Photoacoustic spectroscopy of Anacystis nidulans. III. Detection of photosynthetic activities

Photosynthetic activities of Anacystis nidulans can be detected by photoacoustic spectroscopy. Algae treated by a photosynthetic inhibitor are used to provide the signal from the photochemically inactive sample. The results of these measurements correspond well with the activities which can be monitored by conventional biochemical assays. Acoustic data from A. nidulans are used to obtain the action spectrum for photochemical energy storage. It is concluded that phycocyanin harvests light for both photoreactions but that chlorophyll alpha molecules convey most of their excitation energy to photoreaction I. As judged from the relationship between the modulation frequency and the acoustic signal intensity, at least 60% of the photons absorbed at 630 nm perform photochemical work and about half of the useful energy is stored at stable products. Although it cannot be separated from the purely thermal effect, the contribution of modulated oxygen evolution to the acoustic signal of algae is estimated to be relatively small. Due to structural peculiarities, the opposite situation predominates in low frequency measurements performed with leaves from Impatiens petersiana.     

Characterization of Sulphate Uptake in Anacystis nidulans

Sulphate uptake in the blue-green alga Anacystis nidulans appears based upon an active mechanism with a Km of 0.75 μM and V_max of 0.7 pmol/min × 10⁶ cells. Sulphate uptake is competitively inhibited by thiosulphate and sulphite. The sulphate uptake has a pH optimum at 8 and a temperature optimum at 40°C. By increasing the extracellular sulphate concentration from 0.1 to 10 μM the sulphate pool in Anacystis was altered from 8.3, 10^?5M to 5.9, 10^?4M.     

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.     

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.     

Spontaneous pigment mutants of Anacystis nidulans selected by growth under far-red light

Under far-red (>650 nm) illumination Anacystis nidulans grows poorly and develops a low chlorophyll content. During continued culture over many generations there are increases in growth rate and in the chlorophyll/phycocyanin ratio, usually occurring in concomitant and stepwise fashion. From such selection cultures six clones have been established which differ from the parent in pigment content and show improved growth rate in far-red light. From the evidence at hand the six clones are presumed to be spontaneous mutants selected under the photosynthetically restrictive condition of far-red illumination.     

Characterization of phycobilisome glycoproteins in the cyanobacterium Anacystis nidulans R2.

Concanavalin A-reactive linker and anchor subunits of phycobilisomes from Anacystis nidulans R2 (H. C. Riethman, T. P. Mawhinney, and L. A. Sherman, FEBS Lett. 215:209-214, 1987) were purified electrophoretically and analyzed for carbohydrate composition and quantity. Different quantities of glucose and N-acetylgalactosamine were found on the concanavalin A-reactive subunits analyzed. Proteolytic analysis of the purified subunits suggested that small regions of the 33- and 27-kilodalton linker polypeptides previously shown to be important for in vitro phycobilisome assembly contained the concanavalin A-reactive carbohydrates present on these subunits. The linker and anchor subunits from the morphologically different phycobilisome of Synechocystis sp. strain PCC6714 were also shown to be concanavalin A reactive. Membranes from iron-starved Anacystis nidulans, which lack assembled phycobilisomes and are associated with glycogen deposits, were shown to be depleted of linker and anchor proteins and to accumulate very large quantities of a concanavalin A-reactive, extrinsic membrane glycoprotein. We suggest that this iron stress-induced glycoprotein is associated with the glycogen deposits on the thylakoid surface and that the glycosylation of phycobilisome linker and anchor subunits is involved in the physiological regulation of phycobilisome assembly and degradation.     

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.     

Excitation energy transfer in Anacystis nidulans.

Several natural acyclic sesquiterpenes with capacity for insect growth regulation have been shown to uncouple oxidative phosphorylation in mouse-liver mitochondria. These agents stimulate succinate oxidation, reverse oligomycin-inhibited state 3 respiration, activate ATP-hydrolysis, induce loss of respiratory control and abolish $\text{ADP}\text{O}$ ratio. Permeability of the inner membrane to potassium, sodium, ammonium and chloride ions as well as to protons is also enhanced. Since the structure of these agents precludes protonophoric activity, the possible mechanism of uncoupling by these juvenile hormones is discussed.     

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.     

Labeling the deoxyribonucleic acid of Anacystis nidulans.

Analysis of cell-free extracts of Anacystis nidulans disclosed the absence of both thymidine phosphorylase (EC 2.4.2.4) and thymidine kinase (EC 2.7.1.21) activities. Thymine and thymidine were incorporated inefficiently by intact cells of A. nidulans either in the presence or absence of deoxyguanosine (250 mug/ml). Deoxythymidine monophosphate incorporation was also inefficient. Radioactive deoxyadenosine, at a minimally toxic level (3 mug/ml), was incorporated effectively into the deoxyribonucleic acid (DNA). A cesium chloride-ethidium bromide gradient analysis of the DNA revealed that both the plasmid DNA and the principal DNA of the A. nidulans genome were labeled effectively in cells exposed to [8-14C]deoxyadenosine.     

Two simple procedures for isolation of allophycocyanin II from Anacystis nidulans

Allophycocyanin II purification using initial extraction of phycobiliproteins by acetone treatment is introduced. An additional fast method using Al2O3 is described. Both extraction procedures are followed up by conventional hydroxylapatite chromatography.     

Isolation and Characterization of Ultraviolet Light-Sensitive Mutants of the Blue-Green Alga Anacystis nidulans

Three independently isolated ultraviolet light-sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 was most sensitive to UV in the absence of photoreactivation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, indicating the presence of excision enzymes involved in dark repair. Under "black" and "white" illumination, strain uvs-1 displays photoreactivation properties nearly comparable to wild-type culture. Mutants uvs-35 and uvs-88 appeared to have partial photorecovery capacities. Upon pretreatment with chloramphenicol, photoreactivation properties of strains uvs-1 and uvs-88 were not evident although the partial photoreactivation characteristics of strain uvs-35 remained the same. Data indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.     

An iron-containing superoxide dismutase from Anacystis nidulans.

Superoxide dismutase (SOD) was isolated and purified from Anacystis nidulans to near electrophoretic homogeneity. The enzyme has a molecular weight of 37,500, as determined by gel filtration and SDS-gel electrophoresis. The enzyme molecule consists of two subunits of identical molecular weight. Proton-induced X-ray elemental analysis (PIXE) showed that the SOD of A. nidulans is an iron-containing enzyme; the Fe:enzyme mol ratio was found to be 1. The EPR spectra indicated that the active center contains high-spin ferric ion. Based on quantitative EPR data, we conclude that eseentially all iron ions were detected in the EPR experiments and were present in the Fe3+ active center. Effective g'-values were calculated from computer-simulated spectra and analysis of the g'-value anisotropy of the +/-3/2 Kramers doublet made the calculation of crystal field parameters possible. The symmetry of the Fe3+ ion in the SOD molecule was found to be close to rhombic (E/D=0.240).     

Phycobilisomes in Spheroplasts of Anacystis nidulans

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

Leucine biosynthesis in the blue-green bacterium Anacystis nidulans.

Leucine-requiring auxotrophs of the unicellular blue-green bacterium Anacystis nidulans have been isolated. Extracts of these mutants were deficient in alpha-isopropylmalate synthetase (EC 4.1.3.12). In wild-type cells, this enzyme was subject to feedback inhibition by leucine. However, formation of the enzymes of leucine biosynthesis was little affected by exogenous leucine in either wild-type or mutant strains. Cultures of the latter subjected to extreme leucine deprivation showed no change in specific activity of beta-isopropylmalate isomerase (EC 4.2.1.33) and at most a 50% increase in the specific activity of beta-isopropylmalate dehydrogenase (EC 1.1.1.85). These results are compared with others bearing on the evolution of the control of amino acid biosynthesis in blue-green bacteria.