Phycocyanin synthesis and degradation in the blue-green bacterium Anacystis nidulans.

Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits: (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Photosynthetic Membrane System in Anacystis nidulans

Cultures of Anacystis nidulans were grown under conditions of varying light intensity and temperature. Changes in pigment content were compared with changes in the fine structure of these cells. Pigment concentration and lamellar content varied inversely with the light intensity in cells grown with 100 and 1,000 foot candles of fluorescent light. Estimations of the relative area and volume of lamellae in cells showed that the amount of double membrane was directly proportional to the chlorophyll content of whole cells. Continuity of double membranes with cytoplasmic membrane was observed.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Potassium requirement for cell division in Anacystis nidulans.

A potassium requirement for growth can be readily demonstrated in the autotrophic blue-green bacterium Anacystis nidulans strain TX20 equivalent to 0.7% of the cellular dry weight. Starvation of this organism for potassium partially dissociates growth from cell division, thereby inducing 50% of the population to form filaments.     

Cold Shock Syndrome in Anacystis nidulans

The phenomenon of cold shock in Anacystis nidulans has been explored further in terms of loss of viability and immediate and subsequent metabolic effects. Cold shock was observed also in two closely related strains in which unsaturated fatty acid contents are also known to be low and temperature-dependent. Loss of viability was maximum for cells grown at temperatures above 40 C (<10⁻⁴ survivors after 5 min at 0 C) but became negligibly small for cells grown below 34 C. Development of the cold-sensitive condition after transfer 25 → 39 C was slow and comparable to rate of growth; development of the insensitive condition after transfer 39 → 25 C was rapid, implying rapid in situ alteration. An immediate metabolic effect, observed as a decrease in rate of photosynthetic O₂ evolution measured at growth temperature, was less severe than loss of viability. Continued light incubation under growth conditions led to slow decay in rate of O₂ evolution accompanied by loss of membrane chlorophyll. The multiple effects which comprise the cold shock syndrome appear to be membrane-related phenomena and thereby provide an experimental probe of normal membrane function.     

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.     

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).     

The estimation of turnover of spermidine in Anacystis nidulans.

The turnover of spermidine in Anacystis nidulans was studied using [2-¹⁴C]methionine to prelabel intracellular spermidine. It was found that there is essentially neither excretion nor degradation of spermidine in exponentially growing Anacystis nidulans. Spermidine was degraded rapidly in stationary phase cells. The half-life of specific activity of spermidine in exponential phase was 8.3 h, a period similar to that of the doubling time (7.5 h) of the bacterium. The rate of synthesis of spermidine was calculated to be 0.04 nmol/10⁸ cells/h.     

The biosynthesis of phycocyanobilin in Anacystis nidulans

Aminolevulinic acid, the common precursor of linear and cyclic tetrapyrroles, can arise either by condensation of succinate and glycine or from the ent