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.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.     

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.     

集胞藻PCC 6803高产精氨酸藻株的紫外诱变选育

精氨酸在医药和食品工业上具有广泛用途。集胞藻PCC 6803是单细胞蓝藻, 能利用工业废气(主要成分是氮氧化物NO_x)与水反应生成的硝酸盐和亚硝酸盐合成氨基酸等化合物, 因而选育高产精氨酸藻株, 不仅能提高精氨酸产量, 而且能去除工业废气中的NO_x, 具有潜在的应用前景。研究在集胞藻PCC 6803中利用紫外诱变, 筛选抗0.8 g/L D-精氨酸和抗0.2 g/L 6-氮尿嘧啶的突变株, 选育到了一株精氨酸产量显著提高的突变株#13807-111-55, 它每OD₇₃₀值细胞的胞外精氨酸产量相比出发株提高了62.3倍, 达到(0.76±0.1) mg/(L·OD₇₃₀), 总精氨酸产量相比出发株提高了6.0倍, 达到(0.82±0.08) mg/(L·OD₇₃₀)。该突变株每OD₇₃₀值细胞的胞外精氨酸产量明显高于胞内, 表明该突变藻株是精氨酸分泌型, 因而具有潜在的应用前景。     

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

Phototactic motility in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 is a unicellular motile cyanobacterium that shows positive and negative phototaxis on agar plates under lateral illumination. Recent studies on the molecular mechanisms of the phototactic motility of Synechocystis have revealed that a number of genes are responsible for its pilus-dependent motility and phototaxis. Here we describe what is known about these genes. We also discuss the novel spectral properties of the phytochrome-like photoreceptor PixJ1 in Synechocystis, that is essential for positive phototaxis and which has revealed the existence of a new group of chromophore-binding proteins in cyanobacteria.     

Phycobilin biosynthetic reactions in extracts of cyanobacteria

Phycobilins are the chromophores of phycobiliproteins, the light-harvesting pigments of cyanobacteria, red algae and cryptophytes. Phycobilins are biosynthesized from heme by the action of heme oxygenase, which converts heme to biliverdin, followed by the action of other enzymes that convert biliverdin to the phycobilins. We previously reported on the enzymes and biosynthetic intermediates of phycobilin formation in extracts of the unicellular red alga Cyanidium caldarium. Heme oxygenase activity has now been obtained from extracts of the cyanobacterium Synechocystis sp. PCC 6701. The reaction requirements are similar to those for the C. caldarium enzyme: heme substrate, reduced ferredoxin, and a second reductant such as ascorbate or Trolox. The enzymatic nature of the reaction was verified by two criteria in addition to the requirement for cell extract: production of only the IX isomer of the bilin product and inhibition by the substrate analog Sn-protoporphyrin IX. The enzyme was partially purified by high-speed centrifugation, 35–75% differential (NH4)2SO4 precipitation, and DEAE-cellulose anion exchange chromatography. In addition, extract capable of converting biliverdin IX to phycobilins has been obtained from Synechocystis sp. PCC 6701 and another cyanobacterium, Synechocystis sp. PCC 6803. Only the (3Z) isomers of the phycobilins accumulated in the incubations containing unfractionated cell extracts, in contrast to incubations with unfractionated C. caldarium extracts which produce both the (3Z) and (3E) isomers. Phycocyanobilin and phycoerythrobilin were produced in comparable amounts by Synechocystis sp. PCC 6701 extracts, but only phycocyanobilin accumulated in Synechocystis sp. PCC 6803 extracts. This difference in in vitro product accumulation correlates with the phycobilins that are found in vivo in these two cell types.     

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Proteomic study of the soluble proteins from the unicellular cyanobacterium Synechocystis sp. PCC6803 using automated matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting

The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigating environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5-5.5) zoom gels, automated matrix-assisted laser desorption/ionization mass spectrometry acquisition, spectral processing and database searching. The work sets the foundation for investigations of proteomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not previously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism.     

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Effects of selected electron transport chain inhibitors on 24-h hydrogen production by Synechocystis sp. PCC 6803

One factor limiting biosolar hydrogen (H(2)) production from cyanobacteria is electron availability to the hydrogenase enzyme. In order to optimize 24-h H(2) production this study used Response Surface Methodology and Q2, an optimization algorithm, to investigate the effects of five inhibitors of the photosynthetic and respiratory electron transport chains of Synechocystis sp. PCC 6803. Over 3 days of diurnal light/dark cycling, with the optimized combination of 9.4 mM KCN (3.1 μmol 10(10) cells(-1)) and 1.5 mM malonate (0.5 μmol 10(10) cells(-1)) the H(2) production was 30-fold higher, in EHB-1 media previously optimized for nitrogen (N), sulfur (S), and carbon (C) concentrations (Burrows et al., 2008). In addition, glycogen concentration was measured over 24 h with two light/dark cycling regimes in both standard BG-11 and EHB-1 media. The results suggest that electron flow as well as glycogen accumulation should be optimized in systems engineered for maximal H(2) output.     

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.     

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.     

Comparison of tryptophan fluorescence lifetimes in cyanobacterial photosystem I frozen in the light and in the dark

Photosynthesis Research - The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the...     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

Visualisation of cyanobacteria in aqueous solution by atomic force microscopy

Methodical approaches for studying of living cells in aqueous solutions by atomic force microscopy (AFM) are demonstrated. Images of intact cyanobacteria Synechocystis PCC 6803 in TES buffer were captured in tapping mode using aminomodified mica as AFM substrate. Modification of freshly cleaved mica has been done in 3-aminopropyltri-ethoxysilane vapours. The average size of cyanobacteria was determined from AFM images. The linear size of Synechocystis PCC 6803 in TES buffer was equal to 70 x 90 nm and their height was about 20 nm. Possible causes of insufficiently high resolution of the cyanobacteria AFM images in aqueous solutions and possible ways for gaining molecular resolution in studies of structural, functional and micromechanical properties of living cells are discussed.