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.     

Evidence for two functionally distinct hydrogenases in Anacystis nidulans

The effect of growth conditions on aerobic and anaerobic hydrogenase activities of Anacystis nidulans was studied. It was found that the two hydrogenase activities both of which were confined to the particulate fraction of cell-free extracts correlated in an opposite way with growth temperature: The algae were always grown photoautotrophically in presence of H₂ but after growth at 25° C a significant oxyhydrogen reaction contrasted with negligible photoreduction rates while the opposite was true after growth at 40°C. A similar correlation between incubation temperature and induction of the respective hydrogenase activity was also observed with resting cells.Kinetic analysis of the two different types of hydrogenase — catalysed reactions with Anacystis membranes yielded the following Michaelis-Mentenparameters: K _M=55 M H₂ and v _max=0.12 mol H₂ per min and mg protein for the oxyhydrogen reaction, and K _M=170 M H₂ and v _max=0.3 mol H₂ per min and mg protein for the photoreductions. Also the dependences of oxyhydrogen and of photoreduction activities on pH and on temperature were measured; both pH and temperature profiles were found to be markedly different for each type of H₂-supported reaction.The results are discussed as pointing to the possible occurrence of two functionally distinct hydrogenase enzymes which can be synthesized by Anacystis in response to the conditions of induction.Abbreviations BO p-benzoquinone - CAP chloramphenicol - chl chlorophyll - cytc horse heart cytochrome c - DCMU 3-(34-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - fd ferredoxin - FeCy ferricyanide - MB methylene blue - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethan     

Genetic transformation of the blue-green bacterium,Anacystis nidulans

Evidence is presented which confirms the existence of genetic transformation in the blue-green bacterium, Anacystis nidulans. This process has been demonstrated for three mutations: streptomycin resistance, a phenylalanine requirement and an ornithine requirement. The optimal conditions under which transformation occurs are also investigated, and the potential of this system for genetic mapping is discussed.This work was submitted by K. G. O. in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology, Bryn Mawr College.     

Redox modulation of a phosphatase from Anacystis nidulans

Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0–7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn²⁺ ions and partially restored by Co²⁺ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co²⁺ ions only. Zn²⁺ ions did not activate this enzymatically inactive protein. The Co²⁺-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.Abbreviations AA ascorbic acid - DEAE diethylamino ethyl - DHAA dehydroascorbic acid - EDTA ethylene-diaminetetra-acetate - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - HMP hexose monophosphate - P _i inorganic phosphorus - pNPP p-nitrophenylphosphate - pNP p-nitrophenol - Tris Tris(hydroxymethyl)-aminomethane     

Synthesis of DNA during the cell cycle of synchronous Anacystis nidulans

Cells of Anacystis nidulans strain 1402-1 incorporate [methyl-³H]thymidine or [8-³H]adenine into DNA; in synchronous cultures (21/2 h full light, 1/2 h weak light, 5 h dark), this incorporation occurs in the dark to different extents according to the labeled precursor offered or to its specific activity. The specific activity of in vivo, uniformly labeled DNA decreases to half the initial value when the cells are grown in the absence of radioactive DNA precursors during the light phase; it does not decrease during the following dark phase. If unlabeled thymidine is given during the dark phase, the specific activity of the DNA starts to decrease at the onset of the next light phase. The time course of the decrease supports the hypothesis that all cells start their DNA replication immediately after illumination and that the first cells have completed if after 1.25 h. The slowest cells then need 3.75 h for completion of DNA replication. It is discussed whether the incorporation during the dark might be due to pool size effects.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

The regulation of the energy-dependent phosphate uptake by the blue-green alga Anacystis nidulans

Investigations of the energy-dependent accumulation of orthophosphate by the blue-green alga Anacystis nidulans have established: 1. The transport through the cell membrane is the rate-limiting step in the incorporation of phosphate.-2. This transport is facilitated by a carrier that can be activated by Ca²⁺ and Mg²⁺ and inhibited by EDTA.-3. The activation of the carrier in the light is associated with changes of the cytoplasmic Mg²⁺ content.-4. Intracellular phosphate is shown to be present in bound form.-5. The energy-dependent accumulation of orthophosphate within the cell depends strictly on the cytoplasmic pH and not on the energy conversion at the thylakoid membrane which is responsible for the energy supply. The cytoplasmic pH is different in the light, in the dark, and in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Orthophosphate accumulation can most readily be explained in terms of a pH dependent precipitation into a complex with bivalent cations rather than by an active transport against a concentration gradient.Abbreviation CCCP Carbonyl cyanide m-chlorophenylhydrazone     

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.     

The developmental genetics of pigment mutants in the Mexican axolotl

The Mexican axolotl (Ambystoma mexicanum) provides a well-defined set of color genes which are useful for various types of analyses. These include the a (albino), m (melanoid), ax (axanthic), and d (white) genes. In addition, various combinations of these genes and a number of as yet undescribed mutants also exist. Three of these mutants (a, ax, and m) have defects associated with specific neural-crest-derived pigment cell types. The fourth mutant (d) appears to provide an unsuitable environment for the migration and maintenance of pigment cells. In one case (m), detailed information concerning the specific nature of the genetic defect is available. The goal of this article is to demonstrate ways in which the existing information on the axolotl color genes can best be utilized in terms of understanding not only the mutant phenotypes, but basic concepts in the cell and developmental biology of pigmentation as well. Thus, an attempt has been made to sort through the genetic and biochemical data relevant to these mutants in order to stimulate renewed interest in a more detailed pursuit of such studies.     

Reduced sulfur and nitrogen compounds and molecular hydrogen as electron donors for anaerobic CO2 photoreduction in Anacystis nidulans

Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O₂ evolution and CO₂ fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O₂ evolution much more strongly than CO₂ fixation, indicating ability to supply electrons for CO₂ photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO₂ fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO₂ fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O₂. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H₂ and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone - DCMU dichlorophenyl dimethyl urea - DSPD disalicylidenepropane diamine-(1,3) - EDAC 1-ethyl-3(3-dimethylaminopropyl-) carbodiimide     

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.     

Information about previous phosphate fluctuations is stored via an adaptive response of the high-affinity phosphate uptake system of the cyanobacterium Anacystis nidulans

The high-affinity uptake system of phosphatelimited cyanobacterium Anacystis nidulans [Synechococcus leopoliensis (Raciborski) Komarek] is characterized by a threshold value below which uptake cannot occur. Here it is shown that, if phosphate-limited cyanobacteria are challenged with a short pulse of high phosphate concentration that appreciably exceeds this threshold value, the uptake system undergoes an adaptive response, leading to the attainment of new kinetic properties and a new threshold value. These new properties are maintained for several hours after the pulse. A notable characteristic of this new state is a wide linear dependence of the uptake rate on the external phosphate potential that is a function of the driving force of the uptake process. According to theoretical arguments it is shown that this “linear operation mode” can be explained by the simultaneous operation of several uptake systems with different, staggered threshold values and kinetic properties. Moreover, the new linear uptake properties, in turn, reflect the prehistory of phosphate supply experienced by the population. The consequences of this result with regard to environmental fluctuations of the phosphate concentration in lakes are discussed.     

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.     

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     

Functional organization and plasticity of the photosynthetic unit of the cyanobacterium Anacystis nidulans

Anacystis nidulans grown under high and low light, 100 and 10 μE m^?2 s^?1, respectively, was analyzed with respect to chlorophyll/P700, phycobiliproteins/P700, chlorophyll/cell, and oxygen evolution parameters. The photosynthetic unit sizes of this cyanobacterium, measured as the ratio of total chromophores (chlorophyll and bilin) to P700, were shown to be similar to those of higher plants and green algae. High light grown cells possessed a photosynthetic unit consisting of a core of 157 ± 6 chlorophyll a molecules per P700 associated with a light harvesting system of 95 ± 3.5 biliprotein chromophores. Low light grown cells had substantially more biliprotein chromophores per P700 (125 ± 3.1) than high light cells, but showed no significant difference in the numbers of chlorophyll a molecules per P700 (149 ± 4). Analyses of aqueous biliprotein extracts indicate that low light grown cells produce proportionately more phycocyanin relative to allophycocyanin than high light cells. Calculations of the molecular weight of biliproteins per P700 suggest that there is less than one phycobilisome per reaction center I under both growth conditions. Differences in chlorophyll/cell ratios and oxygen evolution characteristics were also observed. High light cells contain 6.3 × 10^?12 mg chlorophyll cell^?1, while low light grown cells contain 12.8 × 10^?12 mg chlorophyll cell^?1. Photosynthetic oxygen evolution rate vs. light intensity curves indicate that high light grown cells reach maximal levels of oxygen evolution at higher light intensity than low light grown cells. Maximal rates of oxygen evolution were 16.6 μmol oxygen min^?1 (mg chlorophyll)^?1 for high and 8.4 μmol oxygen min^?1 (mg chlorophyll)^?1 for low light cells. Maximal oxygen evolution rates per cell were equivalent for both cell types, although the amount of P700 per cell was lower in high light cells. High light grown cells are therefore capable of producing more oxygen per reaction center I than low light grown cells.     

Enzymes of glycogen mobilization in the photosynthetic procaryote,Anacystis nidulans

Glycogen, the principal storage compound of assimilatory products in Anacystis nidulans, is synthesized in the light and degraded in the dark. ¹⁴C-labelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude homogenate of cells kept in the dark contained the following enzymes: glycogen phosphorylase (EC 2.4.1.1.) that is firmly bound to glycogen, a debranching enzyme that hydrolyzes 1,6--glucosidic bonds, and an -glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable Using ion exchange chromatography on DEAE-cellulose, -glucosidase and the debranching enzyme could be partly separated from each other and completely from the phosphorylase-glycogen complex. On the basis of their known substrate specificities, the cooperation of these 3 enzymes is sufficient to account for the complete conversion of glycogen into glucose and glucose 1-phosphate.     

Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803

Mutants affected in their pigment content and in the structure of their phycobilosomes (PBS) were isolated in the cyanobacterium Synechocystis PCC 6803 by enriching a population with the inhibitor p-hydroxymercuribenzoate. Three of these mutants, PMB 2, PMB 10 and PMB 11, with original phenotypes, are described. Applying several criteria of analysis (77K absorption and fluorescence, protein electrophoretic patterns, electron microscopy), it was possible to assign the component polypeptides to each substructure of the phycobilisome. The model structure obtained fits with those described in other species PMB 10 and PMB 11, completely lacking PC, are the first source of pure PBS cores available, in which no contamination by residual PC can be feared, and are thus particularly interesting for further biochemical studies. The capacity of genetic transformation of Synechocystis PCC 6803 by chromosomal DNA makes this system very convenient for the analysis of the regulation of synthesis of the PBS constituents.Abbreviations PSI, PSII photosystems I, II - PBS phycobilisomes - PC phycocyanin - APC allophycocyanin - APC-B alophycocyanin B - PE phycoerythrin - PEC phycoerythrocyanin - WT wind type - Chl chlorophyll Present address: Service de Physiologie Microbienne Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15, France     

Alterations in the structure of phycobilisomes of the cyanobacterium,Spirulina platensis in response to enhanced Na+ level

Incubation of Spirulina platensis at enhanced Na⁺ concentrations resulted in 30% increased intracellular accumulation of Na⁺ ions. This accumulation of Na⁺ ions intracellularly altered the phycobilisome organization as revealed by transmission electron microscopic data and changes in the absorption spectrum. The room temperature emission peak at 638 nm in the control sample was blue-shifted by 7 nm in the treated samples suggesting phycobilisome disorganization. SDS-PAGE of phycobilisome polypeptides showed a significant increase in a 66.2 kDa polypeptide. This is the first report that the enhanced concentration of intracellular Na⁺ ion alters the structure of the phycobilisome in S. platensis. This revised version was published online in November 2006 with corrections to the Cover Date.