Identification of novel targets of cyanobacterial glutaredoxin

Glutaredoxins (Grxs) are small ubiquitous glutathione-disulfide oxidoreductase that reduce disulfide bonds of target proteins and maintain the redox homoeostasis of cells. Disruption of ssr2061 reduced the viability of cells indicated Grx2061 has a protective role against oxidative stress in Synechocystis sp. PCC 6803. To understand the function of Grx2061 in cyanobacteria and its difference from plant, Grx targets were retained specifically on an affinity media coupled with a mutated monocysteinic Grx and identified by mass spectra. Among 42 identified targets, 26 of them are novel ones compared with those known in higher plants. These proteins are supposed to be involved in 12 cellular processes including oxidative stress response, Calvin cycle, protein synthesis, and etc. Biochemical tests highlighted four of them which showed a Grx-dependent activation of peroxiredoxin and deactivation of catalase. Oxidized Grx2061 could keep redox equilibrium with another probable Grx and be reduced by thioredoxin reductase, indicating that Grx2061 can accept electrons from either glutathione or thioredoxin reductase. Our studies suggest Grx2061 in cyanobacteria plays an important role in redox network and its targets are as extensive as that in other organisms.     

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli

The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.     

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.     

Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp. PCC 6803 in vivo in ssr2016 deletion mutants generated either in a wild-type background or in a ndhB deletion mutant. Our results indicate that ssr2016 is required for FQR and that it operates in a parallel pathway to the NDH1 complex. The ssr2016 deletion mutants are high light sensitive, suggesting that FQR might be important in controlling redox poise under adverse conditions.     

phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.     

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria

Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.     

Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity. They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP. KtrABE neither mediated Na(+) uptake nor Na(+) efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore. A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K(+) from the cells. The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level. These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.     

小鼠金属硫蛋白在聚胞藻中的金属诱导表达与纯化

应用蓝藻类金属硫蛋白基因启动子(smt O-P)的金属诱导性,在单细胞的聚胞藻PCC 6803中表达小鼠金属硫蛋白结构基因(mMT-1 cDNA)。在大肠杆菌HB 101中构建含有smt O-P和mMT1 cDNA的穿梭表达载体pKT-MRE,经质粒转移,链霉素筛选,Southern和Western杂交分析鉴定得稳定的转基因工程藻落。同时,做小批量锌诱导表达,并纯化了外源蛋白,5L培养液含鲜藻重5.0g,得到3.5mg mMT-1;转基因藻在高金属浓度下的耐受性测定表明,外源基因的表达提高了蓝藻对金属离子的抗性,约为野生藻的2倍。     

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.     

A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular Cyanobacterium synechocystis sp. strain PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF sll1575, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain sll1575 was part of a gene (designated spkA) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing spkA into ORFs sll1574 and sll1575. The structural integrity of spkA agreed well with the motility phenotype, determined by colony morphology on agar plates. The spkA gene was expressed in Escherichia coli as a His-tagged protein, which was purified by Ni2+ affinity chromatography. With [gamma-32P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction of Synechocystis cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in Synechocystis.     

The thioredoxin reductase-glutaredoxins-ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds ( Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis -like selenate reductase.     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

集胞藻PCC6803膜脂循环关键基因酶学性质和生理功能

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索,发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969,但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用,加深对集胞藻PCC6803脂肪酸代谢途径的了解,文中将sll1969基因在大肠杆菌中过表达和体外纯化,得到重组蛋白Sll1969,并对其酶学性质进行初步分析。在30℃条件下,测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为(1.16±0.01)mmol/L和(332.8±10.0)/min;该脂肪酶的最适反应温度为55℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化,发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关,但Sll1969不是细胞中唯一的脂肪酶。     

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.     

Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803

The biosynthetic pathway for the pteridine moiety of cyanopterine, as well as tetrahydrobiopterine, has been investigated in Synechocystis sp. PCC 6803. Open reading frames slr0426, slr1626, slr0078 and sll0330 of the organism putatively encoding GTP cyclohydrolase I, dihydroneopterine aldolase, 6-pyruvoyltetrahydropterine synthase and sepiapterine reductase, respectively, have been cloned into T7-based vectors for expression in Escherichia coli. The recombinant proteins have been purified to homogeneity and demonstrated to possess expected genuine activities except that of sll0330. Our result is the first direct evidence for the functional assignment of the open reading frames in Synechocystis sp. PCC 6803. Furthermore, the 6-pyruvoyltetrahydropterine synthase gene is demonstrated for the first time in prokaryotes. Based on the result, biosynthesis of cyanopterine is discussed.     

Engineering desiccation tolerance in Escherichia coli

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.