Chloride transport in Anacystis nidulans

Summary Anacystis nidulans will take up and accumulate chloride ions. When the external concentration was 0.2 mM Cl^- the level in the cells was 2.8 mM Cl^- and under these conditions the flux across the cell surface was in the region of 10^-13equiv Cl^-·sec^-1·cm^-2. It is suggested that this Cl^- influx is active and operates against an electrochemical potential gradient estimated to be 117 mV or 2.68 kcal/mole. The uptake of ³⁶Cl was inhibited by low temperatures and there was a net loss of Cl^- from the cells with the level decreasing towards the equilibrium value as estimated from K⁺ distribution. Although the active influx of Cl^- was often stimulated by light this was not always the case. Dark storage treatment and regulation of the chlorophyll a/phycocyanin ratios as well as total pigment content of the cells did not clarify the conditions which brought about light stimulation. Moreover, the metabolic inhibitors CCCP and CMU and also the use of anaerobic conditions did not clearly indicate the relationship between the influx mechanism and light-dark metabolism and no firm conclusions could be made about the nature of the energy source. The variation in the degree of light stimulation probably reflects the fact that in this procaryotic organism the photosynthetic and respiratory units are located on the same membrane systems and are in very close proximity to the probable site of the Cl^- pump, the plasmalemma.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - CMU 3-(4-chlorophenyl)-1,1-dimethylurea     

Sodium chloride stimulated respiration of Anacystis nidulans.

With certain salts a stimulation of respiration of the blue-green alga Anacystis nidulans was found in the dark. The stimulation was observed only at high concentrations (10(-2)M--10(-1)M). NaCl or LiCl are the most effective salts and on addition the increase of the respiration is about 2.5fold. Li is assumed to function as a substitute for Na. Potassium salts, except KCl, are ineffective. The order for the effectiveness is: NaCl greater than NaNO3, Na2SO4 greater than KCl greater than KNO3, K2SO4 (=zero). Accordingly, the cation Na+, and to a less degree the anion Cl- are responsible for the stimulatory effect. K, which is ineffective, is passively accumulated by Anacystis according to the membrane potential. Na is actively extruded. At 0.1 M external NaCl, the passive influx of Na is high, but even then it is balanced by an active efflux. This increases the energy consumption of the cells and leads to a stimulated respiration. With DCCD (N,N'-dicyclohexylcarbodiimide) or NEM (N-ethylmaleimide), the Na efflux is inhibited, simultaneously the stimulation of respiration is abolished and the passive influx of Na becomes detectable. At 0.1 M NaCl, the passive influx of Na measured in presence of DCCD is 5 x 10(-6) moles Na/min and ml packed cells. In absence of DCCD on addition of 0.1 M NaCl the extra oxygen consumption is 2 x 10(-6) moles O2/min and ml cells. This may prove that the stimulation of respiration is mainly caused by the active Na extrusion.     

Methylammonium transport in Anacystis nidulans R-2

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 +/- 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3+ required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Km, 7.2 microM; Vmax, 12.5 nmol min-1 mg of protein-1 at 15 degrees C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 14CH3NH3+ for entry, and addition of 5 microM NH4+ or 100 microM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4+ and 14CH3NH3+, indicating that soluble periplasmic components are not involved in the translocation. Some significant differences between the translocation of 14CH3NH3 and that of NH4+ were observed: growth with NH4+ in place of NO3- repressed 14CH3NH3+ accumulation ability without affecting the NH4+ uptake rate Na+ was not required for NH4+ uptake, and concentration of KCl inhibitory with 14C3NH3+ did not reduce NH4+ uptake.     

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.     

Synchronization of Anacystis nidulans

A new technique of short alternating lightdark periods was successfully used to synchronize the blue-green alga Anacystis nidulans. Oxygen evolution during the cell cycle is characterized by a maximum in the middle of the cycle and by a minimum at the time of division, a pattern very similar to that found in synchronized green algae.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

DCCD induced sodium uptake by Anacystis nidulans

The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na⁺/H⁺-antiport system. The necessary H⁺-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H⁺-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10^-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10^-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD N,N-dicyclohexylcarbodiimide - NEM N-ethylmaleimide - CCCP carbonylcyanide m-chlorophenylhydrazone - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Phycobilisomes in Anacystis nidulans

Phycobilisomes were demonstrated in Anacystis nidulans by chemical and morphological studies on cells grown in red light. These cells showed a marked reduction in the chlorophyll-phycocyanin ratio owing to a decreased chlorophyll content. Granular structures of approximately 35 nm were observed throughout red light-grown cells, but were most distinct in the peripheral region. The presence of phycobilisomes in cells grown in red light as well as in cells grown in white light is supported by experiments in which glutaraldehyde was used to stabilize the attachment between the phycobiliprotein and the thylakoids, allowing the isolation of both in the same fraction by sucrose density gradient centrifugation.     

A Highly Active Oxygen-Evolving Photosystem II Preparation from the Cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem (PS)-II fraction has been isolated from the cyanobacterium, Anacystis nidulans R2, using an isolation buffer containing high concentrations of sucrose and salts and subsequent solubilization of the thylakoid membranes with the detergent Triton X-100. The isolated fraction had very high PSII activity (2500 micromoles O₂ per milligram chlorophyll per hour) and was largely depleted of PSI activity. Fluorescence emission spectra (77 K) and polypeptide analysis indicated that this preparation is highly enriched in PSII, but almost completely devoid of Cyt b₆-f and PSI complexes.     

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

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Spectral Changes in Anacystis nidulans Induced by Chilling

When Anacystis nidulans, strain TX 20 was grown at 39 C, then rapidly chilled to 0 C, a pigment with a carotenoid-like spectrum was bleached. This effect was not seen when cells which had been grown at 25 C were chilled. The effect seen in 39 C-grown cells was not reversible except under extreme conditions such as heating to near boiling for several minutes. Bleaching could be prevented by prior exposure of cells to glutaraldehyde, but could not be reversed by glutaraldehyde treatment following chilling. The effect occurred upon chilling 39 C-grown cells even after extensive heating at 85 C, a treatment which destroys phycocyanin and metabolic activities. 25 C-grown cells were induced to bleach by chilling when suspended in 50% glycerol. The results are interpreted as indicating a chill-induced change in aggregation state of a carotenoid, which changes its specific absorbance.     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Cation regulation in Anacystis nidulans

Summary Anacystis nidulans accumulates K⁺ in preference to Na⁺. The majority of the internal K⁺ exchanges with ⁴²K by a first order process at rates of about 1.3 pequiv·cm^-2·sec^-1 in the light and 0.26 pequiv·cm^-2·sec^-1 in the dark. Although the K⁺/K⁺ exchange was stimulated by light and inhibited by 10^-4 M CCCP and 10^-5 M DCMU there are several indications that this cation is passively distributed in Anacystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K⁺ fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K⁺ leak. Assuming K⁺ is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na⁺ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Phycobilisomes in Spheroplasts of Anacystis nidulans

Phycobilisomes in Anacystis nidulans can be seen more readily in spheroplasts than in cells with intact walls.     

Polysaccharide Produced by Anacystis nidulans: Its Ecological Implication

An extracellular polysaccharide from Anacystis nidulans was extracted from cell-free medium. Analysis showed that the polysaccharide consisted of glucose, galactose, and mannose in the ratio of 60:14:20. The production of polysaccharide depends on the age of culture, the growth temperature, and the form of nitrogen available.     

Polyphosphate-deficient mutants of Anacystis nidulans.

Polyphosphate-deficient mutants of Anacystis nidulans have been isolated by either ethyl methanesulfonate (EMS) or N-methyl nitrosoguanidine (NTG) mutagenesis and penicillin-enrichment techniques. Mutagenised stock was preincubated in a medium lacking sulfate, then transferred to a phosphate-lacking medium before penicillin treatment. Many single-colony isolates, in contrast to wild-type, show little growth in absence of phosphate, and have altered polyphosphate, and have altered polyphosphate kinase levels indicating that the lesions affect either the activity or the expression of this enzyme. In these same mutants radioactive phosphate incorporation is severely retarded. Electron micrographs confirm the absence of polyphosphate granules in some mutants.