Sonia Cortassa

Chaotic dynamics and fractal space in biochemistry: simplicity underlies complexity

In this work we attempt to analyze the coupling between the dynamics of biochemical reactions (especially chaotic dynamics), and the geometry of cytoarchitecture (especially fractal ultrastructure), because of its importance and consequences for the ultradian dynamic behaviour of cells. Fractal geometry in intracellular macromolecular assemblies suggests that chaotic dynamics occur during their organization. Non‐linear interactions in and between spatial and temporal domains and over wide ranges of scales underlie the emergent properties of complex biological systems.

On the fractal nature of cytoplasm

On the basis of a quantification of the fractal dimension, D, in micrographs of cytoskeleton components or microtrabecular lattice, we propose that the cellular cytoplasm can be described as a percolation cluster, a sort of ‘random fractal’. Our hypothesis deals with: (i) the existence of the percolation threshold — a remarkable property of percolation processes; and (ii) the reactivity increase — when enzymes, or targets, and substrates, or effectors, coexist in the same topological dimension.

Coherent and robust modulation of a metabolic network by cytoskeletal organization and dynamics

In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the models steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

Abstract The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.

Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behavior in glucose-limited chemostat cultures

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates (D) and respiro-fermentative metabolism at high D. We have studied the onset of ethanol production and cell cycle behavior in glucose-limited chemostat cultures of the wild type S. cerevisiae strain CEN.PK122 (WT) and isogenic mutants, snf1 (cat1) and snf4 (cat3) defective in proteins involved in catabolite derepression and the mutant in glucose repression mig1 (cat4). The triggering of fermentative metabolism was dependent upon catabolite repression properties of yeast and was coincident with a significant decrease of G1 length. WT cells of the strain CEN.PK122 displayed respiratory metabolism up to a D of 0.2 h-1 and exhibited longer G1 lengths than the snf1 and snf4 mutants that started fermenting after a D of 0.1 and 0.15 h-1, respectively. The catabolite derepression mutant snf4 showed a significant decrease in the duration of G1 with respect to the WT. An increase of 300% to 400% in the expression of CDC28 (CDC28-lacZ) with a noticeable shortening in G1 to values lower than approximately 150 min, was detected in the transformed wild type CEN.SC13-9B in glucose-limited chemostat cultures. The expression of CDC28-lacZ was analyzed in the wild type and isogenic mutant strains growing at maximal rate on glucose or in the presence of ethanol or glycerol. Two- to three-fold lower expression of the CDC28-lacZ fusion gene was detected in the snf1 or snf4 disruptants with respect to the WT and mig1 strains in the presence of all carbon sources. This effect was further shown to be growth rate-dependent exhibiting apparently, a threshold effect in the expression of the fusion gene with respect to the length of G1, similar to that shown in chemostat cultures. At the onset of fermentation, the control of the glycolytic flux was highly distributed between the uptake, hexokinase, and phosphofructokinase steps. Particularly interesting was the fact that the snf1 mutant exhibited the lowest fluxes of ethanol production, the highest of respiration and correspondingly, the branch to the tricarboxylic acid cycle was significantly rate-controling of glycolysis.

The onset of fermentative metabolism in continuous cultures depends on the catabolite repression properties of saccharomyces cerevisiae

Abstract In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates ( D ) and respirofermentative metabolism at high D . We hypothesized that the onset of fermentative metabolism is related with the catabolite repression or glucose repression effect. To test this hypothesis, we have investigated the physiological behavior in glucose-limited continuous cultures of S. cerevisiae strain CEN.PK122 and isogenic mutants, snf1 ( cat1 ) and snf4 ( cat3 ), defective in proteins involved in the release from glucose repression and the mutant in glucose repression mig1 . We analyzed the behavior of the wild type and mutant strains at steady state in chemostat cultures as a function of D . Wild-type cells displayed respiratory metabolism up to a D of 0.2 h −1 . snf1 and snf4 mutants started fermenting after a D of 0.1 and 0.15 h −1 , respectively. The latter behavior was not due to an impairment of respiration since their specific rate of oxygen consumption was similar or even higher than that shown by the wild type. The snf1 strain displayed much lower yields than the wild type and the other mutants in the whole range of D studied. We conclude that the onset of fermentative metabolism in yeast growing in chemostat cultures is related with glucose repression.

A method for quantifying rates of O2 consumption and CO2 production in soil

Considering the soil as a heterogeneous bioreactor, a method is developed that permits a quantitative evaluation of O2 and CO2 levels in the soil atmosphere. Mathematical modeling allowed us to quantitate the rate at which soil consumes O2 or produces CO2 under close-to-field conditions. Values of qCO2 and qO2 ranging between 0.2 and 8 kmol CO2-C ha−1 day−1, and 0.4 to 35 kmol O2 ha−1 day−1, respectively, were measured in a soil recently subjected to agriculture in the El Salado river basin (Buenos Aires, Argentina). The mathematical model discriminates between purely physical (diffusion, advection) and biological (e.g., microbial activity) processes. The gas measurements were sensitive to the crop stage in a soil recently subjected to agriculture. We conclude that qO2 reflects more accurately than qCO2 the contribution of microbial activity to the rate of gas exchanges in soil. Results are discussed in terms of soil biogeochemical processes.

ULTRASENSITIVITY IN (SUPRA)MOLECULARLY ORGANIZED AND CROWDED ENVIRONMENTS

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis‐Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up‐ or downmodulate ultrasensitivity under physico‐chemical conditions resembling those predominant in cells.

Altered topoisomerase activities may be involved in the regulation of DNA supercoiling in aerobic-anaerobic transitions in Escherichia coli

This study uncovers a new mechanism of regulation of DNA supercoiling operativein vivo upon an aerobic-anaerobic transition inEscherichia coli. Exponentially growing aerobic batch cultures were subjected to a shift to anaerobic conditions. The ratio [ATP]/[ADP] remained essentially constant at 8.5 in the aerobic culture and after a transition to anaerobiosis while DNA supercoiling increased noticeably upon anaerobiosis. This result indicated that the mechanism of regulation of DNA supercoiling by the [ATP]/[ADP] ratio was not operative. The increase in DNA supercoiling was followed by a large decrease in the DNA-relaxing activity of topoisomerase I while gyrase activity remained relatively constant. This decrease in the activity of topoisomerase I is likely to be responsible for the increase in DNA supercoiling.

Quantitation of the Effects of Disruption of Catabolite (De)Repression Genes on the Cell Cycle Behavior of Saccharomyces cerevisiae

Abstract. We have studied the effect of disrupting catabolite (de)repression genes SNF1, SNF4, and MIG1 on the cell cycle behavior of the CEN.PK122 wild type (WT) strain of Saccharomyces cerevisiae by flow cytometry in glucose-limited chemostat cultures or batch growth in the presence of different carbon sources. Through a combination of flow cytometry of propidium iodide–stained cells and mathematical modeling we showed that the deletion of the SNF4 gene provoked a decrease in the length of G1 with respect to the WT strain along with a smaller difference in the cell cycle length of parent and daughter cells. snf1 and mig1 mutants exhibited slightly shorter G1 respect to the WT. Additionally, in the mig1 mutant the cell cycle length of parent and daughter cells was slightly altered. The results obtained are in agreement with the view that the SNF4 gene is involved in the regulation of cell cycle in yeast.