Growth and gas-vacuole development in vegetative cells of Anabaena flos-aquae

Summary Gas-vacuoles consisting of collections of gas-cylinders, and continuity of the plasma membrane with lamellae, are observed at all stages of growth of Anabaena flos-aquae. In day+4 cells, increased numbers of invaginations of the plasma membrane are observed and the lamellae tend to lie parallel to the cell wall. Some evidence is presented which suggests an associated synthesis of -granules and gas-cylinders by lamellae. Features of older cells are increased numbers of gascylinders, -granules, structured granules, and large intralamellar vesicles which appear to correlate with the presence of pinkish vacuoles as observed with the light microscope. The mean percentage of the volume of cells occupied by gas-vacuoles is about 20% during exponential growth when the doubling time is 56.5 hours, increasing to 34% in day+24 cells when growth is stationary.     

Modeling culture profiles of the heterocystous N2-fixing cyanobacterium Anabaena flos-aquae

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.     

Effect of Anabaena flos-aquae on the abilities of Daphnia and Keratella to feed and reproduce on unicellular algae

SUMMARY.

• 1 This study tests the hypothesis that the filamentous cyanobacterium, Anabaena flos-aquae, inhibits Daphnia's ability to feed and reproduce on unicellular algae more than it does Keratella's. Double-label radiotracer experiments using whole and sonieally disrupted Anabaena filaments showed that even low concentrations of Anabaena (5 × 10³ cells ml^?1) inhibited the abilities of Daphnia pulex and D. galeata mendotae to feed on Chlamydomonas and that this inhibition was due to both mechanical interference with feeding and increased food availability. A single-label radiotracer experiment showed that Anabaena (5 × 10³ and 2 × 10⁴ cells ml^?l) reduced the abilities of neonate, juvenile, and adult D. pulex to feed on Chlamydomonas but had a much more pronounced effect on adults.
• 2 Single-label radiotracer experiments using screened (25 μm mesh) and unscreened suspensions of whole Anabaena filaments showed that only very high concentrations of Anabaena (10⁵ cells ml^?1) reduced the ability of Keratella cochlearis to feed on Cryptomonas and that this inhibition probably was due to mechanical interference with feeding, since Keratella ate Anabaena extremely inefficiently - especially when single cells and short filaments were removed by screening.
• 3 Experiments comparing the survivorship and fecundity of individuals fed Anabaena, Chlamydomonas, or no food showed that the Anabaena was not toxic to either Keratella or D. galeata mendotae, was not utilized by Keratella, and was utilized by Daphnia, although not as well as Chlamydomonas was.
• 4 Anabaena (5 × 10³ or 10⁴ cells ml^?1) did not affect, or slightly increased, the population growth rate (r_m) of K. cochlearis on Cryptomonas, while it substantially increased that of D. pulex, Duphnia's ability to utilize Anabaena probably more than offset its reduced ability to feed on Cryptomonas. The presence of cyanobacterial filaments in natural plankton communities could adversely affect Daphnia and decrease its ability to competitively suppress Keratella and other rotifers if the filaments were of little nutritional value to the Daphnia, greatly interfered

     

Uptake and Utilization of Sugar Phosphates by Anabaena flos-aquae

The effect of various sugar phosphates on CO₂ fixation in Anabaena flos-aquae was investigated and found to be very similar to that found for isolated spinach chloroplasts. One exception, glucose 6-phosphate, has a stimulatory effect on CO₂ fixation in Anabaena but not in isolated chloroplasts.     

Regulation of glutamine synthetase in the blue-green alga Anabaena flos-aquae

Glutamine synthetase (GS) was isolated from log phase cells and purified to a single protein as evidenced by gel electrophoresis. Protamine and ammonium sulfate precipitation and chromatography on DEAE-cellulose and Bio-Gel resulted in 380-fold purification. The enzyme was most sensitive to alanine (85% inhibition at 0.1 mM) but was also inhibited by glycine, arginine and serine. Combinations of inhibitory amino acids or nucleotides (AMP, ADP, ATP) exhibited cumulative inhibition. Cooperative inhibition was noted with CTP and any single nucleotide. Inhibition by CTP alone was uncompetitive with respect to glutamine. The enzyme was also regulated by the energy charge of the cell.     

Chemical analysis of the phenol-water-extractable materials from Anabaena flos-aquae.

The high molecular-weight carbohydrate substances extracted in the aqueous and phenol phases by phenol-water extraction of Anabaena flos-aquae A-37 were found to be polysaccharides without lipid attached.     

Some Effects of Sodium on Nitrate Assimilation and N(2) Fixation in Anabaena cylindrica

Anabaena cylindrica grown with nitrate required higher levels of sodium (0.4 meq/l NaCl) to prevent chlorosis than when grown without combined nitrogen (0.004 meq/l NaCl). Nitrite accumulated in sodium-deficient cultures containing nitrate. Amounts of nitrite similar to those found in deficient cultures when added to normal cultures resulted in a chlorosis of the cells. Thus loss of chlorophyll was caused by nitrite toxicity.     

Growth characteristics and toxin production in batch cultures of Anabaena flos-aquae: effects of culture media and duration

The effects of media and culture duration on growth, macromolecular composition and toxicity of an anatoxin- a-producing freshwater cyanobacterium Anabaena flos-aquae (UTEX 2383) were evaluated. The four media A₃M₇, CB, MA and B-12 influenced growth in terms of cell number, chlorophyll-a content and specific growth rate. A₃M₇ medium supported the best growth. The macromolecular composition of cultured cells, viz. total carbohydrate, protein and lipid content varied with media and culture duration reaching maximum concentration at various growth periods. The differences were significant due to interaction of the culture medium and duration. Toxicity of cells grown in different media was compared by Artemia salina bioassay and mouse units. The cells grown in A₃M₇ medium showed highest toxicity and the optimum culture duration was 5 weeks. In terms of both growth characteristics and toxicity the media can be ranked as A₃M₇, MA, CB and B-12 in decreasing order.     

Evidence for the production of extracellular herbivore deterrents by Anabaena flos-aquae

SUMMARY. 1. Daphnia pulex grown in the cell-free filtrate of a log phase culture of Anabaena flos-aquae showed lower fitness as measured by filtering rate, survivorship and reproduction than did individuals in a control treatment. This suggests that the extracellular metabolites of some algae may have considerable adaptive value as a herbivore deterrent.
2. Heating of the cell-free filtrate neutralized the inhibitory effect on filtering rate, but increased the depression of reproduetive rate. This suggests the presence of a variety of bioactive compounds which may inhibit specific herbivore functions.     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae

Ammonium suppresses nitrogenase activity in Anabaena flos-aquae (Lyng) Breb. at all pH values tested. l-Methionine-dl-sulfoximine at 1 millimolar totally inhibited glutamine synthetase, and 10 micromolar partially inhibited. Both concentrations protected nitrogenase activity from ammonium-induced suppression at pH 7.1 and 8.1. At pH 9.3 and 10.2, methionine sulfoximine did not alleviate the suppression of nitrogenase by ammonium. This pH-dependent protection of nitrogenase activity is a result of the noncompetitive inhibition of the ammonium transporter by methionine sulfoximine. At pH 7.1 and 8.2, ammonium is protonated and methionine sulfoximine inhibits its entry into the cell. At pH 9.3 and 10.2, unprotonated ammonia is abundant and may enter the cell independent of the transport system. The effects of ammonium are closely mimicked by the ammonium analog methylamine. These results suggest that ammonium per se is an important in vivo regulator of nitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re-evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter.     

溶藻细菌L7对水华鱼腥藻氮代谢的影响

【目的】进一步探明藻菌关系,研究溶藻细菌对藻类氮代谢的影响及其作用机制。【方法】将水华鱼腥藻和溶藻细菌L7按两种比例接种入BG11培养液中,在室内进行共培养(藻细胞初始密度为1.21×108cells/L;溶藻细菌L7初始密度分别为1.75×107、1.75×108CFU/mL)。连续7 d测定藻细胞数、异形胞频率和藻细胞内的硝酸还原酶(NR)活性、谷氨酰胺合成酶(GS)活性、谷氨酸合成酶(GOGAT)活性、蛋白质含量、丙二醛(MDA)含量。【结果】低密度溶藻细菌L7能够促进藻生长(第7天藻细胞密度是对照组的1.58倍),增加异形胞频率(第7天高于对照组66.67%);高密度则会抑制藻生长(第7天藻细胞密度相比对照组下降98.84%),降低异形胞频率(第7天为0)。在藻细胞内氮代谢关键酶活性方面,接种后2 5 d,两处理组中藻细胞内NR和GOGAT活性均极显著高于对照组(P<0.01);接种后0 5 d,高密度处理组的GS活性极显著高于对照组(P<0.01),而低密度处理组的则在大部分时间内极显著低于对照组(P<0.01)。在整个实验期内,低密度处理组中藻细胞内蛋白质含量一直极显著高于对照组(P<0.01);而在高密度处理组中,除第5天外,细胞内蛋白质含量则全部极显著低于对照组(P<0.01)。接种后2 4 d,高密度处理组中藻细胞内MDA含量呈现上升趋势,并极显著高于其余两组(P<0.01)。【结论】低密度溶藻细菌L7能够提高水华鱼腥藻对氮源的需求,加速蛋白质合成,促进氮代谢;而高密度溶藻细菌L7会对藻细胞产生过氧化伤害,阻碍蛋白质合成和氮代谢过程。     

Crystalline structure of the gas vesicle wall from Anabaena flos-aquae.

The gas vesicles isolated from Anabaena flos-aquae have been studied by X-ray diffraction. Electron microscopy has previously shown that the gas vesicles are elongated shapes, with a thin wall having regular striations (ribs) at right-angles to the long axis. The X-ray diffraction pattern from a specimen of oriented, intact vesicles includes a number of sharp reflections which are attributed to regular structure in the plane of the wall. After correcting for the imperfect alignment of the long axes of the vesicles, the in-plane reflections are all seen to lie on a few, regularly spaced lines parallel to the long axis. This result shows for the first time that there are subunits regularly spaced along each rib, one subunit every 11 Å. The spacing of the in-plane reflections along each line is consistent with a rib periodicity of 46 Å. The 11 Å repeat, together with the 46 Å repeating distance from rib to rib and the average wall thickness of about 20 Å, define a volume for the subunit. Assuming a reasonable value for the density of the protein making up the wall, the molecular weight of the subunit indicated is about 8000 g/mol.The X-ray data also indicate that a large part of the protein is in the β-sheet conformation. In this structure there are parallel, or anti-parallel, polypeptide chains which are hydrogen-bonded to one another in a regular way to form a thin sheet. Assuming the wall contains β-sheet in two layers, one on top of the other and with the chains in each layer tilted at 35 ° to the long axis of the vesicle, we can explain a number of the X-ray observations: (1) oriented arcs with a Bragg spacing of 4.7 Å, which is the distance between the axes of neighbouring chains in each layer; (2) diffraction oriented in the direction of the chains at a spacing of 6 to 7 Å, which is the repeating distance of the dipeptide unit along the chain; (3) the 11 Å repeat, which is the repeating distance of pairs of chains along each rib; and (4) a broad band of diffraction at right-angles to the plane of the wall and centred at a spacing of 10 Å, which is a reasonable value for the distance between the mid-planes of the two sheets. Moreover, we can also find the remaining lattice parameter, the angle relating the centres of the subunits in neighbouring ribs. Thus the shortest line joining the centres makes an angle of 86 ° with the direction of the ribs.     

Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture

Summary Anabaena flos-aquae was grown in chemostats with phosphate-limiting growth and dilution rate of 0.015–0.03 h^-1. The yields of cells were dependent on dilution rate and a two-fold increase obtained by growth in the presence of 15 mM KNO₃. Alkaline phosphatase activity varied 20-fold, lowest activity with excess phosphate light-limited cells and the highest activity with cells grown in the presence of 15 mM KNO₃. There was no correlation between hot water soluble phosphate of cells and alkaline phosphatase activity.     

Determination of Cu Environments in the Cyanobacterium Anabaena flos-aquae by X-Ray Absorption Spectroscopy

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.     

Some Observations on the Sodium and Potassium Interactions in the Blue-green Alga Anabaena flos-aquae A-37

The growth of Anabaena flos-aquae A-37 has been shown to be severely limited by the absence of either sodium or potassium from the culture medium. Neither element is capable of replacing the other. The addition of sodium to sodium-starved cells restores growth while potassium-starved cells are not affected by the addition of potassium.     

The fine structure of gas-vacuoles released from cells of the blue-green alga Anabaena flos-aquae

Summary Cells of the blue-green alga Anabaena flos-aquae were ruptured and the gas-cylinders, which comprise the gas-vacuoles, were released. The gas-cylinders released retained their same shape and structure: 70 m or more wide, 0.2–0.9 long, with conical ends. Staining with phosphotungstic acid showed the membrane to be made up of particles arranged in rows, about 5 m apart, running round the cylinder at right angles to the long axis. Some of the cylinders became flattened during the preparation. Rounding-off of the gas-cylinders on isolation, described by others, was not encountered. However, some of the isolated gas-cylinders were considerably wider than 70 m and it is possible that they tend to shorten and widen on isolation from the cell.     

Kinetics of synthesis of nitrogenase in batch and continuous culture of Anabaena flos-aquae

Summary Nitrogenase of Anabaena flos-aquae was inactivated by oxygen and recovery of activity was measured in batch and iron, phosphate and urea-limited continuous cultures. In batch culture, canavanine, chloramphenicol, methylamine, proflavine, puromycin and urea inhibited the recovery process. The rate of recovery of nitrogenase activity in continuous cultures was dependent on light intensity, concentration of urea, ammonium salts and nitrate, and independent of growth rate. Oxygen and urea caused an inactivation of nitrogenase in continuous cultures.     

Determination of Cu environments in the cyanobacterium Anabaena flos-aquae by X-ray absorption spectroscopy

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.