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.     

Determination of guanosine tetraphosphate (ppGpp) and adenosine pentaphosphate (pppApp) in various microorganisms by radioimmunoassay

The intracellular levels of ppGpp and pppApp in various microorganisms were determined by radioimmunoassay, with was of more accuracy and convenience than previous ³²P-labeling method. ppGpp was detected in bacteria, actinomycetes, yeasts and fungi. pppApp was found in non-spore forming bacteria such as the genera Pseudomonas and Escherichia, fungi and actinomycetes, but not in yeasts. In conclusion, ppGpp and pppApp are present in various prokaryotes and lower eukaryotes.     

Ultraviolet light-induction and photoreactivation of thymine dimers in a cyanobacterium,Anacystis nidulans

Partially photoreactivable mutant of Anacystis nidulans demonstrates partial photorepair of thymine dimers. The wild type which is completely photoreactivable at the conditions studied shows higher level of thymine dimer photolysis.Abbreviations UV ultraviolet light, peak intensity at 254 nm - PR photoreactivation - Dm D medium of Kratz and Myers modified by van Baalen - WT wild type     

The DNA,RNA and protein composition of the cyanobacterium Anacystis nidulans grown in light- and carbon dioxide-limited chemostats

The DNA, RNA and protein content of the cyanobacterium Anacystis nidulans was determined in light-limited and carbon dioxide-limited chemostat cultures over the dilution rate range, D=0.02 h^-1 to 0.19 h^-1. The macromolecular contents as a percentage of the dry weight and on a per cell basis varied significantly as a function of organism growth rate and the nature of the growth conditions. For both limitations the RNA content per cell increased [20–55 fg RNA (cell)^-1] with increasing dilution rate and also showed an increase as a percentage of the dry weight. The DNA content as a percentage of the dry weight showed a 2-fold decrease with increasing dilution rate over the range examined. On a per cell basis DNA reached a peak at D=0.1 h^-1 [4.5 fg DNA (cell)^-1] for light-limited organisms and at D=0.08 h^-1 [8.0 fg DNA (cell)^-1] for carbon dioxide-limited organisms. The q _RNA increased with increasing dilution rates over the complete growth rate range examined whilst q _DNA reached a maximum at D=0.09 to 0.10 h^-1. The protein content as a percentage of the dry weight was greater in CO₂-limited organisms than light-limited organisms but in both cultures declined as the dilution rate was increased above D=0.10 h^-1.     

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.     

Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality.In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase(GS/GOGAT) cycle.How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RES...     

Drifts in DNA level in the cyanobacterium Anacystis nidulans during synchrony induction and in synchronous culture

Cultures of the cyanobacterium Anacystis nidulans were synchronized by using alternating light-dark cycles. The DNA level in the cells was determined, at intervals, during pre-synchrony treatment and subsequent synchronous growth. The DNA content/cell gradually increased during synchrony induction and reached a maximum value after about 9–10 dark-light cycles, coinciding with the minimum length of pre-synchrony treatment necessary for obtaining good synchrony of cell division in our system. DNA synthesis was found to be discontinuous in the synchronous cultures. The results suggest two gaps in DNA synthesis, one occurring before and one after cell division. The results are compared with the relevant data published on the life cycle of other prokaryotic microorganisms.     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.     

Bioenergetic characterization of transient state phosphate uptake by the cyanobacterium Anacystis nidulans

A force flow relationship based on nonequilibrium thermodynamics was derived to analyze the variable transient state phosphate uptake phenomena of cyanobacteria seen under different growth conditions and external phosphate concentrations. This relationship postulates the following basic properties of the uptake system: First, a threshold value exists, below which incorporation is energetically impossible. Second, threshold values are influenced by the activity of the phosphate uptake system, such that a decrease of the activity increases the threshold level. Third, near the thermodynamic equilibrium the uptake rate is linearly dependent on the free energy of polyphosphate formation and the pH-gradient at the thylakoid membrane. Experiments performed with Anacystis nidulans showed that phosphate uptake characteristics conformed to the properties predicted by the linear force-flow relationship. Linearity extented into regions far form thermodynamic equilibrium, e.g. to high phosphate concentrations, when algae were preconditioned to high phosphate levels. Under phosphate limited growth linearity was confined to a small concentration range, threshold values decreased below 10 nM, and the external concentration approached threshold. The data suggest that the uptake system responds to changes in the external phosphate concentration in the same way as sensory systems to input stimuli by amplifying signals and adapting to them.Abbreviations chl chlorophyll - H _e ⁺ , H _C ⁺ , H _T ⁺ protons in the external medium, the cytoplasmic and thylakoid space respectively - P_c phosphate in the cytoplasmic space - P_e phosphate in the external medium - P_n, P_n+1 polyphosphates - pH_T pH-gradient across the thylakoid membrane     

Deposition of condensed phosphate as an effect of varying sulfur deficiency in the cyanobacterium Synechococcus sp. (Anacystis nidulans)

Uptake of orthophosphate and deposition of condensed phosphate were investigated in cells of Synechococcus sp. (Anacystis nidulans) deficient in phosphorus or sulfur. When phosphorus was restored to phosphorus-starved cells, uptake was rapid and immediate, with the greatest accumulation occurring within the first hour. Uptake was optimum in the pH 7.5–8.5 range. Long-term (6-day) studies of uptake and deposition with cells exposed to a wide range of sulfur deficiency showed that both processes were greatest when the level of exogenous sulfur was reduced to zero. The increase in cellular phosphorus as determined chemically was in agreement with the increased number and size of polyphosphate bodies at the ultrastructural level. Possible mechanisms for the control of phosphorus uptake and condensed phosphate formation by exogenous sulfur are discussed.     

Mutation of the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301): Improved conditions for the isolation of auxotrophs

Previous attempts to isolate auxotrophic mutants of Anacystis nidulans produced only a limited range of phenotypes. The frequency of recovery of auxotrophic mutants has been quantified following different mutagenic and selective treatments, and their yield has been improved by using (1) a complete medium, (2) additional mutagens, (3) multiple cycles of penicillin enrichment and (4) altered pre-enrichment starvation conditions. These modified induction and selection conditions permitted the isolation of mutants defective in nitrate reductase, nitrite reductase or malate dehydrogenase, unable to reduce sulphate, or deficient in the synthesis of biotin, thiamine, paminobenzoate, serine, glutamate, adenine or uracil.     

Isolation and properties of an isocitrate dehydrogenase from Anacystis nidulans

A NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) was isolated and purified over 400-fold from Anacystis nidulans. The enzyme activity responded slowly to rapid changes in ligand (NADP⁺, isocitrate, Mg²⁺-ions) or enzyme concentration as well as to rapid changes in temperature. These are properties characteristic of the hysteretic enzymes. In addition, the enzyme activity was subject to product (-ketoglutarate) inhibition. ATP, ADP and CDP also inhibited the enzyme. Unlike several other cyanobacterial enzymes, the isocitrate dehydrogenase of Anacystis is not under redox control.     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

Effect of gibberellic acid on photosynthesis and glycollate dehydrogenase in Anacystis nidulans

Gibberellic acid at 10^-4 Mxxx was optimal for enhancement of growth, O₂ evolution, photosystem II and I and the activity of glycollate dehydrogenase of Anacystis nidulans. A stimulatory effect was observed on photosystem II. Other concentrations of gibberellic acid were inhibitory to O₂ evolution and photosystem I. Syntheses of phycocyanin, phycoerythrin and -carotene were significantly enhanced after 48 h incubation with gibberellic acid at 10^-3 Mxxx but the chlorophyll content began to increase 3 h after adding 10^-4 Mxxx gibberellic acid.The author is with the Department of Biological Sciences, Faculty of Science, University of Science and Technology, Irbid, Jordan.     

Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria

Ammonium at low concentrations caused a rapid and effective inhibition of nitrate utilization in the light by the cyanobacterium Anacystis nidulans without affecting the cellular level of nitrate reductase activity. The inhibition was reversible, and the ability of the cells to utilize nitrate was restored immediately after ammonium had been exhausted. The inhibitory effect was dependent on consumption by the cells of the added ammonium which was rapidly incorporated into amino acids. In the presence of L-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase pathway, ammonium did not exhibit any inhibitory effect on nitrate utilization. Ammonium assimilation, rather than ammonium itself, seems to regulate nitrate utilization in A. nidulans. Short-term inhibition by ammonium of nitrate utilization and its prevention by MSX were also demonstrated in the filamentous cyanobacteria Anabaena and Nostoc.Abbreviations MSX L-Methionine-d-l-sulfoximine     

Oxidation of exogenous c-type cytochromes by intact spheroplasts of Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize reduced c-type cytochromes derived from horse heart, tuna, Saccharomyces oviformis, Candida krusei, Rhodocyclus purpureus, Rhodopseudomonas plustris and Paracoccus denitrificans with characteristics similar to those observed with isolated membranes. Rates of cytochrome c oxidation by the spheroplasts were only 10% of those measured with isolated membranes in which thylakoid-bound cytochrome oxidase contributes to the overall rates. Small amounts of an endogenous c-type cytochrome were released upon lysozyme treatment of the cells. The results appear to indicate the presence of cytochrome oxidase in the cytoplasmic membrane of A. nidulans.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - cyt cytochrome(s)     

Physiological performances of the blue-green alga Anacystis nidulans in continuous turbidostat fermenter culture

A sterile continuous turbidostat culture in a 2-1 fermenter was used to systematically measure the gas exchange rates of Anacystis nidulans in a highly turbulent system under strictly controlled environmental conditions. An extensive physiological characterization of Anacystis is given in terms of photosynthesis rates (CO₂ uptake and O₂ evolution) and dark respiration rates as function of different parameters such as stirrer speed, temperature, CO₂ and O₂ concentration, light intensity, culture density and pH. Steady state ATP levels and apparent photophosphorylation rates complete the performance data. The dependence of the photosynthetic quotient from the parameters enables a physiological characterization of the light dependent nitrate assimilation.     

Changes in intracellular amino acids and organic acids induced by nitrogen starvation and nitrate or ammonium resupply in the cyanobacterium Phormidium laminosum

In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) This work has been supported by grants from the Spanish Ministry of Education and Science (DGICYT and PB92-0464) and the University of the Basque Country (042.310-EC203/94) M.I.T. and J.A.G. were the recipients of fellowships from the Basque Government.     

Coupling between the initiation of DNA replication and cell division in the blue-green alga Anacystis nidulans

The effect of mitomycin C on cell mass increase, cell division, RNA synthesis and DNA synthesis in the blue-green alga Anacystis nidulans has been examined. Data suggests that the initiation of DNA replication, rather than its termination was the necessary event for cell division to occur.     

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol