Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Nucleotide sequence of psbC,the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.     

Isolation of two thioredoxins from the cyanobacterium Synechococcus 6301

Two different thioredoxins designated as thioredoxin A and B have been isolated from the cyanobacterium Synechococcus 6301. Methods for large scale purification of these thioredoxins were developed. Thioredoxin B has been purified to homogeneity; it has a molecular weight of 11,800 and an isoelectric point of 4.6. The following K _m data were obtained for this thioredoxin; a) in the PAPS-sulfotransferase assay of Synechococcus 6301: 10.7 M; b) in the fructose-1-6-bisphosphatase assay of Synechococcus 6301: 1.7 M; c) in the APS-sulfotransferase assay of Chroococcidiopsis 7203: 5.4M. Thioredoxin A has an isoelectric point of 4.1 and it is active in the PAPS-sulfotransferase and fructose-1-6-bisphosphatase of Synechococcus 6301; it is not active in the APS-sulfotransferase of Chroococcidiopsis 7203.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Amino acid sequence of photosystem I subunit IV from the cyanobacterium Synechocystis PCC 6803

We describe here the complete amino acid sequence of photosystem I subunit IV from Synechocystis 6803. The molecular mass of 8.0 kDa is lower than in higher plants and Chlamydomonas, due to the lack of a characteristic, proline-rich, N-terminal sequence. The remaining sequence exhibits a good conservation, with a hydrophilic and strongly basic N-tenninal head followed by two hydrophobic domains. There is no possibility of classical membrane-spanning alpha helices. This component is likely to be one of the most stroma accessible subunits of photosystem I.     

Oxygen evolving membranes and particles from the transformable cyanobacterium Synechocystis sp. PCC6803

Membranes and PS II particles retaining high rates of O₂-evolving activity have been isolated from the transformable cyanobacterium, Synechocystis sp. PCC6803. Membranes from cells grown under red light exhibit rates of O₂-evolution ranging from 500–700 mole O₂/mg chl/h. PS II particles are prepared by a simple procedure involving DEAE column chromatography of detergent extracts obtained by simultaneous treatment of membranes with octylglucoside and dodecylmaltoside. The isolated PS II fraction is enriched in polypeptides immunologically cross-reactive with polypeptides present in core reaction center preparations of spinach, exhibits 77 K fluorescence emission maxima at 685 and 696 nm, but not emission and absorption due to phycobilines and is capable of rates of O₂-evolution exceeding 1000 mole O₂/mg chl/h.Abbreviations DM dodecyl--D-maltoside - OG octyl--D-glucoside     

Isolation,sequence and expression in Escherichia coli of the nitrite reductase gene from the filamentous,thermophilic cyanobacterium Phormidium laminosum

The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N₂-fixing cyanobacterium Phormidium laminosum. Putative promoter-like and Shine-Dalgarno sequences appear at the 5 end of the 1533 bp long nir-coding region. The deduced amino acid sequence of NiR from P. laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR. This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 from the E. coli lac promoter and probably from the P. laminosum NiR promoter.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - NiR nitrite reductase - NR nitrate reductase - NT nitrate transport - SiR sulfite reductase     

Absence of the psbH gene product destabilizes the Photosystem II complex and prevents association of the Photosystem II-X protein in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PS II-H is a small hydrophobic protein that is universally present in the PS II core complex of cyanobacteria and plants. The role of PS II-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The psbH disruptant could grow photoautotrophically; however, its growth was much slower than that of the wild type cell. Chromatography enabled the isolation of active oxygen-evolving PS II complexes from both the mutant and the wild type. The mutant yielded a relatively large amount of inactive PS II complex that lacked the following extrinsic proteins: the 33-kDa protein, the 12-kDa protein, and cytochrome c ₅₅₀ . There were differences between the psbH disruptant and the wild type in terms of the oxygen evolution activities of the cells, thylakoids, and PS II complexes. At high concentrations of 2,6-DCBQ, the activity was much lower in the mutant than in the wild type. Gel filtration chromatography of the PS II complexes showed that both active and inactive PS II complexes isolated from the mutant were mostly in the monomeric form, while the active PS II complex from the wild type was in the dimeric form. The polypeptide composition of both active and inactive PS II complexes from the mutant showed the absence of another small polypeptide, PS II-X. These results suggest that the PS II-H protein is essential for stable assembly of native dimeric PS II complex containing PS II-X.     

The cyanobacterium Synechococcus modulates Photosystem II function in response to excitation stress through D1 exchange

In this minireview we discuss effects of excitation stress on the molecular organization and function of PS II as induced by high light or low temperature in the cyanobacterium Synechococcus sp. PCC 7942. Synechococcus displays PS II plasticity by transiently replacing the constitutive D1 form (D1:1) with another form (D1:2) upon exposure to excitation stress. The cells thereby counteract photoinhibition by increasing D1 turn over and modulating PS II function. A comparison between the cyanobacterium Synechococcus and plants shows that in cyanobacteria, with their large phycobilisomes, resistance to photoinhibition is mainly through the dynamic properties (D1 turnover and quenching) of the reaction centre. In contrast, plants use antenna quenching in the light-harvesting complex as an important means to protect the reaction center from excessive excitation.Abbreviations D1 reaction center protein of Photosystem II - P₆₈₀ the reaction center of Photosystem II - Q_A the primary quinone acceptor of Photosystem II - Tyr_Z tyrosine electron donor to P₆₈₀     

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.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.