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.Abbreviations ORF open reading frame - PS I Photosystem I - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This paper is dedicated to commemorate the late Professor D.I. Arnon with whom the senior author (T.H.) spent five years from 1974 to 1979 as his last postdoctoral fellow at the Department of Cell Physiology, University of California, Berkeley.The sequence data presented here have been submitted to DDBJ/EMBL/GenBank under the accession number D17355.     

Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.     

Structural implications for a phycobilisome complex from the thermophilic cyanobacterium Thermosynechococcus vulcanus

Phycobilisomes (PBSs) are huge, water-soluble light-harvesting complexes used by oxygenic photosynthetic organisms. The structures of some subunits of the PBSs, including allophycocyanin (APC) and phycocyanin (PC), have been solved by X-ray crystallography previously. However, there are few reports on the overall structures of PBS complexes in photosynthetic organisms. Here, we report the overall structure of the PBS complex isolated from the cyanobacterium Thermosynechococcus vulcanus, determined by negative-staining electron microscopy (EM). Intact PBS complexes were purified by trehalose density gradient centrifugation with a high-concentration phosphate buffer and then subjected to a gradient-fixation preparation using glutaraldehyde. The final map constructed by the single-particle analysis of EM images showed a hemidiscoidal structure of the PBS, consisting of APC cores and peripheral PC rods. The APC cores are composed of five cylinders: A1, A2, B, C1, and C2. Each of the cylinders is composed of three (A1 and A2), four (B), or two (C1 and C2) APC trimers. In addition, there are eight PC rods in the PBS: one bottom pair (Rb and Rb'), one top pair (Rt and Rt'), and two side pairs (Rs1/Rs1′ and Rs2/Rs2′). Comparison with the overall structures of PBSs from other organisms revealed structural characteristics of T. vulcanus PBS.     

Multiple forms of chlorophyll-protein complexes from a thermophilic cyanobacterium Synechococcus sp

Eight chlorophyll-proteins were resolved from the thylakoid membranes, or digitonin particles, of a thermophilic cyanobacterium Synechococcus sp. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six chlorophyll-proteins with slower electrophoretic mobilities were shown to be P700-chlorophyll a-protein complexes (CP1), whereas faster-moving proteins (CP2) were related to photosystem 2. Extraction of CP1 complexes from the membranes with different detergent/chlorophyll ratios and reelectrophoresis of extracted CP1 complexes indicated that the chlorophyll-proteins are closely interrelated with each other; any CP1 complex could be transformed to other CP1 complexes with faster electrophoretic mobilities. This, together with the Ferguson plot and the polypeptide composition, showed that six CP1 complexes are different in terms of polypeptide composition, oligomerization, SDS-binding, or conformation of the proteins but represent, in the order of increasing electrophoretic mobility, increasing degree of modification of the native P700-chlorophyll a-protein.     

Structure of c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 A: structural implications for thermal stability in phycobilisome assembly

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has been determined by molecular replacement to 2.5 A resolution. The crystal belongs to space group R32 with cell parameters a=b=188.43 A, c=61.28 A, alpha=beta=90 degrees, gamma=120 degrees, with one (alphabeta) monomer in the asymmetric unit. The structure has been refined to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 A. The crystals were grown from phycocyanin (alphabeta)(3) trimers that form (alphabeta)(6) hexamers in the crystals, in a fashion similar to other phycocyanins. Comparison of the primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella diplsiphon and the thermophilic Mastigocladus laminosus were performed. We show that each level of assembly of oligomeric phycocyanin, which leads to the formation of the phycobilisome structure, can be stabilized in thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each association interface. In addition, a significant shift in the position of ring D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin, enables the formation of important polar interactions at both the (alphabeta) monomer and (alphabeta)(6) hexamer association interfaces.     

Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.     

Effects of phospholipase and lipase treatments on photosystem II core dimer from a thermophilic cyanobacterium

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A(2) (PLA(2)) and lipase, and examined their effects on PSII structure and function. PLA(2)-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA(2)- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the Q(B)-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA(2)- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA(2)- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA(2)- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.     

Identification of photosystem I components from the cyanobacterium, Synechococcus vulcanus by N-terminal sequencing

The photosystem I core complex isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, is composed of eight low-molecular-mass proteins of 18, 14, 12, 9.5, 9, 6.5, 5 and 4.1 kDa in addition to the PS I chlorophyll protein. N-terminal amino acid sequences of all these components were determined and compared with those of higher plants. Clearly, the 9.5 kDa component corresponds to the protein which carries the non-heme iron-sulfur centers A and B. This protein is so poorly visualized by staining that it has probably been overlooked in gel electrophoresis analyses. The 18, 14, 12 and 9 kDa components show appreciable homology with respective subunits of higher plant PS I. In contrast, the 6.5, 5 and 4.1 kDa components do not correspond to any known proteins except that the sequence of the 4.1 kDa component matches an unidentified open reading frame (ORF) 42 (liverwort) or ORF44 (tobacco) of chloroplast DNA.     

The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus.

Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes. The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined. The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species. We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene. The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs. The S. vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles.     

Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein.

A 16-kDa protein, one of the major proteins that accumulates upon heat-shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity. The N-terminal and internal amino acid sequences of the protein exhibited a homology to the alpha-crystallin-related, small heat shock proteins from other organisms. The protein was designated HspA. Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo-oligomer consisting of 24 subunits. It prevented the aggregation of porcine malic dehydrogenase at 45 degrees C and 50 degrees C and citrate synthase at 50 degrees C. The activity of the malic dehydrogenase, however, was not protected under these heat-shock conditions or reactivated after a shift in temperature from 45 or 50 degrees C to 21 degrees C. HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP-independent manner. A homologue to the 16-kDa protein was also found to be induced upon heat-shock treatment in the mesophilic cyanobacterium Synechocystis sp. PCC 6803.     

Prokaryotic triterpenoids: Bacteriohopanetetrol glycuronosides from the thermophilic cyanobacterium Synechococcus PCC 6907

Abstract Diploptene and composite triterpenoids of the hopane series, 35-(0-β-galacturonosyl)-2β-methylbacteriohopanetetrol and 35-(0-α-glucuronosyl)-2β-methylbacteriohopanetetrol, a novel hopanoid, as well as their non-methylated equivalents, were isolated from the temperature resistant cyanobacterium Synechococcus PCC 6907. This is the first report of rare bacteriohopanetetrol glycosides containing glycuronic acid moieties from a cyanobacterium.     

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C₅) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C₂₀) diphosphate synthase, another encodes a farnesyl (C₁₅) diphosphate synthase whose optimal reaction temperature is 60 °C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 °C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C₂₅), hexaprenyl (C₃₀), and heptaprenyl (C₃₅) diphosphates from dimethylallyl (C₅) diphosphate, geranyl (C₂₀) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).     

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion. Received: 28 July 1997 / Accepted: 26 November 1997     

Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas. The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability. ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120 nmol X min-1 X mg protein-1, respectively; the former was strongly stimulated by an uncoupler. ATP hydrolysis induces membrane energization as monitored by membrane-potential- and surface-potential-indicating probes and by different pH indicators trapped inside the vesicles. The probes used were a membrane-bound fluorescent aminoacridine, which monitors surface charge-density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red. The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane-localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space. Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.     

Bipolar localization of putative photoreceptor protein for phototaxis in thermophilic cyanobacterium Synechococcus elongatus

We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp. PCC6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The mRNA of S. elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon. SePixJ was detected exclusively in the membrane fraction after cell fractionation. Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell; probably bound with the cytoplasmic membrane.     

The 1.45 A three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 A resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.     

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.