Gene map for the Cyanophora paradoxa cyanelle genome.

The genes for the following proteins were localized by hybridization analysis on the cyanelle genome of Cyanophora paradoxa: the alpha and beta subunits of phycocyanin (cpcA and cpcB); the alpha and beta subunits of allophycocyanin (apcA and apcB); the large and small subunits of ribulose-1,5-bisphosphate carboxylase (rbcL and rbcS); the two putative chlorophyll alpha-binding apoproteins of the photosystem I-P700 complex (psaA and psaB); four apoproteins believed to be components of the photosystem II core complex (psbA, psbB, psbC, and psbD); the two apoprotein subunits of cytochrome b-559 which is also found in the core complex of photosystem II (psbE and psbF); three subunits of the ATP synthase complex (atpA and atpBE); and the cytochrome f apoprotein (petA). Eighty-five percent of the genome was cloned as BamHI, BglII, or PstI fragments. These cloned fragments were used to construct a physical map of the cyanelle genome and to localize more precisely some of the genes listed above. The genes for phycocyanin and allophycocyanin were not clustered and were separated by about 25 kilobases. Although the rbcL gene was adjacent to the atpBE genes and the psbC and psbD genes were adjacent, the arrangement of other genes encoding various polypeptide subunits of protein complexes involved in photosynthetic functions was dissimilar to that observed for known chloroplast genomes. These results are consistent with the independent development of this cyanelle from a cyanobacterial endosymbiont.     

Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.     

The immunologically conserved phycobilisome-thylakoid linker polypeptide

We have isolated phycobilisomes from two classes of red algae, several subdivisions of the cyanobacteria, and the cyanelles of Cyanophora paradoxa. In addition to the major light harvesting biliproteins, these phycobilisomes also contain several other polypeptides, the largest of which ranges from 75 to 120 kilodaltons in the different species surveyed. This protein, previously isolated and characterized from three species, was shown to be the final emitter of excitation energy in phycobilisomes and is also thought to be involved in the attachment of the phycobilisomes to the thylakoid membrane. We have obtained polyclonal antibodies to the 95 kilodalton polypeptide isolated from phycobilisomes of the cyanobacterium, Nostoc sp. This protein shares no common antigenic determinants with either the α or β subunits of allophycocyanin, or any of the other biliproteins, as determined by the sensitive Western immunoblotting technique. However, this antiserum cross-reacts with the highest molecular weight polypeptide of all the rhodophytan and cyanobacterial phycobilisomes tested. That these proteins are immunologically related, but are unrelated to other biliproteins, is reminiscent of previous immunological studies of biliproteins which showed that while the three major spectroscopically distinct classes of biliproteins (phycoerythrin, phycocyanin, and allophycocyanin) shared no common antigenic determinants, there was a strong antigenic determinant to specific biliprotein classes which crossed taxonomic divisions.     

坛紫菜别藻蓝蛋白α亚基基因的原核表达与鉴定

通过PCR的方法从重组质粒pMD-apcAB中扩增坛紫菜别藻蓝蛋白α亚基基因(apcA),并将其克隆到高效表达外源基因的原核表达质粒pTO-T7。将构建好的质粒导入表达型大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行Western-blot和质谱鉴定。结果显示:apcA全长486bp,表达的α亚基(apcA)为带有原核表达载体T7g10的12个起始氨基酸的融合蛋白,其分子量约为19.7KD。0.5mmol/L的IPTG在37℃诱导6h时,apcA的表达量达到最大,达菌体总蛋白50%以上。Western-blot和质谱鉴定的结果表明获得的融合蛋白为重组坛紫菜别藻蓝蛋白α亚基。       

Complementary chromatic and far-red photoacclimations in <Emphasis Type="Italic">Synechococcus</Emphasis> ATCC 29403 (PCC 7335). I: The phycobilisomes,a proteomic approach

Synechococcus ATCC 29403 (PCC 7335) is a unicellular cyanobacterium isolated from Puerto Peñasco, Sonora Mexico. This cyanobacterium performs complementary chromatic acclimation (CCA), far-red light photoacclimation (FaRLiP), and nitrogen fixation. The Synechococcus PCC 7335 genome contains at least 31 genes for proteins of the phycobilisome (PBS). Nine constitutive genes were expressed when cells were grown under white or red lights and the resulting proteins were identified by mass spectrometry in isolated PBS. Five inducible genes were expressed under white light, and phycoerythrin subunits and associated linker proteins were detected. The proteins of five inducible genes expressed under red light were identified, the induced phycocyanin subunits, two rod linkers and the rod-capping linker. The five genes for FaRLiP phycobilisomes were expressed under far-red light together with the apcF gene, and the proteins were identified by mass spectrometry after isoelectric focusing and SDS-PAGE. Based on in silico analysis, Phylogenetic trees, and the observation of a highly conserved amino acid sequence in far-red light absorbing alpha allophycoproteins encoded by FaRLiP gene cluster, we propose a new nomenclature for the genes. Based on a ratio of ApcG2/ApcG3 of six, a model with the arrangement of the allophycocyanin trimers of the core is proposed.     

Effect of Light Quality on Phycobilisome Components of the Cyanobacterium Spirulina platensis

Phycobilisomes from the nonchromatic adapting cyanobacterium Spirulina platensis are composed of a central core containing allophycocyanin and rods with phycocyanin and linker polypeptides in a regular array. Room temperature absorption spectra of phycobilisomes from this organism indicated the presence of phycocyanin and allophycocyanin. However, low temperature absorption spectra showed the association of a phycobiliviolin type of chromophore within phycobilisomes. This chromophore had an absorption maximum at 590 nanometers when phycobilisomes were suspended in 0.75 molar K-phosphate buffer (pH 7.0). Purified phycocyanin from this cyanobacterium was found to consist of three subparticles and the phycobiliviolin type of chromophore was associated with the lowest density subparticle. Circular dichroism spectra of phycocyanin subparticles also indicated the association of this chromophore with the lowest density subparticle. Absorption spectral analysis of α and β subunits of phycocyanin showed that phycobiliviolin type of chromophore was attached to the α subunit, but not the β subunit. Effect of light quality showed that green light enhanced the synthesis of this chromophore as analyzed from the room temperature absorption spectra of phycocyanin subparticles and subunits, while red or white light did not have any effect. Low temperature absorption spectra of phycobilisomes isolated from green, red, and white light conditions also indicated the enhancement of phycobiliviolin type of chromophore under green light.     

Biogenesis of phycobiliproteins: I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phyococyanobilin lyase specific for beta-phycocyanin and allophycocyanin subunits

Phycobilin lyases covalently attach phycobilin chromophores to apo-phycobiliproteins (PBPs). Genome analyses of the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 identified three genes, denoted cpcS-I, cpcU, and cpcV, that were possible candidates to encode phycocyanobilin (PCB) lyases. Single and double mutant strains for cpcS-I and cpcU exhibited slower growth rates, reduced PBP levels, and impaired assembly of phycobilisomes, but a cpcV mutant had no discernable phenotype. A cpcS-I cpcU cpcT triple mutant was nearly devoid of PBP. SDS-PAGE and mass spectrometry demonstrated that the cpcS-I and cpcU mutants produced an altered form of the phycocyanin (PC) beta subunit, which had a mass approximately 588 Da smaller than the wild-type protein. Some free PCB (mass = 588 Da) was tentatively detected in the phycobilisome fraction purified from the mutants. The modified PC from the cpcS-I, cpcU, and cpcS-I cpcU mutant strains was purified, and biochemical analyses showed that Cys-153 of CpcB carried a PCB chromophore but Cys-82 did not. These results show that both CpcS-I and CpcU are required for covalent attachment of PCB to Cys-82 of the PC beta subunit in this cyanobacterium. Suggesting that CpcS-I and CpcU are also required for attachment of PCB to allophycocyanin subunits in vivo, allophycocyanin levels were significantly reduced in all but the CpcV-less strain. These conclusions have been validated by in vitro experiments described in the accompanying report (Saunée, N. A., Williams, S. R., Bryant, D. A., and Schluchter, W. M. (2008) J. Biol. Chem. 283, 7513-7522). We conclude that the maturation of PBP in vivo depends on three PCB lyases: CpcE-CpcF, CpcS-I-CpcU, and CpcT.     

Characterization of the biliproteins of Gloeobacter violaceus chromophore content of a cyanobacterial phycoerythrin carrying phycourobilin chromophore

The biliproteins of the unicellular, thylakoid-less cyanobacterium Gleobacter violaceus were resolved by chromatography on hydroxylapatite and DEAE-cellulose into five components: phycoerythrin I and II, phycocyanin I and II, and allophycocyanin. Allophycocyanin B was not detected. Three of these components, phycoerythrin II, phycocyanin II, and allophycocyanin, were purified to homogeneity. Phycoerythrin II crystallized as hexagonal prisms. G. violaceus allophycocyanin crystallized as thin plates; unter similar conditions other cyanobacterial allophycocyanins crystallize as needles. The biliproteins in the phycoerythrin I and phycocyanin I components were present in polydisperse, high molecular weight aggregates, which may represent incompletely dissociated substructures of the phycobilisome.Both phycoerythrin components from G. violaceus carry phycoerythrobilin and phycourbilin groups in the ratio of 6:1. Separation of the and subunits of these biliproteins revealed that the phycoerythrobilins were equally distributed between the two subunits, and that the subunit alone carried the phycourobilin. These phycoerythrins are the first cyanobacterial phycobiliproteins found to carry a phycourobilin prosthetic group.Abbreviations used PE poycoerythrin - PC phycocyanin - AP allophycocyanin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - B Bangiophycean - R Rhodophytan - C Cyanobacterial     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta)

The genes encoding the and subunits of allophycocyanin, phycocyanin and phycoerythrin from the red alga Aglaothamnion neglectum were isolated and characterized. While the operons containing the different phycobiliprotein genes are dispersed on the plastid genome, the genes encoding the and subunits for each phycobiliprotein are contiguous. The subunit gene is 5 for both the phycocyanin and phycoerythrin operons, while the subunit gene is 5 for the allophycocyanin operon. The amino acid sequences of A. neglectum phycobiliproteins, as deduced from the nucleotide sequences of the genes, are 65–85% identical to analogous proteins from other red algae and cyanobacteria. The conserved nature of the plastid-encoded red algal and cyanobacterial phycobiliprotein genes supports the proposed origin of red algal plastids from cyanobacterial endosymbionts.Many environmental factors effect phycobilisome biosynthesis. The effect of both nutrient availability and light quantity on the level of A. neglectum phycobiliprotein subunits and the mRNA species encoding those subunits is described.     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Protein translocation into and within cyanelles (review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.