Rubidium transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 is a unicellular blue-green alga. The cells will grow on Rb⁺ as a substitute for K⁺ but at a slower rate (t₂～ 15 h versus 12 h). Potassium is not, strictly speaking, an essential element for Synechococcus. Rubidium duxes (using ⁸⁶Rb⁺) are much slower than those of potassium, about 1 nmol m^?2 s^?1 in the light (0.35 mol m^?3 Rb⁺). ⁸⁶Rb⁺ fluxes in the dark are about 0.1 nmol m^?2 s^?1. These fluxes are very slow compared to those of Na⁺ and other ions. Isotopic influx of Rb⁺ can supply sufficient Rb⁺ to keep up with the demands for growth, but the net dux needed to keep up with growth in the light is a large proportion of the total observed dux. Kinetic studies of Rb⁺ uptake versus [Rb⁺] show two uptake phases consistent with a high-affinity and a low-affinity system. Both systems appear to be light-activated. Transport of Rb⁺ appears to be passive at pH_o 10 in the light and dark. There is no case for active transport of Rb⁺ at pH_o 7.5 in the light, but a marginal case for active uptake in the dark (about 3 kJ mol^?1). There is only a small effect of Na⁺ upon Rb⁺ transport. ⁸⁶Rb⁺ should not be used in place of ⁴²K⁺ in K⁺ nutrition studies as the details of Rb⁺ transport are different to those of K⁺ transport.     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Nitrate transport in the cyanobacterium Anacystis nidulans

A significant progress in the knowledge of different aspects of nitrate transport in the unicellular cyanobacterium Anacystis (Synechococcus ) has been achieved in the last few years. The main contributions of our group are summarized in this article and discussed in relation to other information available. Endergonic accumulation of nitrate into the cells, indicative of the operation of an active nitrate transport system, has been experimentally substantiated and methods established to evaluate and analyze the activity of the system. Nitrate transport activity is sensitive to regulation exerted by products of both ammonium and CO₂ assimilation, thus providing evidence that photosynthetic nitrate assimilation in cyanobacteria is primarily controlled at the level of substrate supply to the cell. The expression of nitrate transport was also shown to be under nitrogen control, being repressed when ammonium is used as the nitrogen source. A 47-kDa polypeptide, which is a major plasma membrane component in nitrate-grown cells but is virtually absent in ammonium-grown cells, was identified as an essential component of the nitrate transporter. More recently, evidence of a strict Na'-dependence of active nitrate transport has been obtained, Δμ(Na⁺) appearing as the driving force of a sodium-nitrate symport system. Kinetic studies indicate also that the nitrate transporter may transport nitrite into the cell.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Uptake of Thallium, a Toxic Heavy-Metal, in the Cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. Leopoliensis) PCC 7942

Uptake of the toxic heavy-metal, thallium, was studied in thecyanobacterium Synechococcus R-2 (PCC 7942) using clinicallyavailable ²⁰¹Tl ⁺. Thallium was found to distribute across theplasmalemma passively, and so the accumulation ratio of theion ([Tl⁺]_i/[Tl⁺]_o) could be used to calculate the apparentmembrane potential (­_i,o) of the cells (^ETI⁺_i,o = ­_i,o).The permeability of the plasmalemma to TI⁺ (^PTI⁺ 1 to 5 nms^–1)is higher than that of K⁺. Valinomycin does not increase thepermeability of TI⁺. Transient changes in the ­_i,o of cells,because of electrogenic transport of ions, could be detectedfrom its effects upon the uptake rate of TI⁺. HCO^–₃ hyperpolarizedSynechococcus cells, whereas NH⁺₄, CH₃NH⁺, and K⁺ led to depolarization.The use of TI⁺ as a reporter of ­_i,o has some inherent limitations.Tl⁺ is toxic at very low concentrations (inhibitory effectsare apparent after about 6 h at concentrations as low as 1 mmolm^–3). The rate of equilibration is slow (t_1/25 to 20 min).Equilibration of TI⁺ takes about 2 h, which limits its valueas a membrane potential probe. Large amounts of TI⁺ bind tothe surface of the cells making the method impracticable formeasuring accumulation ratios of less than about 10 (­_i,o)values smaller than about –60 mV). Cultures continuouslyexposed to Tl⁺ (10 mmol m^–3) eventually become TI⁺ resistantby actively extruding TI⁺ (µTI⁺_i,o= –3±0.2kJ mol^–1) and so thallium cannot be used as a ­_i,oprobe in such cells. (Received October 28, 1997; Accepted August 31, 1998)     

A Unique Ascorbate Peroxidase Active Component in the Cyanobacterium Synechococcus PCC 7942 (R2)

Ascorbate peroxidase active component (APAC) was purified and characterized in Synechococcus PCC 9742 (R2) cells. APAC was isolated from freshly harvested cells, by ion exchange chromatography on DEAE cellulose, ultrafiltration through a 3000 dalton cut off filter and high pressure liquid chromatography through a reversed phase C-18 column. APAC was found to be extremely stable to harsh treatments of boiling water for 30 min, acidification to pH 2.0 and proteolytic digestion. A close correlation between activity and iron content of APAC was observed throughout the purification steps. E.S.R. spectrum of APAC showed a resonance line at g = 4.3 in the oxidized from. Peroxide reduction by ascorbate decreased the E.S.R. signal, which reappeared upon reoxidation by H₂O₂. The affinities of APAC to H₂O₂ and ascorbate were high (0.38 mM and 0.2 mM, respectively). Amino acid composition analysis of APAC revealed the presence of glutamic acid: glycine: cysteine residues at 2: 1: 1 ratio.     

Light adaptation of cyclic electron transport through Photosystem I in the cyanobacterium Synechococcus sp. PCC 7942

Photosystem I-driven cyclic electron transport was measured in intact cells of Synechococcus sp PCC 7942 grown under different light intensities using photoacoustic and spectroscopic methods. The light-saturated capacity for PS I cyclic electron transport increased relative to chlorophyll concentration, PS I concentration, and linear electron transport capacity as growth light intensity was raised. In cells grown under moderate to high light intensity, PS I cyclic electron transport was nearly insensitive to methyl viologen, indicating that the cyclic electron supply to PS I derived almost exclusively from a thylakoid dehydrogenase. In cells grown under low light intensity, PS I cyclic electron transport was partially inhibited by methyl viologen, indicating that part of the cyclic electron supply to PS I derived directly from ferredoxin. It is proposed that the increased PSI cyclic electron transport observed in cells grown under high light intensity is a response to chronic photoinhibition.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES energy storage - MV methyl viologen - PA_m photoacoustic thermal signal with strong non-modulated background light added - PA_s photoacoustic thermal signal without background light added CIW/DPB Publication No. 1205.     

Carbon-free production of 2-deoxy-scyllo-inosose (DOI) in cyanobacterium Synechococcus elongatus PCC 7942

Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.     

Driving Forces for Bicarbonate Transport in the Cyanobacterium Synechococcus R-2 (PCC 7942)

Air-grown Synechococcus R-2 (PCC 7942) cultures grown in BG-11 medium are very alkaline (outside pH is 10.0) and use HCO3- as their inorganic carbon source. The cells showed a dependence on Na+ for photosynthesis, but low Na+ conditions (1 mol m-3) were sufficient to support saturating photosynthesis. The intracellular dissolved inorganic carbon in the light was greater than 20 mol m-3 in both low-Na+ conditions and in BG-11 medium containing the usual [Na+] (24 mol m-3, designated high-Na+ conditions). The electrochemical potential for HCO3- in the light was in excess of 25 kJ mol-1, even in high-Na+ conditions. The Na+-motive force was greater than -12 kJ mol-1 under both Na+ conditions. On thermodynamic grounds, an Na+-driven co-port process would need to have a stoichiometry of 2 or greater ([greater than or equal to]2Na+ in/HCO3-1 in), but we show that Na+ or K+ fluxes cannot be linked to HCO3- transport. Na+ and K+ fluxes were unaffected by the presence or absence of dissolved inorganic carbon. In low-Na+ conditions, Na+ fluxes are too low to support the observed net 14C-carbon fixation rate. Active transport of HCO3- hyperpolarizes (not depolarizes) the membrane potential.     

Mutation of the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301): Improved conditions for the isolation of auxotrophs

Previous attempts to isolate auxotrophic mutants of Anacystis nidulans produced only a limited range of phenotypes. The frequency of recovery of auxotrophic mutants has been quantified following different mutagenic and selective treatments, and their yield has been improved by using (1) a complete medium, (2) additional mutagens, (3) multiple cycles of penicillin enrichment and (4) altered pre-enrichment starvation conditions. These modified induction and selection conditions permitted the isolation of mutants defective in nitrate reductase, nitrite reductase or malate dehydrogenase, unable to reduce sulphate, or deficient in the synthesis of biotin, thiamine, paminobenzoate, serine, glutamate, adenine or uracil.     

The cyanobacterium Synechococcus sp. PCC 7942 possesses a close homologue to the chloroplast ClpC protein of higher plants

The Clp family consists of large, ubiquitous proteins that function as molecular chaperones and/or regulators of ATP-dependent proteolysis. A single copy gene coding for one of these proteins, ClpC, was cloned from the unicellular cyanobacterium Synechococcus sp. PCC 7942. The predicted polypeptide is most similar (ca. 88%) to the chloroplast-localized ClpC protein from higher plants. Using degenerate PCR primers specific for the two distinct ATP-binding domains characteristic of all ClpA-C proteins, partial sequences homologous to clpC from Synechococcus were also identified in five other cyanobacterial strains. The Synechococcus clpC gene is transcribed under standard growth conditions as a monocistronic message of around 2.7 kb. The level of this message, however, decreases slightly after a shift from 37 to 47.5°C for 2 h, similar to expression previously observed for clpC mRNA from heat-shocked higher plants. At the protein level, the amount of ClpC remains relatively unchanged during the high temperature shift, while that of the known heat shock protein GroEL rises considerably. In contrast, the constitutive level of ClpC in Synechococcus increases considerably under conditions of rapid growth, both with increasing light intensities or CO₂ concentrations. This, and the fact that attempts to inactivate clpC expression fail to produce a viable phenotype, suggest that ClpC activity is essential for growth in this obligate photoautotrophic cyanobacterium.     

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     

Factors influencing the phycobilisome rod composition of the cyanobacterium Synechococcus sp. PCC 7942: Effects of reduced phycocyanin content, lack of rod-linkers, and over-expression of the rod-terminating linker

Four novel mutants with altered phycobilisomes were constructed in the cyanobacterium Synechococcus 7942 to study factors influencing the rod length and composition. These mutants show (1) reduced phycocyanin content, (2) reduced phycocyanin content combined with loss of the 33 kDa linker, (3) loss of the 30 kDa rod-linker and (4) overexpression of the 9 kDa rod terminating linker. For these mutants we determined the 33 to 27 kDa and 30 to 27 kDa linker ratios in the isolated phycobilisomes and compared these ratios with those in the wild type. The 30 kDa linker can be incorporated into the rods in absence of the 33 kDa linker. The incorporation of the 30 kDa linker is lower in absence of the 33 kDa linker. When the 30 kDa linker is missing, an increase in the level of the 33 kDa linker is seen, indicating that there could be an excess of the 33 kDa linker in the cells. Our results also show that a reduction in the phycocyanin content causes a decrease in the rod length simultaneously with a reduction of the 30/27 linker ratio, without altering the 33/27 ratio. Reduced phycocyanin content and absence of the 33 kDa linker cause a dramatic reduction in the incorporation of the 30 kDa linker into the rods in the mutant B2SMIKM. Over-expression of the 9 kDa linker results in a decreased incorporation of both the 33 and 30 kDa linkers into the rods, the effect being more pronounced for the 30 kDa linker. This result indicates that the level of the 9 kDa linker relative to those of the 33 and the 30 kDa linkers may be an important determinant of the phycobilisome rod length.     

人铜锌超氧化物歧化酶基因改良及在聚球藻中表达

应用PCR定点突变技术把质粒pESOD中人铜锌超氧化物歧化酶基因(hCu,Zn-SOD)的Cys111密码子突变为Ala111密码子,再构建重组子,通过随机同源重组将突变后的hCu,Zn-SOD整合入聚球藻Synechococcussp.PCC7942,并实现表达。表达产物用SDS-PAGE、Western blot、酶活等方法测定均为阳性反应;热稳定性测定显示,hCu,Zn-SOD在80℃保温30min后仍具有95%的活力,耐热能力比天然hCu,Zn-SOD有了较大的提高。蛋白扫描结果显示目的蛋白占可溶性蛋白的3.61%。     

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.