Red antenna states of photosystem I from Synechococcus sp. PCC 7002

Absorption, fluorescence and single-molecule spectroscopy at low temperatures were used to elucidate spectral properties, heterogeneities and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacterium Synechoccocus sp. PCC 7002. The 77 K absorption spectrum indicates the presence of 2–3 red-shifted Chla’s absorbing at about 708 nm. The fluorescence emission spectrum is dominated by a broad band at 714 nm. The emission spectra of single PSI complexes show zero-phonon lines (ZPLs) as well as a broad intensity distribution without ZPLs. The spectral region below 710 nm often shows ZPLs, they form a spectral band with a maximum at 698 nm (F698). The region above 710 nm is dominated by broad intensity distributions and the observation of ZPLs is less frequent. The broad distributions are due to the emission of the C708 Chla’s and the emission from F698 stems from a Chla species absorbing at the blue side of P700. The properties of these two emissions show a close relation to those of the C708 and C719 pools observed in T. elongatus. Therefore an assignment of F698 and C708 to Chla-species with similarities to C708 and C719 in T. elongatus is proposed.     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002

Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.     

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.     

聚球藻7002在光生物反应器中的光自养培养

通过对聚球藻7002在光生物反应器中的培养,研究了光强在聚球藻7002培养液中的衰减规律,得到了培养过程光强随藻细胞浓度和光程距离变化的关系式,即I=I₀exp［-(-0.0239+0.0777OD₇₅₀)·L］。并对培养过程特性及培养温度、外加CO₂浓度和光照强度对藻细胞生长的影响进行了较为详细的研究,得到了反应器中较为适宜的聚球藻7002的培养条件,藻细胞培养密度达到3.4g/L(干重),体积产率达到0.57g/(L·d)的较高水平。     

FNRD在聚球藻7002中的表达及其性质研究

置于Lac启动子和Kan启动子控制之下的petHL基因分别转化蓝细菌Synechococcussp.PCC7002,从Southern blot分析结果推断,petHL已整合到蓝细菌染色体DNA上。Western blot分析表明,转入蓝细菌体内的petHL基因得到了表达,且Kan启动子启动该基因表达的效率高于Lac启动子。内源FNRD表现出与FNR全酶相同的稳定性。Triton X-114分相实验结果显示,部分FNRD可进入Triton X-114相,推测这些分子可能发生了脂酰化修饰。同时FNRD在体内可能参与了光合电子传递而使光合放氧速率增加。     

A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl--maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A molecular weight of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents.The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P₇₀₀. The full set of Fe-S clusters (F_A, F_B and F_X) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy.Abbreviations BA benzamidine - CAS 6-amino-n-caproic acid - C₈-G octyl--D-glucopyranoside - C₁₂-M lauryl--D-maltoside - C₁₀-M decyl--D-maltoside - C₈-TG octyl--D-thioglucoside - Chl a chlorophyll a - EPR electron paramagnetic resonance - F_A, F_B, F_X iron-sulfur centers - HPLC high perfomance liquid chromatography - kDa kilodalton(s) - LDAO lauryldimethylamine oxide - MES 2-(N-morpholino)ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - P₇₀₀ primary electron donor - SB12 sulfobetain 12 - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tris tris(hydroxymethyl)-aminomethane     

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q_A, gave a decreased O₂ evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Binding affinity of bicarbonate and formate in herbicide-resistant D1 mutants of Synechococcus sp. PCC 7942

We examined the effects of mutations at amino acid residues S264 and F255 in the D1 protein on the binding affinity of the stimulatory anion bicarbonate and inhibitory anion formate in Photosystem II (PS II) in Synechococcus sp. PCC 7942. Measurements on the rates of oxygen evolution in the wild type and mutant cells in the presence of different concentrations of formate with a fixed bicarbonate concentration and vice versa, analyzed in terms of an equilibrium activator-inhibitor model, led to the conclusion that the equilibrium dissociation constant for bicarbonate is increased in the mutants, while that of the formate remains unchanged (11±0.5 mM). The hierarchy of the equilibrium dissociation constant for bicarbonate (highest to lowest, ±2 M) was: D1-F255L/S264A (46 M)>D1-F255Y/ S264A (31 M)D1-S264A (34 M)D1-F255Y (33 M)>wild type (25 M). The data suggest the importance of D1-S264 and D1-F255 in the bicarbonate binding niche. A possible involvement of bicarbonate and these two residues in the protonation of Q_B ^-, the reduced secondary plastoquinone of PS II, in the D1 protein is discussed.Abbreviations Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - MES 2-[N-morpholino]ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - Q_A bound plastoquinone, a one-electron acceptor in Photosystem II - Q_B another bound plastoquinone, a two-electron acceptor in Photosystem II This paper is dedicated to the memory of my dear friend Robin Hill-Govindjee.     

Transformation of the fresh water cyanobacterium Synechococcus PCC7942 with the shuttle-vector pAQ-EX1 developed for the marine cyanobacterium Synechococcus PCC7002

The transformation of the fresh water cyanobacterium Synechococcus PCC7942 with the shuttle-vector pAQ-EX1 developed for the marine cyanobacterium S. PCC7002 was examined. The S. PCC7942 cells were successfully transformed with the pAQ-EX1 vector, and the vector was stably maintained in the transformant cells.     

聚球藻7002嗜铁素的检测与分离

【目的】探究聚球藻7002嗜铁素的检测和分离方法,为深入研究海洋嗜铁素提供科学依据。【方法】在缺铁MediumA中培养聚球藻7002,利用双层平板法、混合平板法和传统铬天青S(CAS)平板法定性检测嗜铁素,用CAS蓝色液体检测液定量检测嗜铁素。采用大孔树脂XAD-2和固定化金属离子亲和层析(IMAC)对嗜铁素进行分离,IMAC采用降低pH和竞争性洗脱两种洗脱方式。【结果】混合平板定性检测法更快速、高效、便捷。缺铁培养的聚球藻7002发酵液中,嗜铁素的相对含量高达93.50%。大孔树脂分离,上样液pH调为2.0时,嗜铁素吸附充分,分离效果较好。试验发现,分离得到的聚球藻7002嗜铁素在254nm紫外下具有明显的荧光特性。【结论】试验得到了聚球藻7002嗜铁素定性检测和分离的有效方法。     

Reconstitution of iron-sulfur center FB results in complete restoration of NADP+ photoreduction in Hg-treated Photosystem I complexes from Synechococcus sp. PCC 6301

The F_B iron-sulfur cluster is destroyed preferentially by treating Photosystem I complexes with HgCl₂(Kojima Y, Niinomi Y, Tsuboi S, Hiyama T and Sakurai H (1987) Bot Mag 100: 243–53). When F_B is 95% depleted but F_Ais quantitatively retained in cyanobacterial PS I complexes, the reduction potential of F_A remains highly electronegative (E_m=–530 mV, n=1), the EPR spectral and spin relaxation properties of F_A and F_Xremain unchanged, but NADP⁺ photoreduction rates decline from 552 to 72 mol mg Chl^–1 h^–1.When F_B is reconstituted with FeCl₃, Na₂S and -mercaptoethanol, NADP⁺photoreduction rates recover to 528 mol mg Chl^–1 h^–1. The correlation between the presence of F_Band NADP⁺ photoreduction provides direct experimental evidence that this iron-sulfur cluster is required for electron throughput from cytochromec ₆ to flavodoxin or ferredoxin in Photosystem I.Abbreviations Chl chlorophyll - DPIP dichlorophenolindophenol - PS I Photosystem I Published as Journal Series #11091 of the University of Nebraska Agricultural Research Division. This paper is dedicated to the memory of the late Professor Daniel Arnon, who is remembered for his gracious and generous encouragement of the senior author's early career.     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

人尿激酶原基因在聚球藻7002中的克隆和表达

将人尿激酶原基因连在ＰｐｓｂＡ启动子之后,再将启动子连同基因克隆入整合载体ｐＴＺ１８中。ｐＴＺ１８－８中含有一段来源于集胞藻６８０３的ｐｓｂＢ基因片段作为整合平台。将整合表达载体用直接转化的方法转聚球藻Ｓｙｎｅ－ｃｈｏｃｏｃｃｕｓ ｓｐ．ＰＣＣ７００２中。经氨苄青霉素选前扩大培养后的转化进行ＤＮＡ斑点印迹及Ｗｅｓｔｅｒｎ印迹, 基因的存在及表达,菌体破碎后的上清液有较高的溶解纤维蛋白的活性,说明表     

Cloning and Expression of Human Pro-urokinase Gene in the Cyanobacterium Synechococcus sp. PCC 7002

Pro-urokinase (pro-UK) gene was ligated with promoter PpsbA and cloned into the integrative vector pTZ18-8, which contained a psbB gene fragment from Synechocystis sp. PCC 6803 as the integrative platform. The expression vector was transferred into Synechococcus sp.PCC 7002 via natural transformation. Transformants conferring ampicillin resistance were amplified and then analyzed. DNA dot blot and Western blot demonstrated the existence and expression of pro-UK gene. The supernatant from crude cell extract showed thrombolytic activity, indicating that the expression product did not form inclusion bodies. According to the results of ELISA, expression of pro-UK was about 2×10 -5 -3×10 -5 g per gram of wet cells.     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

A novel plasmid recombination mechanism of the marine cyanobacterium Synechococcus sp. PCC7002.

We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria.     

Phycobilisome Mobility in the Cyanobacterium Synechococcus sp. PCC7942 is Influenced by the Trimerisation of Photosystem I

We have constructed a mutant of the cyanobacterium Synechococcus sp. PCC7942 deficient in the Photosystem I subunit PsaL. As has been shown in other cyanobacteria, we find that Photosystem I is exclusively monomeric in the PsaL(-) mutant: no Photosystem I trimers can be isolated. The mutation does not significantly alter pigment composition, photosystem stoichiometry, or the steady-state light-harvesting properties of the cells. In agreement with a study in Synechococcus sp. PCC7002 [Schluchter et al. (1996) Photochem Photobiol 64: 53-66], we find that state transitions, a physiological adaptation of light-harvesting function, occur significantly faster in the PsaL(-) mutant than in the wild-type. To explore the reasons for this, we have used fluorescence recovery after photobleaching (FRAP) to measure the diffusion of phycobilisomes in vivo. We find that phycobilisomes diffuse, on average, nearly three times faster in the PsaL(-) mutant than in the wild-type. We discuss the implications for the mechanism of state transitions in cyanobacteria.