrin IX. The enzyme was partially purified by high-speed centrifugation, 35–75% differential (NH4)2SO4 precipitation, and DEAE-cellulose anion exchange chromatography. In addition, extract capable of converting biliverdin IXα to phycobilins has been obtained from Synechocystis sp. PCC 6701 and another cyanobacterium, Synechocystis sp. PCC 6803. Only the (3Z) isomers of the phycobilins accumulated in the incubations containing unfractionated cell extracts, in contrast to incubations with unfractionated C. caldarium extracts which produce both the (3Z) and (3E) isomers. Phycocyanobilin and phycoerythrobilin were produced in comparable amounts by Synechocystis sp. PCC 6701 extracts, but only phycocyanobilin accumulated in Synechocystis sp. PCC 6803 extracts. This difference in in vitro product accumulation correlates with the phycobilins that are found in vivo in these two cell types.

A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny.

Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen. We have analyzed one of the genes essential for this process, secY. A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid. Unlike the situation seen in bacteria the C. caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons. A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet. In this analysis plastid secY genes fall into two classes. One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart. Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga. All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues. We postulate that the two classes of secY genes are paralogous, i.e. their gene products are involved in different protein translocation processes. Based on this assumption a model for the evolution of the plastid sec apparatus is presented.

Nucleotide sequence and expression of the genes for the alpha and beta subunits of phycocyanin in Cyanidium caldarium.

The nucleotide sequence of the plastid-encoded operon containing genes for the alpha (cpcA) and beta (cpcB) subunits of phycocyanin in the unicellular red alga Cyanidium caldarium is described. cpcB is located 5' to cpcA and the two genes are separated by a 102-bp spacer region. The transcription start site of cpcBA was mapped to 80 bp upstream of the ATG initiation codon of cpcB. Promoter-like elements similar to the -10 (TATAAT) and -35 (TTGACA) consensus promoters in bacteria were found 6 and 31 bp upstream of the transcription initiation site. Northern blotting revealed an abundant 1.3-kb cpcBA transcript in illuminated cells, but this transcript was undetectable in dark-grown cells. Expression levels of cpcBA in cells incubated with 10(-6) M heme in the dark were similar to those in cells illuminated for 24 h. Cells illuminated with 150 microM gabaculine (an inhibitor of delta-aminolevulinate synthesis) or 10 mM levulinic acid (an inhibitor of delta-aminolevulinate dehydrase) lacked detectable cpcBA transcripts. In cells illuminated with 200 microM N-methyl-mesoporphyrin IX (an inhibitor of ferrocheletase), inhibition of cpcBA expression and phycocyanin synthesis was similar. These results provide strong evidence that light induction of the cpcBA operon is dependent on synthesis of heme.

Physical map of the plastid genome of the unicellular red alga Cyanidium caldarium strain RK-1

The physical map of the plasmid genome of the unicellular red alga Cyanidium caldarium strain RK-1 was constructed. The 150-kbp genome was circular and had an inverted repeated region (IR) which contained the genes for 16 s and 23 s ribosomal RNAs, as is usually seen in most plastid genomes. Since C. caldarium is a very "primitive" alga, the results suggest that the ancestral cyanobacteria lost most of its genome as an endosymbiont comparatively early in the process of plastid formation. After that, several genes seem to have been lost from plastid genomes, step by step, during the course of evolution.

The trpA gene on the plastid genome of Cyanidium caldarium strain RK-1.

The trpA gene (for the alpha subunit of tryptophan synthase) was found on the plastid genome of the "primitive" unicellular red alga Cyanidium caldarium strain RK-1. This is the first example of an actively-transcribed gene for tryptophan synthase encoded on a plastid genome. In contrast to trpA, trpB (the gene for the beta subunit of tryptophan synthase) was encoded in the cell nucleus. Considering the primitive characteristics of C. caldarium, trpB must have been lost from the plastid genome before trpA.

Organization and expression of a phycobiliprotein gene cluster from the unicellular red alga Cyanidium caldarium.

We have sequenced a plastid gene cluster from the unicellular red alga Cyanidium caldarium which is located downstream from the psbA gene and contains, in the following order, genes for a beta-allophycocyanin-like protein (apcB'), a putative 9.5 kDa allophycocyanin linker protein (apcL9.5) and a putative 29 kDa phycocyanin linker protein (cpcL29). The apcB' and apcL9.5 genes are organized in the form of an operon. The cpcL29 gene is transcribed monocistronically from the opposite strand of DNA. Both transcription units are probably terminated at a 25 bp inverted repeat 3 and 5 bp downstream of the stop codons of the apcL9.5 and cpcL29 genes, respectively. The levels of both transcripts are greatly reduced in the dark as is the psbA transcript. Downstream from the phycobiliprotein gene cluster two open reading frames (ORFs) were found which are homologous to ORFs from plastid DNAs and cyanelle DNA of Cyanophora paradoxa. Sequence homologies between genes analysed in this study and corresponding genes from cyanobacteria, chlorophytic plastids and cyanelles point to a large phylogenetic distance between the plastids of Cyanidium and cyanobacteria and other plastid types.

A 16 kb small single-copy region separates the plastid DNA inverted repeat of the unicellular red alga Cyanidium caldarium: physical mapping of the IR-flanking regions and nucleotide sequences of the psbD-psbC, rps16, 5S rRNA and rpl21 genes.

The four inverted repeat (IR) flanking regions of the Cyanidium caldarium plastid DNA were cloned. Southern blotting, transcript and sequence analyses of the border regions revealed the psbD-psbC operon and the rps16 gene within the large single-copy region upstream of the 16S rDNA gene and the rpl21 gene downstream of the 5S rDNA within the 16 kb small single-copy region. The size of the IR is ca. 5 kb. The nucleotide sequences of the psbD-psbC, rps16, rpl21 and 5S rRNA genes with the corresponding alignments and physical maps of the regions are presented. Northern analysis revealed a less complex psbD-psbC transcription pattern than has been found in higher plants. Comparisons to other red algal data point to structural diversity within red algal plastid DNA.

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA(Ile)(GAU) and tRNA(Ala)(UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by 'small-sized' rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.

Structure of the Rubisco operon from the unicellular red algaCyanidium caldarium: Evidence for a polyphyletic origin of the plastids

The genes for both subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) were located on the plastid DNA (ptDNA) of the unicellular red alga Cyanidium caldarium. Both genes are organized together in an operon. The sequence homology of both genes to the corresponding genes from the unicellular red alga Porphyridium aerugineum is remarkably high, whereas homology to Rubisco genes from chloroplasts and two recent cyanobacteria is significantly lower. These data provide strong evidence for a polyphyletic origin of chloroplasts and rhodoplasts. In addition the genes for the small subunit of Rubisco (rbcS) from red algae show about 60% homology to rbcS genes from cryptophytes and chromophytes. Thus, homologies in the rbcS gene indicate a close phylogenetic relationship between rhodoplasts and the plastids of Chromophyta.

The psbA-gene from a red alga resembles those from Cyanobacteria and Cyanelles

Plastid DNA (ptDNA) from the unicellular red alga Cyanidium caldarium was isolated. A 5.8 kb Eco RI, fragment containing the entire psbA-gene was cloned and the nucleotide sequence of the psbA-gene determined. At the carboxyl terminus the encoded protein (D1) contains the seven amino acid-insertion which was found to be typical of the cyanobacteria and the cyanelles of Cyanophora paradoxa. However, the overall sequence homology does not support a direct relationship between the plastids of Cyanidium, cyanelles and the cyanobacteria. As in other photosynthetic organisms the psbA-gene is transcribed as a monocistronic mRNA. The ribosomal RNA operon was located 4 kb upstream of the psbA-gene.

The effect of gabaculine on tetrapyrrole biosynthesis and heterotrophic growth in Cyanidium caldarium.

Pigment synthesis in four strains of the unicellular red alga Cyanidium caldarium with different pigment-synthesizing patterns was inhibited in the presence of gabaculine (3-amino-2,3-dihydrobenzoic acid). Parallel inhibition of light-induced chlorophyll and phycocyanin synthesis was observed in strain III-D-2, which only synthesizes pigments in the light. Similar parallel inhibition was observed in the dark in mutant CPD, which is able to synthesize chlorophyll and phycocyanin in the absence of light. Inhibition of pigment synthesis in all strains was overcome by addition of 5-aminolaevulinic acid. Inhibition of phycocyanin synthesis in mutant GGB (unable to synthesize chlorophyll) and inhibition of chlorophyll synthesis in mutant III-C (unable to synthesize phycocyanin) were also observed. Gabaculine also inhibited the heterotrophic growth of C. caldarium in the dark. However, inhibition was overcome after an extended lag period, following which cell growth proceeded at a similar rate to that of control cells not exposed to gabaculine. Heterotrophic growth in cells pre-exposed to gabaculine was not inhibited by subsequent exposure. Possible mechanisms for this adaptation are discussed.

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components.

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z-ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.

Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium

In the unicellular non-vacuolate red alga Cyanidium caldarium nitrate uptake occurs through two specific permease systems which, on the basis of kinetic constants can be defined as low affinity system and high affinity system. The high affinity system is saturated at very low nitrate concentrations (<1 μM), whereas the low affinity system is saturated only at high nitrate concentrations (Km=0.45±0.10 mM). The low affinity system is present in cells growing under conditions of nitrogen limitation as well as in cells growing in excess nitrate. In contrast, the high affinity system is present only in cells growing under conditions of nitrogen limitation. The high affinity system works only at acid pH and is inactive at neutral pH. The low affinity system is active both at acid and at neutral pH.

Investigations on the apoprotein of phycocyanin from Cyanidium caldarium

Cultures of the unicellular red alga Cyanidium caldarium were transferred from heterotrophic growth conditions to photoautotrophic growth. During photoautotrophic growth, the biliprotein phycocyanin is synthesized de novo. In the presence of 2–5 mM/l levulinic acid which inhibits the biosynthesis of tetrapyrrole chromophores, phycocyanin biosynthesis is suppressed by a factor of 29. Immunoprecipitation yields small amounts of “apoprotein” i.e. phycocyanin which lacks all or part of its chromophore(s). In various experiments the ratio apoprotein/residual holoprotein (phycocyanin) was determined as 2–6 to one. Incubation with [3H]leucine leads to labelled immunoprecitable material: apoprotein (18,300–19,600 Mr) and larger poly-peptides (50,000–52,000 Mr) of unknown nature. The apoprotein was separated from residual phycocyanin by chromatography on DEAE-cellulose and preparative isoelectric focusing (IEF). The significance of the results for further studies on the last steps of phycocyanin biosynthesis is discussed.

Glutamine synthetase activity, ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.