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.     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

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.     

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.     

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     

Pattern of inhibition of nitrate utilization by ammonium in the acidophilic thermophilic unicellular alga Cyanidium caldarium

Ammonium-induced inhibition of nitrate utilization was monitored in cell suspensions of the unicellular alga Cyanidium caldarium. It was found that the inhibition followed an exponential pattern with a t _1/2 value of about 1.5 min in cells previously grown under conditions of excess nitrate, and of about 15 min in cells grown under conditions of severe nitrate limitation. In the latter cells only, a pretreatment with cycloheximide greatly increased the t _1/2 value of inhibition. Also the resumption of nitrate utilization when ammonium was depleted followed an exponential pattern with a t _1/2 value of about 4.5 min.Our results are consistent with the hypothesis that inhibition of nitrate utilization occurs at the level of nitrate reductase activity.     

Effect of some environmental factors on cyanophage AS-1 development in Anacystis nidulans

The development cycle of the cyanophage AS-1 was studied in the host blue-green alga, Anacystis nidulans, under conditions that impair photosynthesis and under various light/dark regimes. Under standard conditions of incubation the 16-h development cycle consisted of a 5-h eclipse period and an 8-h latent period. Burst size was decreased by dark incubation to 2% of that observed in the light. An inhibitor of photosystem II, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), reduced the burst size to 27% of that of the uninhibited control, whereas cyanophage production was completely abolished by carbonyl-cyanide m-chlorophenyl hydrazone (CCCP), an inhibitor of photosynthetic electron transport. Dark incubation of infected cells decreased the latent period by 1–2 h and the eclipse period by 1 h, once the cultures were illuminated. This suggests that adsorption took place in the dark. Intracellular growth curves indicated that light is necessary for viral development. Infected cells must be illuminated at least 13 h to produce a complete burst at the same rate as the continuously illuminated control. Low light intensities retarded the development cycle, and at lowest light intensities no phage yield was obtained. AS-1 is highly dependent on host cell photophosphorylation for its development.List of Abbreviations CCCP Carbonyl-cyanide m-chlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - m.o.i. multiplicity of infection - O.D. optical density - PFU plaque-forming unit Dedicated to Prof. Roger Y. Stanier on the occasion of his 60th birthday     

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     

Endogenous ammonia production by Anacystis nidulans R-2 induced by methionine sulfoximine

Anacystis nidulans R-2 produced ammonia from endogenous sources for at least 6 h when illuminated without external nitrogen source but with CO₂ in the presence of 50 M methionine sulfoximine. The onset of ammonia release coinciding with complete inhibition of glutamine synthetase. The total quantity of ammonia which could be released exceeded the nitrogen content of small molecule pools, and suggested protein degradation as the most likely source of the nitrogen. Ammonia release was not accompanied by leakage of carbon compounds from the cells. Methionine sulfoximine-induced ammonia release was energy requiring, and was barely detectable under dark anaerobic conditions, or in the presence of 10 M carbonyl cyanide m-chlorophenylhydrazone in light. Phenyl methyl sulfonylfluoride, an inhibitor of serine proteases, eliminated ammonia release, and the rate of release was reduced to one-third of control values, after a lag, in the presence of 50–75 g/ml chloramphenicol. The rate of NH ₊ ⁴ release was maximal (1.4 nmol·min^-1·mg^-1 protein) if suspensions were bubbled with 100% O₂, but could not be reduced below 0.6 nmol·min^-1·mg^-1 protein in air: CO₂, suggesting that release was at most only partly due to photorespiration.Abbreviations used MSX L-methionine D,L-sulfoximine - PMSF phenylmethylsulfonylfluoride - CAP chloramphenicol - CCCP carbonyl cyanide-m-chlorophenyl hydrazone     

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.     

Shift in carbon flow and stimulation of amino-acid turnover induced by nitrate and ammonium assimilation in Anacystis nidulans

The influence of nitrate and ammonium assimilation on the flow of recently fixed carbon has been determined in intact Anacystis nidulans cells actively fixing CO₂. Assimilation of nitrate or ammonium resulted in substantial increases in the incorporation of carbon into acid-soluble metabolites, the magnitude of the effect being dependent on the irradiance. The radiolabel in sugar phosphate was virtually unaffected by nitrogen assimilation, whereas that in organic acids and, in particular, in amino acids was markedly increased. Enhancement of carbon incorporation into amino acids induced by nitrogen assimilation was not accompanied by parallel increases in the size of the amino acid pools. This resulted in an appreciable increase of the specific radioactivity of most amino acids under conditions of nitrogen assimilation. The data indicate that nitrate and ammonium assimilation induce an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to oxaloacetate and α-ketoglutarate, as well as a stimulation of amino-acid turnover. These effects were more pronounced at saturating irradiance. We thank the Dirección General de Investigación Científica y Técnica, Spain (research grant PB88-0019) and the Plan Andaluz de Investigación (grupo 3101) for financial support, and P. Pérez de León for excellent secretarial assistance.     

Production of ammonium dependent on basicL-amino acids byAnacystic nidulans

The incubation of the cyanobacteriumAnacystis nidulans withL-Arg,L-Lys orL-Orn, but neither with the correspondingD-isomers nor with other twentyL-amino acids, resulted in the production of large amounts of ammonium which accumulated in the outer medium. Relevant properties of thisin vivo ammonium production activity have been studied in cell suspensions treated with the glutamine synthetase inactivatorL-methionine-D,l-sulfoximine (MSX) to prevent assimilation by the cells of the resulting ammonium. In addition to its specificity for the basicL-amino acids, the system exhibited a set of properties (K _m value for substrates, requirement of oxygen which is taken up stoichiometrically with the production of ammonium, inhibition by o-phenanthroline and divalent cations) all of which are shared by a peculiarL-amino acid oxidase recently isolated fromA. nidulans. The data strongly suggest the participation of this enzyme in the production of ammonium from basic amino acids byA. nidulans, an activity that could account for the ability of this cyanobacterium to use arginine as a nitrogen source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - MSX L-methionine-D,l-sulfoximine     

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.     

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.     

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.     

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.     

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)     

DCCD induced sodium uptake by Anacystis nidulans

The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na⁺/H⁺-antiport system. The necessary H⁺-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H⁺-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10^-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10^-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD N,N-dicyclohexylcarbodiimide - NEM N-ethylmaleimide - CCCP carbonylcyanide m-chlorophenylhydrazone - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Effect of ammonium and ferricyanide on nitrate utilization by Chlorella vulgaris

It has been shown previously that added ammonium salts cause a cessation of nitrate utilization in some Chlorella species. It has also been shown that Chlorella vulgaris can form an inactivated nitrate reductase which is an HCN complex. In the present study, a comparison has been made of the rate of nitrate utilization and the rate of nitrate reductase inactivation in Chlorella vulgaris in response to the addition of ammonium salts and light-dark changes. The rate of formation of HCN-inactivated enzyme is too slow to account for the prompt inhibition of nitrate utilization caused by adding ammonium. In contrast, when nitrate utilization is inhibited by addition of ferricyanide to intact cells, the HCN-inactivated enzyme is promptly formed in vivo, and might account for the inhibition of nitrate utilization, though inhibition of nitrate uptake can not be excluded.     

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.