Comparative physiological and transcriptional analysis of weak organic acid stress in Bacillus subtilis.

The advent of 'omics' techniques bears significant potential for the assessment of the microbiological stability of foods. This requires the integration of molecular data with their implication for cellular physiology. Here we performed a comparative physiological and transcriptional analysis of Bacillus subtilis stressed with three different weak organic acids: the commonly used food preservatives sorbic- and acetic-acid, plus the well-known uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). The concentration of each compound needed to cause a similar reduction of the growth rate negatively correlated with their membrane solubility, and positively with the concentration of undissociated acid. Intracellular acidification was demonstrated by expressing a pH-sensitive GFP derivative. The largest drop in intracellular pH was observed in CCCP-stressed cells and was accompanied by the transcriptional induction of the general stress response (GSR) and SigM regulon, responses known to be induced by acidification. The GSR was induced by acetate, but not by sorbate in mildly-stressed cells. Microarray analysis further revealed that all three acids activate transcriptional programs normally seen upon nutrient limitation and cause diverse responses indicative of an adaptation of the cell envelope. Based on the responses observed and the utilized pH measurements, the inhibitory effect of sorbic acid seems to be more focused on the cell membrane than that of acetic acid or CCCP.

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 cell's 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.

Quantitative analysis of the modes of growth inhibition by weak organic acids in Saccharomyces cerevisiae.

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 (pH(i)) 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 cell's ability to restore pH(i), was a determinant for growth inhibition. This pH(i) 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 pH(i), 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 pH(i) upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.

Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.

The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using Saccharomyces cerevisiae as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

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.

Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pH(c) in Saccharomyces cerevisiae.

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

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

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.

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 V(max) 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.

In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.

The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pHcyt) and mitochondrial pH (pHmit) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pHcyt of 7.2 and a constant pHmit of 7.5 in cells exponentially growing on glucose. We show that pHcyt and pHmit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pHi decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between pHi and growth.