Cyanobacterium Anacystis Nidulans Research Articles

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: Thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of −350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between −240 and −320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H + ionophores) and F 0F 1ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary Δψ (−100 mV at pH o 9, preceding the acid jumps) and a favorable stationary ΔpH (+2 units at pH o 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of Δψ and ΔpH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of −200 to −250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to −400 mV in dark anaerobic i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H +-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H + and Na + from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na + concentration ( Na o + Na i + at 150 mM external NaCl) and proton electrochemical potential (Δ μ H + ~) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F 0F 1-ATPase inhibitors dicyclohexylcarbodiimide (F 0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F 1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 ± 0.15 to 0.7 ± 0.1 nmol/ mg dry wt while overall O 2 uptake and Δ μ H + ~ were much less affected. H + efflux was inhibited only 60 to 75%. Aerobic Na o + Na i + ratios (5.9 ± 0.6) under these conditions remained 50% above the anaerobic level (2.1 ± 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H + efflux and O 2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H + O ratios from 4.0 ± 0.3 to 1.9 ± 0.2. Similarly, with KCN and CO the Na o + Na i + ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 m m KCN. Measurements of the proton motive force (Δ μ H + ~) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na + H + antiporter for the active exclusion of Na + from the cell interior.

Presence of a flavoprotein in O 2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O 2-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O 2-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl 2 on the two examined reactions could also be demonstrated with this O 2-evolving Photosystem II complex: CaCl 2 stimulated photosynthetic O 2 evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.

Organization of the genes for protein synthesis elongation factors Tu and G in the cyanobacterium Anacystis nidulans.

The genes for protein synthesis elongation factors Tu and G were cloned from the cyanobacterium Anacystis nidulans. The locations of these genes were mapped within the cloned DNA fragment by hybridization with Escherichia coli probes. The organization of the cloned fragment and the DNA flanking it in the A. nidulans chromosome was also determined. The elongation factor Tu and G genes are adjacent to one another and in the same 5'-to-3' orientation. In contrast to other gram-negative bacteria, A. nidulans contains only one gene for elongation factor Tu.

Immunological identification of aa 3-type cytochrome oxidase in membrane preparations of the cyanobacterium [formula omitted][formula omitted

Membranes were isolated from the cyanobacterium Anacystis nidulans by French press extrusion of lysozyme-treated cells. The membranes were solubilized with sodium dodecylsulfate and subjected to denaturing polyacrylamide gel electrophoresis. Separated polypeptides were transferred to nitrocellulose by Western blotting, and incubated with antibodies against aa 3-type cytochrome oxidase of Paracoccus denitrificans ; antibodies against subunits I and II, and against the holoenzyme, were used and gave pronounced complementary cross reaction with two of the Anacystis membrane polypeptides corresponding to molecular weights of approximately 55,000 and 32,000, respectively. From this we conclude that an aa 3-type cytochrome oxidase is present in Anacystis nidulans as was previously suggested from spectral evidence (G.A. Peschek, Biochim. Biophys. Acta 635 (1981) 470–475), and that this enzyme is composed of at least two subunits with apparent homology to subunits I and II of the corresponding Paracoccus cytochrome oxidase.

Phenylalanyl-tRNA synthetase from chloroplasts of a higher plant (Phaseolus vulgaris). Purification and comparison of its structural, functional, and immunological properties with those of the enzymes from the corresponding cytoplasm, the cyanobacterium Anacystis nidulans, and the photosynthetic green sulfur bacterium Chlorobium limicola.

Chloroplastic phenylalanyl-tRNA synthetase from bean leaves is purified under optimal protective conditions over 4,900-fold. Its apparent molecular weight is 78,000, as determined by gel filtration, with a dimeric subunit structure of alpha beta (alpha = 33,000 and beta = 42,000), as determined by sodium dodecyl sulfate gel electrophoresis. This indicates a drastic size reduction of 40% for each subunit compared to the corresponding cytoplasmic enzyme and a unique quaternary structure. Heterologous aminoacylation and substrate properties of ATP analogs indicate substantial differences in the topographies of the substrate binding domains of these two heterotopic intracellular plant enzymes. No common antigenic determinants with the bean cytoplasmic enzyme were detected by polyclonal antibodies against the chloroplastic enzyme. The same negative result applies to the immunological comparison with the partially purified enzymes from the cyanobacterium Anacystis nidulans and the photosynthetic green sulfur bacterium Chlorobium limicola that both have a molecular weight of 260,000.

Respiratory electron transport in plasma and thylakoid membrane preparations from the cyanobacterium Anacystis nidulans

Plasma and thylakoid membranes were separated and purified from cell-free extracts of Anacystis nidulans by discontinuous sucrose density gradient centrifugation. Plasma membranes contained less than 0.05% (w/w) chlorophyll per protein. Both plasma and thylakoid membranes oxidized horse heart ferrocytochrome c (10 ± 4 and 3 ± 1 nmol mm per mg protein, respectively), with strong inhibition by low concentrations of cyanide, sulfide, azide, carbon monoxide, and salicyl aldoxime. The activity was stimulated 125% by 3.5% Tween 80 and totally suppressed by 100 mM NaCl. Oxidized cytochrome c was reduced by both types of membranes in the presence of NAD(P)H. The results indicate the occurrence of a respiratory chain including cytochrome- c oxidase in both plasma and thylakoid membranes of A. nidulans.

Organization of the genes for protein synthesis elongation factors Tu and G in the cyanobacterium Anacystis nidulans.

Organization of the genes for protein synthesis elongation factors Tu and G in the cyanobacterium Anacystis nidulans.

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.

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.

Comparison of amino acid sequences between phosphoenolpyruvate carboxylases from [formula omitted] (allosteric) and [formula omitted] (non-allosteric): Identification of conserved and variable regions

Amino acid sequences of phosphoenolpyruvate carboxylases of Escherichia coli (allosteric) and a cyanobacterium Anacystis nidulans (non-allosteric) were aligned. The pattern of homology suggests that the enzyme molecule is comprised of two distinct regions, namely, a conserved region (C-terminal half) and a variable region (N-terminal half). Among the amino acid residues which have previously been presumed essential for the catalytic activity, three histidine residues were found to be conserved, but cysteine residues were not. Furthermore, the conserved sequence unique to the enzyme was identified by comparison of the enzyme sequence with amino acid sequences in our data bank.

Photoinhibition and Reactivation of Photosynthesis in the Cyanobacterium Anacystis nidulans

The susceptibility of photosynthesis to photoinhibition and its recovery were studied on cultures of the cyanobacterium Anacystis nidulans. Oxygen evolution and low temperature fluorescence kinetics were measured. Upon exposure to high light A. nidulans showed a rapid decrease in oxygen evolution followed by a quasi steady state rate of photosynthesis. This quasi steady state rate decreased with increasing photon flux density of the photoinhibitory light. Reactivation of photosynthesis in dim light after the photoinhibitory treatment was rapid: 85 to 95% recovery occurred within 2 hours. In the presence of the translation inhibitor, streptomycin (250 micrograms per milliliter), no reactivation occurred. We also found that the damage increased dramatically if the high light treatment was done with streptomycin added. A transcription inhibitor, rifampicin, did not inhibit the reactivation process. Based on these data we conclude that the photoinhibitory damage observed is the net result of a balance between the photoinhibitory process and the operation of the repairing mechanism(s).

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.

Organization of pigment proteins in the photosystem II complex of the cyanobacterium Anacystis nidulans R2.

Two chlorophyll-protein complexes associated with photosystem II (PSII) of the cyanobacterium Anacystis nidulans R2 have been detected. The larger of the two complexes, CPVI-1, contained a 71-kDa and a 42-kDa protein. The 71-kDa protein was determined to be the anchor protein of the phycobilisomes (the light-harvesting complex of A. nidulans PSII), since it was recognized by an antibody raised against a similar protein from another cyanobacterium. The second complex, CPVI-4, contained a previously unobserved 36-kDa chlorophyll-binding protein. Additionally, two other PSII chlorophyll-protein bands were characterized. CPVI-2 contained a 52-kDa band that was recognized by an antibody raised against the presumptive PSII reaction center protein of Chlamydomonas reinhardtii. It gave rise to a fluorescence emission peak (77K) at 695 nm, indicating that this chlorophyll-protein complex may harbor the reaction center of PSII. Finally, CPVI-3 was found to have a 45-kDa protein and to be immunologically related to the presumptive immediate-antenna protein of the C. reinhardtii PSII.

Changes in the Polypeptide Composition of the Cytoplasmic Membrane in the Cyanobacterium Anacystis nidulans during Adaptation to Low CO2 Conditions

Changes in the Polypeptide Composition of the Cytoplasmic Membrane in the Cyanobacterium Anacystis nidulans during Adaptation to Low CO2 Conditions

Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits.

The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans were subcloned into plasmid pUC9. After induction, both genes were expressed in Escherichia coli and the subunits were assembled into an active holoenzyme. The enzyme was purified from E. coli to high specific activity and was found to contain equimolar amounts of large and small subunits. The assembly of the hexadecameric ribulose bisphosphate carboxylase/oxygenase in E. coli should provide the basis for studies on the mechanism of assembly and the role of small subunits in catalysis.

Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation

Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution.

Microinjected photoreactivating enzymes from Anacystis and Saccharomyces monomerize dimers in chromatin of human cells

Photoreactivating enzymes (PRE) from the yeast Saccharomyces cerevisiae and the cyanobacterium Anacystis nidulans have been injected into the cytoplasm of repair-proficient human fibroblasts in culture. After administration of photoreactivation light, PRE-injected cells displayed a significantly lower level of UV-induced unscheduled DNA synthesis (UDS) than non-injected cells. This indicates that monomerization of the UV-induced pyrimidine dimers in the mammalian chromatin had occurred as a result of photoreactivation by the injected PRE at the expense of repair by the endogenous excision pathway. Purified PRE from yeast is able to reduce UDS to 20–25% of the UDS found in non-injected cells, whereas the in vitro more active PRE from A. nidulans gives a reduction to only 70%. This suggests that the eukaryotic enzyme is more efficient in the removal of pyrimidine dimers from mammalian chromatin than its equivalent purified from the prokaryote A. nidulans.

Retardation of electron donation to Photosystem I in aged cyanobacteria and its reversal by metal cations

The dark reduction of photooxidized P-700 in the cyanobacterium Anacystis nidulans becomes slower as a consequence of aging. In cells, of which the envelope has been enzymatically permeabilized to electrolytes, the reduction of P-700 by endogenous electron donors is accelerated by KCl plus valinomycin, but not by KCl alone. The K +-valinomycin system is ineffective, however, in the case of aged but unpermeabilized cells. These results suggest that a consequence of aging in Anacystis is an increase in the negative electric potential at the inner thylakoid surface, which makes electron donation to P-700 by acidic donors (cytochrome c-553 and plastocyanin) less probable. This situation is reversed by the permeant K +-valinomycin system, which suppresses the inner surface potential and collapses the inside-outside potential difference, but not by K + alone, since the thylakoid membrane is impermeable to it.

Genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase constitute a single operon in a cyanobacterium Anacystis nidulans 6301

The structure and transcription of the genes for the large (LS) and small subunits (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of Anacystis nidulans 6301 were studied. The SS gene is located 93 base pairs (bp) downstream from the LS gene on the same DNA strand. Northern hybridization analysis revealed that the LS and SS genes are transcribed as a 2.2 kilobase (kb) mRNA. The 5′ end of the mRNA was mapped 158–160 bp upstream from the ATG initiation codon of the LS gene and its 3′ end 224–230 bp downstream from the TAA termination codon of the SS gene. These observations indicate that the LS and SS genes constitute a single operon. Upstream from the LS gene, we found two open reading frames on the same DNA strand. These are overlapped by 40 nucleotides and portions of their amino acid sequences resemble each other