Thermophilic Cyanobacterium Synechococcus Research Articles

Cloning, characterization and functional analysis of groEL-like gene from thermophilic cyanobacterium Synechococcus vulcanus, which does not form an operon with groES

A gene encoding 57 102 Da polypeptide homologous to groEL of Escherichia coli but accompanying no groES, has been cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus. The amount of the gene transcript increased several folds by heat shock. The gene was expressed as a minor component of two types of HSP60, and designated as groEL2. Although expressed and induced well upon heat shock treatment in the E. coli, introduction of the cloned groEL2 gene of S. vulcanus into an E. colis groEL-less mutant did not result in the complementation of heat sensitivity.

Structure of Synechococcus elongatus [Fe2S2] ferredoxin in solution.

Ferredoxins of the [Fe2S2] type function in photosynthetic electron transport as essential electron acceptors of photosystem I. The solution structure of the 97 amino acid ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus was determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. The structure consists of a four-stranded parallel/ antiparallel beta-sheet, a short two-stranded antiparallel beta-sheet, and three short helices. The overall structure is similar to the structure of the ferredoxin from Anabaena. In contrast to related ferredoxins from mesophilic organisms, this thermostable protein contains a salt bridge inside a 17-amino acid hydrophobic core.

Nucleotide sequence of the psaD gene from the thermophilic cyanobacterium Synechococcus vulcanus

The nucleotide sequence was determined for the psaD gene of a thermophilic cyanobacterium, Synechococcus vulcanus, which encoded the PsaD subunit (Subunit II) of the Photosystem I reaction center complex. Except for some differences in the peripherals, the nucleotide sequence of the gene encoding PsaD was identical to that of another thermophilic cyanobacterium Synechococcus elongatus reported previously. Relationship between these primary structures and thermostability was also discussed.

Functional and structural changes of the photosystem II complex induced by high irradiance in cyanobacterial cells.

A gradual disintegration of the photosystem II (PSII) complex, initiated by a release of the chlorophyll-protein CP43, was identified during low-temperature illumination of Synechococcus cells. This process was slower compared to the decline of the PSII primary charge separation activity, and much slower than the photoinactivation of oxygen evolution. All three processes were slowed down in the presence of diuron. The results indicate that when the PSII repair was blocked, the inactivation of charge separation activity and the release of CP43 preceded the degradation of the D1 protein. In contrast, a much faster degradation of D1 connected to its rapid exchange was triggered by inactivation of oxygen evolution, and no disassembly of PSII was needed. We propose the existence of two different mechanisms of D1 degradation in the cells of the thermophilic cyanobacterium Synechococcus elongatus.

Photosystem I from Synechococcus elongatus: preparation and crystallization of monomers with varying subunit compositions

Monomers of Photosystem I (PS I) from the thermophilic cyanobacterium Synechococcus elongatus were prepared and characterized. Using both the zwitterionic sulfobetain 12 and the nonionic β-dodecyl maltoside as detergents, PS I was extracted in trimeric form from the thylakoid membrane. The trimers dissociate into monomers during gel-filtration, apparently due to a change in the osmotic pressure. The PS I monomer prepared in β-dodecyl maltoside exhibits the same subunit composition as the trimeric PS I. The preparation in sulfobetain 12, however, leads to the removal of the two hydrophobic subunits K and L from the monomer. Further treatment of this sample with sulfobetain 12 detaches the subunits F, J and M, associated with the disappearance of a 706 nun band in the absorption spectrum. Both the PS I monomer with complete subunit composition and the monomer lacking subunits K and L were crystallized using batch and vapor diffusion techniques with poly(ethylene glycol) as the precipitating agent.

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu(2+) ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO4 and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (ΔA of 430-440 nm) and by the flash induced formation of P680(+) (ΔA at 820 nm). The relatively slow relaxation kinetics of the latter signal (t1/2 ≈ 1 ms) may suggest that in a substantial fraction of the RCs QA remains bound to the complex.

Supramolecular structure of the photosystem II complex from green plants and cyanobacteria.

Photosystem II (PSII) complexes, isolated from spinach and the thermophilic cyanobacterium Synechococcus elongatus, were characterized by electron microscopy and single-particle image-averaging analyses. Oxygen-evolving core complexes from spinach and Synechococcus having molecular masses of about 450 kDa and dimensions of approximately 17.2 x 9.7 nm showed twofold symmetry indicative of a dimeric organization. Confirmation of this came from image analysis of oxygen-evolving monomeric cores of PSII isolated from spinach and Synechococcus having a mass of approximately 240 kDa. Washing with Tris at pH 8.0 and analysis of side-view projections indicated the possible position of the 33-kDa extrinsic manganese-stabilizing protein. A larger complex was isolated that contained the light-harvesting complex II (LHC-II) and other chlorophyll a/b-binding proteins, CP29, CP26, and CP24. This LHC-II-PSII complex had a mass of about 700 kDa, and electron microscopy revealed it also to be a dimer having dimensions of about 26.8 and 12.3 nm. From comparison with the dimeric core complex, it was deduced that the latter is located in the center of the larger particle, with additional peripheral regions accommodating the chlorophyll a/b-binding proteins. It is suggested that two LHC-II trimers are present in each dimeric LHC-II-PSII complex and that each trimer is linked to the reaction center core complex by CP24, CP26, and CP29. The results also suggest that PSII may exist as a dimer in vivo.

Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach (Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light‐harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen‐evolving complex was found to be independent of phosphorylation.The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.

Isolation and Partial Characterization of a Manganese and Chloride Binding Protein Present in Highly Purified Photosystem II Complexes of the Thermophilic Cyanobacterium Synechococcus sp.: The Protein Being Detected by Its L -Arginine Metabolizing Activity

Photosystem II complexes were solubilized with the detergent sulfobetaine 12 from thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. and purified by two sucrose gradient centrifugations and by chromatography on a Mono Q column. In such photosystem II complexes having a photosynthetic O2, evolving activity of 2938 μmol O2 evolved/mg chlorophyll x h, an ʟ-arginine metabolizing activity leading to ornithine and urea as major products, could be shown to be present. Besides ornithine and urea, a product (or products) of yet unknown structure is formed in addition - especially under aerobic conditions. This activity remained associated with photosystem II complexes even after substantial additional treatments to remove loosely bound proteins. On chlorophyll basis the maximal activity obtained under optimal assay conditions corresponded to 94 μmol ornithine formed/mg chlorophyll x h. This PS II associated, ʟ-arginine metabolizing enzyme was isolated (utilizing a manganese charged chelating Sepharose 6 B column) and partially characterized. It could be shown that this enzyme requires manganese and chloride for its ʟ-arginine metabolizing activity and that manganese becomes totally lost during purification indicating that manganese is bound to a fairly exposed site on the protein. Since it is rather unlikely that two different manganese and chloride binding proteins are present in such highly purified photosystem II complexes, the possibility of this protein being the water oxidizing enzyme will be discussed. Whether the manganese and chloride requiring ʟ-arginine metabolizing activity of this protein which provided a suitable assay for its isolation from photosystem II complexes, has any physiological significance, can not be answered at the present time.

Spectroscopic characterization of PS I core complexes from thermophilic Synechococcus sp : Identical reoxidation kinetics of A −1 before and after removal of theiron-sulfur-clusters F A and F B

Monomeric and trimeric PS I complexes missing the three stromal subunits E,C and D (termed PS I core complexes) were prepared from the thermophilic cyanobacterium Synechococcus sp. by incubation with urea. The subunits E,C and D are sequentially removed. In the monomeric PS I the subunit C is removed with a half life of approx. 5 min. This is about eight times faster than in the trimeric PS I complex. In parallel with the removal of the F A B containing subunit C the reduction kinetics of P700 + changed from a half life of about 25 ms to about 750 μs. The partner of P700 + in the 750 μs charge recombination was identified to be F X by the difference spectrum of this phase. There are some minor differences in the spectra of trimeric and monomeric PS I core complexes. At 77K the forward electron transfer from A − 1 to F X is blocked in the major fraction of the PS I core complexes and P700 +A − 1 recombines with a half life of about 220 μs. In the remaining fraction P700 +F X − is formed and decays with a half life of approx. 10 ms at 77 K. The kinetics of the forward electron transfer from A − 1 to the iron-sulfur-clusters was measured in the native PS I and the corresponding core complexes. The reoxidation kinetics of A − 1 are identical in both cases ( t 1 2 = 180 ns). We conclude that Fx is an obligatory intermediate in the normal forward electron transfer.

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase fromSynechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacterium Synechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6-8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min-1 at 40 degrees C. The activation energy was 12.4 Kcal for t > 29 degrees C and 20.6 Kcal for t < 29 degrees C. The specific absorption coefficient, A 1% 1cm 280 mm of the pure enzyme in phosphate buffer at pH 6.8 was 15.2. By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer. When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.

The genes in the thermophilic cyanobacterium Synechococcus vulcanus encoding cytochrome- c oxidase

It is still controversial whether cyanobacteria (blue-green algae) contain an aa 3-type cytochrome- c oxidase. We have approached this problem using DNA analysis. Using a DNA probe coding for the most conserved part of subunit I of the Bacillus enzymes, structural genes for the oxidase of a thermophilic cyanobacterium Synechococcus vulcanus were cloned and sequenced. We found genes for subunits II, I, III and IV of this order like those of the Bacillus enzymes, and a terminator structure after the gene for subunit IV. The deduced protein sequences for the subunits II, I and III showed consensus amino-acid residues atevery important portion, suggesting that these genes are operating. However, the S. vulcanus oxidase lacked a cytochrome- c-moiety fused to subunit II, the 13th and 14th hydrophobic segments of subunit I which are lacking in the Paracoccus enzyme, and the 1st and 2nd ones of subunit III which are lacking in the Bacillus enzyme, were not found. A gene homologous to ctaB gene, which locates at the 5′-upstream region of the gene for subunit II and co-transcribed in Bacillus subtilis, was not found. Comparison of protein sequences showed that S. vulcanus cytochrome oxidase is closer to Bacillus cytochrome oxidases than the mitochondrial and Paracoccus enzymes, or quinol oxidases from B. subtilis and Escherichia coli.

Cellular localization of cytochrome c550. Its specific association with cyanobacterial photosystem II.

Cellular localization of cytochrome (cyt) c550, a low potential, c-type monoheme cytochrome, in a thermophilic cyanobacterium Synechococcus vulcanus was investigated by systematic fractionation of the cells followed by its enzymatic and immunological detection. While cyt c-553, a soluble cyt that donates an electron to P700, was detected in the supernatant of osmotic disruption of lysozyme-treated cells, cyt c550 was detected only in the thylakoid membrane fraction, being tightly bound to thylakoids, and its removal required sonication in the presence of 1 M CaCl2. Upon further fractionation of the thylakoids into photosystem (PS) I and PSII by lauryl dimethylamine N-oxide solubilization, cyt c550 was exclusively concentrated in the crude PSII fraction together with cyt f, with no significant amount being detected in any of the soluble and PSI fractions. Upon further fractionation of the crude PSII by n-dodecyl beta-D-maltoside solubilization followed by column chromatography, cyt c550 was detected exclusively in the purified PSII core complex fraction but not in any other fractions. A 12-kDa protein, one of the extrinsic components of cyanobacterial PSII, exhibited completely the same behavior as that of cyt c550 during these fractionation procedures. These results, coupled with our previous results that cyt c550 binds stoichiometrically to the cyanobacterial PSII core complex and enhances O2 evolution (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832), indicate that there is only one species of cyt c550 in cyanobacterial cells and that this cyt is exclusively associated with PSII as a functional component for O2 evolution.

Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716.

The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences. They are arranged at three separate loci. Six of them are in one cluster in the order a: c: b': b: delta: alpha, and those for the beta and epsilon subunits form a second and separate cluster. The gene for the gamma-subunit is at a third site. As in other bacteria, the gene for subunit a is immediately preceded by a gene coding for a small hydrophobic protein of unknown function, known as uncI in Escherichia coli. The gene orders in Synechococcus 6716 are related to the orders of ATP synthase genes in the plastid genomes of higher plants, and particularly of a red alga and a diatom. The sequences of the subunits are similar to those of chloroplast ATP synthase, the alpha, beta and c subunits being particularly well conserved. Differences in the primary structures of the Synechococcus 6716 and chloroplast gamma subunits probably underlie different mechanisms of activation of ATP synthase. The nucleotide sequences that are presented also contain 12 other open reading frames. One of them encodes a protein sequence related to the E. coli DNA repair enzyme, photolyase, and another codes for a protein that contains internal repeats related to sequences in the myosin heavy chain.

Protein and substrate coordination to the manganese cluster in the photosynthetic water oxidizing complex: nitrogen 15 and proton ENDOR spectroscopy of the S2 state multiline signal in the thermophilic cyanobacterium Synechococcus elongatus

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProtein and substrate coordination to the manganese cluster in the photosynthetic water oxidizing complex: nitrogen 15 and proton ENDOR spectroscopy of the S2 state multiline signal in the thermophilic cyanobacterium Synechococcus elongatusX. S. Tang, M. Sivaraja, and G. C. DismukesCite this: J. Am. Chem. Soc. 1993, 115, 6, 2382–2389Publication Date (Print):March 1, 1993Publication History Published online1 May 2002Published inissue 1 March 1993https://doi.org/10.1021/ja00059a037RIGHTS & PERMISSIONSArticle Views139Altmetric-Citations47LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

Nucleotide sequence of the Mn-stabilizing protein gene of the thermophilic cyanobacterium Synechococcus elongatus

The nucleotide sequence of the psbO gene encoding the extrinsic 33 kDa protein (the Mn-stabilizing protein) from the thermophilic cyanobacterium Synechococcus elongatus was determined. The deduced amino acid sequence consisted of 272 residues; 26 for the signal peptide and 246 for the mature protein. The amino acid sequences of nine proteolytic peptides from the isolated protein completely agreed with the deduced amino acid sequence. Several unique variations of amino acids were found in the primary structure, of which some may be related to the high thermostability of the protein.

Activation of the H +-ATP synthase in thylakoid vesicles from the cyanobacterium Synechococcus 6716 by [formula omitted]. Including a comparison with chloroplasts, and introducing a new method to calibrate light-induced [formula omitted

Activation of ATP hydrolysis activity in well-coupled membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 has been studied. In addition to a basal (dark) activity dependent on culture age, the cyanobacterial thylakoid ATP synthase is activated by Δ \\ ̄ gm H + . Activation of this enzyme has been studied quantitatively and the results are compared with literature data regarding activation of the chloroplast ATP synthase. We found that activation behaviour both in chloroplasts and in cyanobacterial membrane vesicles may be characterized by the value of Δ \\ ̄ gm H + where 50% activation occurs ( Δ \\ ̄ gm a ) and a Hill-type coefficient ( p) that estimates the number of protons involved. The activating Δ \\ ̄ gm H + was induced either by the acid-base pulse method, or by illumination. To calibrate the size of light-induced Δ \\ ̄ gm H + , a method has been developed whereby at each light intensity the value of ΔG p is determined where the ATP synthase reaction is in equilibrium. Δ \\ ̄ gm H + at equilibrium then is calculated from this ΔG p and the H + ATP ratio. The value of p for the cyanobacterial ATP synthase is similar to that of the chloroplast enzyme (between 1 and 2). The cyanobacterial enzyme resembles the thiol-modulated (reduced) form of the chloroplast ATP synthase in that Δ \\ ̄ gm a is low (14.1 kJ mol −1) and that the Δ \\ ̄ gm H + -activated state is quite stable. As in the thiol-modulated chloroplast ATP synthase, decay of the activated state is accelerated by ADP. We conclude that the Synechococcus 6716 membrane vesicles are an excellent system in which to study Δ \\ ̄ gm H + activation of ATP synthase with no need for thiol modulation.

Binding and functional properties of two new extrinsic components, cytochrome c-550 and a 12-kDa protein, in cyanobacterial photosystem II.

Cytochrome c-550, a low-potential c-type cytochrome, and a 12-kDa protein were recently shown to be associated extrinsically and stoichiometrically with purified photosystem II (PSII) complex of the thermophilic cyanobacterium Synechococcus vulcanus [Shen, J.-R., Ikeuchi, M., & Inoue, Y. (1992) FEBS Lett. 301, 145-149]. The binding and functional properties of these two extrinsic components in PSII were studied by means of release-reconstitution and thermoluminescence techniques. The following results were obtained: (i) cyt c-550 rebound appreciably to cyanobacterial PSII in the absence of the 33- and 12-kDa extrinsic proteins, but the presence of these two proteins facilitated the rebinding, affording a full level of binding equal to that in native PSII. (ii) The 12-kDa protein did not rebind to PSII at all unless the 33-kDa protein or cyt c-550 was present. It rebound only partially in the presence of either of these two proteins, but it rebound maximally when reconstituted together with both of them. (iii) Reconstitution with cyt c-550 or the 12-kDa protein alone in the absence of the 33-kDa protein did not restore the O2-evolving activity of CaCl2-washed PSII. Reconstitution with cyt c-550 in combination with the 33-kDa protein appreciably enhanced the activity, but the activity restoration was much more marked and reached a level close to that of the original activity when all three extrinsic proteins were included.(ABSTRACT TRUNCATED AT 250 WORDS)

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. I. Is the psaF gene product required for oxidation of cytochrome c-553?

Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides. Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products. Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product. Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide. The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553. Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide. Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553. Mg 2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not. The function of this subunit polypeptide is discussed.

Co-reconstitution of the H +-ATP synthase and cytochrome [formula omitted] complex from a thermophilic cyanobacterium : High ATP yield and mutual effects on the enzymatic activities

The H +-ATP synthase and the cytochrome b-563 c-554 (Q bc) complex were selectively solubilized and isolated from the thylakoid membranes of the thermophilic cyanobacterium Synechococcus 6716. The enzymes were co-reconstituted into proteoliposomes with the native cyanobacterial lipids by detergent dialysis. After (co-)reconstitution of the protein complexes in variable stoichiometries, the different enzymatic activities were analyzed. Optimization of the functionally coupled enzymes led to a compromise for the weight ratios of 20 mg lipid and 0.5 mg of both complexes per ml of reconstitution medium. The average diameter (around 300 nm) and zeta potential of these proteoliposomes were determined. The oxidoreductase activity of reconstituted Q bc complex was found to be stimulated by the presence of co-reconstituted (inactive) ATP synthase. Conversely, ATP hydrolysis was inhibited or stimulated by the protonmotive force generated by the Q bc complex, co-reconstituted in the right-side out or inside-out orientation, respectively. In proteoliposomes containing trapped horse-heart cytochrome c the proton movement induced by the Q bc complex resulted in an ATP production of about 1 μmol ATP per mg of ATP synthase within 1 min. The mutual stimulation and high ATP synthesis activity may indicate improved incorporation and/or some kind of functional association of these proteins in the membrane, although as yet aspecific protein-stabilizing effects cannot be excluded.