Nucleotide sequence of the cyanelle genome fromCyanophora paradoxa

The complete nucleotide sequence of the cyanelle genome ofCyanophora paradoxa Pringsheim strain LB 555 was determined (accession number U30821). The circular molecule is 135,599 base pairs in length. The physical map of this DNA molecule is shown along with identified genes and open reading frames.     

RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.     

Nucleotide sequence of the genes encoding cytochrome b-559 from the cyanelle genome of Cyanophora paradoxa

Cyanophora paradoxa is a flagellated protozoan which possesses unusual, chloroplast-like organelles referred to as cyanelles. The psbE and psbF genes, which encode the two apoprotein subunits of cytochrome b-559, have been cloned from the cyanelle genome of C. paradoxa. The complete nucleotide sequences of these genes and their flanking sequences were determined by the chain-termination, dideoxy method. The psbE gene is composed of 75 codons and predicts a polypeptide of 8462 Da that is seven to nine residues smaller than most other psbE gene products. The psbF gene consists of 43 codons and predicts a polypeptide of 4761 Da. Two open reading frames, whose sequences are highly conserved among cyanobacteria and numerous higher plants, were located in the nucleotide sequence downstream from the psbF gene. The first open reading frame, denoted psbI, is composed of 39 codons, while the second open reading frame, denoted psbJ, is composed of 41 codons. The predicted amino acid sequences of the psbI and psbJ gene products predict proteins of 5473 and 3973 Da respectively. These proteins are probably integral membrane proteins anchored in the membrane by a single, transmembrane alpha helix. The psbEFIJ genes are probably co-transcribed and constitute an operon as found for other organisms. Each of the four genes is preceded by a polypurine sequence which resembles the consensus ribsosome binding sequences for Escherichia coli.     

Isolation of the plastid FtsZ gene from Cyanophora paradoxa (Glaucocystophyceae, Glaucocystophyta)

Plastids of glaucocystophytes are termed cyanelles and retain primitive features, such as a peptidoglycan wall. We isolated a full‐length prokaryotic plastid division gene, FtsZ, from the glaucocystophyte alga Cyanophora paradoxa Korshikov (CpFtsZ‐cy). CpftsZ‐cy has a chloroplast‐targeting signal at the N‐teminus. Immunofluorescence microscopy showed that CpFtsZ‐cy forms a ring‐like structure at the division plane of cyanelles.     

Photomovement in Cyanophora paradoxa

Photomovement has been studied in the symbiontic association of the colorless flagellate, Cyanophora paradoxa Korschikoff with the cyanelles, Cyanocyta korschikoffiana. There is no phototactic orientation in this organism, but a photokinetic effect. In addition, the cells show a pronounced step-up photophobic response (however no or only a weak step-down response). The phobic response is mediated by a subset of the photosynthetic pigments located in the symbiontic cyanelles. It is linked to the noncyclic photosynthetic electron transport chain but it is independent of the photosynthetic generation of a proton gradient and the ATP synthesis linked to it.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzo quinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The central part of the cyanelle rDNA unit of Cyanophora paradoxa: Sequence comparison with chloroplasts and cyanobacteria

The 287-bp spacer and the flanking 3-end of the 16S- and 5-end of the 23S-rRNA genes of the cyanelles from Cyanophora paradoxa have been sequenced and compared with the corresponding regions of cyanobacteria and chloroplasts. The spacer contains the uninterrupted genes for tRNA^ile and tRNA^ala. All coding regions show high homology to their prokaryotic counterparts. At the 3-end of the 16S-rDNA a CCTCCTTT sequence has been identified which is complementary to putative ribosome binding sites observed immediately upstream of the coding region of cyanelle protein genes.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

Nucleotide sequences ofCyanophora paradoxa cellular and cyanelle-associated 5S ribosomal RNAs: The cyanelle as a potential intermediate in plastid evolution

Summary The 5S ribosomal RNAs from the cell cytoplasm and cyanelle (photosynthetic organelle) ofCyanophora paradoxa have been isolated and sequenced. The cellular and cyanelle 5S rRNAs were 119 and 118 nucleotides in length, respectively. Both RNAs exhibited typical 5S secondary structure, but the primary sequence of the cellular species was clearly eukaryotic in nature, while that of the organellar species was prokaryotelike. The primary sequence of the cyanellar 5S rRNA was most homologous to cyanobacterial 5S sequences, yet possessed secondary-structural features characteristic of higher-plant chloroplast 5S rRNAs. Both sequence comparison and structural analysis indicated an evolutionary position for cyanelle 5S rRNA intermediate between blue-green alga and chloroplast 5S rRNAs.Contribution from the Department of Biochemistry, School of Agriculture and Life Sciences and School of Physical and Mathematical Sciences, North Carolina State University, Raleigh, North Carolina. This is paper no. 10259 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601, USA     

Effects of solar and artificial radiation on motility and pigmentation in Cyanophora paradoxa

The effects of solar radiation and artificial UV irradiation on motility and pigmentation were studied in the flagellate system Cyanophora paradoxa. Both percentage of motile cells and average velocity decreased drastically after a solar exposure of a few hours. This effect was not due to an overheating since the cells were exposed under temperaturecontrolled conditions. Partial reduction of the UV-B radiation by cut-off filters or by insertion of an artificial ozone layer increased the tolerated exposure times. Artificial UV radiation also induced the same effects. Under both solar and artificial UV irradiation the photosynthetic pigments within the cyanelles were bleached also within short exposure times. Kinetics of pigment destruction showed that the accessory phycobilins are lost with a half life of 1.3 h while the chlorophylls had a half life of 33 h and a carotenoid with an absorption maximum at 480 nm of 17.3 h.     

Identification of Photosystem I components from a glaucocystophyte,Cyanophora paradoxa: The PsaD protein has an N-terminal stretch homologous to higher plants

Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive ‘plastids’, and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both ‘green’ lineages and cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro synthesis of the cyanelle proteins of Cyanophora paradoxa by isolated cyanelles and cyanelle RNA

Cyanelles isolated from the alga Cyanophora paradoxa Korschikoff synthesized cyanelle proteins in vitro. This synthesis was stimulated by light and totally inhibited by chloramphenicol. Cycloheximide had only a small inhibitory effect. Electrophoretic separation of the labelled soluble cyanelle proteins yielded at least 20 discrete polypeptides. The RNA isolated from the cyanelles and the whole cells was successfully translated in a rabbit reticulocyte-lysate system.Abbreviations poly(A)^-RNA, poly(A)⁺RNA nonadenylated, polyadenylated RNA; - SDS sodium dodecyl sulfate     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.     

Zur osmotischen Beeinflussung der Photosynthese von Cyanophora paradoxa Korsch. durch Saccharose

Zusammenfassung Die Geschwindigkeit der photosynthetischen Sauerstoffentwicklung des sich recht langsam vermehrenden Cyanoms Cyanophora paradoxa ist nur geringfügig niedriger als diejenige anderer Algen (in bezug auf die Chlorophyllkonzentration). Merkwürdigerweise stellt man eine starke Abhängigkeit photosynthetischer Reaktionen (O₂-Entwicklung, CO₂ Fixierung und Hill-Reaktion mit 2,6-Dichlorphenolindophenol) von der im Medium vorgegebenen Konzentration eines Osmotikums fest. Unter bestimmten Voraussetzungen können steigende Saccharosekonzentrationen (bis ca. 0,1 M) die assimilatorische Sauerstoffproduktion zunächst beschleunigen, höhere Saccharose-Konzentrationen (über 0,1 M) hemmen dagegen. Der Hemmeffekt ist unspezifisch und kann auch in Abhängigkeit von Sorbit und Natriumchlorid beobachtet werden. Die bisherigen Versuche sprechen dafür, daß der Hemmeffekt bis zu einer Konzentration von ca. 0,1 M Saccharose während kurzer Einwirkungszeit reversibel ist, bei höheren Konzentrationen jedoch schon irreversible Zellschädigungen anzeigt.

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Sucrose as an effector of photosynthesis in Cyanophora paradoxa korsch

Cyanophora paradoxa, a fresh-water alga, was found to evolve oxygen photosynthetically with activities similar to other plants (50–300 mol O₂/mg chlorophyll·h). In spite of this the doubling time was 10–30 days. The photosynthetic activity turned out to react very sensitively to external osmotic concentrations. In lower concentrations as conventionally used for chloroplast preparations, sucrose—partially irreversibly—inhibited both photosynthetic O₂ evolution and Hill reaction with 2,6-Dichlorophenolindophenol progressively between 0.1 and 0.5 M. Sorbitol and sodium chloride had similar effects. In a medium of low osmolarity (0.025–0.1 M sucrose) the photosynthetic activity was increased. These results were interpreted as consequence of osmotic interactions between the cell and its environment. When the cells are unable to correct the external osmotic pressure, an irreversible degradation of intracellular membranes is initiated, which finally leads to a total decomposition of the eukaryotic cell.

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Abkurzungen EDTA Athylendiamintetraessigsäure - DCPIP 2,6-Dichlorphenolindophenol     

Substitutional bias confounds inference of cyanelle origins from sequence data

Summary Available molecular and biochemical data offer conflicting evidence for the origin of the cyanelle of Cyanophora paradoxa. We show that the similarity of cyanelle and green chloroplast sequences is probably a result of these two lineages independently developing the same pattern of directional nucleotide change (substitutional bias). This finding suggests caution should be exercised in the interpretation of nucleotide sequence analyses that appear to favor the view of a common endosymbiont for the cyanelle and chlorophyll-b-containing chloroplasts. The data and approaches needed to resolve the issue of cyanelle origins are discussed. Our findings also have general implications for phylogenetic inference under conditions where the base compositions (compositional bias) of the sequences analyzed differ. Offprint requests to: C.J. Howe     

Identification of two different glyceraldehyde-3-phosphate dehydrogenases (phosphorylating) in the photosynthetic protist Cyanophora paradoxa

Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NAD- and NADP-dependent, have been found in cell extracts of the cyanelle-bearing photosynthetic protist Cyanophora paradoxa. Whereas the two G3P dehydrogenase activities were detected with similar specific activity levels (0.1 to 0.2 U/mg of protein) in extracts of the photosynthetic organelles (cyanelles), only the NAD-dependent activity was found in the cytosol. Thus, a differential intracellular localization occurred. The perfect overlapping of the two G3P dehydrogenase activity peaks of the cyanelle in both hydrophobic interaction chromatography and subsequent FPLC (fast protein liquid chromatography) gel filtration indicated that the two activities were due in fact to a single NAD(P)-dependent G3P dehydrogenase (EC 1.2.1.-) with a molecular mass of 148,000. SDS-PAGE of active fractions from FPLC gel filtration showed that the intensity of the major protein band (molecular mass, 38,000) of the enzyme preparation clearly paralleled the activity elution profile, thus suggesting a tetrameric structure for the cyanelle dehydrogenase. On the other hand, FPLC gel filtration analysis of the cytoplasmic fraction revealed a NAD-dependent G3P dehydrogenase with a native molecular mass of 142,000, being equivalent to the classical glycolytic enzyme (EC 1.2.1.12) present in the cytosol of all the organisms so far studied. The significance of these results is discussed taking into account that the cyanobacteria, photosynthetic prokaryotes which share many structural and biochemical features with cyanelles and are considered as their ancestors, have a similar NAD(P)-dependent G3P dehydrogenase.Abbreviation FPLC Fast protein liquid chromatography     

The flagellar developmental cycle in algae: flagellar transformation inCyanophora paradoxa (Glaucocystophyceae)

Summary Flagellar development during cell division was studied inCyanophora paradoxa using agarose-embedded cells, Nomarski optics and electronic flash photography. The cells bear two heterodynamic and differently oriented (anterior and posterior) flagella. Prior to cell division, cells produce two new anterior flagella while the parental anterior flagellum transforms into a posterior flagellum. The parental posterior flagellum remains a posterior flagellum throughout this and subsequent cell divisions. The development of a single flagellum thus extends through at least two cell cycles and flagellar heterogeneity is achieved by semiconservative distribution of the flagella during cell division. Based on these principles a universal numbering system for basal bodies and flagella of eukaryotic cells is proposed.     

Metabolic activities in Cyanophora paradoxa and its cyanelles

Isolated cyanelles of Cyanophora paradoxa perform photosystem I and II dependent Hill reactions. The photosynthetic electron transport of the cyanelles does not show special features uncommon in cyanobacteria or chloroplasts of red algae. A preparation of cyanelles performs photosynthetic O₂-evolution with approximately 1/3 of the rate of intact Cyanophora, in only, however, the first three minutes of the experiment. All attempts to stabilize the CO₂-fixation activity of isolated cyanelles failed. Isolated cyanelles do not perform KCN-sensitive O₂-uptake, indicating that respiratory cytochrome oxidase is lacking in cyanelles. O₂-consumption by crude extracts from Cyanophora is inhibited by KCN when N-tetramethyl-p-phenylenediamine/ascorbate or NADH but not NADPH are supplied as the electron donors in contrast to the situation in cyanobacteria. These findings suggest that cyanelles do not respire. It is concluded that cyanelles are not so much related to cyanobacteria as formerly believed, but share many properties with chloroplasts of eukaryotic cells.Abbreviations Chl chlorophyll - DCPIP dichlorophenol-indophenol - TMPD N-tetramethyl-p-phenylenediamine To whom correspondence should be addressed     

Synechocystis sp. PCC6803 fusB gene,located outside of the str operon,encodes a polypeptide related to protein synthesis factor EF-G

Synechocystis sp. PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon. The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results). There is no evidence for the presence of a fusB homologue in other bacteria. The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes.