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     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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     

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.     

Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rRNA and S7 mRNA

Summary A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe. Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S6 and elongation factors EF-G and EF-Tu in this DNA region. The arrangement is rps12 (124 codons)-167 bp spacer-rps7 (156 codons)-77 bp spacer-fus (694 codons)-26 bp spacer-tufA (409 codons), which is similar to that of the Escherichia coli str operon. The deduced amino acid sequences of the A. nidulans S12 and EF-Tu show high homology (72%–82%) with the E. coli and chloroplast counterparts while those of the A. nidulans S7 and EF-G give low homology (51%–59%). Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A. nidulans.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Chemical and biological studies on the lipopolysaccharide (O-antigen) of Anacystis nidulans

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 13 linked and of 25% 14 linked D-mannose units.Lipid A of A. nidulans is phosphate-free. The main fatty acid, -hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are -hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium.The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.     

Effect of sulfur starvation on the morphology and ultrastructure of the cyanobacterium Gloeothece sp. PCC 6909

Gloeothece sp. PCC 6909 is a unicellular, nitrogen-fixing cyanobacterium that accumulates sulfate in its sheath. An ultrastructural study of sulfate-deficient and normal Gloeothece sp. PCC 6909 cells was carried out. The physiological alterations, caused by sulfur starvation, were related to important morphological alterations in the cell: a structureless sheath, accumulation of cyanophycin, polyhydroxybutyrate and glycogen granules, and disintegration of thylakoidal membranes. Most of these changes were reversed by the addition of sulfate to the culture medium. The important role of sulfate in the sheath structure was demonstrated.     

The effects of nitrogen limitation on the ultrastructure of the cyanobacterium Agmenellum quadruplicatum

The effects of nitrogen limitation on the ultrastructure of the unicellular cyanobacterium, Agmenellum quadruplicatum, were studied by thin sectioning transmission electron microscopy. Nitrogen became limiting for growth 14–15 h after transfer to nitrogen-limiting medium, but cultures retained full viability for at least 45 h. The c-phycocyanin: chlorophyll a ratio and cellular nitrogen content of the culture dropped rapidly after 14–15 h, as a progressive deterioration of major cell structures took place. Phycobilisomes were degraded first, followed by ribosomes and, then, thylakoid membranes. These structures were virtually depleted from the cells within 26 h. Intracellular polysaccharide accumulated in place of the normal cell structures throughout this period. Nitrogen limitation did not affect polyphosphate bodies, carboxysomes, lipid granules, the cell envelope, or the extra-cellular glycocalyx. All of the ultrastructural changes resulting from nitrogen limitation were reversed upon addition of nitrate to a starved culture. Most cell structures were restored within 3 h, and restoration was complete within 9 h.