Effect of chloride ions on the S-state distribution and deactivation kinetics in preparations of the cyanobacterium Anacystis nidulans

The roles of Ca²⁺ and Cl⁻ on the photosynthetic O₂ yield under flash illumination have been examined in EDTA-washed preparations of the cyanobacterium Anacystis nidulans. Especially the effect of Cl⁻ deficiency on the O₂ yield and on the S-state distribution was analyzed. As the results show, omission of both Ca²⁺ and Cl⁻ (Mn²⁺ present) almost totally inhibited O₂ evolution. When Ca²⁺ was replaced by Na⁺, a substantial reduction of the O₂ yield was observed, but only a minor change in the S-state distribution occurred. However, when Cl⁻ was displaced by NO⁻₃, which is equivalent to Cl⁻ deficiency of the water-splitting complex, a substantial reduction of the O₂ yield and in addition a significant change in the S-state distribution was observed. The comparison of deactivation kinetics in NO⁻₃ containing samples with those in control samples indicated that Cl⁻ deficiency allowed accumulation of oxidizing equivalents up to the S₃ state but modified the final step of O₂ evolution. Moreover, those centers which advanced to the S₃ state in the absence of Cl⁻ deactivated in a special way which involved a faster deactivation of S₂ and an increased formation of S₋₁.     

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l-methionine dl-sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO₂, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l-methionine dl-sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO₂-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.     

Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2

Linear-dichroism spectra of Anacystis nidulans at 77 K were determined for whole cells chemically fixed in light State 1 and light State 2. Whole cells were oriented by the squeezed gel technique using 5% gelatin 2.2 M sucrose gels. Peaks with positive dichroism were observed at 638 nm and 688 nm with shoulders at approx. 650 nm and 700 nm. The amplitude of the 650 nm shoulder was greater for cells in State 2 than those in State 1, and the State-2-minus-State 1 difference spectrum had a single peak at 656 nm. The linear dichroism spectrum of phycobilisomes isolated from A. nidulans showed peaks at 635 nm (phycocyanin) and 656 nm (allophycocyanin). The spectrum for thylakoid membranes free of phycobilisomes had one peak at 685 nm with a shoulder at 698 nm. We suggest that the change in dichroism at 656 nm between cells in State 1 and State 2 results from a change in orientation of the allophycocyanin core of the phycobilisome. This result is discussed in the context of our model for the light-state transition in phycobilisome-containing organisms.     

Oscillations of the NAD(P)H pool size and of the redox state of a cytochrome b during dark respiration of the blue-green alga, Anacystis nidulans

Oscillations of the oxygen uptake rate of the blue-green alga (cyanobacterium) Anacystis nidulans were induced by light pulses. The pool size of NAD(P)H and the redox state of a cytochrome b showed oscillations of similar shape and frequency. Phase diagrams revealed that these three oscillations were presumably linked. The cytochrome b should be a part of the respiratory chain of this blue-green alga. The oscillations were inducible only in a limited physiological state of the alga.     

Self-cloning in the cyanobacterium Anacystis nidulans R2: Fate of a cloned gene after reintroduction

Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self-cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid.     

Protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Anacystis nidulans R2

The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of ³⁵S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with ⁵⁹Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b₆ and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b₆ appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

Oxidation and reduction of plastoquinone by photosynthetic and respiratory electron transport in a cyanobacterium Synechococcus sp.

The I-D dip, an early transient of the fluorescence induction, was examined as a means to monitor redox changes of plastoquinone in cells of a cyanobacterium, Synechococcus sp. That the occurrence of the dip depends upon the reduced state of the plastoquinone pool was indicated by observations that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not affect the initial rise to I but abolished the subsequent decline from I to D and that illumination of the cells with light 1, prior to fluorescence measurements, eliminated the transient. The I-D dip was prominent in freshly harvested cells containing abundant endogenous substrates, disappeared slowly as the cells were starved by aeration but reappeared on addition of fructose to the starved cells in the dark. The dip that had been induced by a brief illumination of the starved cells with light 2 was rapidly diminished in the dark and KCN inhibited the dark decay of the transient. The results indicate that plastoquinone is reduced with endogenous as well as exogenous substrates and oxidized by a KCN-sensitive oxidase in the dark, thus providing strong support for the view that plastoquinone of photosynthetic electron transport also functions in respiration. In addition, the occurrence of a cyclic pathway of electrons from Photosystem I to plastoquinone, possibly via ferredoxin or NADP, was suggested. Several lines of evidence indicate that, under a strong light 2, Photosystem I-dependent oxidation of plastoquinone predominates over Photosystem II-dependent reduction of the quinone in the cyanobacterium which contains Photosystem I more abundantly than Photosystem II.     

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

Regulation of nitrate reductase in cyanobacteria. Repression-derepression control of nitrate reductase apoprotein in the cyanobacterium Nostoc muscorum

The repression-derepression control of Nostoc muscorum nitrate reductase was studied with regard to the Mo-cofactor and apoprotein levels. It was found that the synthesis of Mo-cofactor is constitutive but the apoprotein is subject to the repression-derepression control. In NH₄⁺ medium apoprotein synthesis was repressed and in N₂ and NO₃^? media apoprotein synthesis was derepressed. The apoprotein levels were similar in NO₃^? and N₂ media; however, the nitrate reductase activity was lower in N₂ medium due to lower Mo-cofactor activity. The lower Mo-cofactor activity in N₂-fixing conditions as compared to that in non-N₂-fixing conditions was consistent with the earlier view that the Mo-cofactor of nitrate reductase may be a precursor for FeMo-cofactor of nitrogenase.     

Electron-transport reactions in cytoplasmic and thylakoid membranes prepared from the cyanobacteria (blue-green algae) Anacystis nidulans and Synechocystis PCC 6714

Cytoplasmic and thylakoid membranes prepared from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6714 were compared for their electron-transport activities. High cytochrome oxidase activity, which was sensitive to cyanide and azide, was found only in the thylakoid membranes of both strains. Activities of NAD(P)H-cytochrome c and succinate-cytochrome c oxidoreductase were low in both membranes from both strains. The NADH-cytochrome c oxidoreductase activity of the cytoplasmic membranes from Synechocystis was markedly stimulated by quinones, among which 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) was the most effective. High NADH-DBMIB oxidoreductase activity, which was relatively resistant to salt washing, was found in the cytoplasmic membranes from Synechocystis.     

The quenching of chlorophyll a fluorescence as a consequence of the transport of inorganic carbon by the cyanobacterium Synechococcus UTEX 625

The chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625 decreased upon the initiation of inorganic carbon transport. The fluorescence yield recovered upon the depletion of inorganic carbon from the medium or upon the addition of DCMU. The inhibition of photosynthetic CO₂ fixation by iodoacetamide did not prevent this reduction of fluorescence yield. Similar results were obtained for both Na⁺-stimulated HCO₃⁻ transport and for the transport (presumably of CO₂) that is stimulated by carbonic anhydrase. A transient lowering of the fluorescence yield was also observed when cell suspensions were pulsed with CO₂. In cells not inhibited with iodoacetamide, a very close quantitative relationship existed between the net rate of O₂ evolution and the maximum extent of fluorescence quenching seen as a function of the inorganic carbon concentration. The fluorescence quenching, however, was not due to CO₂ fixation but rather to the transport of inorganic carbon or the accumulation of the internal pool of inorganic carbon. If quenching is due to the latter it is not surprising that the extent of quenching corresponds to the maximum rate of photosynthesis as the rate of photosynthesis also depends on the size of the internal pool. The results with DCMU suggest that the quenching is Q quenching and transport must provide a mechanism for the oxidation of Q other than CO₂ fixation.     

Relationship between nitrogen fixation and nitrate metabolism in the Nodularia strains M1 and M2

Nitrogen fixation and nitrate-reduction activities were determined in photoautotrophic cultures of two wild-type strains of cyanobacterium Nodularia, spp. M1 and M2. Air could support growth of the two strains at a similar rate in the presence or absence of exogenous nitrate, ammonium and/or bicarbonate. Nitrogenase activity in air-grown cultures varied with culture age, and totally disappeared after 6 h of darkness. Recovery took place upon culture re-illumination. Ammonium at a concentration of 1 mM resulted in the total disappearance of nitrogenase activity and of heterocysts. In contrast, 20 mM nitrate hardly affected nitrogenase activity and heterocyst formation after ten generations. Under the same conditions, either ammonium or nitrate completely abolished nitrogenase activity and heterocyst formation in Anabaena sp. PCC 7119, a typical heterocystous strain. The inefficiency of nitrate in inhibiting nitrogen fixation in Nodularia M1 and M2 seemed to be caused by a low nitrate-reductase activity, and not by an impairment of nitrate-uptake activity. On the other hand, the presence of nitrate was not required for uptake activity to be expressed in Nodularia.Abbreviation NR nitrate reductase We thank C. Fernández-Cabrera (Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain) for technical assistance, and Dr. G. Pérez-Silva (CSIC) for his collaboration in the Anabaena NR assays. This work was supported by grants from Spanish CI-CyT (PB 87-0204 and PB 92-0497).     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.