Information Processing by Cyanobacteria during Adaptation to Environmental Phosphate Fluctuations

Phosphate limited grown Anabaena variabilis has the capability of processing information about external phosphate fluctuations by means of interconnected adaptive events. Adaptive events are physiological processes that are characterized by two opposite manifestations, namely adapted states and adaptive operation modes. In adapted states the energy-converting constituents of the uptake system operate under the prevailing external conditions in a coherent manner with least energy dissipation. Adaptive operation modes take place when adapted states are disturbed by persistent changes in phosphate supply. In this mode the outcome of former adaptations to elevated phosphate levels guides the emergence of a new adapted state. The influence of antecedent adapted states on subsequent adaptations was studied experimentally and characteristic examples for such information processing are given. The theory of self-referential systems allowed analyzing these examples. For this purpose adaptive events had to be considered as elements of a communicating network, in which, along a historic succession of alternating adapted states and adaptive operation modes, information pertaining to the self-preservation of the organism is transferred from one adaptive event to the next: the latter “interprets” environmental changes by means of distinct adaptive operation modes, aimed at preservation of the organism. The result of this interpretation is again leading to a coherent state that is passed on to subsequent adaptive events. A generalization of this idea to the adaptive interplay of other energy converting subsystems of the cell leads to the dynamic view of cellular information processing in which the organism recreates itself in every new experience.Key Words: adaptation, cyanobacteria, information processing, phosphate uptake, self-referential systems     

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.     

Phosphate Uptake by Eukaryotic Algae in Cultures and by a Mixed Phytoplankton Population in a Lake: Analysis by a Force-Flow Relationship*

The phosphate uptake behaviour of monospecific cultures of green algae in the laboratory and of mixed phytoplankton populations in a mesotrophic lake has been analyzed with the aid of a force-flow relationship. This analysis yields two parameters:

• 1 A conductivity coefficient, that characterizes the activity of the phosphate uptake system.
• 2 An external threshold phosphate concentration, below which uptake of phosphate is excluded on energetic grounds.

When the phosphate concentration lies below the threshold value, the algae show an activation of the uptake system, reflected in an increase in the conductivity coefficient. Correspondingly, excess phosphate above the threshold value leads to a diminuation of the conductivity. Using this simple analysis, phosphate discharge into lake water may be readily monitored.     

Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes

The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs-Ringer bicarbonate buffer, become essential for insulin action in bicarbonate-free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post-binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed.     

Siliciumstoffwechsel bei Mikroorganismen

Summary Soil bacteria which have been used in earlier experiments to demonstrate an active uptake of silicon, loose phosphate during silicon uptake when cultured in P-free medium. This could be shown by comparable determinations of the phosphate and silicon concentration of the cells. Under the conditions given in our experiments the exchange of Si for P lies in the range of 1:2. By addition of rising P-concentrations to media with constant concentration of Si, it was shown that about 100 P/ml will completely inhibit the uptake of silicon within 24 hours. Increasing concentrations of phosphate going along with decreasing concentrations of silicate showed to cause a linear decrease of Si-uptake intensity within the first 24 hours in the range of 20–100 P/ml. Above these concentrations (and the proportion of Si/P=1:4) silicon uptake is completely inhibited independent of phosphate concentrations. About 10% of the silicon incorporated can be extracted from the cells with ethanol in the form of instable, easily hydrolysable complexes. The entire silicon of the cells is completely exchanged against phosphate when silicon containing cells are cultured in Si-free phosphate medium, whereas cells adapted to silicon will not extrude the silicon taken up before, when incubated in a medium containing both elements. References to the possible synthesis of organic silicon compounds resulting from these experiments are discussed.     

The bioenergetic coordination of a complex biological system is revealed by its adaptation to changing environmental conditions

The properties of the phosphate uptake system of the cyanobacterium Anacystis nidulans have been studied during the transition from a phosphate-deficient non-growing state to a non-deficient growing state. In the phosphate-deficient state the high affinity phosphate transport system in the cell membrane is extremely adaptive. As a result of these adaptive features the phosphate transport system cannot be described by determinate, fixed parameters, because the transport system is influenced by the measurement of the uptake process itself. When the growing state has been initiated by a persisting phosphate pulse, the transport system rapidly loses its adaptive features and can then be characterized by determinate parameters that remain unchanged for a long period of time, even if no uptake occurs in that time. Depending on the amount of phosphate stored during a pulse the cell makes a choice between slow or fast growth. In the latter case the light harvesting and energy converting machinery is completely reorganized before growth commences. Thereby the components of this machinery conform to each other and to the stable properties of the phosphate transport system. It is suggested that the mutual adjustment of these adaptive energy converting subunits is guided by attractors that function as the final cause for the development of the whole system.An application of this model to an analysis of the selforganization of aquatic ecosystems is discussed.     

Serum-stimulated phosphate uptake and initiation of fibroblast proliferation

Previous studies have shown that initiation of proliferation of density-inhibited fibroblasts by fresh serum is accompanied by a rapid increase in phosphate uptake. This increase might be a key event in the initiation of DNA synthesis. The present studies examined this possibility. Mouse 3T3, secondary chick embryo, or human diploid foreskin cultures were grown to quiescence in medium containing varying levels of serum. When proliferation of the cultures was initiated by addition of fresh serum, the changes in phosphate uptake were inversely related to the final increases in cell number. Additional experiments showed that the change in phosphate uptake following serum addition was determined by the level of phosphate uptake prior to serum addition. Addition of dexamethasone to quiescent 3T3 cultures caused them to proliferate but did not increase phosphate uptake. Similarly, trypsin or insulin stimulated proliferation of quiescent secondary chick embryo cultures, but caused little or no change in phosphate uptake. Quiescent 3T3 cultures switched to medium containing fresh serum and reduced levels of phosphate showed a decrease in both phosphate uptake and intracellular phosphate pool size. Cell proliferation in these cultures, however, was stimulated to the same degree as cultures switched to medium containing fresh serum and the normal amount of phosphate. In addition, quiescent secondary chick embryo cultures switched to medium containing fresh serum and no phosphate showed a decrease in the intracellular phosphate pool size. Thymidine incorporation and final cell number in these cultures, however, was stimulated to the same or higher degree than in cultures switched to medium containing fresh serum and the normal amount of phosphate. These results demonstrate that the rapid increase in phosphate uptake following addition of fresh serum to quiescent fibroblasts is not a necessary event for the initiation of proliferation.     

Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species

Acinetobacter has been found to be the major species responsible for mediating biological phosphate removal. The growth kinetics and phosphate uptake were investigated for an isolated Acinetobacter strain growing in a defined medium. The phosphate uptake is dependent on growth rate, temperature, and pH. Polyphosphate granules occurred in a balanced growth stage. The maximum phosphorus content in cells was 4.8% at the dilution rate of 12 day(-1). The specific phosphate uptake rate was found to be a quadratic polynomial function of the dilution rate. Increased calcium (up to 36 mg/L) and magnesium (up to 15 mg/L), and the addition of yeast extract (100 mg/L), primary effluent (20%), and fluoride (10 mg/L) did not affect phosphate uptake. Anaerobic conditioning (N(2) stripping), low pH (CO(2) stripping), and addition of sodium acetate under anaerobic conditions failed to stimulate immediate phosphate release. Nevertheless, After 21-24 h, the phosphate release was ca. 3, 5, and 15 mg P/g cell, respectively, for N(2) purging, the addition of acetate, and CO(2) purging. For two-stage completely stirred reactor operation, there was negligible phosphate overplus at the second reactor when phosphate was added, when the first reactor was subjected to phosphate limitation. When both phosphate and carbon limited the growth in the first reactor, there was slight phosphate accumulation under endogenous respiration conditions in the second reactor.     

Growth optimization of the invasive cyanobacterium Cylindrospermopsis raciborskii in response to phosphate fluctuations

The role of ecophysiological traits in the success and expansion of the toxic cyanobacterium Cylindrospermopsis raciborskii is still under debate. One key factor appears to be the high physiological flexibility of this organism when obtaining limiting resources. Recent studies have found that filamentous bloom-forming cyanobacteria are able to optimize their growth by adjusting phosphate uptake during fluctuating nutrient conditions. We investigated the growth response of two phosphate-deficient C. raciborskii isolates (MVCC19 from Uruguay and CCMP1973 from USA) to short-term fluctuations in phosphate supply. These isolates were exposed to five phosphate concentrations which were provided in two supply modes: a single pulse (SingleP) versus the same amount divided in 10 pulses (TenP), with one pulse applied every 6 min. Morphological traits and changes in chlorophyll a and phycocyanin fluorescence were also evaluated. Growth rates of CCMP1973 and MVCC19 almost doubled and tripled, respectively, when exposed to multiple rather than single phosphate additions. Different growth rates were observed with the same total added resource, thus contradicting the classical model of dependence of growth rate on absolute external concentration. Phosphate-deficient C. raciborskii showed a remarkable physiological flexibility in adapting to phosphate availability on a timescale from minutes to hours. The TenP mode provided an extension of phosphate exposure time that allowed the energetic optimization of uptake and growth. The morphological plasticity of the species in response to phosphate supply mode was also shown by differences in trichome length and individual size. Our results are the first evidence of growth optimization of phosphate-deficient C. raciborskii to short-term nutrient fluctuations, revealing its physiological flexibility. This adaptive behaviour may help to explain the invasive success of this diazotrophic cyanobacterium in a wide range of aquatic ecosystems where phosphorus is frequently the limiting resource.     

Adaptation of Pseudomonas aeruginosa to 2,2'-methylenebis (4-chlorophenol)

A culture of Pseudomonas aeruginosa, isolated from a cooling water system, was grown in the presence of sub-inhibitory concentrations of 2,2'-methylenebis(4-chlorophenol) (MBC). It adapted to increasing concentrations from an initial minimum inhibitory concentration of 36 μg ml^-1 to the highest, 80 μg ml^-1. Resistant cultures exhibited a higher survival rate when exposed to 320 μg ml^-1 than did the original strain. Lipopolysaccharide and outer membrane protein profiles were determined by SDS PAGE. No changes were detected in lipopolysaccharide profiles. The quantity of OprP, the phosphate uptake protein in the outer membrane, decreased to a low level correlating with decreased phosphate (P_i) uptake during growth. It is proposed that OprP is the place of entry for MBC and that the cell can adapt by decreasing the level of OprP in the outer membrane.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Root cortical aerenchyma inhibits radial nutrient transport in maize (Zea mays)

Background and Aims

Formation of root cortical aerenchyma (RCA) can be induced by nutrient deficiency. In species adapted to aerobic soil conditions, this response is adaptive by reducing root maintenance requirements, thereby permitting greater soil exploration. One trade-off of RCA formation may be reduced radial transport of nutrients due to reduction in living cortical tissue. To test this hypothesis, radial nutrient transport in intact roots of maize (Zea mays) was investigated in two radiolabelling experiments employing genotypes with contrasting RCA.

Methods

In the first experiment, time-course dynamics of phosphate loading into the xylem were measured from excised nodal roots that varied in RCA formation. In the second experiment, uptake of phosphate, calcium and sulphate was measured in seminal roots of intact young plants in which variation in RCA was induced by treatments altering ethylene action or genetic differences.

Key Results

In each of three paired genotype comparisons, the rate of phosphate exudation of high-RCA genotypes was significantly less than that of low-RCA genotypes. In the second experiment, radial nutrient transport of phosphate and calcium was negatively correlated with the extent of RCA for some genotypes.

Conclusions

The results support the hypothesis that RCA can reduce radial transport of some nutrients in some genotypes, which could be an important trade-off of this trait.     

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction.     

Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with alpha-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT1 in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by alpha TC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.     

Kinetics of uptake of phosphates and nitrates by marine multicellular algae <Emphasis Type="Italic">Gelidium latifolium</Emphasis> (Grev.) Born. et Thur.

We studied nonstationary kinetics of the uptake of phosphates and nitrates by the red marine algae Gelidium latifolium (Grev.) Born et Thur. and calculated constants of the Michaelis-Menten equation for these elements. In the area of 0–3 μM, the kinetics of phosphate consumption had the following coefficients: maximum rate of uptake 0.8 μmol/(g h), constant of half-saturation 1.745 μM. For nitrate nitrogen at 0–30 μM, an adaptive strategy of uptake kinetics was noted with change of the equation parameters with time: after 1 h, the maximum rate of uptake was 5.1 μmol/(g h) and constant of half-saturation 19 μM, while within 2 h, the maximum rate of uptake significantly increased. This could be related to the synthesis of nitrate reductase. Coupled with the uptake of nitrates, nonstationary kinetics of the release of nitrates in the surrounding medium had a one-peak pattern: the maximum concentration of nitrites in the medium and the time of its achievement increased with the initial concentration of nitrates. The maximum concentration of nitrites was 6 to 14% of the initial concentration in the medium.     

Interaction of lambda bacteriophages with mammalian cells. II. Elucidation of the role of interacting components

The study of 3H-thymidine labelled bacteriophage lambda C185757 uptake by HeLa, RH and Chinese hamster cell revealed the lack of cells or phage specificity in the phage interaction with cells. The phage uptake is shown to be an active process depending on the cell state. The mechanism of "protective" action of calcium chloride is found to be as follows: the calcium phosphate precipitate formed in phosphate-containing media absorbs the phage, thus increasing its concentration on the cell surface, which makes the pinocytosis more effective.     

Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

Background

A number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood.

Methodology/Principal Findings

Here we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2.

Conclusions/Significance

The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.     

Reversibility and partial reactions of the Na(+)-K+ pump of rat erythrocytes

An assay was developed to characterize the kinetic parameters of the Na(+)-K+ pump of rat erythrocytes under conditions as physiological as possible. Changes in the red cell Na+ and Rb+ content were determined in Na+ media (containing 2.5 mM inorganic phosphate (PO4) as a function of cell Na+ (2-8 mmol/l) and extracellular Rb+ (0.2-5 mM). Evaluation of the data revealed that under these conditions the Na(+)-K+ pump mediates, in addition to forward running 3 Nai+: 2 Rbo+ exchange, 1 Ki+:Rbo+ exchange and pump reversal (3 Nao+:2 Ki+ exchange). The two latter modes of Na(+)-K+ pump operation are accelerated by PO4 and lowering of cell Na+. At physiological cation and PO4 concentrations, 1Ki+:Rbo+ exchange contributes by 30-60% to total ouabain-sensitive Rb+ uptake. Thereby, the stoichiometry of ouabain-sensitive Na+ net-extrusion to Rb+ uptake is reduced to values between 1.0 and 0.5. Only at cell Na+ contents above 20 mmol/l the Na+:Rb+ stoichiometry approaches the value of 3:2 = 1.5. At certain constellations of Nai+ and Rbo+ the Na(+)-K+ pump cannot perform any net-transport of Na+ and K+ (Rb+). These equilibrium points are not far from those expected from thermodynamic considerations. The results demonstrate that in normal rat erythrocytes the reversible reaction cycle of the Na(+)-K+ pump runs in several modes of operation. The "abnormal" modes complicate the interpretation of unidirectional fluxes mediated by the Na(+)-K+ pump.