Gertien J. Smits

Cell wall dynamics in yeast

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism

Intracellular pH and Lipid Metabolism Intracellular pH regulates metabolism by poorly understood mechanisms, but biosensors are likely to be important in this process. Young et al. (p. 1085) took a systems-biology approach in yeast to identify in excess of 200 genes that regulate phospholipid metabolism. They found that the signaling lipid, phosphatidic acid, appeared to act as a cytosolic biosensor via the pH-dependent binding of protein effectors to phosphatidic acid. This pH-dependent mechanism directly affects gene expression and is involved in a pathway in which nutrient availability regulates phospholipid metabolism to control production of membranes. Lipid signaling in yeast is regulated by intracellular pH. Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

Development of the Pacemaker Tissues of the Heart

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues.

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal conditions.

Intracellular pH is a tightly controlled signal in yeast

BACKGROUND Nearly all processes in living cells are pH dependent, which is why intracellular pH (pH(i)) is a tightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pH(i) responds to extracellular conditions such as the availability of nutrients. This raises the question of how pH(i) dynamics affect cellular function. SCOPE OF REVIEW We discuss the control of pH(i,) and the regulation of processes by pH(i), focusing on the model organism Saccharomyces cerevisiae. We aim to dissect the effects of pH(i) on various aspects of cell physiology, which are often intertwined. Our goal is to provide a broad overview of how pH(i) is controlled in yeast, and how pH(i) in turn controls physiology, in the context of both general cellular functioning as well as of cellular decision making upon changes in the cells environment. MAJOR CONCLUSIONS Besides a better understanding of the regulation of pH(i), evidence for a signaling role of pH(i) is accumulating. We conclude that pH(i) responds to nutritional cues and relays this information to alter cellular make-up and physiology. The physicochemical properties of pH allow the signal to be fast, and affect multiple regulatory levels simultaneously. GENERAL SIGNIFICANCE The mechanisms for regulation of processes by pH(i) are tightly linked to the molecules that are part of all living cells, and the biophysical properties of the signal are universal amongst all living organisms, and similar types of regulation are suggested in mammals. Therefore, dynamic control of cellular decision making by pH(i) is therefore likely a general trait. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Genome-wide analysis of yeast stress survival and tolerance acquisition to analyze the central trade-off between growth rate and cellular robustness

A genome-wide analysis of the acquisition of stress cross-tolerance shows that reduction of growth rate is an important determinant of severe stress survival. Cellular functions important for the coupling of growth rate to stress resistance are identified, as are those required for cross-tolerance acquisition independent of growth rate reduction.

Quantitative Analysis of the Modes of Growth Inhibition by Weak Organic Acids in Saccharomyces cerevisiae

ABSTRACT Weak organic acids are naturally occurring compounds that are commercially used as preservatives in the food and beverage industries. They extend the shelf life of food products by inhibiting microbial growth. There are a number of theories that explain the antifungal properties of these weak acids, but the exact mechanism is still unknown. We set out to quantitatively determine the contributions of various mechanisms of antifungal activity of these weak acids, as well as the mechanisms that yeast uses to counteract their effects. We analyzed the effects of four weak organic acids differing in lipophilicity (sorbic, benzoic, propionic, and acetic acids) on growth and intracellular pH (pHi) in Saccharomyces cerevisiae. Although lipophilicity of the acids correlated with the rate of acidification of the cytosol, our data confirmed that not initial acidification, but rather the cells ability to restore pHi, was a determinant for growth inhibition. This pHi recovery in turn depended on the nature of the organic anion. We identified long-term acidification as the major cause of growth inhibition under acetic acid stress. Restoration of pHi, and consequently growth rate, in the presence of this weak acid required the full activity of the plasma membrane ATPase Pma1p. Surprisingly, the proposed anion export pump Pdr12p was shown to play an important role in the ability of yeast cells to restore the pHi upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.

Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pHc in Saccharomyces cerevisiae

BackgroundBecause protonation affects the properties of almost all molecules in cells, cytosolic pH (pHc) is usually assumed to be constant. In the model organism yeast, however, pHc changes in response to the presence of nutrients and varies during growth. Since small changes in pHc can lead to major changes in metabolism, signal transduction, and phenotype, we decided to analyze pHc control.ResultsIntroducing a pH-sensitive reporter protein into the yeast deletion collection allowed quantitative genome-wide analysis of pHc in live, growing yeast cultures. pHc is robust towards gene deletion; no single gene mutation led to a pHc of more than 0.3 units lower than that of wild type. Correct pHc control required not only vacuolar proton pumps, but also strongly relied on mitochondrial function. Additionally, we identified a striking relationship between pHc and growth rate. Careful dissection of cause and consequence revealed that pHc quantitatively controls growth rate. Detailed analysis of the genetic basis of this control revealed that the adequate signaling of pHc depended on inositol polyphosphates, a set of relatively unknown signaling molecules with exquisitely pH sensitive properties.ConclusionsWhile pHc is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter. The coupling of pHc to growth rate is even more robust to genetic alteration. Changes in pHc control cell division rate in yeast, possibly as a signal. Such a signaling role of pHc is probable, and may be central in development and tumorigenesis.

Transcriptome Analysis of Sorbic Acid-Stressed Bacillus subtilis Reveals a Nutrient Limitation Response and Indicates Plasma Membrane Remodeling

The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts, and molds. We have used genome-wide transcriptional profiling of Bacillus subtilis cells during mild sorbic acid stress to reveal the growth-inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid-stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong derepression of the CcpA, CodY, and Fur regulon and the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response. The fatty acid biosynthesis (fab) genes and BkdR-regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity toward the fab inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of B. subtilis to sorbic acid stress.

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate. Growth at increased temperature (38 °C versus 30 °C) resulted in a strongly increased glycolytic flux, accompanied by a switch from respiration to a partially fermentative metabolism. We observed an increased flux through all enzymes, ranging from 5- to 10-fold. We quantified the contributions of direct temperature effects on enzyme activities, the gene expression cascade and shifts in the metabolic network, to the increased flux through each enzyme. To do this we adapted flux regulation analysis. We show that the direct effect of temperature on enzyme kinetics can be included as a separate term. Together with hierarchical regulation and metabolic regulation, this term explains the total flux change between two steady states. Surprisingly, the effect of the cultivation temperature on enzyme catalytic capacity, both directly through the Arrhenius effect and indirectly through adapted gene expression, is only a moderate contribution to the increased glycolytic flux for most enzymes. The changes in flux are therefore largely caused by changes in the interaction of the enzymes with substrates, products, and effectors.